Bacillus subtilis

FEBS Lett, 1990 Nov 26, 275(1-2), 61 - 4
Reversible thermal unfolding of Bacillus subtilis levansucrase is modulated by Fe3+ and Ca2+; Chambert R et al.; The equilibrium transition curves for thermal unfolding of levansucrase were established at several pH values . At pH 7 and within the temperature range of bacterial growth, the unfolded form is predominant . However, under such conditions, refolding is promoted by the only addition of Ca2+ or Fe3+ . We propose that the tertiary structure flexibility of levansucrase plays a key role in its secretion process.

FEBS Lett, 1990 Nov 26, 275(1-2), 107 - 10
Detoxification of the macrolide toxin brefeldin A by Bacillus subtilis; Kneusel RE et al.; The macrolide toxin brefeldin A is a determinant of Alternaria leaf blight disease in safflower, which causes severe economic losses worldwide . Soilborne bacteria, classified as Bacillus subtilis spp., were isolated and shown to readily metabolize brefeldin A in laboratory culture to one major product . This product was identified by high resolution 2D 1H NMR and FAB mass spectroscopies as the acid resulting from hydrolysis of the macrolide ring in brefeldin A . In contrast to brefeldin A, the acid completely lacked phytotoxic activity in the standard leaf bioassay . Detoxification of brefeldin A by the lactonase activity from Bacillus subtilis may be exploited in the future to introduce resistance to Alternaria leaf blight in safflower.

Biochim Biophys Acta, 1990 Nov 15, 1041(2), 207 - 15
Low temperature EPR and MCD studies on cytochrome b-558 of the Bacillus subtilis succinate: quinone oxidoreductase indicate bis-histidine coordination of the heme iron; Friden H et al.; Bacillus subtilis cytochrome b-558 was expressed in high amounts in Escherichia coli, solubilized from membranes with detergent and purified free from other hemoproteins . The cytochrome possibly contains two heme groups . To determine the axial ligands to the low-spin heme and the heme rhombicity, the cytochrome was analyzed using low-temperature electron paramagnetic resonance (EPR) and magnetic circular dichroism (MCD) spectroscopy . The combined results exclude bis-methionine, bis-lysine and histidine-methionine coordination . Bis-histidine coordination of the heme(s) with a near perpendicular orientation of the imidazole planes is strongly suggested by the highly axial low-spin EPR signals and the intense near infrared MCD spectrum (delta epsilon = 380 M-1.cm-1 at 4.2 K and 5 T) of the charge-transfer band at 1600 nm.

Biochem J, 1990 Nov 15, 272(1), 93 - 7
Identification of autodigestion target sites in Bacillus subtilis neutral proteinase; van den Burg B et al.; Autocatalytic degradation of purified Bacillus subtilis neutral proteinase was examined under various conditions . At elevated temperatures, under non-inhibitory conditions, mature protein was rapidly degraded, but no accumulation of specific breakdown products occurred . However, by incubating purified neutral proteinase on ice during extended periods of time, specific peptides accumulated . These peptides were analysed by SDS/PAGE and Western blotting, and the N-terminal sequences were determined for the four major peptides, which had sizes of 30, 22, 20 and 15 kDa . Sequence data identified five fission sites in the neutral proteinase, three of which were identical with autodigestion target sites in thermolysin, a thermostable neutral proteinase . Comparison of the identified fission sites of the B . subtilis neutral proteinase with the known substrate-specificity of the enzyme indicated that they were in agreement, showing a preference for the generation of fissions at the N-terminal side of large hydrophobic residues, such as leucine, isoleucine and methionine . These results suggest a high degree of similarity in the three-dimensional structures of B . subtilis neutral proteinase and thermolysin.

J Biol Chem, 1990 Nov 15, 265(32), 20000 - 6
Isolation and phosphorylation of the Bacillus subtilis degS and degU gene products; Mukai K et al.; The Bacillus subtilis sacU locus consists of two genes, degS and degU, which positively regulate the synthesis of several extracellular enzymes including the neutral and alkaline proteases . Both the DegS and DegU proteins have been purified from overproducing Escherichia coli strains harboring degS or degU gene-carrying plasmids, and the following results were obtained . DegS was autophosphorylated in the presence of {gamma-32P}ATP, and transferred the phosphoryl group to DegU . The transfer reaction was rapid in contrast to the autophosphorylation reaction . The phosphoryl groups incorporated into DegS and DegU were released at their own specific rates, the latter being twice faster than the former . The linkage between DegS and the phosphoryl moiety was unstable at acidic pH, whereas reverse was the case for the linkage between DegU and its phosphoryl group, suggesting that His and Asp are involved in the formation of DegS-phosphate and DegU-phosphate, respectively . Deletion of degS resulted in the reduced expression of the exocellular alkaline protease gene, aprE . These results suggest that phosphorylation of DegS by its own kinase activity and subsequent transfer of the phosphoryl group to DegU play a role in the activation of the aprE gene.

Biochim Biophys Acta, 1990 Nov 9, 1036(2), 101 - 6
Studies on the biosynthesis of iturin, an antibiotic of Bacillus subtilis, and a lipopeptide containing beta-hydroxy fatty acids; Besson F et al.; The biosynthesis of iturin, an antibiotic containing a beta-amino fatty acid, was studied by incubating Bacillus subtilis in the presence of various 14C-labelled precursors . Sodium acetate or palmitic acid were incorporated into the beta-amino acids of iturin . Among the alpha-amino acids (asparagine, glutamine, serine, proline and tyrosine) in the peptidic part of iturin, asparagine appears to be the best precursor . In the presence of sodium {14C}acetate or {14C}asparagine, there was a synthesis of radioactive compound (compound X) before the synthesis of radioactive iturin . Compound X contained asparagine and/or aspartic acid, glutamine and/or glutamic acid and beta-hydroxy fatty acids.

Curr Genet, 1990 Nov, 18(4), 287 - 91
Cloning and sequencing of a human cDNA coding for a multifunctional polypeptide of the purine pathway by complementation of the ade2-101 mutant in Saccharomyces cerevisiae; Minet M et al.; A HeLa cell cDNA library on a yeast expression vector was used to complement auxotrophic markers of Saccharomyces cerevisiae . Clones complementing the ade2-101 mutation harbor a 1.5 kb poly(A)+ tailed insert with a 425 amino acid open reading frame hybridizing with two human mRNAs of 1.5 kb and 3.1 kb . Its 5' half is homologous to Bacillus subtilis SAICAR synthetase (E.C.6.3.2.6.) and its 3' terminal half corresponds to the catalytic subunit of Escherichia coli and B . subtilis AIR carboxylase (E.C.4.1.1.21) . In agreement with these homologies, pADE2H1 clones complement both ade1 and ade2 mutants of S . cerevisiae, as was also recently reported for a 3.1 kb cDNA isolated from human hepatocytes.

Mikrobiologiia, 1990 Nov-Dec, 59(6), 1046 - 9
{Intraspecific and interspecific transmission of plasmid and chromosomal DNA during natural transformation in Bacillus subtilis and Escherichia coli}; Kosovich PV et al.; The transmission of plasmids from Escherichia coli to Bacillus subtilis cells was studied in the course of natural transformation in a mixed liquid culture of the two bacteria . The frequency of the transmission was 2-3 times lower than between B . subtilis cells under the same conditions.

Mol Microbiol, 1990 Nov, 4(11), 1801 - 6
Cascades of sigma factors revisited; Stragier P et al.; Programmed gene expression during the process of endospore formation in Bacillus subtilis is governed by the successive appearance of five developmental sigma factors . These sigma factors are encoded by genes in which mutations arrest sporulation at a defined stage . These genes are turned on sequentially and depend for their own transcription on the activity of a previously synthesized sigma factor . Superimposed on the regulation of synthesis of the sigma factors are post-transcriptional control mechanisms that couple the activation of the developmental sigma factors to the course of sporulation . Here we review evidence indicating that these developmental transcription factors comprise a regulatory cascade in the order sigma H----sigma F----sigma E----sigma G----sigma K in which the activity of each sigma factor depends on the action of the preceding sigma factor in the cascade.

J Gen Microbiol, 1990 Nov, 136 ( Pt 11), 2267 - 73
A bacteriolytic muramidase from the basidiomycete Schizophyllum commune; Grant WD et al.; The basidiomycete Schizophyllum commune produces an extracellular bacteriolytic enzyme when grown on heat-killed cells of Bacillus subtilis as sole C, N and P source . The enzyme catalyses the dissolution of isolated B . subtilis cell walls at an optimum pH of 3.2-3.4, releasing muramyl reducing groups, which indicates that it is a muramidase . Although low levels of enzyme activity are present when the fungus is grown in the absence of bacteria, full enzyme production appears to be induced by bacterial cells and repressed by glucose . Whole bacteria are not lysed by the enzyme at pH 3.3, but are rendered osmotically fragile, and lyse when the pH is raised to 7 or higher . The muramidase is effective against several Gram-positive bacteria but did not lyse any of the Gram-negative species tested.

J Gen Microbiol, 1990 Nov, 136 ( Pt 11), 2209 - 16
Cloning, sequencing and genetic mapping of a Bacillus subtilis cell wall hydrolase gene; Kuroda A et al.; We have cloned DNA fragments from Bacillus subtilis 168S into Escherichia coli, which produced a lytic zone on an agar medium containing B . subtilis cell wall . Sequencing of the fragments showed the presence of an open reading frame (ORF) which encodes a polypeptide of 272 amino acids with a molecular mass of 29919 Da . The deduced amino acid sequence showed considerable homology with that of the cell wall hydrolase gene of Bacillus sp . (Potvin, C., Leclerc, D., Tremblay, G., Asselin, A . & Bellemare, G . (1988) . Molecular and General Genetics 214, 241-248) . Accordingly, the gene was designated cwlA, for cell wall lysis . The N-terminal amino acid sequence of cwlA gene product prepared from a E . coli clone was AIKVVKNLVSKSKYGLKCPN, which is consistent with that of the deduced sequence starting from Ala at second position from the initiation codon of the cwlA gene . A presumed sigma A promoter and a rho-independent terminator were found upstream and downstream of the ORF, respectively . A chloramphenicol-resistance determinant integrated into the ORF was mapped by PBS1 transduction, which indicated the gene sequence dnaE-aroD-cwlA.

Izv Akad Nauk SSSR Biol, 1990 Nov-Dec, (6), 928 - 31
{The combined action of N-methyl-N'-nitro-N-nitrosoguanidine and UV light on Bacillus subtilis}; Lotareva OV; The mutagenic interaction between ultraviolet irradiation and the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine was studied in repair-competent and excision-deficient strains of Bacillus subtilis . Pre-exposure to low doses of MNNG with following treatment by low and intermediate doses of UV light increase the resistance of Bac . subtilis to UV radiation (antagonistic effect) . Probably pre-exposition with MNNG leads to induction of enzymes reparation, UV damages being controlled with adaptive response genes.

Genetics, 1990 Nov, 126(3), 487 - 96
Genetic analysis of fusion recombinants in Bacillus subtilis: function of the recE gene; Ftouhi N et al.; Bacillus subtilis protoplast fusion allows the study of the genetic recombination of an entire procaryotic genome . Protoplasts from bacterial strains marked genetically by chromosomal mutations were fused using polyethylene glycol and the regenerated cells analyzed . Recombinants represent 19.3% of heterozygotic cells; they are haploids . Individual characterization of clones show a unique particular phenotype in each colony suggesting that recombination takes place immediately after fusion, probably before the first cellular division . Recombination occurs in the whole chromosome; in one-third of the cases both reciprocal recombinants could be shown in the colony . The genetic interval that includes the chromosome replication origin shows the highest recombination level . Our results suggest that the RecE protein accounts for most of the fused protoplast recombination; however, some "replication origin-specific" recombination events were independent of the recE gene product.

Proc Natl Acad Sci U S A, 1990 Nov, 87(22), 8726 - 30
The mtr locus is a two-gene operon required for transcription attenuation in the trp operon of Bacillus subtilis; Gollnick P et al.; We have cloned and characterized the mtr operon of Bacillus subtilis . This operon encodes a presumed RNA-binding regulatory protein that is required for attenuation control of the trp operon . We have shown that the mtr operon consists of two structural genes, mtrA and mtrB, predicted to encode 22-kDa and 8-kDa polypeptides, respectively . MtrB shows homology with RegA, an RNA-binding regulatory protein of bacteriophage T4 . The lesions in several mtr mutants were localized to mtrB or the putative mtr promoter . Several mtrB alleles were dominant to mtr+, suggesting that the regulatory factor is a multimeric protein . The in vivo action of the mtrA and mtrB gene products was analyzed in an E . coli strain containing a trpE-lacZ gene fusion under control of the B . subtilis trp promoter/attenuator region . Both MtrA and MtrB were necessary for regulation of beta-galactosidase production.

J Bone Joint Surg Br, 1990 Nov, 72(6), 1036 - 7
Dose-dependent reduction of bone inductive properties by ethylene oxide; Aspenberg P et al.; Sterilisation of demineralised bone matrix with ethylene oxide has been claimed to destroy the ability of bone matrix to induce new bone formation on intramuscular implantation . Other workers have routinely used ethylene oxide sterilised bone matrix for assays in rodents without detrimental effects . We studied the effects of various lengths of exposure to ethylene oxide gas, and found that bone induction properties are destroyed in a dose-dependent manner . After a short exposure, bone induction properties were moderately diminished . However, this short ethylene oxide treatment did not kill Bacillus subtilis spores . A sterilisation procedure that killed these spores rendered the implants incapable of bone-induction.

J Bacteriol, 1990 Nov, 172(11), 6380 - 5
Bacillus subtilis mutants with alterations in ribosomal protein S4; Henkin TM et al.; Two mutants with different alterations in the electrophoretic mobility of ribosomal protein S4 were isolated as spore-plus revertants of a streptomycin-resistant, spore-minus strain of Bacillus subtilis . The mutations causing the S4 alterations, designated rpsD1 and rpsD2, were located between the argGH and aroG genes, at 263 degrees on the B . subtilis chromosome, distant from the major ribosomal protein gene cluster at 12 degrees . The mutant rpsD alleles were isolated by hybridization using a wild-type rpsD probe, and their DNA sequences were determined . The two mutants contained alterations at the same position within the S4-coding sequence, in a region containing a 12-bp tandem duplication; the rpsD1 allele corresponded to an additional copy of this repeated segment, resulting in the insertion of four amino acids, whereas the rpsD2 allele corresponded to deletion of one copy of this segment, resulting in the loss of four amino acids . The effects of these mutations, alone and in combination with streptomycin resistance mutations, on growth, sporulation, and streptomycin resistance were analyzed.

J Bacteriol, 1990 Nov, 172(11), 6282 - 90
Complementarity of Bacillus subtilis 16S rRNA with sites of antibiotic-dependent ribosome stalling in cat and erm leaders; Rogers EJ et al.; Inducible cat and erm genes are regulated by translational attenuation . In this regulatory model, gene activation results from chloramphenicol- or erythromycin-dependent stalling of a ribosome at a precise site in the leader region of cat or erm transcripts . The stalled ribosome is believed to destabilize a downstream region of RNA secondary structure that sequesters the ribosome-binding site for the cat or erm coding sequence . Here we show that the ribosome stall sites in cat and erm leader mRNAs, designated crb and erb, respectively, are largely complementary to an internal sequence in 16S rRNA of Bacillus subtilis . A tetracycline resistance gene that is likely regulated by translational attenuation also contains a sequence in its leader mRNA, trb, which is complementary to a sequence in 16S rRNA that overlaps with the crb and erb complements . An in vivo assay is described which is designed to test whether 16S rRNA of a translating ribosome can interact with the crb sequence in mRNA in an inducer-dependent reaction . The assay compares the growth rate of cells expressing crb-86 with the growth rate of cells lacking crb-86 in the presence of subinhibitory levels of inducers of cat-86, chloramphenicol, fluorothiamphenicol, amicetin, or erythromycin . Under these conditions, crb-86 retarded growth . Deletion of the crb-86 sequence, insertion of ochre mutations into crb-86, or synonymous codon changes in crb-86 that decreased its complementarity with 16S rRNA all eliminated from detection inducer-dependent growth retardation . Lincomycin, a ribosomally targeted antibiotic that is not an inducer of cat-86, failed to selectively retard the growth of cells expressing crb-86 . We suggest that cat-86 inducers enable the crb-86 sequence in mRNA to base pair with 16S rRNA of translating ribosome . When the base pairing is extensive, as with crb-86, ribosomes become transiently trapped on crb and are temporarily withdrawn from protein synthesis to the extent that growth rate declines . Site-specific positioning of an antibiotic-stalled ribosome is a hallmark of the translational attenuation model . The proposed rRNA-mRNA interaction may precisely position the ribosome on the stall site and perhaps contributes to stabilizing the ribosome leader mRNA complex.

Mol Microbiol, 1990 Nov, 4(11), 1881 - 9
The importance of the 5'-region in regulating the stability of sdh mRNA in Bacillus subtilis; Melin L et al.; The decay of the polycistronic Bacillus subtilis sdh mRNA was analysed using probes specific for each of the component cistrons, sdhC, sdhA and sdhB . In exponentially growing cells, the entire sdh mRNA seems to decay with an 'all or nothing' mechanism and with a uniform half-life of 2-3 min for all cistrons . In stationary-phase cells, the half-life of the 5'-part had dropped to about 0.6 min whereas that of the 3'-part was about 1.2 min . Decay of sdh mRNA was also measured in exponentially growing cells containing a 'down-mutation' in the ribosomal binding site preceding sdhC which decreases the expression of sdhC by about 90% . The mutation has a moderate effect on expression of the downstream cistron sdhA . In this mutant, the half-life of the 5'-part of sdh mRNA was about 0.5 min (i.e . the same as in stationary phase wild-type cells) and the half-life of the 3'-part about 1.3 min . Also, analysis of the decay of an sdh-cat fusion transcript revealed that the sdh (5') part decayed more rapidly than the cat part and this difference was more pronounced in stationary-phase cells compared to exponentially growing cells . The results of these experiments demonstrate the importance of the 5'-segment of sdh mRNA in controlling the stability of the transcript under different growth conditions.

Proc Natl Acad Sci U S A, 1990 Nov, 87(21), 8355 - 9
Changes in the stability of specific mRNA species in response to growth stage in Bacillus subtilis; Resnekov O et al.; In this study we compared the cellular concentrations and stability of the mRNA transcribed from the aprE (subtilisin) gene (a gene preferentially expressed in stationary growth phase) with those of a vegetative mRNA, succinate dehydrogenase (SDH) mRNA . The subtilisin transcript was shown to be at least 3 times more stable in early stationary phase than it is 2 hr further into stationary phase . When cells were shifted from maximum expression of the subtilisin transcript in stationary phase to physiological conditions, which allowed for the resumption of vegetative growth, the cellular concentration of the subtilisin mRNA decreased rapidly . We conclude that mRNA degradation is one of the means by which the cellular concentrations of the SDH and subtilisin transcripts are adjusted in response to growth stage.

J Bacteriol, 1990 Nov, 172(11), 6372 - 9
Cloning and analysis of the Bacillus subtilis rpsD gene, encoding ribosomal protein S4; Grundy FJ et al.; The rpsD gene, encoding ribosomal protein S4, was isolated from Bacillus subtilis by hybridization with oligonucleotide probes derived from the S4 amino-terminal protein sequence . Sequence analysis of the cloned DNA indicated that rpsD is likely to be monocistronic, in contrast to Escherichia coli rpsD, which is located in the alpha operon and is the translational regulator for alpha operon ribosomal protein gene expression in E . coli . The cloned gene was shown to map at position 263 degrees on the B . subtilis chromosome, at the position to which mutations conferring alterations in the electrophoretic mobility of protein S4 were localized . A promoter was identified upstream of the rpsD coding sequence; initiation of transcription at this promoter would result in a transcript containing a leader region 180 bases in length . Immediately downstream of the rpsD coding region were two sequences resembling transcriptional terminators . An open reading frame homologous to tyrosyl-tRNA synthetase (tyrS) genes was identified downstream of rpsD but in the opposite orientation . The leader region of rpsD mRNA is predicted to have extensive secondary structure, resembling a region of B . subtilis 16S rRNA where S4 is likely to bind; similar mRNA features have been found to be important in ribosomal gene regulation in E . coli . These results provide the first steps toward analysis of the regulation of rpsD gene expression in B . subtilis.

Gene, 1990 Oct 30, 95(1), 137 - 41
Efficiency of transcriptional terminators in Bacillus subtilis; Hess GF et al.; To promote more efficient synthesis of heterologous gene products in a Bacillus subtilis host, we have developed a system for rapidly testing the effect of a putative terminator on in vivo gene expression . Terminator structures from the Bacillus amyloliquefaciens amyE gene, the Bacillus licheniformis penP gene, the B . subtilis bglS gene, and the Bacillus thuringiensis cry gene were subcloned and inserted into a vector in such a way as to disrupt expression of the cat-86 gene . Comparisons are made between gene expression levels and the stabilities of the respective stem-loop structures.

FEBS Lett, 1990 Oct 29, 273(1-2), 75 - 8
Complementation of the protein transport defect of an Escherichia coli secY mutant (secY24) by Bacillus subtilis secY homologue; Nakamura K et al.; Bacillus subtilis SecY homologue shares 41.3% homology with that of E . coli and remarkably higher homologous regions (more than 80%) are present in the four cytoplasmic regions {(1990) J . Biochem . 107, 603-607} . Based on the formation of the mature form of OmpA in E . coli, we have shown that the protein transport defect of the E . coli secY mutant (secY24) is complemented by the gene product from the B . subtilis secY homologue, which is expressed under the lac promoter control . However, B . subtilis SecY could not restore growth of the E . coli mutant at nonpermissive temperature.

J Biol Chem, 1990 Oct 25, 265(30), 18581 - 9
The glucose permease of Bacillus subtilis is a single polypeptide chain that functions to energize the sucrose permease; Sutrina SL et al.; Biochemical, immunological, and sequence analyses demonstrated that the glucose permease of Bacillus subtilis, the glucose-specific Enzyme II of the phosphoenolpyruvate-dependent phosphotransferase system, is a single polypeptide chain with a C-terminal Enzyme III-like domain . A flexible hydrophilic linker, similar in length and amino acid composition to linkers previously identified in other regulatory or sensory transducing proteins, functions to tether the Enzyme IIIGlc-like domain of the protein to the membrane-embedded Enzyme IIGlc . Evidence is presented demonstrating that the Enzyme IIIGlc-like domain of the glucose permease plays a dual role and functions in the transport and phosphorylation of both glucose and sucrose . The sucrose permease appears to lack a sucrose-specific Enzyme III-like domain or a separate, soluble IIIScr protein . Enzyme IIScr was capable of utilizing the IIIGlc-like domain of the glucose permease regardless of whether the IIIGlc polypeptide was provided as a purified, soluble protein, as a membrane-bound protein within the same membrane as Enzyme IIScr, or as a membrane-bound protein within membrane fragments different from those bearing Enzyme IIScr . These observations suggest that the IIIGlc-like domain is an autonomous structural unit that assumes a conformation independent of the hydrophobic, N-terminal intramembranal domain of Enzyme IIGlc . Preferential uptake and phosphorylation of glucose over sucrose has been demonstrated by both in vivo transport studies and in vitro phosphorylation assays . Addition of the purified IIIGlc-like domain strongly stimulated the phosphorylation of sucrose, but not that of glucose, in phosphorylation assays that contained the two sugars simultaneously . The results suggest that the preferential uptake of glucose over sucrose is determined by competition of the corresponding sugar-specific permeases for the common P approximately IIIGlc/Scr domain.

J Mol Biol, 1990 Oct 20, 215(4), 559 - 66
RNA dependence of the bacteriophage phi 29 DNA packaging ATPase; Grimes S et al.; The activity of the DNA packaging adenosine triphosphatase (ATPase) of the Bacillus subtilis bacteriophage phi 29 is dependent upon prohead RNA . The 174 nucleotide viral-encoded RNA is positioned on the head-tail connector at the portal vertex of the phi 29 precursor shell (prohead) . Here, the RNA interacts with the ATP-binding gene 16 product (gp16) to constitute the DNA-packaging ATPase and initiate DNA packaging in vitro . Both the prohead connector (gene 10 product, gp10) and gp16 may utilize an RNA recognition motif characteristic of a number of RNA-associated proteins, and the binding of gp16 by proheads shields the prohead RNA from RNase A . The ATPase activity of gp16 is stimulated fourfold by RNA and tenfold by proheads with RNA . RNA is needed continuously for the gp16/RNA ATPase activity and is essential for the gp16/prohead ATPase activity . The prohead, with its connector, RNA and associated gp16 in an assembly-regulated configuration, hydrolyzes ATP and drives phi 29 DNA translocation.

J Mol Biol, 1990 Oct 5, 215(3), 359 - 72
Novel mutations that alter the regulation of sporulation in Bacillus subtilis . Evidence that phosphorylation of regulatory protein SpoOA controls the initiation of sporulation; Olmedo G et al.; Sporulation in Bacillus subtilis is a complex developmental process that occurs in response to nutrient deprivation . To identify components of the mechanism that allows cells to monitor their nutritional status and to understand how this sensory information is transduced into a signal to activate specific sporulation genes, we have isolated mutants that are able to sporulate efficiently under nutritional conditions that strongly inhibit sporulation in wild-type bacteria, a phenotype we refer to as Coi (control of initiation) . Four coi mutations were found to be within the coding sequence of spoOA, a gene in which null mutations prevent the initiation of sporulation and a gene whose product shares a domain of homology with phosphorylation-activated proteins that play signal transduction roles in bacteria . All four of the spoOA mutations were within this conserved domain and in close proximity to the presumptive phosphoacceptor site . The wild-type and one of the mutant SpoOA proteins were purified and shown to be competent to accept phosphoryl groups from a phosphohistidine within a bacterial signal transduction kinase (CheA) . The mutant SpoOA protein exhibited enhanced phosphoacceptor activity compared with the wild-type . This property of the mutant protein, together with additional genetic information, supports a model for regulation of sporulation initiation by control of the phosphorylation level of SpoOA.

Eur J Biochem, 1990 Oct 5, 193(1), 61 - 7
Purification and characterization of an exo-1,4-beta-galactanase from a strain of Bacillus subtilis; Nakano H et al.; An arabinogalactan 4-beta-D-galactanohydrolase was purified to a homogeneous state from the culture filtrate of a strain of Bacillus subtilis . The enzyme have a molecular mass of 36 kDa and an isoelectric point of pH 7.9 . The enzyme is most active at around pH 6.5-7 and at 60 degrees C, and is stable between pH 6-10 and below 55 degrees C . Hg2+ and Cu2+ inhibit the activity . The enzyme hydrolyze soybean arabinogalactan which contains beta-1,4-galactosidic linkages in its main chain structure, but not other polysaccharides with beta-1,3-galactosidic linkages . The hydrolysis products from soybean arabinogalactan are predominantly galactobiose with a small amount of galactotetraose . The enzyme is an exo-enzyme and the ability to transfer galactobiose to other galactobiose molecules is indicated by the formation of galactotetraose.

J Bacteriol, 1990 Oct, 172(10), 5575 - 85
Similar organization of the sigB and spoIIA operons encoding alternate sigma factors of Bacillus subtilis RNA polymerase; Kalman S et al.; Bacillus subtilis sigma-B is an alternate sigma factor implicated in controlling stationary-phase gene expression . We characterized the genetic organization and regulation of the region containing the sigma-B structural gene (sigB) to learn which metabolic signals and protein factors govern sigma-B function . sigB lay in an operon with four open reading frames (orfs) in the order orfV-orfW-sigB-orfX, and lacZ gene fusions showed that all four frames were translated in vivo . Experiments with primer extension, S1 nuclease mapping, and lacZ transcriptional fusions found that sigB operon transcription initiated early in stationary phase from a site 32 nucleotides upstream of orfV and terminated 34 nucleotides downstream of orfX . Fusion expression was abolished in a strain carrying an in-frame deletion in sigB, suggesting that sigma-B positively regulated its own synthesis, and deletions in the sigB promoter region showed that sequences identical to the sigma-B-dependent ctc promoter were essential for promoter activity . Fusion expression was greatly enhanced in a strain carrying an insertion mutation in orfX, suggesting that the 22-kilodalton (kDa) orfX product was a negative effector of sigma-B expression or activity . Notably, the genetic organization of the sigB operon was strikingly similar to that of the B . subtilis spoIIA operon, which has the gene order spoIIAA-spoIIAB-spoIIAC, with spoIIAC encoding the sporulation-essential sigma-F . The predicted sequence of the 12-kDa orfV product was 32% identical to that of the 13-kDa SpoIIAA protein, and the 18-kDa orfW product was 27% identical to the 16-kDa SpoIIAB protein . On the basis of this clear evolutionary conservation, we speculate these protein pairs regulate their respective sigma factors by a similar molecular mechanism and that the spoIIA and sigB operons might control divergent branches of stationary-phase gene expression.

Biochimie, 1990 Oct, 72(10), 725 - 34
Structure and nucleotide sequence of the Bacillus subtilis phenylalanyl-tRNA synthetase genes; Brakhage AA et al.; The nucleotide sequence of the Bacillus subtilis pheST genes coding for the 2 subunits of phenylalanyl-tRNA synthetase has been determined . The pheS gene corresponds to 1029 bp and the pheT gene to 2412 bp . The encoded proteins have Mrs of 38,947 (343 amino acids, alpha-subunit) and 87,916 (804 amino acids, beta-subunit), respectively . The genes are adjacent on the chromosome separated by only 15 nucleotides . The pheT gene is immediately followed by a hairpin structure typical of a rho-independent transcription terminator . S1 nuclease mapping and primer extension analysis of pheST mRNA revealed a major start of transcription 318 nucleotides upstream of the pheS gene, and 6 nucleotides downstream of a E sigma 43 promoter consensus sequence . Within the 5'-noncoding region several potential secondary structures have been noted.

Mol Microbiol, 1990 Oct, 4(10), 1785 - 8
Direction of DNA entry in competent cells of Bacillus subtilis; Vagner V et al.; Direction of DNA entry in Bacillus subtilis competent cells was studied using molecules in which only one of the two strands was radioactively labelled . The label was either distributed homogeneously or was localized in a small region of the strand, in the centre or at one of the ends . Regardless of the distribution and the position of the label, similar amounts of radioactivity were taken up by the cells exposed to the labelled molecules . This suggests that DNA enters B . subtilis either by two different uptake systems having opposite polarities, or by a single non-polar system.

Protein Eng, 1990 Oct, 4(1), 99 - 104
Contribution of the C-terminal amino acid to the stability of Bacillus subtilis neutral protease; Eijsink VG et al.; The role of the C-terminal Leu300 in maintaining thermal stability of the neutral protease of Bacillus subtilis was investigated . From model building studies based on the three-dimensional structure of thermolysin, the neutral protease of B . thermoproteolyticus, it was concluded that this residue is located in a hydrophobic pocket composed of residues located in the C-terminal and the middle domain . To test the hypothesis that Leu300, by contributing to a stabilizing interaction between these domains, is important for enzyme stability, several neutral protease mutants were constructed and characterized . The thermostability of the enzyme was lowered by deleting Leu300 or by replacing this residue by a smaller (Ala), a polar (Asn) or a sterically unfavourable (Ile) amino acid . Thermostability was increased upon replacing Leu300 by Phe . These results are in agreement with model-building studies . The effects on thermostability observed after mutating the corresponding Val318 in the thermostable neutral protease of B.stearothermophilus were less pronounced.

Appl Environ Microbiol, 1990 Oct, 56(10), 3191 - 203
Remobilization of toxic heavy metals adsorbed to bacterial wall-clay composites; Flemming CA et al.; Significant quantities of Ag(I), Cu(II), and Cr(III) were bound to isolated Bacillus subtilis 168 walls, Escherichia coli K-12 envelopes, kaolinite and smectite clays, and the corresponding organic material-clay aggregates (1:1, wt/wt) . These sorbed metals were leached with HNO3, Ca(NO3)2, EDTA, fulvic acid, and lysozyme at several concentrations over 48 h at room temperature . The remobilization of the sorbed metals depended on the physical properties of the organic and clay surfaces and on the character and concentration of the leaching agents . In general, the order of remobilization of metals was Cr much less than Ag less than Cu . Cr was very stable in the wall, clay, and composite systems; pH 3.0, 500 microM EDTA, 120-ppm {mg liter-1} fulvic acid, and 160-ppm Ca remobilized less than 32% (wt/wt) of sorbed Cr . Ag (45 to 87%) and Cu (up to 100%) were readily removed by these agents . Although each leaching agent was effective at mobilizing certain metals, elevated Ca or acidic pH produced the greatest overall mobility . The organic chelators were less effective . Lysozyme digestion of Bacillus walls remobilized Cu from walls and Cu-wall-kaolinite composites, but Ag, Cr, and smectite partially inhibited enzyme activity, and the metals remained insoluble . The extent of metal remobilization was not always dependent on increasing concentrations of leaching agents; for example, Ag mobility decreased with some clays and some composites treated with high fulvic acid, EDTA, and lysozyme concentrations . Sometimes the organic material-clay composites reacted in a manner distinctly different from that of their individual counterparts; e.g., 25% less Cu was remobilized from wall- and envelope-smectite composites than from walls, envelopes, or smectite individually in 500 microM EDTA . Alternatively, treatment with 160-ppm Ca removed 1.5 to 10 times more Ag from envelope-kaolinite composites than from the individual components . The particle size of the deposited metal may account for some of the stability changes; those metals that formed large, compact aggregates (Cr and Ag) as seen by transmission electron microscopy were less likely to be remobilized . In summary, it is apparent that remobilization of toxic heavy metals in sediments, soils, and the vadose zone is a complicated issue . Predictions based on single inorganic or organic component systems are too simplistic.

Anal Biochem, 1990 Oct, 190(1), 116 - 9
Fluorometric in vivo determination of Bacillus subtilis and phage 2C DNA; Daxhelet GA et al.; The validity of in vivo fluorometric assays was ascertained for phage and bacterial DNA measurements . The following parameters were determined by this simple technique . The DNA content of dividing cells of Bacillus subtilis 168/2 was 2.65 times higher than in resting cells . Assuming that resting cells harbor 1 genomic equivalent, its Mr was estimated to be 4.4 x 10(9) Da . A polymerization rate during growth of 788,000 bp min-1/cell is accounted for by a multifork replication mechanism . Both phage and host DNA could be measured accurately during the lytic cycle . Phage 2C DNA synthesis proceeded at a linear rate of 5.2 genome equivalents min-1.

FEMS Microbiol Lett, 1990 Oct, 60(1-2), 55 - 8
Promoters of major Escherichia coli heat shock genes seem non-functional in Bacillus subtilis; Wetzstein M et al.; To find out whether Escherichia coli heat shock promoters are recognized in Bacillus subtilis, the regulatory regions including the heat shock promoters of the main heat shock genes dnaKJ, lon, groES, and htpG were fused to the indicator genes lacZ and cat . Whereas all transcriptional fusions were expressed in E . coli at low temperature and transient increases of beta-galactosidase activity could be measured at the inducing temperature, no enzymatic activity was found in B . subtilis . This indicates that E . coli heat shock promoters are nonfunctional in B . subtilis.

FEMS Microbiol Lett, 1990 Oct, 60(1-2), 173 - 6
A pleiotropic mutation affecting purine metabolism in Bacillus subtilis; Maznitsa II et al.; A pleiotropic mutation (cpm) which is localized in the vicinity of the spoOA gene is described in Bacillus subtilis . The mutation inhibits spore formation, rendering bacteria auxotrophic for adenine and tyrosine, enhances sensitivity to antibiotics, decreases cell motility, inhibits the ability to grow on pentoses and to maintain bacteriophage multiplication, induces severalfold the activities of alkaline proteinase and alpha-amylase . At the same time, the cpm mutant starts to excrete inosine into the growth medium . This excretion most probably is explained by a 50-fold increase in the activity of inosine monophosphate: 5'-nucleotidase and a 10-fold decrease in the activity of purine nucleoside phosphorylase . The inosine production and Ade- phenotype of the cpm mutant is not accompanied by the change in the activity of succinyl adenosine monophosphate synthetase . The nature of the mutation is discussed.

Am J Infect Control, 1990 Oct, 18(5), 307 - 15
A new perspective of microbial survival and dissemination in a prospectively contaminated air-fluidized bed model; Winters WD; A major concern of users of air-fluidized beds has been the possibility that such beds might be a source of microbial contamination . The purpose of this series of prospective, controlled experiments was to measure quantitatively the dissemination and survival of Bacillus subtilis, a species of bacterium that forms desiccation-resistant spores, as it was associated with circulating and clumped microbeads after challenge in an air-fluidized bed operating under decontamination conditions of heating at 48 degrees C and microbead agitation by an air flow of 100% at 110 cu ft/min . Microbead samples collected after B . subtilis challenge from predesignated depths and locations within the air-fluidized bed at 0.25, 1, 2, 4, 24, and 48 hours were assayed for colony-forming units (CFU) of challenge bacteria by end point dilution and streak-plate assays . Results of three experiments with an average challenge of 1110 CFU of B . subtilis per gram of microbeads indicated that no more than 0.46 CFU of B . subtilis per gram of circulating microbeads and 0.78 CFU per gram of clumped microbeads were detected after 24 to 48 hours at decontamination conditions . Few microbes were detected during three control (sterile water) challenges of the air-fluidized bed operating at 33 degrees C with 95% to 100% air flow . This study has demonstrated that an air-fluidized bed operating at the described decontamination conditions for 24 to 48 hours caused significant decreases, averaging a thousandfold per gram, in levels of microbead-associated challenge bacteria.(ABSTRACT TRUNCATED AT 250 WORDS)

Artif Organs, 1990 Oct, 14(5), 361 - 8
Chlorine dioxide gas sterilization of oxygenators in an industrial scale sterilizer: a successful model; Jeng DK et al.; Artificial organ oxygenators, with large surface areas and complicated structures, were sterilized using chlorine dioxide gas in an industrial scale sterilizer . Bacillus subtilis var . niger ATCC 9372 biological indicators (BI) (10(6) spores/BI) planted in the artificial organs were reproducibly sterilized in a designed cycle schedule with a 30-min dwell time with a chlorine dioxide gas concentration of approximately 30 mg/L in 80 to 85% relative humidity at 30 degrees C . The D value (time required for 90% spore inactivation under specific conditions) was estimated to be 4.4 min . Chlorine dioxide was not detected after post-sterilization aeration . The intravenous median lethal dose (LD50) of chlorine dioxide derivatives, chlorite and chlorate, in rats was found to be 112.8 and 2,228.6 mg/kg, respectively . In an immediate hypersensitivity test, chlorine dioxide gas-treated ovalbumin and bovine serum albumin, unlike ethylene oxide gas-treated proteins, did not cause sterilant-specific IgE-mediated hypersensitivity reactions in rats . Results of an Ames mutagenicity test on chlorine dioxide and on the extracts of the chlorine dioxide gas-exposed oxygenators were negative.

J Bacteriol, 1990 Oct, 172(10), 5892 - 900
Replication genes of plasmid pE194-cop and repF: transcripts and encoded proteins; Byeon WH et al.; In vivo transcription of the replication region of plasmid pE194 yeidls two classes of mRNAs that encode Cop and RepF proteins, respectively . These transcripts are oriented 5' to 3' exclusively in the clockwise direction on the standard map . The cop region contains an open reading frame capable of encoding a 55-amino-acid protein that was demonstrated electrophoretically as a 6-kilodalton product synthesized in Bacillus subtilis minicells and chemically by N-terminal sequencing of a 116-kilodalton fusion protein with Escherichia coli beta-galactosidase . Four transcripts derived from the repF region were found, of which the longest, approximately 720 nucleotides, had the length, orientation, and transcription start site necessary to code for the full-length RepF protein (216 amino acid residues), deduced from the DNA sequence . The 5' ends of the shorter repF transcripts fall within the repF open reading frame . We propose that (i) cop specifies a protein rather than an RNA countertranscript, (ii) the Cop protein functions as a negative-acting element in pE194 replication by regulating synthesis of both RepF and of itself, and (iii) increased plasmid copy number can be explained in terms of cop region mutations that either reduce the intrinsic activity of Cop protein or the rate of its synthesis.

Electrophoresis, 1990 Oct, 11(10), 795 - 801
Analysis of the conformational transitions of proteins by temperature-gradient gel electrophoresis; Birmes A et al.; Temperature-gradient gel electrophoresis (TGGE) is a technique for studying the structural transitions of nucleic acids and proteins . A temperature gradient is formed in a horizontal slab gel perpendicular to the direction of the electric field . Whereas the principle of the TGGE method has previously been applied to proteins, we describe in this report the systematic optimization of TGGE as a routine technique for the quantitative analysis of conformational transitions in proteins . Using alpha-amylase as an example we show the kinds of results which may be obtained from such measurements . Buffers suitable for use in gel electrophoresis were analyzed with respect to the dependence of their pH value upon temperature . The correct pH range for TGGE of a given protein is determined by electrophoretic titration curves . The protein bands are detected by silver and/or activity staining . The thermal denaturation of alpha-amylase from Aspergillus oryzae showed a discontinuous transition into the denatured conformation, which exhibited much slower electrophoretic mobility . The discontinuity is due to an irreversible denaturation process under the gel conditions . The transition temperature was measured as a function of several parameters, e.g., the concentration of Ca(+)+, dithiotreithol, urea and the pH value . The structural transition of alpha-amylase is accompanied by a loss of enzymatic activity as determined by activity staining or by an activity assay carried out in solution . The structural transitions of two other alpha-amylases from Bacillus subtilis and Bacillus licheniformis were also studied . The results show that the TGGE method is simple to perform and allows the analysis of conformational transitions of proteins in a wide variety of conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

FEMS Microbiol Lett, 1990 Oct, 60(1-2), 41 - 6
A new defective phage containing a randomly selected 8 kilobase-pairs fragment of host chromosomal DNA inducible in a strain of Bacillus natto; Tsutsumi Y et al.; A new defective phage, designated PBND8, was induced in Bacillus natto strain IAM1207 with bleomycin and mitomycin C . PBND8 particles contained a randomly selected 8 kilobase-pairs (kbp) fragment of the host chromosomal DNA . Electron microscopy showed that PBND8 has a small head with a complex tail structure like PBSX, a defective phage of Bacillus subtilis 168 . The PBND8 head, however, is clearly smaller than that of PBSX which contains 13-kbp fragments of the host chromosomal DNA . SDS-polyacrylamide gel electrophoretic analysis revealed that the structural proteins of PBND8 are distinct from those of PBSX and PBSY (PBSZ) of B . subtilis W23 . PBND8 exhibited a bacteriocin-like killing activity to the other Bacillus cells.

J Bacteriol, 1990 Oct, 172(10), 5724 - 31
Characterization of a multienzyme complex derived from a Bacillus subtilis DNA-membrane extract that synthesizes RNA and DNA precursors; Laffan JJ et al.; The activity of a variety of enzymes involved in the synthesis of RNA and DNA precursors was found to copurify with initiation of DNA replication activity . These enzymes included ribo- and deoxyribonucleoside kinases, kinases for their phosphorylated intermediates, and ribonucleoside diphosphate reductase . This precursor-synthesizing complex is part of a Bacillus subtilis DNA-membrane extract originally shown to contain all of the enzymes and template necessary for initiation of DNA replication (J . Laffan and W . Firshein, J . Bacteriol . 169:2819-2827, 1987) . Although the complex incorporated deoxyribonucleoside triphosphates into DNA, deoxyribonucleosides were incorporated even faster, suggesting catalytic facilitation . Both ribonucleosides and deoxyribonucleosides were found by thin-layer chromatography separation to be converted by the complex into their mono-, di-, and triphosphate derivatives . Ribonucleotides were incorporated into DNA via the action of ribonucleoside diphosphate reductase . Some regulatory mechanisms of the kinase system may also be retained by the complex . Electron microscope studies revealed that the precursor-synthesizing-initiation subcomplex is contained within a particulate fraction consisting of different-size vesicles resembling liposomes and that these particles may be structurally important in maintaining the synthetic activity of the subcomplex.

J Bacteriol, 1990 Oct, 172(10), 5631 - 6
Localization of a second SigH promoter in the Bacillus subtilis sigA operon and regulation of dnaE expression by the promoter; Qi FX et al.; The presence of a second SigH promoter in the sigA operon of Bacillus subtilis was demonstrated by use of a promoter probe plasmid, a sigH deletion mutant, primer extension studies, and in vitro transcription with E sigma H holoenzyme . Both SigH promoters were expressed at low levels even during the growth phase but were expressed at higher levels during the early stationary phase . Expression from the upstream SigH promoter allowed the expression of both dnaE and sigA genes; however, expression from the downstream SigH promoter, which was located in the ribosome-binding site of the dnaE gene, resulted only in the expression of the sigA gene, since the truncated dnaE ribosome-binding site could not be used for initiating translation . Thus, promoter switching during the early stationary phase resulted not only in expression from SigH promoters but also in differential expression of the genes in the sigA operon.

Agric Biol Chem, 1990 Oct, 54(10), 2585 - 91
Structure of di-O-alpha-maltosyl cyclodextrins produced from alpha-maltosylfluoride and cyclodextrins; Yoshimura Y et al.; The structures of di-O-alpha-maltosyl beta-cyclodextrins ((G2)2-beta-CDs), which were produced from alpha-maltosylfluoride (alpha-G2F) and cyclodextrin (CD) by the transfer action of debranching enzymes, were examined by the enzymic method using Bacillus subtilis saccharifying alpha-amylase (BSA) . (G2)2-beta-CD was converted to (G1)2-beta-CD by treatment with glucoamylase before the examination . BSA completely hydrolyzed (G1)2-beta-CD to produce glucose, 6(3)-O-alpha-glucosylmaltotriose, and 6(3),6(5)-di-O-alpha-glucosyl maltopentaose . (G2)2-beta-CD was the mixture of 6A,6C-di-O-alpha-maltosyl beta-CD and 6A,6D-di-O-alpha-maltosyl beta-CD . The ratio of A,C/A,D in (G2)2-beta-CD synthesized with Pseudomonas isoamylase and Aerobacter pullulanase were 40:60-45:55 and 30:70, respectively . The content of 6A,6C-di-O-alpha-maltosyl gamma-CD in (G2)2-gamma-CD synthesized by isoamylase was about 35%.

J Ind Microbiol, 1990 Oct, 6(2), 91 - 100
Regulation of biosynthesis of bacilysin by Bacillus subtilis; Ozcengiz G et al.; Production of the dipeptide antibiotic bacilysin by Bacillus subtilis 168 was growth associated and showed no evidence of repression by glucose or sucrose . Carbohydrates other than glucose and sucrose yielded lower specific titers of bacilysin . Bacilysin production in three such carbon sources (maltose, xylose, ribose) was delayed until growth slowed down . Ammonium salts were poor for bacilysin production when used as the sole nitrogen source . When added to the standard medium containing glutamate, they suppressed antibiotic production . Aspartate was slightly better than glutamate for antibiotic production as sole nitrogen source . No other nitrogen source tested, including inorganic, organic or complex, approached the activity of glutamate or aspartate . When added to glutamate, casamino acids, phenylalanine and alanine (a substrate of bacilysin synthetase) suppressed bacilysin production while stimulating growth . Phosphate provided for optimum growth and production at 7.5 mM and both processes were inhibited at higher concentrations . Ferric citrate stimulated growth and inhibited bacilysin production, the effects being due to both the iron and the citrate components . Elimination of ferric citrate stimulated production as did increasing the concentration of Mn to its optimum concentration of 6.6 x 10(-4) M.

Gene, 1990 Sep 28, 94(1), 45 - 51
Site-directed mutagenesis of the YCDTDS amino acid motif of the phi 29 DNA polymerase; Bernad A et al.; The Bacillus subtilis phage phi 29 DNA polymerase, involved in protein-primed viral DNA replication, contains amino acid consensus sequences common to other alpha-like DNA polymerases . Using site-directed mutagenesis we have studied the functional significance of the most conserved C-terminal segment mainly represented by the YCDTDS motif . A series of single point mutants has been constructed and the corresponding proteins have been overproduced and characterized . Measurements, on crude fractions, of the activity of the mutant proteins in the formation of the protein p3-dAMP initiation complex and in an in situ DNA polymerase assay, indicate that the YCDTDS domain is involved both in initiation and in elongation reactions.

Gene, 1990 Sep 28, 94(1), 125 - 8
CUG as a mutant start codon for cat-86 and xylE in Bacillus subtilis; Ambulos NP Jr et al.; The cat-86 gene specifies chloramphenicol acetyltransferase (CAT) . The cat-86 start codon is UUG, although related genes have AUG as the start codon . Changing the start codon to AUG increased expression of cat-86 by 36% in Bacillus subtilis . Changing the start codon to GUG and CUG decreased expression to 65% and 30%, respectively, of the level obtained when AUG was the start codon . CUG has not been previously shown to function as a start codon in B . subtilis . N-terminal sequencing of purified CAT protein specified by the CUG mutant, revealed that CUG was indeed the start codon and specified methionine . The gene xylE, which specifies catechol 2,3-dioxygenase, has AUG as its start codon . Changing the start codon for xylE to CUG decreased expression by 98% . However, when the ribosome-binding site sequence for xylE was optimized and the spacing between it and the start codon was increased to 8 nucleotides, xylE activity increased to 13% of the activity observed for AUG . CUG did not function efficiently as a start codon for cat-86 in Escherichia coli . These data suggest conditions under which CUG can function, with modest efficiency, as a start codon in B . subtilis.

Gene, 1990 Sep 28, 94(1), 121 - 4
Identification of a low-copy-number mutation within the pUB110 replicon and its effect on plasmid stability in Bacillus subtilis; Leonhardt H; A mutation (cop1) within the minimal replicon of plasmid pUB110, reducing its copy number from 48 +/- 2 to 11 +/- 2 in a Bacillus subtilis host, was isolated . This mutation is a G----T transversion within the translation control region of the replication initiation gene (repU) . The cop1 mutation, when present in the recombinant plasmid, increases both its structural and segregational stability.

Gene, 1990 Sep 28, 94(1), 115 - 9
Analysis of the tet gene of plasmid pCIS7 isolated from Bacillus subtilis; Ives CL et al.; We have previously shown that plasmid pCIS7, which contains 11.5 kb of Bacillus subtilis DNA isolated from a tetracycline-sensitive (TcS) strain, confers Tc resistance when integrated and amplified in the chromosome of TcS B . subtilis 168trpC2 {Ives and Bott, J . Bacteriol . 171 (1989) 1801-1810} . Here, we report that the number of integrated plasmid sequences required to confer Tc resistance is greater than the 20 copies seen with increasing chloramphenicol selection and, by dot-blot analysis, exceeds 100 copies per cell . The amplification is accompanied by a corresponding increase in mRNA encoding the tet gene . The tet gene sequence of pCIS7 has been compared to B . subtilis tetGSY908 {Sakaguchi et al., Biochim . Biophys . Acta . 94 (1988) 49-57} and other Gram-positive tet genes . The tet gene of pCIS7 is a member of the class L TcR determinants, and probably confers Tc resistance by increasing the efflux of Tc from the bacterial cell.

Nucleic Acids Res, 1990 Sep 25, 18(18), 5473 - 80
Bacillus subtilis ada operon encodes two DNA alkyltransferases; Morohoshi F et al.; By prophage transformation and subcloning, we have obtained Bacillus subtilis DNA fragments that could complement the hypersensitivity of ada (adaptive response deficient) mutants to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) . The nucleotide sequence contained two open reading frames that were assigned to the genes adaA and adaB, encoding methylphosphotriester-DNA methyltransferase and O6-methylguanine-DNA methyltransferase, respectively . These two genes overlap by 11 bp and comprise a small operon . The 1.6 Kb transcripts derived from the operon were detected in ada+ cells cultured in the presence of MNNG but not in control ada+ cells . From analysis of the syntheses of DNA alkyltransferases in the ada mutant cells harboring the plasmid carrying the complete or partial fragment, we conclude that the adaA gene product functions as a transcriptional activator of the ada operon, while the adaB gene product specializes in repair of mutagenic O6-methylguanine residues . Comparison with Escherichia coli ada operon showed that the two genes correspond to portions of the E . coli ada gene, implicating gene fusion or splitting as the origin of the difference in the organizations of the genes.

Biochemistry, 1990 Sep 18, 29(37), 8872 - 8
Monofunctional chorismate mutase from Bacillus subtilis: kinetic and 13C NMR studies on the interactions of the enzyme with its ligands; Gray JV et al.; The interaction of the monofunctional chorismate mutase from Bacillus subtilis with chorismate and prephenate has been studied kinetically and by NMR spectroscopy with 13C specifically labeled substrates . Prephenate dominates the population of enzyme-bound species, and the "off" rate constant (approximately 60 s-1) obtained from line-broadening experiments is close to the value of kcat for chorismate (50 s-1) determined kinetically . The calculated "on" rate constant for prephenate (8 x 10(5) M-1 s-1) is similar to the value of kcat/Km for chorismate (5 x 10(5) M-1 s-1) . The kinetic parameters of the Bacillus mutase are remarkably insensitive to pH over a wide range and display no solvent isotope effect . These results suggest that the enzyme-catalyzed reaction may be encounter controlled (slowed from the diffusion limit by some feature of the enzyme's active site) and that kcat for chorismate is determined by the product off rate . There is now no evidence to suggest that the skeletal rearrangement on the enzyme surface occurs by a pathway other than a pericyclic process.

FEBS Lett, 1990 Sep 17, 270(1-2), 41 - 4
Purification and site-specific immobilization of genetically engineered glucose dehydrogenase on thiopropyl-Sepharose; Persson M et al.; The gene encoding glucose dehydrogenase (EC 1.1.1.47) from Bacillus subtilis was inserted in a plasmid 1.0 kb downstream from a lac promoter, resulting in a 70-fold higher production of the enzyme when expressed in Escherichia coli . A glucose dehydrogenase mutant containing a cysteine residue at position 44 could also be expressed at the same high level . This single cysteine residue was used as an 'affinity tag' to simplify the purification procedure as well as for site-specific immobilization of glucose dehydrogenase on Thiopropyl-Sepharose . This enzyme was purified to homogeneity with a final recovery of 65% and a specific activity of 240 U/mg . The oriented immobilization resulted in increased thermal stability.

FEBS Lett, 1990 Sep 17, 270(1-2), 147 - 51
Bacillus subtilis holo-cytochrome c-550 can be synthesised in aerobic Escherichia coli; von Wachenfeldt C et al.; Bacillus subtilis membrane-bound holo-cytochrome c-550 was found to be expressed from the structural gene cloned on a plasmid vector in aerobically grown Escherichia coli and exhibited normal biochemical properties . This occurs despite the lack of endogenous cytochrome c and suggests that cytochrome c-heme lyase activity is also present in aerobic E . coli . The membrane topology of B . subtilis cytochrome c-550 was studied using fusions to alkaline phosphatase (PhoA) . The results show that the heme domain (at least when fused to PhoA) can be translocated as apo-cytochrome and confirm that the N-terminal part of the cytochrome functions as both export signal and membrane anchor for the C-terminal heme domain . A model for the organisation of B . subtilis cytochrome c-550 in the cytoplasmic membrane is presented.

J Biol Chem, 1990 Sep 5, 265(25), 14947 - 55
Aspartokinase II from Bacillus subtilis is degraded in response to nutrient limitation; Graves LM et al.; Aspartokinase II from Bacillus subtilis was shown by immunochemical methods to be regulated by degradation in response to starvation of cells for various nutrients . Ammonium starvation induced the fastest aspartokinase II decline (t1/2 = 65 min), followed by amino acid starvation (t1/2 = 80 min) and glucose limitation (t1/2 = 120 min) . Loss of enzyme activity was closely correlated with the disappearance of the alpha subunit; degradation of the beta subunit was somewhat delayed or slower under some conditions . Pulse-chase experiments demonstrated that aspartokinase II was stable during exponential growth; the synthesis of the enzyme rapidly declined in response to nutrient exhaustion . The degradation of aspartokinase II was interrupted by inhibitors of energy production and protein synthesis but was not changed in a mutant lacking a major intracellular protease . Mutants lacking a normal stringent response displayed only a slight decrease in the rate of aspartokinase II degradation, even though aspartate transcarbamylase was degraded more slowly in the same mutant cells . These results indicate that although energy-dependent degradation of biosynthetic enzymes is a general phenomenon in nutrient-starved B . subtilis cells, the degradation of specific enzymes probably involves different pathways.

J Allergy Clin Immunol, 1990 Sep, 86(3 Pt 1), 393 - 9
ELISA for human IgE antibody to subtilisin A (Alcalase): correlation with RAST and skin test results with occupationally exposed individuals; Sarlo K et al.; An ELISA was developed to detect specific IgE antibody to the Bacillus subtilis-derived proteolytic detergent enzyme, subtilisin A (Alcalase), in sera from exposed detergent workers . Workers in the detergent industry are exposed via inhalation to low levels of the enzyme dust in the presence of detergent dust . Chemically inactivated Alcalase was used as the test antigen . Significant binding of IgE antibody to the immobilized enzyme was detected in the ELISA . The binding of allergic antibody to Alcalase was specifically inhibited in a dose-dependent manner by preincubating sera with 0.1 to 100 micrograms of inactivated Alcalase . Binding of IgE antibody to Alcalase could not be inhibited by two other inactivated bacterial proteases, Savinase and Esperase, derived from different Bacillus species . ELISA and skin test results demonstrated total agreement for 27 of 31 samples (87%), whereas RAST and skin test results demonstrated total agreement for only 24 of 31 samples (77%), indicating that the ELISA was more sensitive than the RAST . We conclude that the ELISA is a sensitive, fast, alternative to the RAST for detection of Alcalase-specific IgE antibody in detergent enzyme-exposed workers.

J Bacteriol, 1990 Sep, 172(9), 5147 - 53
Nutrient-dependent methylation of a membrane-associated protein of Escherichia coli; Young CC et al.; Starvation of a mid-log-phase culture of Escherichia coli B/r for nitrogen, phosphate, or carbon resulted in methylation of a membrane-associated protein of about 43,000 daltons (P-43) in the presence of chloramphenicol and {methyl-3H}methionine . The in vivo methylation reaction occurred with a doubling time of 2 to 5 min and was followed by a slower demethylation process . Addition of the missing nutrient to a starving culture immediately prevented further methylation of P-43 . P-43 methylation is not related to the methylated chemotaxis proteins because P-43 is methylated in response to a different spectrum of nutrients and because P-43 is methylated on lysine residues . The characteristics of P-43 are similar to those of a methylated protein previously described in Bacillus subtilis and B . licheniformis (R . W . Bernlohr, A . L . Saha, C . C . Young, B . R . Toth, and K . J . Golden, J . Bacteriol . 170:4113-4118, 1988; K . J . Golden and R . W . Bernlohr, Mol . Gen . Genet . 220:1-7, 1989) and are consistent with the proposal that methylation of this protein functions in nutrient sensing.

J Bacteriol, 1990 Sep, 172(9), 4861 - 9
Cloning and nucleotide sequences of the Bacillus stearothermophilus neutral protease gene and its transcriptional activator gene; Nishiya Y et al.; Both the neutral protease gene (nprS) and its transcriptional activator gene (nprA) from Bacillus stearothermophilus TELNE were cloned in Bacillus subtilis by using pTB53 as a vector plasmid . The presence of the nprA gene enhanced protease synthesis by about fivefold . The nucleotide sequences of nprS and its flanking regions were determined . nprS was composed of 1,653 base pairs and 551 amino acid residues . A Shine-Dalgarno (SD) sequence was found 9 bases upstream from the translation start site (ATG) . The deduced amino acid sequence was very similar to that of another thermostable neutral protease gene, nprM (M . Kubo and T . Imanaka, J . Gen . Microbiol . 134:1883-1892, 1988) . the amino acid sequence of the extracellular neutral protease NprS was completely identical to that of NprM . By deletion analysis and substitution of the original promoter with a foreign promoter, it was found that the nprA gene existed upstream of nprS . It was also found that a possible target region (palindromic sequence) of the gene product of nprA existed near the promoter sequence of nprS . The nucleotide sequences of nprA and its flanking regions were determined . The DNA sequence revealed only one large open reading frame, composed of 1,218 base pairs (406 amino acids; molecular weight, 49,097) . The SD sequence was found 4 bases upstream from the translation start site (GTG) . A possible promoter sequence (TTGAAG for the -35 region and AATTTT for the -10 region) was also found about 20 bases upstream of the SD sequence . The nprA gene was separated from nprS by a typical terminator sequence . By constructing an in-frame fusion between the lacZ gene and the 5' region of the nprA gene, it was demonstrated that the coding region of nprA was indeed translated in vivo . Three palindromic sequences, which were highly homologous with a possible target region by NprA, were also found in the 5' region of the nprA gene . This suggests that eh expression of nprA is autoregulated . From the time course of the production of NprA-LacZ fusion protein, it was indicated that nprA was expressed in late log phase, whereas nprS was expressed in the stationary phase . The NprA protein had consensus regions homologous to the DNA recognition domains of DNA-binding proteins but showed no sequence homology with any other regulatory proteins for protease production . It is inferred that NprA protein binds to the upstream region of nprS promoter and activates transcription of nprS . A new regulatory mechanism by the nprA-nprS genes is discussed.

EMBO J, 1990 Sep, 9(9), 2905 - 10
Isolation of a dnaA mutant of Bacillus subtilis defective in initiation of replication: amount of DnaA protein determines cells' initiation potential; Moriya S et al.; The dnaA gene is essential for initiation of chromosomal replication in Escherichia coli . A gene homologous with the E . coli dnaA was found in the replication origin region of the Bacillus subtilis chromosome . We have now isolated a temperature sensitive mutant of the B . subtilis dnaA by in vitro mutagenesis of the cloned gene . At a nonpermissive temperature, 49 degrees C, DNA replication stops completely after 60% increase in a rich medium, while cell mass continues to increase exponentially at 2.5 times the rate at 30 degrees C . A ratio of gene frequency between purA (origin marker) and metB (terminus marker) changes gradually from 2.7 at 30 degrees C to 1.0 in 45 min at 49 degrees C, indicating completion of the ongoing replication cycle . Upon the temperature shift down to 30 degrees C after the incubation at 49 degrees C for 60 min, DNA replication resumes without delay, and the purA/metB ratio increases rapidly to 6, i.e . consecutive initiation of more than two rounds of replication . Addition of chloramphenicol at the time of the temperature shift down did not inhibit the increase in the purA/metB ratio, while rifampicin inhibited the re-initiation completely . The mutation is a single base change from C to T in the dnaA gene resulting in an amino acid substitution from Ser to Phe in the DnaA protein . The mutation was responsible for both temperature sensitive growth and the defect in initiation of chromosomal replication . We observed a remarkable correlation between the amount of DnaA protein and the amount of initiation potential accumulated during incubation at the non-permissive temperature.

Mol Biol (Mosk), 1990 Sep-Oct, 24(5), 1381 - 92
{Analysis of the structure of Bacillus brevis neutral proteinase and its biosynthesis in Bacillus subtilis cells}; Kaidalova NV et al.; Amino acid sequence of neutral metalloprotease from Bac . brevis has been compared with that of Bac . amyloloquefaciens, Bac . cereus, Bac . subtilis, Bac . stearothermophilis, Bac . thermoproteolyticus (thermolysine) . A sequence region from N-40 to N-1 with a significant degree of homology allowed to predict the processing site of the propart of Bac . brevis enzyme . The sequence comparison allows to put Bac . brevis enzyme within the evolutionary branch of enzymes, which includes thermolysin and proteases of Bac . cereus and Bac . stearothermophilus . Using automated Edman degradation the N-terminal sequence of Bac . brevis protease has been determined . It does not differ from the sequence predicted from the nucleotide sequence of the gene . It was shown that, when Bac . brevis gene coding for thermostable protease is expressed on a plasmid vector in Bac . subtilis cells at 37 degrees C, enzyme forms possessing low activity are secreted . The enzyme may be significantly activated without an additional cleavage or processing and the activation includes numerous conformation transition states of the protein molecule.

FEMS Microbiol Lett, 1990 Sep 1, 59(1-2), 129 - 32
Temperature-dependent activation of Bacillus brevis metalloprotease expressed in mesophilic Bacillus subtilis; Kostrov SV et al.; When the Bacillus brevis secretory metalloprotease is expressed from the npr gene on a plasmid vector in the mesophile B . subtilis, grown at 37 degrees C, the enzyme was found to be properly processed, but secreted into the culture medium in a low-active conformation . Secreted metalloprotease can by heat-treatment (70 degrees C for 30 min) be converted into fully active enzyme.

Mol Gen Mikrobiol Virusol, 1990 Sep, (9), 29 - 31
{Plasmid and chromosomal gene transfer in Bacillus subtilis and Escherichia coli in natural transformation}; Kosovich PB et al.; The transfer of hDHFR gene by natural transformation between Escherichia coli and Bacillus subtilis cells has been studied, as well as intrageneric gene transfer in Bacillus subtilis . The gene was transferred by natural transformation in bacterial cells and included into the chromosome or the plasmid.

Mol Gen Genet, 1990 Sep, 223(2), 268 - 72
Gene-directed mutagenesis on the chromosome of Bacillus subtilis 168; Itaya M et al.; We have devised a method whereby any mutagenized cloned DNA from Bacillus subtilis can be reinserted at the original site on the B . subtilis chromosome . The procedure depends on the accuracy and high frequency of homologous recombination between the B . subtilis chromosome and the DNA taken up by the cell . The method makes use of two drug resistance selection markers (the chloramphenicol resistance gene and the neomycin resistance gene) and a marker gene which functions as a catalyst . The utility of the method has been demonstrated using leuB and pro of B . subtilis as target gene and catalyst, respectively, and mutations such as leuB::cat, leuB-, and pro::neo constructed in vitro on the cloned DNA fragments . Transformation in sequential steps as (leuB+ pro+)----(leuB::cat pro+)----(leuB- pro::neo)----(leuB- pro+) resulted in a leuB- single mutant without affecting other regions of the B . subtilis chromosome (gene-directed mutagenesis) . We also demonstrate that other single mutations such as metD- and pro-, as well as the double mutation leuB- pro- can be introduced by the same procedure . In principle, true isogenies with multiple mutations can be constructed by the method described in this paper . Furthermore, the procedure should be generally applicable to any organisms in which homologous recombination is proficient.

Mol Gen Genet, 1990 Sep, 223(2), 185 - 91
Novel plasmid marker rescue transformation system for molecular cloning in Bacillus subtilis enabling direct selection of recombinants; Haima P et al.; A versatile plasmid marker rescue transformation system was developed for homology-facilitated cloning in Bacillus subtilis . It is based on the highly efficient host-vector system 6GM15-pHPS9, which allows the direct selection of recombinants by means of beta-galactosidase alpha-complementation . The system offers several advantages over previously described cloning systems: (1) the convenient direct selection of recombinants; (2) the ability to effectively transform B . subtilis competent cells with plasmid monomers, which allows the forced cloning of DNA fragments with high efficiency; (3) the availability of 6 unique target sites, which can be used for direct clone selection, SphI, NdeI, NheI, BamHI, SmaI and EcoRI; and (4) the rapid segregational loss of the helper plasmid from the transformed cells.

Gene, 1990 Sep 1, 93(1), 41 - 7
An improved beta-galactosidase alpha-complementation system for molecular cloning in Bacillus subtilis; Haima P et al.; The recently described beta-galactosidase alpha-complementation system for molecular cloning in Bacillus subtilis {Haima et al., Gene 86 (1990) 63-69}was optimized in several ways . First, the efficiency of translation of the lac Z delta M15 gene was improved . Second, the plasmid-borne lacZ delta M15 gene was segregationally stabilized by integration into the B . subtilis chromosome . Third, a new lacZ alpha complementing cloning vector was constructed, containing more unique target sites . It was shown that large heterologous DNA fragments (up to at least 29 kb) could be cloned with lacZ alpha-complementing vectors carrying the replication functions of the cryptic B . subtilis plasmid pTA1060, and that these inserts were structurally stably maintained for at least 100 generations of growth.

J Bacteriol, 1990 Sep, 172(9), 5482 - 5
The Bacillus subtilis outB gene is highly homologous to an Escherichia coli ntr-like gene; Albertini AM et al.; The Bacillus subtilis outB gene was found to have strong similarities to an Escherichia coli gene complementing ntr-like mutations in Rhodobacter capsulatus . The deduced gene products had 52% identical amino acids (65% similar residues) . The phenotype of strains affected in the OutB function indicates that this B . subtilis gene may be involved in nitrogen utilization.

J Bacteriol, 1990 Sep, 172(9), 5432 - 9
Temporal regulation of the Bacillus subtilis early sporulation gene spo0F; Bai U et al.; The initiation of sporulation in Bacillus subtilis depends on seven genes of the spo0 class . One of these, spo0F, codes for a protein of 14,000 daltons . We studied the regulation of spo0F by using spo0F-lacZ translational fusions and also measured Spo0F protein levels by immunoassays . spo0F-lacZ and Spo0F levels increased as the cells entered the stationary phase, and this effect was repressed by glucose and glutamine . Decoyinine, which lowers GTP levels and allows sporulation in the presence of normally repressing levels of glucose, induced spo0F-lacZ expression and raised Spo0F levels . The expression of spo0F-lacZ was dependent on spo0A, -0B, -0E, -0F, and -0H genes, a spo0H deletion causing the strongest effect . In most respects, the spo0F gene was regulated in a manner similar to that of spoVG . However, the presence of an abrB mutation did not relieve the dependence of spo0F gene expression on spo0A, as it does with spoVG (P . Zuber and R . Losick, J . Bacteriol . 169:2223-2230, 1987).

J Bacteriol, 1990 Sep, 172(9), 5408 - 15
Multiple regulatory sites in the Bacillus subtilis citB promoter region; Fouet A et al.; The aconitase (citB) gene of Bacillus subtilis is repressed during growth in a medium that contains a rapidly metabolizable carbon source and a source of 2-ketoglutarate . It is derepressed when either of these nutrient sources becomes limiting . Repression by rapidly metabolizable carbon sources was shown previously to depend at least in part on a DNA sequence located 67 to 84 base pairs upstream of the start point of citB transcription . In the present work, this region and surrounding DNA were mutagenized to identify more precisely the target for carbon catabolite repression . Mutations in a symmetric sequence located between positions -73 and -59 led to constitutive transcription from the citB promoter in media that normally provoke catabolite repression . By gel mobility shift assays, it was shown that at least one protein in extracts of B . subtilis binds to the symmetric sequence and that DNA of constitutive mutants binds to this protein much less effectively . A second sequence located near position -45 was also implicated in this regulation . A second form of regulation of citB was also investigated . This gene is known to be derepressed when cells are induced to sporulate by exhaustion of a nutrient broth medium or limitation of guanine nucleotide synthesis . The mutations that led to constitutivity with respect to the carbon source had no effect on citB expression in nutrient broth medium, indicating that control by catabolite repression and control by components of nutrient broth (presumably amino acids) act by different mechanisms.

J Bacteriol, 1990 Sep, 172(9), 5011 - 9
Structural alterations in the Bacillus subtilis Spo0A regulatory protein which suppress mutations at several spo0 loci; Spiegelman G et al.; Secondary site mutations that restore sporulation to sporulation-defective spo0F or spo0B deletion mutants were found to reside in the spo0A gene . Sequence analysis of 23 such sof mutants showed that the sof mutations fell into six classes of missense codon changes, primarily in the conserved amino-terminal domain of the response regulator Spo0A protein . Changes were observed in codons 12, 14, 60, 92, and 121 . The residues affected were predominantly located in the potential turn regions at one end of the amino-terminal conserved domain on the same topological face as the active site aspartate residues . The ability of sof mutations to suppress deficiencies in the transmitter kinases, KinA and KinB, of two-component regulatory systems was tested . All of the sof mutations suppressed the sporulation deficiency of kinA mutants but only two classes among five tested suppressed kinB mutations . sof mutants segregated Spo- colonies at high frequency . Five of these Spo- mutants were found to result from mutations in the spo0A locus that reversed the effect of the sof mutatation . One of these was sequenced and found to have the original sof mutation and a new mutation, sos, at codon 105 . The accumulation of sos mutations in sof strains suggested that the sof mutations have a subtle, yet deleterious, effect on the growth of the cell . The results suggested that the sof mutations increase the avidity for or reactivity with transmitter kinases in an allele-specific manner, although in some cases it is possible that the sof mutations obviate the need for phosphorylation to activate the Spo0A protein . An alternative hypothesis is presented in which the sof mutations play the role of bypass mutations for kinases.

J Bacteriol, 1990 Sep, 172(9), 4901 - 8
Molecular cloning, nucleotide sequencing, and expression of the Bacillus subtilis (natto) IAM1212 alpha-amylase gene, which encodes an alpha-amylase structurally similar to but enzymatically distinct from that of B . subtilis 2633; Emori M et al.; An alpha-amylase gene of Bacillus subtilis (natto) IAM1212 was cloned in a lambda EMBL3 bacteriophage vector, and the nucleotide sequence was determined . An open reading frame encoding the alpha-amylase (AMY1212) consists of 1,431 base pairs and contains 477 amino acid residues, which is the same in size as the alpha-amylase (AMY2633) of B . subtilis 2633, an alpha-amylase-hyperproducing strain, and smaller than that of B . subtilis 168, Marburg strain . The amino acid sequence of AMY1212 is different from that of AMY2633 at five residues . Enzymatic properties of these two alpha-amylases were examined by introducing the cloned genes into an alpha-amylase-deficient strain, B . subtilis M15 . It was revealed that products of soluble starch hydrolyzed by AMY1212 are maltose and maltotriose, while those of AMY2633 are glucose and maltose . From the detailed analyses with oligosaccharides as substrates, it was concluded that the difference in hydrolysis products of the two similar alpha-amylases should be ascribed to the different activity hydrolyzing low-molecular-weight substrates, especially maltotriose; AMY1212 slowly hydrolyzes maltotetraose and cannot hydrolyze maltotriose, while AMY2633 efficiently hydrolyzes maltotetraose and maltotriose . Further analyses with chimeric alpha-amylase molecules constructed from the cloned genes revealed that only one amino acid substitution is responsible for the differences in hydrolysis products.

J Bacteriol, 1990 Sep, 172(9), 4758 - 65
Regulation of histidine and proline degradation enzymes by amino acid availability in Bacillus subtilis; Atkinson MR et al.; The first enzymes of the histidine (hut) and proline degradative pathways, histidase and proline oxidase, could not be induced in Bacillus subtilis cells growing in glucose minimal medium containing a mixture of 16 amino acids . Addition of the 16-amino-acid mixture to induced wild-type cells growing in citrate minimal medium repressed histidase synthesis 25- to 250-fold and proline oxidase synthesis 16-fold . A strain containing a transcriptional fusion of the hut promoter to the beta-galactosidase gene was isolated from a library of Tn917-lacZ transpositions . Examination of histidase and beta-galactosidase expression in extracts of a hut-lacZ fusion strain grown in various media showed that induction, catabolite repression, and amino acid repression of the hut operon were mediated at the level of transcription . This result was confirmed by measurement of the steady-state level of hut RNA in cells grown in various media . Since amino acid repression was not defective in B . subtilis mutants deficient in nitrogen regulation of glutamine synthetase and catabolite repression, amino acid repression appears to be mediated by a system that functions independently of these regulatory systems.

Mol Microbiol, 1990 Sep, 4(9), 1419 - 23
Triple post-transcriptional control; Bechhofer DH; The ermC gene confers resistance to MLS antibiotics in a Bacillus subtilis host . Synthesis of the ermC gene product, a ribosomal RNA methylase, is inducible by the addition of subinhibitory concentrations of erythromycin . Regulation of ermC gene expression occurs at the post-transcriptional level in three ways: translational attenuation, translational autoregulation, and messenger RNA stabilization.

Gene, 1990 Sep 1, 93(1), 35 - 40
Temperature-inducible gene expression in Bacillus subtilis mediated by the cI857-encoded repressor of bacteriophage lambda; Breitling R et al.; An efficient system to control the expression of cloned genes in Bacillus subtilis was established by introducing the Escherichia coli bacteriophage lambda cI857 repressor-pR promoter system into this host . A staphylokinase reporter gene (sak42D), which was fused to the lambda pR promoter was constitutively expressed in B . subtilis even when the cI857 gene was present on the same plasmid . S1 nuclease mapping of the transcription start point confirmed that the pR promoter was active in B . subtilis . Constitutive expression under pR-control in B . subtilis was, therefore, likely to result from a lack of repressor formation caused by the inefficiency of cI857 expression signals in the Gram+ host . This lack of repressor synthesis was overcome by fusing the cI857 gene to sak42D transcription and translation signals which have previously been shown to function efficiently in B . subtilis . Plasmids carrying the cI857 gene together with an alpha-amylase-encoding gene (amy) under pR-control mediated temperature-inducible amy expression at 37 degrees C and 42 degrees C . The high repression factor (greater than or equal to 1400) was comparable to the OR efficiencies reported in E . coli.

J Bacteriol, 1990 Sep, 172(9), 5052 - 63
Secretory S complex of Bacillus subtilis: sequence analysis and identity to pyruvate dehydrogenase; Hemila H et al.; We have cloned the operon coding for the Bacillus subtilis S complex, which has been proposed to be a component in protein secretion machinery . A lambda gt10 library of B . subtilis was screened with antiserum directed against the Staphylococcus aureus membrane-bound ribosome protein complex, which is homologous to the B . subtilis S complex . Two positive overlapping lambda clones were sequenced . The S-complex operon, 5 kilobases in size, was shown to contain four open reading frames and three putative promoters, which are located upstream of the first, the third, and the last gene . The four proteins encoded by the operon are 42, 36, 48, and 50 kilodaltons in size . All of these proteins were recognized by antisera separately raised against each protein of the S . aureus membrane-bound ribosome protein and B . subtilis S complexes, thus verifying the S-complex identity of the lambda clones . Sequence analysis revealed that all four proteins of the B . subtilis S complex are homologous to the four subunits of the human pyruvate dehydrogenase (PDH) . Also, the N terminus of the 48-kilodalton protein was found to have 70% amino acid identity with the N-terminal 211 amino acids, determined so far, from the E2 subunit of B . stearothermophilus PDH . Furthermore, chromosomal mapping of the S-complex operon gave a linkage to a marker gene located close to the previously mapped B . subtilis PDH genes . Thus, the S complex is evidently identical to the B . subtilis PDH, which has been shown to contain four subunits with molecular weights very similar to those of the S complex . Therefore, we propose that the S complex is not a primary component of protein secretion.

J Bacteriol, 1990 Sep, 172(9), 4936 - 44
Characterization of chromosomal DNA amplifications with associated tetracycline resistance in Bacillus subtilis; Ives CL et al.; Endogenous chromosomal DNA amplifications with associated tetracycline resistance (Tcr) in Bacillus subtilis were first described by C . R . Wilson and A . E . Morgan (J . Bacteriol . 163:445-453, 1985) . We have confirmed and extended their results, and we show that fusion of protoplasts from Tcs B . subtilis 168 trpC2 with polyethylene glycol and regeneration on medium containing 20 micrograms of tetracycline per ml induces Tcr regenerants that contain amplified DNA . This phenomenon appeared to be recE dependent and requires the addition of polyethylene glycol . Along with three regenerants kindly provided by Wilson and Morgan (RAD1, RAD6, and RAD7), we characterized three strains (CLI20, CLI22, CLI30) isolated in this laboratory . All six contain an amplified region of DNA which was independently cloned on plasmid pCIS7 . Integration of pCIS7 into the wild-type (Tcs) B . subtilis chromosome and amplification of the plasmid sequences generated a Tcr phenotype, even though the DNA on pCIS7 was cloned from Tcs B . subtilis KS162 (Ives and Bott, J . Bacteriol . 171:1801-1810, 1989) . The amplified DNA also showed homology (through hybridization analysis) with pAM alpha 1 delta 1, a gram-positive Tcr plasmid, indicating that B . subtilis normally contains a silent integrated copy of the gene whose amplification confers Tcr . The amplifications were determined to lie between purA and gyrB on the B . subtilis chromosome, and the endpoints were mapped . RAD6 and CLI30 may share the same left-hand endpoint, but the other endpoints are different in each isolate . The amplified DNAs of RAD1, RAD6, CLI20, and CLI30 end near known DNA membrane binding sites . The number of amplified units of DNA was determined through dot blot analysis to do approximately 80 to 100 copies per cell, with corresponding increases in transcription of RAD1, RAD6, CLI20, CLI22, and CLI30.

Appl Microbiol Biotechnol, 1990 Sep, 33(6), 657 - 63
Regulated expression of heterologous genes in Bacillus subtilis using the Tn10 encoded tet regulatory elements; Geissendorfer M et al.; The Escherichia coli-derived tet regulatory elements from Tn10 have been used to construct vectors allowing the regulated, inducible, high-level expression of foreign genes in Bacillus subtilis . While the wild-type tet promoters are inactive in B . subtilis, a synthetic mutant tet sequence with improved promoter consensus sequences and upstream poly A blocks shows activity in B . subtilis . The expression of an indicator cat gene is inducible by sublethal amounts of tetracycline, indicating that the Tet repressor protein and the tet operator sequences are functional . However, the inducibility and maximal expression are not sufficient in this construct . To improve these properties a tet operator sequence was placed between the -35 and -10 boxes of the B . subtilis-derived very strong xyl promoter . In the presence of a tetR gene this construct is about 100-fold inducible and has high promoter strength, but some basal expression . This is avoided by placing a second tet operator downstream resulting in no detectable basal expression at the expense of reduced inducibility . Using the system with a single tet operator inducible expression of glucose dehydrogenase from B . megaterium was obtained at a very high level, and inducible expression of human single-chain urokinase-like plasminogen activator was achieved at the same level as in E . coli . Unlike in E . coli, the product was not degraded up to 4 h after induction in B . subtilis . These results demonstrate that the regulated expression vector described here should be very useful for production of foreign gene products from B . subtilis cultures.

Eur J Biochem, 1990 Aug 28, 192(1), 195 - 200
Purification and properties of phosphoribosyl-diphosphate synthetase from Bacillus subtilis; Arnvig K et al.; Phosphoribosyl-diphosphate (PPRibP) synthetase from Bacillus subtiliis has been purified to near homogeneity from an Escherichia coli delta prs strain bearing the cloned B . subtilis prs gene, encoding PPRibP synthentase, on a plasmid . The Mr of the subunit (34,000) and its amino-terminal amino acid sequence (14 residues) were in complete agreement with expectations from the nucleotide sequence of the prs gene . The Mr of the native enzyme (280,000 +/- 10,000) was consistent with an octameric quaternary structure . No tendency toward multiple states of aggregation of the enzyme was seen . The purified enzyme required Mg2+ and inorganic phosphate for activity; Mn2+ supported only 30% the activity seen with Mg2+ . Michaelis constants for ATP and ribose 5-phosphate (Rib5P) were 0.66 mM and 0.48 mM, respectively . Of several end products tested, only ADP was strongly inhibitory; GDP was a weak inhibitor . ADP inhibition displayed homotropic cooperativity and was enhanced by increasing saturation of the enzyme with ATP . These observations strongly suggest a specific allosteric site for ADP binding . A comparison of physical and kinetic properties of bacterial and mammalian PPRibP synthetases is presented.

Eur J Biochem, 1990 Aug 28, 192(1), 219 - 24
A vitamin-K2-binding factor secreted from Bacillus subtilis; Ikeda H et al.; The synthesis of vitamin K2 by Bacillus subtilis was found to increase in parallel with the number of cells . In the exponential growth phase, most vitamin K2 was found in the bacterial cells . At the end of the growth phase, however, the soluble form of vitamin K2 appeared in the incubation media and amounted to 40% of the total vitamin K2 at the stationary phase . DEAE ion-exchange chromatography of the incubation medium showed a single elution peak corresponding to vitamin K2, suggesting the secretion of vitamin K2 in the form of a soluble complex with a specific acidic binding factor . This factor was partially purified by consecutive DEAE chromatography, and examined by polyacrylamide disc gel electrophoresis . A band stainable with acridine orange, a cationic dye, was found to co-migrate with vitamin K2 . This band was also visualized by periodic acid/Schiff staining . Gel-permeation chromatography showed that the binding factor in its active form has an apparent molecular mass of 100 kDa and is capable of binding exogenous menaquinone but not menadione . Chemical analysis indicated that this binding factor contained about 20% carbohydrate and 80% peptide which consisted mainly of Leu, Glu, Asp and Val . The new acidic glycoconjugate was designated as the vitamin-K2-binding factor.

Biochim Biophys Acta, 1990 Aug 27, 1050(1-3), 80 - 3
Characterization of a 17 kDa protein gene upstream from the small cytoplasmic RNA gene of Bacillus subtilis; Struck JC et al.; The Bacillus subtilis small cytoplasmic RNA (scRNA) is the structural homologue of both the RNA component of the eukaryotic signal recognition particle (SRP) and the Escherichia coli 4.5S RNA, and it can complement the essential function of the latter RNA in vivo . In the course of characterization of the single-copy scRNA gene locus (scr) we identified an open reading frame, termed ORF17, upstream from scr that encodes an acidic 17 kDa protein of unknown function . This analysis involved DNA sequencing, monitoring expression of transcriptional and translational ORF17-cat and ORF17-lacZ fusions, respectively, and purification and sequencing of the ORF17-lacZ fusion protein . Apparently, transcription of ORF17 proceeds into scr . A small portion of the 17 kDa protein shows homology to deoxycytidylate (DCMP) deaminase of bacteriophagphage T2, but no similarity exists to the sequenced SRP-polypeptides or any other known protein sequences.

Nucleic Acids Res, 1990 Aug 25, 18(16), 4651 - 7
The generation of concatemeric plasmid DNA in Bacillus subtilis as a consequence of bacteriophage SPP1 infection; Bravo A et al.; Bacteriophage SPP1 infection of Bacillus subtilis cells bearing plasmids induces the synthesis of multigenome-length plasmid molecules . Two independent pathways can account for this synthesis . In one of those, homology to the phage genome is required, whereas in the other such homology is not a prerequisite . In wild type cells both modes overlap . In dnaB(Ts), at non permissive temperature, or in recE polA strains the main concatemeric plasmid replication mode is the homology-dependent plasmid (hdp) mode . The rate of recombination-dependent concatemeric plasmid DNA synthesis is a consequence of a phage-plasmid interaction which leads to chimeric phage::plasmid DNA . The second mode, which is an homology-independent plasmid (hip) mode seems to be triggered upon the synthesis of a phage encoded product(s) (e.g . inactivation of the exonuclease V enzyme).

J Biol Chem, 1990 Aug 15, 265(23), 13939 - 48
Bacillus subtilis 13-kilodalton cytochrome c-550 encoded by cccA consists of a membrane-anchor and a heme domain; von Wachenfeldt C et al.; Little is known about c-type cytochromes in Gram-positive bacteria in contrast to the wealth of information available on this type of cytochrome in Gram-negative bacteria and in eucaryotes . In the present work, the strictly aerobic bacterium Bacillus subtilis was analyzed for subcellular localization and number of different cytochromes c . In vivo labeling with radioactive 5-aminolevulinic acid, a precursor to heme, showed that the proteins containing covalently bound heme are predominantly found in the membrane fraction . One major membrane-bound cytochrome c of about 15 kDa and with an alpha-band absorption peak in the reduced state at 550 nm was analyzed in more detail . Cytochrome c-550 has the properties of an integral membrane protein . The physiological function of this relatively high redox potential cytochrome is not known . Its structural gene, cccA, was cloned, sequenced, and overexpressed in B . subtilis . The gene maps adjacent to rpoD (sigA) at 223 degrees on the chromosome . The amino acid sequence of cytochrome c-550 as deduced from the DNA sequence consists of 120 residues and contains one heme c binding site (Cys-Ile-Ala-Cys-His) located approximately in the middle of the polypeptide . From the hydropathy distribution and from comparisons to soluble c-type cytochromes of known three-dimensional structure, cytochrome c-550 seemingly consists of two domains; an N-terminal membrane-anchor domain and a C-terminal heme domain . A model for the topography of the cytochrome in the cytoplasmic membrane is suggested in which the N-terminal part spans the membrane in the form of a single segment in an alpha-helical conformation and the C-terminal heme domain is exposed on the extracytoplasmic side of the membrane . Deletion of cccA from the chromosome revealed another membrane-bound cytochrome with absorption maximum at 550 nm in the reduced state . Analysis of cccA deletion mutants demonstrated that the cytochrome c-550 encoded by cccA is not essential for growth of B . subtilis on rich or minimal media.

Biochemistry, 1990 Aug 7, 29(31), 7191 - 200
Sequence-specific 1H NMR resonance assignments of Bacillus subtilis HPr: use of spectra obtained from mutants to resolve spectral overlap; Wittekind M et al.; On the basis of an analysis of two-dimensional 1H NMR spectra, the complete sequence-specific 1H NMR assignments are presented for the phosphocarrier protein HPr from the Gram-positive bacterium Bacillus subtilis . During the assignment procedure, extensive use was made of spectra obtained from point mutants of HPr in order to resolve spectral overlap and to provide verification of assignments . Regions of regular secondary structure were identified by characteristic patterns of sequential backbone proton NOEs and slowly exchanging amide protons . B . subtilis HPr contains four beta-strands that form a single antiparallel beta-sheet and two well-defined alpha-helices . There are two stretches of extended backbone structure, one of which contains the active site His15 . The overall fold of the protein is very similar to that of Escherichia coli HPr determined by NMR studies {Klevit, R . E., & Waygood, E . B . (1986) Biochemistry 25, 7774-7781}.

J Biol Chem, 1990 Aug 5, 265(22), 13206 - 14
Chlamydia trachomatis RNA polymerase major sigma subunit . Sequence and structural comparison of conserved and unique regions with Escherichia coli sigma 70 and Bacillus subtilis sigma 43; Koehler JE et al.; We identified and sequenced the gene for the Chlamydia trachomatis RNA polymerase major sigma subunit . The gene encodes a 66,141-dalton protein (sigma 66), intermediate in size between the major sigma subunits of Escherichia coli (sigma 70) and Bacillus subtilis (sigma 43) . The C . trachomatis sigma 66 subunit had extensive amino acid homology with the sigma 70 and sigma 43 . The sigma subunit regions purportedly involved in core enzyme binding and DNA promoter recognition were also highly conserved, despite the lack of a DNA promoter consensus sequence between E . coli and C . trachomatis promoters and the inability of E . coli holoenzyme to specifically transcribe chlamydial genes . Compared with E . coli sigma 70, there were some major differences in the chlamydial sigma 66 sequence, including a gap of 63 amino acids and an additional 16 amino acids at the carboxyl terminus, which may play some role in modifying the sigma-DNA interaction, such that a promoter sequence unique to C . trachomatis is recognized . Monoclonal antibodies specific for E . coli sigma 70 were used to probe for homologous structures between sigma 70 and sigma 66; only one of seven antibodies bound specifically to sigma 66, suggesting minimal conservation of antigenic sites . The chlamydial sigma 66 was present in elementary bodies and was expressed throughout the developmental cycle, which implied that this gene encodes the major vegetative sigma subunit . Because the ability to study the genetics of C . trachomatis is currently limited, this work provides a tool for more detailed study of chlamydial promoter structure and of coordinate gene expression during the developmental cycle.

J Mol Biol, 1990 Aug 5, 214(3), 657 - 71
Levanase operon of Bacillus subtilis includes a fructose-specific phosphotransferase system regulating the expression of the operon; Martin-Verstraete I et al.; The levanase gene (sacC) of Bacillus subtilis is the distal gene of a fructose-inducible operon containing five genes . The complete nucleotide sequence of this operon was determined . The first four genes levD, levE, levF and levG encode polypeptides that are similar to proteins of the mannose phosphotransferase system of Escherichia coli . The levD and levE gene products are homologous to the N and C-terminal part of the enzyme IIIMan, respectively, whereas the levF and levG gene products have similarities with the enzymes IIMan . Surprisingly, the polypeptides encoded by the levD, levE, levF and levG genes are not involved in mannose uptake, but form a fructose phosphotransferase system in B . subtilis . This transport is dependent on the enzyme I of the phosphotransferase system (PTS) and is abolished by deletion of levF or levG and by mutations in either levD or levE . Four regulatory mutations (sacL) leading to constitutive expression of the lavanase operon were mapped using recombination experiments . Three of them were characterized at the molecular level and were located within levD and levE . The levD and levE gene products that form part of a fructose uptake PTS act as negative regulators of the operon . These two gene products may be involved in a PTS-mediated phosphorylation of a regulator, as in the bgl operon of E . coli.

J Bacteriol, 1990 Aug, 172(8), 4690 - 3
Desensitization of Bacillus subtilis aspartokinase I to allosteric inhibition by meso-diaminopimelate allows aspartokinase I to function in amino acid biosynthesis during exponential growth; Zhang JJ et al.; Strains of Bacillus subtilis deficient in aspartokinases II and III are unable to grow in the absence of lysine, methionine, and threonine, although they have normal levels of aspartokinase I (J.J . Zhang, F.M . Hu, N.Y . Chen, and H . Paulus, J . Bacteriol . 172:701-708, 1990) . Revertants with the ability to grow in the absence of lysine and methionine had an altered aspartokinase I, which was insensitive to feedback inhibition by meso-diaminopimelate . This suggests that inhibition by meso-diaminopimelate limits the ability of aspartokinase I to function in amino acid biosynthesis.

J Gen Microbiol, 1990 Aug, 136 ( Pt 8), 1551 - 8
Artificial insertion of peptides between signal peptide and mature protein: effect on secretion and processing of hybrid thermostable alpha-amylases in Bacillus subtilis and Escherichia coli cells; Itoh Y et al.; To study the effect of inserted peptides on the secretion and processing of exported proteins in Bacillus subtilis and Escherichia coli, pBR322-derived DNA fragments coding for small peptides were inserted between the DNA coding for the 31 amino acid B . subtilis alpha-amylase signal peptide and that coding for the mature part of the extracellular thermostable alpha-amylase of B . stearothermophilus . Most of the inserted peptides (21 to 65 amino acids) decreased the production of the enzyme in B . subtilis and E . coli, the effect of each peptide being similar in the two strains . In contrast, with one peptide (a 21 amino acid sequence encoded by the extra DNA in pTUBE638), the production of alpha-amylase was enhanced more than 1.7-fold in B . subtilis in comparison with that of the parent strain . The molecular masses of the thermostable alpha-amylases in the periplasm of the E . coli transformants varied for each peptide insert, whereas those in the culture supernatants of the B . subtilis transformants had molecular masses similar to that of the mature enzyme . Based on the NH2-terminal amino acid sequence of the hybrid protein from pTUBE638, it was shown that in E . coli, the NH2-terminally extended thermostable alpha-amylase was translocated and remained in the periplasm after the 31 amino acid signal sequence was removed . In the case of B . subtilis, after the removal of a 34-amino acid signal sequence, the hybrid protein was secreted and processed to the mature form.

FEMS Microbiol Lett, 1990 Aug, 58(3), 305 - 9
Efficient cloning in Bacillus megaterium: comparison to Bacillus subtilis and Escherichia coli cloning hosts; Von Tersch MA et al.; Quantitative cloning efficiencies for B . megaterium, B . subtilis, and E . coli were compared . Transformation of B . megaterium is less efficient than transformation of B . subtilis or E . coli . The frequency of recombinant clones was equal in E . coli and B . megaterium; both somewhat higher than in B . subtilis . Equivalent average insert sizes were found in B . megaterium and E . coli clones, but significantly smaller inserts were obtained in B . subtilis clones . Clones obtained and propagated in B . megaterium were structurally stable when grown under plasmid selection.

Biotechnol Appl Biochem, 1990 Aug, 12(4), 427 - 35
Characterization of a thermostable Bacillus stearothermophilus alpha-amylase; Vihinen M et al.; Liquefying-type Bacillus stearothermophilus alpha-amylase was characterized . The coding gene was cloned in Bacillus subtilis and the enzyme was produced in three different host organisms: B . stearothermophilus, B . subtilis, and Escherichia coli . Properties of the purified enzyme were similar irrespective of the host . Temperature optimum was at 70-80 degrees C and pH optimum at 5.0-6.0 . The enzyme was stable for 1 h in the pH range 6.0-7.5 at 80 degrees C . The enzyme was stabilized by Ca2+, Na+, and bovine serum albumin . About 50% of the activity remained after heating at 70 degrees C for 5 days or 45 min at 90 degrees C . Metal ions Cd2+, Cu2+, Hg2+, Pb2+, and Zn2+ were inhibitory, whereas EDTA, ethylene glycol bis(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid, and Tendamistat were without effect . The enzyme was fully active after treatment in acetone or ethanol at 55 or 70 degrees C, respectively, for 30 min . Sodium dodecyl sulfate (1%) did not affect stability, whereas 6 M urea denatured totally at 70 degrees C . The Km value for soluble starch was 14 mg/ml . Mr is 59,000 and pI 8.8 . The only difference between the enzymes produced in different hosts was in signal peptide processing.

Biotechnol Appl Biochem, 1990 Aug, 12(4), 370 - 5
Coproduction of surfactin and iturin A, lipopeptides with surfactant and antifungal properties, by Bacillus subtilis; Sandrin C et al.; Of the 13 strains of Bacillus subtilis tested for the coproduction of the lipopeptide surfactin and the antifungal lipopeptides of the iturin family, only 1 produced both lipopeptides with a high yield . The cultures were made in a synthetic medium . Several L-amino acids and various carbon sources were good substrates for the lipopeptide production . The maximum yield of surfactin was about 110 mg/liter and that of iturin A about 39 mg/liter/absorbance unit for the best strain, B . subtilis S 499.

Genetics, 1990 Aug, 125(4), 703 - 8
Orientation of genes in the Bacillus subtilis chromosome; Zeigler DR et al.; The orientation of 96 genes on the Bacillus subtilis chromosome was deduced by the analysis of published data . Of these genes, 91 were found to be oriented so that their promoters were proximal to the chromosomal replication origin and their transcription termini to the replication terminus . Transcription of these genes would therefore be co-directional with replication . This chromosomal organization is consistent with the hypothesis advanced for Escherichia coli that bacteria avoid head-on collisions between RNA polymerase and DNA replication proteins by the appropriate orientation of their transcription units.

Proc Natl Acad Sci U S A, 1990 Aug, 87(16), 6238 - 42
Site-directed mutagenesis of a catabolite repression operator sequence in Bacillus subtilis; Weickert MJ et al.; Catabolite repression of the Bacillus subtilis alpha-amylase gene (amyE) involves an operator sequence located just downstream of the promoter (amyR), overlapping the transcription start site . Oligonucleotide site-directed mutagenesis of this sequence identified bases required for catabolite repression . Two mutations increased both the 2-fold symmetry of the operator and the repression ratio . Although many mutations reduced the repression ratio 3- to 11-fold, some also caused a 2-fold or greater increase in amylase production . Others caused hyperproduction without affecting catabolite repression . Homologous sequences in other catabolite-repressed B . subtilis promoters suggest a common regulatory site may be involved in catabolite repression.

J Bacteriol, 1990 Aug, 172(8), 4694 - 5
Erythromycin induces expression of the chloramphenicol acetyltransferase gene cat-86; Rogers EJ et al.; The plasmid gene cat-86 specifies chloramphenicol-inducible chloramphenicol acetyltransferase in Bacillus subtilis . This gene, like the erythromycin-inducible erm genes, is regulated by translational attenuation . Here we show that cat-86 is also inducibly regulated by erythromycin . cat-86 does not confer resistance to erythromycin.

J Bacteriol, 1990 Aug, 172(8), 4616 - 23
Effects of antibiotics on synthesis and persistence of sigma E in sporulating Bacillus subtilis; Jonas RM et al.; A potential regulatory link between the activation of a sporulation-specific sigma factor (sigma E) and forespore septum formation was investigated by treating Bacillus subtilis with inhibitors of protein or peptidoglycan synthesis and monitoring the consequences of these treatments on sigma E activation and septation . Western blot (immunoblot) and electron microscopic analyses revealed that both the formation of sigma E and septation were inhibited to a similar degree when either rifampin or chloramphenicol was added at different times before the second hour into sporulation but that penicillin preferentially blocked septation . We interpret these results as indicating that the syntheses of the gene products for both septation and sigma E activation occur at approximately the same time in development but that synthesis of an intact septum is unlikely to be a prerequisite for the formation of sigma E . We observed that penicillin could not only block septation but, depending on the time of its addition, could also inhibit both the activation of sigma E and the synthesis of its precursor . The basis of this effect is unknown, but it is not due to an overall disruption of protein synthesis . The incorporation of {35S} methionine by the sporulating cultures was unaffected by penicillin treatment . A time course study of the effects of rifampin and chloramphenicol treatments on sigma E levels revealed that both the synthesis of sigma E and its disappearance from sporulating cultures is inhibited by these antibiotics . This suggests that ongoing macromolecular synthesis is required for the turnover of sigma E.

J Bacteriol, 1990 Aug, 172(8), 4593 - 602
Independent genes for two threonyl-tRNA synthetases in Bacillus subtilis; Putzer H et al.; With the exception of Escherichia coli lysyl-tRNA synthetase, the genes coding for the different aminoacyl-tRNA synthetases in procaryotes are always unique . Here we report on the occurrence and cloning of two genes (thrSv and thrS2), both encoding functional threonyl-tRNA synthetase in Bacillus subtilis . The two proteins share only 51.5% identical residues, which makes them almost as distinct from each other as each is from E . coli threonyl-tRNA synthetase (42 and 47%) . Both proteins complement an E . coli thrS mutant and effectively charge E . coli threonyl tRNA in vitro . Their genes have been mapped to 250 degrees (thrSv) and 344 degrees (thrS2) on the B . subtilis chromosome . The regulatory regions of both genes are quite complex and show structural similarities . During vegetative growth, only the thrSv gene is expressed.

J Bacteriol, 1990 Aug, 172(8), 4178 - 86
Phenotypes of Bacillus subtilis mutants altered in the precursor-specific region of sigma E; Jonas RM et al.; sigma E is a sporulation-specific sigma factor of Bacillus subtilis that is synthesized from an inactive precursor protein (P31) . The structural gene (sigE) for P31 was reengineered by oligonucleotide-directed mutagenesis to encode sigma E directly . The sequence specifying the first amino acid of sigma E (GGC) was placed immediately downstream of the initiating codon (ATG) of P31 . The resulting sigE allele (sigE delta 84) encodes a sigma E-like protein which differs from the "processed product" by a single Met residue at its amino terminus . B . subtilis strains which carried this allele were Spo- and contained no detectable sigma E . The sigE delta 84 allele generated a product in Escherichia coli which, by quantitative Western immunoblot analysis, was present at 10 to 20% of the level of product (P31) obtained from a wild-type allele . A sigma E-like product was also not detected in two B . subtilis strains with missense mutations in the sequence encoding the processed region of P31 . These results suggest that sigma E is a highly labile protein that is stabilized during its synthesis by an element of the precursor sequence . A mutant allele (sigE delta 48) which made an active sigma E-like protein in B . subtilis was isolated . This gene specified a product in which five amino acids, not derived from the P31 processed region, were joined to P31 at a position eight amino acids upstream of the processing site . The sigE delta 48 product was not processed, but it activated the sigma E -dependent spoIID promoter in vivo . The sigE delta 48 product therefore lost both an essential target for processing and a region which inhibited sigma sigma E activity . Cells which carried sig E delta 48 were Spo- . The basis of the sigE delta 48-dependent defect in sporulation is unknown, but the sigma E delta 48 activity appeared to persist beyond the time in development (4 h after onset sporulation) when wild-type sigma E activity declines . Thus, it may interfere with the proper regulation of late sporulation genes.

J Bacteriol, 1990 Aug, 172(8), 4161 - 70
Energy and calcium ion dependence of proteolysis during sporulation of Bacillus subtilis cells; O'Hara MB et al.; Bacterial cells degrade intracellular proteins at elevated rates during starvation and can selectively degrade proteins by energy-dependent processes . Sporulating bacteria can degrade protein with apparent first-order rate constants of over 0.20 h-1 . We have shown, with an optimized {14C}leucine-labeling and chasing procedure, in a chemically defined sporulation medium, that intracellular protein degradation in sporulating cells of Bacillus subtilis 168 (trpC2) is apparently energy dependent . Sodium arsenate, sodium azide, carbonyl cyanide m-chlorophenylhydrozone, and N,N'-dicyclohexylcarbodiimide, at levels which did not induce appreciable lysis (less than or equal to 10%) over 10-h periods of sporulation, inhibited intracellular proteolysis by 13 to 93% . Exponentially growing cells acquired arsenate resistance . In contrast to earlier reports, we found that chloramphenicol (100 micrograms/ml) strongly inhibited proteolysis (68%) even when added 6 h into the sporulation process . Restricting the calcium ion concentration (less than 2 microM) in the medium had no effect on rates or extent of vegetative growth, strongly inhibited sporulation (98%), and inhibited rates of proteolysis by 60% or more . Inhibitors of energy metabolism, at the same levels which inhibited proteolysis, did not affect the rate or degree of uptake of Ca2+ by cells, which suggested that the Ca2+ and metabolic energy requirements of proteolysis were independent . Restricting the Ca2+ concentration in the medium reduced by threefold the specific activity in cells of the major intracellular serine proteinase after 12 h of sporulation . Finally, cells of a mutant of B . subtilis bearing an insertionally inactivated gene for the Ca2(+)-dependent intracellular proteinase-1 degraded protein in chemically defined sporulation medium at a rate indistinguishable from that of the wild-type cells for periods of 8 h.

Eur J Cell Biol, 1990 Aug, 52(2), 219 - 28
Modified lysosomal compartment as carrier of slowly and non-degradable tracers in macrophages; Tassin MT et al.; A previous immunocytochemical study of macrophages infected with Bacillus subtilis showed that a cell wall antigen could be detected for several days in a population of small vesicles randomly distributed within the cells and apparently distinct from perinuclear lysosomes . These observations suggested the possibility that these vesicles might constitute a "storage" compartment for non-degradable compounds . In the present report we compared in pulse-chase experiments the location and fate of a series of degradable and non-degradable pinocytic tracers within the macrophages . The tracers, detected by fluorescent microscopy, were bovine serum albumin (BSA), hen egg ovalbumin (OVA), horseradish peroxidase (HRP) . Lucifer Yellow, fluorescent dextran, and levan . BSA and OVA remained located in perinuclear lysosomes during the chase period until their disappearance occurring within 3 h . In contrast, the other tracers, although initially located in perinuclear lysosomes, were found after a 3 to 5-h chase in small vesicles homogeneously distributed in the macrophage cytoplasm where they remained visible for 2 to 3 days . The use of markers for different cell organelles indicated that these dispersed vesicles exhibited several of the lysosomal features . They were acidic, they contained the 100 kDa and the 120 kDa lysosomal proteins as well as some acid proteases albeit these markers were in lesser concentrations than in the perinuclear lysosomal compartment . The addition of bacteria to the macrophages previously loaded with fluorescent dextran showed that all dispersed vesicles have the same fusion property as lysosomes and that slowly degraded or non-degradable tracers turn over through the perinuclear lysosomal compartment by using the endocytic pathway . Measurement of the release of some of the tracers into the culture medium suggested that the "dispersed vesicles" were probably not implicated in exocytosis of the tracers.

J Biol Chem, 1990 Jul 25, 265(21), 12686 - 9
The lumazine synthase-riboflavin synthase complex of Bacillus subtilis . Crystallization of reconstituted icosahedral beta-subunit capsids; Schott K et al.; The lumazine synthase-riboflavin synthase complex (heavy riboflavin synthase) of Bacillus subtilis consists of an icosahedral capsid of 60 beta-subunits containing a core of three alpha-subunits . The enzyme has been purified from the derepressed mutant H 94 of B . subtilis by a novel efficient procedure using column chromatography and preparative crystallization . Beta-Subunits were isolated after dissociation of the enzyme at pH 8.0 . Ligand-driven renaturation of beta-subunits yields hollow icosahedral beta 60 capsids which could be crystallized in 1.55 M phosphate, pH 8.7, in three different modifications . A monoclinic modification belongs to space group C2 with unit cell dimensions of a = 235.5, b = 191.2, and c = 165.4 A and alpha = gamma = 90 degrees and beta = 134.4 degrees . The crystals contain two hollow beta 60 particles/unit cell and diffract to approximately 2.8-A resolution . A hexagonal modification has the space group P6(3)22 with unit cell dimensions of a = b = 157.2 and c = 300.8 A and alpha = beta = 90 degrees and gamma = 120 degrees . These cell parameters are similar to the dimensions of hexagonal crystals of native heavy riboflavin synthase (alpha 3 beta 60) . A second hexagonal modification shows unit cell parameters of a = b = 156.3 and c = 622.6 A and alpha = beta = 90 degrees and gamma = 120 degrees . The space group of this modification could not be determined unambiguously.

Biochemistry, 1990 Jul 24, 29(29), 6820 - 6
(Difluoromethylene)phosphates of guanine nucleosides as probes of DNA polymerases and G proteins; Arabshahi L et al.; 5'-Polyphosphates of N2-(p-n-butylphenyl)-2'-deoxyguanosine and -guanosine which contain a difluoromethylene group in place of a phosphoanhydride oxygen have been synthesized . 5'-{beta,gamma-(Difluoromethylene)triphosphates}, including that of 2'-deoxyguanosine, were prepared by reaction of the corresponding 5'-phosphates, activated by 1,1'-carbonyldiimidazole, with difluoromethanediphosphonate . The 5'-{(difluoromethylene)diphosphate} of N2-(p-n-butylphenyl)guanosine was prepared by treatment of a protected 5'-tosyl nucleoside with difluoromethanediphosphonate, followed by deprotection . Condensation of this nucleotide, activated with 1,1'-carbonyldiimidazole, with orthophosphate gave N2-(p-n-butylphenyl)guanosine 5'-{(alpha,beta-difluoromethylene)triphosphate} . Products were characterized by 31P and 19F NMR spectroscopy . The phosphonates were tested for their ability to displace {3H}GDP from the GTP binding proteins cellular (EC) and oncogenic (Leu-61) Ha-ras p21, and for their ability to inhibit DNA polymerase alpha from Chinese hamster ovary cells . The p21s bound weakly to a triphosphonate when the CF2 group was in the beta,gamma position, but not when it was in the alpha,beta position, and they did not bind to the corresponding (difluoromethylene)diphosphate . In contrast, the CF2 group had no effect on inhibition of DNA polymerase alpha by N2-(p-n-butylphenyl)-2'-deoxyguanosine 5'-{(beta,gamma-difluoromethylene)triphospate} . 2'-Deoxyguanosine 5'-{(beta,gamma-difluoromethylene)triphosphate} was found to be a bona fide substrate for several DNA polymerases and had a lower apparent Km than dGTP with Bacillus subtilis DNA polymerase III.

J Mol Biol, 1990 Jul 5, 214(1), 73 - 84
Identification of the replication terminator protein binding sites in the terminus region of the Bacillus subtilis chromosome and stoichiometry of the binding; Lewis PJ et al.; DNase I footprinting of the interaction between the replication terminator protein (RTP) of Bacillus subtilis and the inverted repeat region (IRR) at the chromosome terminus, to which it binds to block the clockwise replication fork, showed that two major regions of 41 base pairs (bp) were protected from cleavage . These regions corresponded approximately to the imperfect inverted repeats (IRI and IRII) identified previously . Band retardation analyses of the interaction between RTP and portions of the IRR established that each inverted repeat (IRI or IRII) contained two RTP binding sites . By sedimentation equilibrium in the ultracentrifuge, RTP was found to exist as a dimer of 29 kDa at neutral pH and concentrations above 0.2 g/l . Quantitative studies of the RTP-IRR interaction using {3H}RTP and {32P}IRR showed that the fully saturated complex contained eight RTP monomers per IRR . It is concluded that a dimer of RTP binds to each of the four sites in IRR . The apparent dissociation constant for the interaction was estimated (in the presence of 50% glycerol) to be 1.2 x 10(-11) M (dimer of RTP) . Glycerol was found to have a marked effect on the affinity of RTP for the IRR and on the relative amounts of the interaction complexes formed; in the absence of glycerol the dissociation constant was approximately 50-fold higher and there was pronounced co-operative binding of RTP dimers to adjacent sites in each inverted repeat . Examination of the DNA sequence in IRI and IRII identified two 8 bp direct repeats in each . The regions protected from DNase I cleavage in each inverted repeat and the protection afforded by a core sequence spanning just one of the 8 bp direct repeats were consistent with each 8 bp repeat representing a recognition sequence for the RTP dimer . A model describing the binding of RTP to the IRR is presented.

FEBS Lett, 1990 Jul 2, 267(1), 71 - 4
Evidence for posttranscriptional regulation of synthesis of the Bacillus subtilis Gnt repressor; Fujita Y et al.; Transcription of the Bacillus subtilis gnt operon results in a polycistronic mRNA that encodes from the 5' end the Gnt repressor, gluconate kinase and permease . The RNA is drastically induced through inactivation of the Gnt repressor by gluconate . The results of deletion analysis of the gnt promoter region upstream of the repressor gene indicated that no other promoter except the gnt promoter was present for expression of the gluconate kinase gene . In contrast to the synthesis of gluconate kinase and permease, which was markedly induced by gluconate, the results of a radioimmunoassay for the Gnt repressor indicated that synthesis of the Gnt repressor from the induced mRNA was posttranscriptionally repressed.

Biokhimiia, 1990 Jul, 55(7), 1279 - 86
{Various physico-chemical and kinetic properties of metalloproteinase from bacteriolytic lysoamidase preparation}; Krupianko VI et al.; The amino acid composition of metalloproteinase was determined . It was shown that the enzyme is made up of four cysteinyl residues which makes it distinct from other known neutral metalloproteinases from Bacillus brevis, Bacillus subtilis and thermolysine that are devoid of cysteinyl residues . The inhibiting effect of amino acids and some di- and tripeptides on the metalloproteinase activity was studied . The pH-dependence of the Michaelis constant (pKm0) of native and diethylpyrocarbonate-modified metalloproteinase (pKm) suggests that the enzyme active center contains two imidazole groups of histidine with pK alpha 1 = 6.75 +/- 0.1 and pK alpha 2 = 5.4 +/- 0.1 . The experimental results are compared to those obtained with other microbial metalloproteinases.

Mol Gen Genet, 1990 Jul, 222(2-3), 441 - 5
Genetic analysis of rec E activities in Bacillus subtilis; Ceglowski P et al.; A recE mutant (recE6) of Bacillus subtilis was constructed by insertion of a selectable marker into the recE coding region . The insertional inactivation of the recE gene renders cells very sensitive to DNA damaging agents and severely impairs intermolecular recombination, but does not markedly affect plasmid interstrand annealing and intramolecular recombination . The recE6 allele was then introduced into a set of DNA repair-deficient strains of B . subtilis . The removal of DNA damage by the recF, addA addB, recH, recL and recP gene products is strictly dependent on an active recE gene product (recE-dependent pathway) . On the other hand, the increased sensitization to purine adducts in the uvrA42 recE6 and polA5 recE6 strains suggests that such lethal lesions may be removed either by the recE-dependent or by the recE-independent pathway.

Mol Microbiol, 1990 Jul, 4(7), 1129 - 34
Mycoplasma pneumoniae DNA gyrase genes; Colman SD et al.; We have identified a clone from a lambda EMBL3 library containing a 19kb insert of Mycoplasma pneumoniae DNA which includes the genes that encode both subunits of DNA gyrase . The gyrB gene and the 5' end of the gyrA gene have been subcloned into M13 . The gyrB gene is 1953bp in length and overlaps the gyrA gene by a single base . The nucleotide sequence of these subclones has significant homology to previously reported gyrase genes . In terms of the size of the gyrB gene and its proximity to the gyrA gene, M . pneumoniae is more similar to Bacillus subtilis than to Escherichia coli.

J Bacteriol, 1990 Jul, 172(7), 3966 - 73
The sacT gene regulating the sacPA operon in Bacillus subtilis shares strong homology with transcriptional antiterminators; Debarbouille M et al.; The expression of the Bacillus subtilis sacPA operon is induced by sucrose . A DNA fragment containing the upstream region of this operon was cloned . This fragment contains a promoter from which the operon is expressed . This upstream region also contains a palindromic DNA sequence very similar to the transcriptional terminator which regulates the induction of the B . subtilis sacB gene . Of 37 nucleotides in a region partially overlapping the sacP palindromic sequence, 34 were identical to the corresponding region of the sacB gene . A similar motif is also present in the bgl operon of Escherichia coli . The sacT locus controlling sacPA expression had been identified by a single constitutive mutation sacT30 which mapped close to the sacPA operon . DNA fragments containing the sacT+ and sacT30 alleles were cloned and sequenced . The sacT gene product is very similar to the B . subtilis sacY and to the E . coli bglG gene products . The constitutive sacT30 mutation was identified . It corresponds to a Asp-96-to-Tyr missense mutation located in a highly conserved region in SacT and SacY . These results strongly suggest that sacT is a specific regulatory gene of the sacPA operon.

J Bacteriol, 1990 Jul, 172(7), 3738 - 44
Insertional mutagenesis of Listeria monocytogenes with a novel Tn917 derivative that allows direct cloning of DNA flanking transposon insertions; Camilli A et al.; To carry out efficient insertional mutagenesis in Listeria monocytogenes and to facilitate the characterization of disrupted genes, two novel derivatives of Tn917 were constructed, Tn917-LTV1 and Tn917-LTV3 . The derivatives (i) transpose at a significantly elevated frequency, (ii) generate transcriptional lacZ fusions when inserted into a chromosomal gene in the appropriate orientation, and (iii) allow the rapid cloning in Escherichia coli of chromosomal DNA flanking transposon insertions . The rapid cloning of DNA flanking insertions is possible because the transposon derivatives carry ColE1 replication functions, a cluster of polylinker cloning sites, and antibiotic resistance genes selectable in E . coli (bla in the case of Tn917-LTV1; neo and ble in the case of Tn917-LTV3) . The enhanced transposition frequency of Tn917-LTV1 and Tn917-LTV3 (about 100-fold in Bacillus subtilis) is believed to be due to the fortuitous placement of vector-derived promoters upstream from the Tn917 transposase gene . In L . monocytogenes, Tn917-LTV3 transposed at a frequency of 8 x 10(-4) when introduced on a pE194Ts-derived vector and generated at least eight different auxotrophic mutations . Two nonhemolytic insertion mutants of L . monocytogenes were isolated, and DNA flanking the transposon insertions was cloned directly into E . coli, making use of the ColE1 rep functions and neo gene carried by Tn917-LTV3 . Both insertions were shown to be within hlyA, the L . monocytogenes hemolysin structural gene . Although Tn917-LTV1 and Tn917-LTV3 were constructed specifically for genetic analysis of L . monocytogenes, their enhanced transposition frequency and convenience for cloning of DNA adjacent to sites of insertions make them the transposon derivatives of choice for insertional mutagenesis in any gram-positive bacteria that support replication of pE194Ts.

Mikrobiyol Bul, 1990 Jul, 24(3), 262 - 71
{Production of polygalacturonase by Bacillus subtilis cultured with waste and residues as carbon sources}; Aksoz E; In the recent study, it was observed that if when the initial pH was 7.0, incubation temperature was 37 degrees C and aeration (150 rpm) was supplied polygalacturonase production by Bacillus subtilis reached to its maximum . Molasses, Vinase and Peach or Apricot pomace were used as carbon sources at differing concentrations and the highest differential rate of enzyme synthesis was detected in the medium prepared with Vinase having a 5% of total sugar content (6.74 delta U/delta O.D.) . It also was detected that the waste or residues mentioned above could be used as carbon sources for the polygalacturonase enzyme production by Bacillus subtilis.

Mol Gen Genet, 1990 Jul, 222(2-3), 467 - 9
Genetic mapping of regulatory mutations of Bacillus subtilis riboflavin operon; Kreneva RA et al.; Seven mutations leading to riboflavin overproduction in Bacillus subtilis were found to be linked to the marker dnaF133 (145 degrees on the B . subtilis genetic map) by transformation . Cotransfer indexes (42.5%-61.7%) suggest that the ribC mutations are alleles of the same locus . Results of transduction and transformation crosses suggest the following order of markers:pyrD26-ts-6-dnaF133-ribC-recA1.

J Gen Microbiol, 1990 Jul, 136 ( Pt 7), 1335 - 42
Characterization and cloning of the gerC locus of Bacillus subtilis 168; Yazdi MA et al.; A Bacillus subtilis gerC spore germination mutant demonstrating a temperature-sensitive response to L-alanine as germinant has been characterized in detail . The gerC58 mutation is 50% cotransformed with aroB in the gene order gerC-aroB-trpC . The mutation is responsible for a severe growth defect which is manifest at all growth temperatures and is most extreme on rich media . A second, unlinked, mutation in the original strain suppressed this growth defect, but spores of the suppressed strain failed to germinate in alanine at 42 degrees C . As this germination defect is dependent on the presence of the gerC58 allele, it is likely to be the direct result of a mutant gerC protein . The gerC gene therefore appears to have a role in both spore germination and vegetative cell growth . A gene library of BclI-digested B . subtilis chromosomal DNA was constructed in phage vector phi 105J27 . A derivative containing the gerC region was obtained by complementation of the growth defect of an unsuppressed gerC58 strain . This phage contained a 6.3 kb insert of bacterial DNA, which is above the reported packaging limit of the phage . It failed to form visible plaques, although it could be handled as a prophage and sufficient phage particles be isolated to allow characterization of the insert . A deletion derivative generated in vitro and carrying only 2.9 kb of insert DNA also complemented the gerC defect . This gerC locus is the second locus to be implicated in alanine-stimulated germination . The first, gerA, is a developmentally controlled operon whose gene products are present only in the spore . This study of gerC, in contrast, reveals a role in spore germination for a normally essential vegetative protein.

J Gen Microbiol, 1990 Jul, 136 ( Pt 7), 1223 - 30
Nucleotide sequences of the Bacillus subtilis flaD locus and a B . licheniformis homologue affecting the autolysin level and flagellation; Sekiguchi J et al.; A DNA fragment containing the flaD locus of Bacillus subtilis, which had been cloned into plasmid pAC3, was subcloned into an M13 phage and sequenced . The sequence contained five open reading frames (ORFs), of which ORF2 was the flaD gene . Unexpectedly, the sequence of the flaD locus was identical to that of sin {sporulation inhibition gene; Gaur, N . K., Dubnau, E . & Smith, I . (1986) . Journal of Bacteriology 168, 860-869} . A B . licheniformis homologue (flaL) of the B . subtilis flaD locus was cloned into pUC19 and identified by colony hybridization . The B . licheniformis DNA was subcloned and sequenced . Two ORFs (ORF1, or L-ORF1; and ORF2, or flaL) were detected, encoding 58 and 111 amino acid residues, respectively . These are almost identical in length to ORF1 (D-ORF1; 57 amino acids) and flaD (111 amino acids) on the fragment of B . subtilis DNA . The overall interspecies differences between the nucleotide sequences of D-ORF1 and L-ORF1, and those of flaD and flaL, were 42% and 11%, respectively, and the differences in the predicted amino acid sequences were 50% and 7%, respectively . The regions 3' of the ORFs (flaL and flaD) in both species resemble rho-independent terminators of transcription . The characteristics of the amino acid sequences are also discussed.

J Bacteriol, 1990 Jul, 172(7), 4056 - 63
Suppression of early competence mutations in Bacillus subtilis by mec mutations; Roggiani M et al.; Although competence normally develops only in glucose-minimal salts media, mecA and mecB mutations permit the expression of competence and of late competence genes in complex media as well (D . Dubnau and M . Roggiani, J . Bacteriol . 172:4048-4055, 1990) . The expression of late competence genes is dependent on the products of the regulatory genes comA, comB, comP, sin, abrB, spo0H, and spo0A . We show here that this list must be extended to include degU, csh-293, and spo0K . mecA and -B mutations bypass most of these requirements, making the expression of late competence genes and of competence itself independent of all of these regulatory genes, with the exceptions of spo0A and spo0K (in the case of mecB) . The expression of late competence genes in mec mutants that are deficient for each of the bypassed regulatory functions is still under growth stage-specific regulation . The implications of these findings are discussed, and a provisional scheme for the flow of information during the development of competence is proposed.

J Bacteriol, 1990 Jul, 172(7), 4048 - 55
Growth medium-independent genetic competence mutants of Bacillus subtilis; Dubnau D et al.; The development of competence in Bacillus subtilis is normally dependent on the growth medium . Expression of late competence genes occurs in glucose-minimal salts-based media but not in complex media . Expression is also inhibited when glutamine is added to competence medium and when glycerol is substituted for glucose . Mutations have been identified in two regulatory loci, mecA and mecB, which render competence development independent of these variables . Although in mec mutants the expression of late competence genes, as well as of competence itself, occurred in all media tested, this expression was still growth stage regulated . Thus at least some forms of medium-dependent and growth stage-specific regulation are genetically separable . One of the mecB mutations (mecB31) conferred oligosporogenicity . The mecB mutations were tightly linked by transformation to rif, lpm, and std markers and were located between rif-2103 and cysA14 . The mecA42 mutant was linked by transduction to argC4.

J Bacteriol, 1990 Jul, 172(7), 3730 - 7
The Bacillus subtilis 168 alkaline phosphatase III gene: impact of a phoAIII mutation on total alkaline phosphatase synthesis; Bookstein C et al.; The first alkaline phosphatase (APase) structural gene mutant of Bacillus subtilis 168 was constructed by using a clone identified by hybridization to a synthetic degenerative oligonucleotide . The design of the probe was based on the first 29 amino acids of the sequenced mature APase III protein, which had been isolated from the secreted fraction of vegetative, phosphate-starved cells . DNA sequencing of the clone revealed the first 80 amino acids of the APase III protein, including a typical procaryotic signal sequence of 32 amino acids preceding the start of the mature protein . The 29 amino acids encoded by the predicted open reading frame immediately following the signal sequence are identical to the first 29 amino acids of the sequenced mature protein . This region shows 80% identity to strand A of the beta sheet that is very well conserved in Escherichia coli and mammalian APases . A phoAIII structural mutant was constructed by insertional mutagenesis with a fragment internal to the coding region . The effects of this mutation on APase production in B . subtilis 168 were analyzed under both phosphate starvation and sporulation conditions . The mutation in APase III reduced the total vegetative APase specific activity by approximately 40% and sporulation APase specific activity by approximately 45% . An APase protein was isolated from sporulating cells at stage III and was identified as APase III by protein sequencing of the amino terminus and by its absence in the phoAIII mutant . The APase III gene has been mapped to approximately 50 degrees on the B . subtilis chromosome.

Vet Med (Praha), 1990 Jul, 35(7), 411 - 8
{Residues of inhibitory agents in the tissues of slaughter-house animals--comparison of microbiological methods of agar diffusion}; Hrdlicka J; Three microbiological methods of agar diffusion were compared which are used to detect the residues of inhibitory substances: method using the strain Bacillus subtilis ATCC 6633 (B.s . 6633), method using the strain Bacillus stearothermophilus v . calidolactis C 953 (B . s . v . c . 953), and four-plate method . Using the compared methods, minimum inhibitory concentrations were determined for standard solutions of antibiotics and sulphadimidine . Inhibitory substances were detected parallely in the samples of the tissues of medicated and emergency-slaughtered animals, applying the three compared methods . In indicated cases inhibitory substances were identified by electrophoresis and chemical methods . The results indicate the following: 1) The B.s . 6633 method is the least sensitive of all to the residues of inhibitory substances . 2) For the purposes of detection of most antibiotics under investigation, the B.s.v.c . 953 method and the four-plate method are replaceable . The B.s.v.c . 953 method is more suitable for the detection of penicillin residues, in the case of tetracycline antibiotics it is the four-plate method . 3) Sulphadimidine residues can be detected only by the four-plate method . 4) The four-plate method enables to make the preliminary group identification of antibiotics and sulphonamides . 5) The detection limit of microbiological methods is not sufficiently sensitive to determine chloramphenicol residues; that is why the physico-chemical methods must be used.

J Struct Biol, 1990 Jul-Sep, 104(1-3), 2 - 8
Three-dimensional reconstruction of the sevenfolded form of Bacillus subtilis Gro EL Chaperonin; Carrascosa JL et al.; Bacillus subtilis grown at 42 degrees C produces a major form of Gro EL-like chaperonin that has been analyzed by electron microscopy . Most of the views show a clear sevenfold symmetry when studied by rotational analysis . The particles were classified into defined families by multivariate analysis and supervised fuzzy-set classification methods, and those belonging to a sevenfold family were averaged to produce a two-dimensional representative projection . These selected particles were then used, when titled by 55 degrees in the microscope goniometer stage, as the starting projections for a three-dimensional reconstruction protocol based on the random conical tilt series method . The resulting reconstruction shows the Gro EL-like chaperonin from B . subtilis as a cylindrical body with seven well defined lobules arranged almost parallel to the longitudinal axis of the particle . There is a channel that is placed along this axis and appears fully open in both sides . The geometry of the channel is polar and presents differences in both faces of the particle.

J Biochem (Tokyo), 1990 Jul, 108(1), 116 - 21
Temperature-sensitive mutant of Bacillus subtilis glutamine synthetase obtained by random mutation; Nakano Y et al.; Random mutations were introduced into the B . subtilis glutamine synthetase gene by using nitrous acid, and a high temperature-sensitive mutant was selected . DNA sequencing of the restriction fragment containing the mutation revealed a single base-pair change resulting in the substitution of Leu 318 with Phe . The mutant enzyme was purified, and its kinetic and physical properties were characterized . The Mg2(+)-dependent activity and Mg2+ plus Mn2(+)-dependent activity of the mutant were less than 5% of those of the wild-type at 37 degrees C, and these activities decreased above 15 degrees C, whereas the Mn2(+)-dependent activity was nearly normal . Affinity of the mutant enzyme for glutamate was extremely decreased although the Km values for NH3 or ATP were almost the same as those of the wild-type . The mutant enzyme was more susceptible than the wild-type enzyme to digestion with chymotrypsin in the presence of glutamate, ATP, and Mg2+, although addition of glutamate, ATP, and Mn2+ completely protected both enzymes . These results and circular dichroism analyses suggested that Leu 318 is at the glutamate-binding site and that the substitution of Leu 318 for Phe reduces the ability of the enzyme to form the enzyme-substrate complex, probably supported by Mg2+.

Mol Gen Genet, 1990 Jul, 222(2-3), 470 - 2
The Bacillus subtilis small cytoplasmic RNA gene and 'dnaX' map near the chromosomal replication origin; Struck JC et al.; The Bacillus subtilis small cytoplasmic RNA (scRNA) has an important, although not yet defined function in protein biosynthesis . Here we describe the mapping of the single copy scRNA gene and the flanking homolog to dnaZX of Escherichia coli, termed 'dnaX' . The scRNA gene region of a B . subtilis wild-type strain was marked with a cat gene and mapped by scoring chromosomal cotransformation rates of various mutant strains to chloramphenicol resistance and loss of the mutant phenotypes, respectively . This analysis, together with an EcoRI map comparison, places the scRNA gene and dnaX in the vicinity of recM near the replication origin region of B . subtilis.

J Bacteriol, 1990 Jul, 172(7), 4064 - 71
Transcriptional regulation of comC: evidence for a competence-specific transcription factor in Bacillus subtilis; Mohan S et al.; comC specifies a protein product that is required for genetic competence in Bacillus subtilis . The probable transcriptional start site of comC has been localized by high-resolution primer extension analysis and shown to be preceded by an appropriately positioned sequence that resembles the consensus promoter for the sigma A form of RNA polymerase . Low-resolution S1 nuclease transcription mapping was used to identify the comC terminator, which is located near a palindromic element recognizable in the DNA sequence . Deletion analysis of the sequence upstream from the likely promoter identified a region required in cis for the expression of comC . An overlapping, and possibly identical, sequence was shown to inhibit the expression of competence and of several late competence genes, when present in multiple copies . This was interpreted as due to the titration of a positively acting competence transcription factor (CTF) by multiple copies of the promoter-bearing fragment . In crude lysates of B . subtilis grown to competence, a DNA-binding activity that appeared to be specific for the comC promoter fragment was detected by gel retardation assays . This activity, postulated to be due to CTF, was detected only following growth in competence medium, only in the stationary phase of growth, and was dependent on the expression of ComA, a known competence-regulatory factor . In the presence of the mecA42 mutation, the ComA requirement for CTF activity was bypassed, and CTF activity could be detected in lysates prepared from a strain grown in complex medium . This behavior suggested that either the expression or the activation of CTF was regulated in a competence-specific manner . Comparison of the putative CTF-binding site defined by deletion analysis with a similarly positioned sequence upstream from the start site of the late competence gene comG revealed that both sequences contained palindromes, with 5 of 6 identical base pairs in each arm . It is suggested that these palindromic sequences comprise recognition elements for CTF binding and that CTF binding must occur for the appropriate expression of late competence genes.

Biochem Biophys Res Commun, 1990 Jun 29, 169(3), 1035 - 9
The Glu residue in the conserved Asn-Glu-Pro sequence of two highly divergent endo-beta-1,4-glucanases is essential for enzymatic activity; Baird SD et al.; We initially aligned 28 different cellulase sequences in pairwise fashion and found half of them have the sequence -Asn-Glu-Pro- located in a region flanked by hydrophobic-rich amino acids . Based on lysozyme as a model, the glutamate residue could be essential for enzyme function . We tested this possibility by site-directed mutagenesis of the genes coding Bacillus polymyxa and Bacillus subtilis endo-beta-1,4-glucanases . The genes and amino acid sequences of these two enzymes show very little similarity . Change of Glu-194 and Glu-169 to the isosteric glutamine form in these respective enzymes resulted in a dramatic loss of CMCase activity which could be restored by reverse mutation . Similar mutations to less-conserved residues, Glu-72 and Glu-147, of the B . subtilis enzyme did not cause any loss of activity.

J Chromatogr, 1990 Jun 27, 510, 213 - 23
Composite affinity sorbents and their cleaning in place; Girot P et al.; Making large-scale affinity sorbents that are reusable under acceptable hygienic conditions implies specific treatments for cleaning in place with known aqueous solutions of chemical agents . However, common agents such as sodium hydroxide are frequently considered too drastic for the stability of macromolecular biologically active immobilized ligands . According to a large series of trials, it was found that only a mixture of sodium hydroxide and ethanol was actually effective in sterilizing a sorbent in a single step . When hydroxide or an ethanol-acetic acid mixture were used alone, they were not totally efficient in the inactivation of sporulated Bacillus subtilis . Conversely, they were efficient when used sequentially . All these solutions were able to remove pyrogens from chromatographic sorbents . As the sterilizing solutions contained a certain amount of ethanol, the most suitable chromatographic affinity sorbents had to be based on an incompressible matrix . When washing an affinity silica sorbent that had proteins as ligands with solutions such as sodium hydroxide, ethanol-acetic acid or ethanol-sodium hydroxide, it was found that certain sorbents were able to tolerate the treatments without a noticeable decrease in their biochemical activity.

Biochemistry, 1990 Jun 26, 29(25), 6033 - 42
Binding of Bacillus subtilis ermC' methyltransferase to 23S rRNA; Su SL et al.; ermC 23S rRNA methyltransferase dimethylates adenine 2085 in Bacillus subtilis 23S rRNA and also regulates its own synthesis by autogenous translational repression . We have characterized the binding of ermC' methyltransferase to 23S rRNA . This protein differs in only five amino acid residues from the ermC product and was chosen for study because of its greater stability and ease of isolation . A filter binding assay was used to study the physical aspects of binding in the absence of methylation . The dissociation equilibrium constant of the binding was found to be 4 x 10(-9) M at 37 degrees C . Kinetic studies of complex formation and dissociation revealed that the kon and koff were 4 x 10(6) M-1 s-1 and 6.8 x 10(-2) s-1 respectively at 16 degrees C . Equilibrium competition experiments showed that the enzyme has varying affinities for a variety of nucleic acids in the order 23S rRNA greater than 16S rRNA greater than M13 DNA, f2 RNA greater than tRNA . One of the end products of methylation, methylated 23S rRNA, had an affinity for the ermC' methyltransferase similar to that of unmethylated 23S rRNA . The binding affinity to 23S rRNA and the kinetics of the interaction were not detectably affected by the presence of AdoMet . The binding of ermC' methyltransferase to 23S rRNA had an unfavorable van't Hoff enthalpy (delta H = +6.2 kcal mol-1) and was driven by entropy (delta S = +56.2 cal mol-1 deg-1) . The interaction between the two ligands involved at most two to three ionic pairings, and nonelectrostatic interactions contributed approximately 85% of the binding energy . The structural aspect of the interaction was investigated by probing with dimethyl sulfate, for ermC' methyltransferase dependent protection of 23S rRNA . A region of protection was detected, in the vicinity of the central loop of rRNA domain V and surrounding the site of methylation.

J Mol Biol, 1990 Jun 20, 213(4), 739 - 47
Expression and cloning of complementary DNA for a human enzyme that repairs O6-methylguanine in DNA; Hayakawa H et al.; A cell line with an increased resistance to alkylating agents and an extremely high level of O6-methylguanine-DNA methyltransferase activity was isolated after transfection of methyltransferase-deficient Mer- cells with a cDNA library, prepared from methyltransferase-proficient human Mer+ (Raji) cells . Sodium dodecyl sulfate/polyacrylamide gel electrophoresis analysis revealed that a protein, with a molecular weight of approximately 25,000, accepted 3H label from DNA that had been treated with {3H}methylnitrosourea . Since the cDNA for methyltransferase was integrated into the chromosomal DNA, it was recovered by using the polymerase chain reaction . When the cDNA placed in an expression vector p500 was introduced into Mer- cells, the cells acquired an increased resistance to alkylating agents and exhibited a high level of O6-methylguanine-DNA methyltransferase activity . From the transformants the cDNA could be recovered as a part of the autonomously replicating plasmid . The nucleotide sequence of the cDNA was determined, and an open reading frame comprising 207 amino acid residues was found . The molecular weight of methyltransferase, calculated from the predicted amino acid sequence, was 21,700 . The predicted amino acid sequence of the human methyltransferase exhibits an intensive homology with those of the bacterial counterparts, Ada and Ogt proteins of Escherichia coli and Dat protein of Bacillus subtilis, especially around possible methyl acceptor sites.

Biochemistry, 1990 Jun 19, 29(24), 5676 - 81
DNA strand scission by the novel antitumor antibiotic leinamycin; Hara M et al.; Leinamycin is a recently discovered antitumor antibiotic with an unusual 1,3-dioxo-1,2-dithiolane structure . It preferentially inhibits the incorporation of {3H}thymidine into the acid-insoluble fraction of Bacillus subtilis . In vitro, leinamycin causes single-strand cleavage of supercoiled double-helical pBR322 DNA in the presence of thiol cofactors . Scavengers of oxygen radical did not supress the DNA-cleaving activity . Thiol-activated leinamycin binds calf thymus DNA at 4 degrees C and thermal treatment of the leinamycin-DNA adduct released a chemically modified leinamycin from the complex . The lack of cytotoxicity and DNA-cleaving activity for S-deoxyleinamycin indicates that the 1,3-dioxo-1,2-dithiolane moiety is essential for the activity of leinamycin . Thus, the primary cellular target of leinamycin appears to be DNA . It binds DNA and causes single-strand break at low concentrations, which may account for the potent antitumor activity.

Biochemistry, 1990 Jun 19, 29(24), 5797 - 806
The role of lysine-234 in beta-lactamase catalysis probed by site-directed mutagenesis; Ellerby LM et al.; Lys-234 has been postulated to participate in beta-lactamase catalysis by acting as an electrostatic anchor for the C3 carboxylate of penicillins {Herzberg, O., & Moult, J . (1987) Science 236, 694-701} . To test this hypothesis, site-directed mutagenesis was used to convert the Lys-234 in Bacillus licheniformis beta-lactamase into Glu-234 or Ala-234 . The wild-type, Glu-234, and Ala-234 beta-lactamases have been expressed in Bacillus subtilis and purified to homogeneity . The wild-type, K234E, and K234A enzymes have virtually identical circular dichroism and fluorescence spectra, similar thermal stabilities at neutral pH, and the same susceptibilities to proteolysis, indicating the lack of significant structural perturbation caused by the mutation . At acidic and basic pH the mutant enzymes have the same native circular dichroism as the wild-type enzyme but the thermal stability is significantly different . The mutations cause perturbations of the pK values of the ionizing groups responsible for the pH dependence of the catalytic reaction in both the free enzyme and the E.S complex . As expected, conversion of Lys-234 to Ala or Glu decreased substrate binding (Km) by 1-2 orders of magnitude for several penicillin and cephalosporin substrates at neutral and higher pH . However, at low pH, Km is essentially the same for the K234E and K234A enzymes as for the wild-type enzyme . Furthermore, decreases of 2-3 orders of magnitude in kcat were also observed, indicating substantial effects on the transition-state binding, as well as on ground-state binding . Surprisingly, changing the C3 carboxylate of phenoxymethylpenicillin to a hydroxymethyl group led to little difference in kinetic properties with the K234E or K234A enzyme . The results of this investigation indicate the Lys-234 is an important active-site residue involved in both ground-state and transition-state binding.

J Bacteriol, 1990 Jun, 172(6), 2979 - 85
A mollicute (mycoplasma) DNA repair enzyme: purification and characterization of uracil-DNA glycosylase; Williams MV et al.; The DNA repair enzyme uracil-DNA glycosylase from Mycoplasma lactucae (831-C4) was purified 1,657-fold by using affinity chromatography and chromatofocusing techniques . The only substrate for the enzyme was DNA that contained uracil residues, and the Km of the enzyme was 1.05 +/- 0.12 microM for dUMP containing DNA . The product of the reaction was uracil, and it acted as a noncompetitive inhibitor of the uracil-DNA glycosylase with a Ki of 5.2 mM . The activity of the enzyme was insensitive to Mg2+, Mn2+, Zn2+, Ca2+, and Co2+ over the concentration range tested, and the activity was not inhibited by EDTA . The enzyme activity exhibited a biphasic response to monovalent cations and to polyamines . The enzyme had a pI of 6.4 and existed as a nonspherical monomeric protein with a molecular weight of 28,500 +/- 1,200 . The uracil-DNA glycosylase from M . lactucae was inhibited by the uracil-DNA glycosylase inhibitor from bacteriophage PBS-2, but the amount of inhibitor required for 50% inhibition of the mycoplasmal enzyme was 2.2 and 8 times greater than that required to cause 50% inhibition of the uracil-DNA glycosylases from Escherichia coli and Bacillus subtilis, respectively . Previous studies have reported that some mollicutes lack uracil-DNA glycosylase activity, and the results of this study demonstrate that the uracil-DNA glycosylase from M . lactucae has a higher Km for uracil-containing DNA than those of the glycosylases of other procaryotic organisms . Thus, the low G + C content of the DNA from some mollicutes and the A.T-biased mutation pressure observed in these organisms may be related to their decreased capacity to remove uracil residues from DNA.

Antimicrob Agents Chemother, 1990 Jun, 34(6), 1220 - 6
Lipoteichoic acid as a new target for activity of antibiotics: mode of action of daptomycin (LY146032); Canepari P et al.; Daptomycin at the MIC allowed the cell mass increase of enterococcal strains and Bacillus subtilis to continue for 2 to 3 h at rates comparable to those of the controls . During this time the cell shape of the former changed to a rod configuration and that of the latter changed to long rods . In these bacteria, in which cell mass continued to increase, the MIC of daptomycin inhibited peptidoglycan synthesis by no more than 20% after 20 min of incubation and by roughly 50% after 2 h of incubation . Other macromolecules, such as DNA, RNA, and proteins, were only slightly affected . In contrast, incorporation of {14C}acetate into lipids was reduced by about 50% in the various strains after 20 min of treatment with daptomycin at the MIC . When the effect of the major lipid-containing polymers on synthesis was evaluated in detail, it was found that under conditions in which peptidoglycan and the other macromolecules mentioned above were inhibited only slightly (20%) and total lipid synthesis was inhibited by 50%, synthesis of teichoic and lipoteichoic acid was inhibited by 50 and 93%, respectively . Daptomycin was not found to enter the cytoplasm of either bacterial or mammalian cells . It bound, in the presence of calcium ions only, to whole bacterial cells, cell walls (both those that contained and those that did not contain membranes), and isolated membranes of bacterial and mammalian cells . Washing with EDTA removed daptomycin from all cells mentioned above and cell fractions except the bacterial membrane . It is concluded that lipoteichoic acid is most likely the primary target of daptomycin.

Photochem Photobiol, 1990 Jun, 51(6), 649 - 52
Hydroxyl radical quenching agents protect against DNA breakage caused by both 365-nm UVA and by gamma radiation; Peak MJ et al.; The ability of hydroxyl radical (.OH) scavengers to reduce DNA breakage in isolated DNA from Bacillus subtilis by either gamma radiation or monochromatic radiation in the UVA region (365 nm) was examined by comparing dose reduction factors (the ratio of dose required to induce n DNA breaks in the absence to the presence of quencher) . Previous data have demonstrated that acetate, formate, azide, and mannitol protect supercoiled DNA against gamma-radiation-induced ssb (single-strand breaks-relaxation of supercoil by first nick) in close agreement with the rate at which their solutions quench .OH . Here we show that these quenchers also protect against 365-nm-induced ssb . The ratios for protection against 365-nm induced DNA ssb in isolated B . subtilis DNA by the four quenchers are also in proportion to their ability to quench .OH . In view of the diverse chemical nature of the quenchers and the wide range of concentrations involved, these findings are evidence that both these radiations may induce ssb in DNA via a common step that might involve .OH.

J Bacteriol, 1990 Jun, 172(6), 3481 - 4
DNA cloning and organization of the Staphylococcus aureus gyrA and gyrB genes: close homology among gyrase proteins and implications for 4-quinolone action and resistance; Hopewell R et al.; Staphylococcus aureus gyrA and gyrB genes, which encode the DNA gyrase A and B proteins, have been isolated and found to map contiguously . DNA sequence analysis revealed close homology between the S . aureus gyrase subunits and their counterparts in Bacillus subtilis and Escherichia coli, including several conserved amino acid residues whose substitution in E . coli confers resistance to 4-quinolones . These results are discussed in regard to quinolone resistance mechanisms in S . aureus.

J Bacteriol, 1990 Jun, 172(6), 3138 - 45
Use of a conditionally lethal gene in Anabaena sp . strain PCC 7120 to select for double recombinants and to entrap insertion sequences; Cai YP et al.; Use of the sacB gene (J . L . Ried and A . Collmer, Gene 57:239-246, 1987) provides a simple, effective, positive selection for double recombinants in Anabaena sp . strain PCC 7120, a filamentous cyanobacterium . This gene, which encodes the secretory levansucrase of Bacillus subtilis, was inserted into the vector portion of a suicide plasmid bearing a mutant version of a chromosomal gene . Cells of colonies in which such a plasmid had integrated into the Anabaena chromosome through single recombination were plated on solid medium containing 5% sucrose . Under this condition, the presence of the sacB gene is lethal . A small fraction of the cells from initially sucrose-sensitive colonies became sucrose resistant; the majority of these sucrose-resistant derivatives had undergone a second recombinational event in which the sacB-containing vector had been lost and the wild-type form of the chromosomal gene had been replaced by the mutant form . By the use of this technique, we mutated two selected genes in the chromosome of Anabaena sp . strain PCC 7120 . The conditionally lethal nature of the sacB gene was also used to detect insertion sequences from this Anabaena strain . Sucrose-resistant colonies derived from cells bearing a sacB-containing autonomously replicating plasmid were analyzed . Five different, presumed insertion sequences were found to have inserted into the sacB gene of the plasmids in these colonies . One of them, denoted IS892, was characterized by physical mapping . It is 1.7 kilobases in size and is present in at least five copies in the genome of Anabaena sp . strain PCC 7120.

Mol Microbiol, 1990 Jun, 4(6), 1045 - 56
Role of His residues in Bacillus subtilis cytochrome b558 for haem binding and assembly of succinate: quinone oxidoreductase (complex II); Friden H et al.; Cytochrome b558 in the cytoplasmic membrane of Bacillus subtilis constitutes the anchor and electron acceptor to the flavoprotein (Fp) and iron-sulphur protein (Ip) in succinate:quinone oxidoreductase, and seemingly contains two haem groups . EPR and MCD spectroscopic data indicate bis-imidazole ligation of the haem . Apo-cytochrome was found in the membrane fraction of haem-deficient B . subtilis, suggesting that during biogenesis of the oxidoreductase the cytochrome b558 polypeptide is embedded into the membrane prior to the incorporation of haem and subsequent binding of Fp and Ip . The six His residues in cytochrome b558 were individually changed to Tyr to attempt identification of residues serving as haem axial ligands and to analyse the role of His residues for assembly and function of the oxidoreductase . From the properties of the mutants, His-47 can be excluded as a haem ligand . The remaining His residues (at positions 13, 28, 70, 113 and 155) are located in or close to four predicted transmembrane segments . The Tyr-28 and Tyr-70 mutant proteins appeared to lack one of the two haems . Only the Tyr-13 and Tyr-47 mutant cytochromes were found to function as anchors for Fp and Ip, but the Tyr-13 mutant cytochrome assembles into an enzymatically defective succinate:quinone oxidoreductase . It is concluded from a combination of the experimental findings, sequence comparisons and membrane topology data that His-28, His-70 and His-155 are probably haem axial ligands in a dihaem cytochrome b558 . His-70 and His-155 may be ligands to the same haem.

Antimicrob Agents Chemother, 1990 Jun, 34(6), 1237 - 43
Induction of the SOS response in Escherichia coli by azidothymidine and dideoxynucleosides; Mamber SW et al.; A number of nucleosides with anti-human immunodeficiency virus (HIV) activity were evaluated in two colorimetric (beta-galactosidase) assays for induction of the SOS response in Escherichia coli . 3'-Azido-3'-deoxythymidine (azidothymidine; AZT), 2',3'-dideoxyadenosine (ddA), 2',3'-dideoxyguanosine (ddG), and 2',3'-dideoxyinosine (ddI) induced cell filamentation (sulA) and prophage lambda in well-agar diffusion and liquid microsuspension assays . AZT was approximately 100 times more potent than the dideoxypurine nucleosides, inducing sulA at less than 100 ng/ml . 2',3'-Dideoxythymidine (ddT) and 2',3'-dideoxy-2',3'-didehydrothymidine (D4T) induced sulA at 100 to 1,000 micrograms/ml, while 2',3'-dideoxycytidine (ddC) weakly induced prophage lambda . Activity relationships thus were AZT greater than ddA greater than or equal to ddI greater than or equal to ddG greater than ddT = D4T greater than ddC . ddA and ddI had equivalent activities in agar diffusion assays, but different activity profiles were observed in liquid microsuspension assays . The differences may be related to drug metabolism . AZT and ddA showed marginal effects in a DNA repair (preferential toxicity) assay in which E . coli WP2 and CM871 uvrA recA lexA were used . Furthermore, none of the agents was able to preferentially inhibit Bacillus subtilis M45 recA relative to wild-type strain H17 . These data suggest that AZT and the dideoxynucleosides do not cause DNA lesions that are repairable by excision repair and/or error-free postreplication repair processes . Rather, the SOS response appears to be induced by DNA chain termination leading to the inhibition of DNA replication . Bacterial assays for induction of the SOS response may be useful as simple, rapid prescreens for the discovery of new anti-HIV agents . Moreover, such assays may provide an additional parameter in the evaluation of agents with demonstrated activity against HIV and other retroviruses.

J Biochem (Tokyo), 1990 Jun, 107(6), 799 - 801
The blasticidin S resistance gene (bsr) selectable in a single copy state in the Bacillus subtilis chromosome; Itaya M et al.; The blasticidin S resistance gene (bsr), originally isolated from Bacillus cereus, was studied in Bacillus subtilis . It was found that a 617 bp fragment including the intact bsr gene and its 5' flanking region could confer BS resistance on B . subtilis when integrated in its chromosome, even in a single copy state . The construction of a bsr gene cassette and its practical application as a novel selection marker for B . subtilis are reported.

J Gen Microbiol, 1990 Jun, 136 ( Pt 6), 1137 - 43
Expression of levansucrase-beta-galactosidase hybrids inhibits secretion and is lethal in Bacillus subtilis; Zagorec M et al.; The lacZ gene of Escherichia coli was fused to several positions downstream from the 5' end of the Bacillus subtilis sacB gene, which encodes levansucrase (LS), a sucrose-inducible extracellular enzyme . Effects of hybrid protein expression in B . subtilis were studied . Several fusions were tested, and two significantly interfered with growth of cells and with LS secretion when induced with sucrose . Chromosomal amplification of the fusions, leading to strong expression of the hybrid proteins, completely blocked LS secretion and was lethal for B . subtilis when expression was induced.

J Bacteriol, 1990 Jun, 172(6), 3503 - 6
Characterization of an inducible oxidative stress system in Bacillus subtilis; Bol DK et al.; Exponentially growing cells of Bacillus subtilis demonstrated inducible protection against killing by hydrogen peroxide when prechallenged with a nonlethal dose of this oxidative agent . Cells deficient in a functional recE+ gene product were as much as 100 times more sensitive to the H2O2 but still exhibited an inducible protective response . Exposure to hydrogen peroxide also induced the recE(+)-dependent DNA damage-inducible (din) genes, the resident prophage, and the product of the recE+ gene itself . Thus hydrogen peroxide is capable of inducing the SOS-like or SOB system of B . subtilis . However, the induction of this DNA repair system by other DNA-damaging agents is not sufficient to activate the protective response to hydrogen peroxide . Therefore, at least one more regulatory network (besides the SOB system) that responds to oxidative stress must exist . Furthermore, the data presented indicate that a functional catalase gene is necessary for this protective response.

J Bacteriol, 1990 Jun, 172(6), 3435 - 43
Studies of sigma D-dependent functions in Bacillus subtilis; Marquez LM et al.; Gene expression in Bacillus subtilis can be controlled by alternative forms of RNA polymerase programmed by distinct sigma factors . One such factor, sigma D (sigma 28), is expressed during vegetative growth and has been implicated in the transcription of a regulon of genes expressed during exponential growth and the early stationary phase . We have studied several functions related to flagellar synthesis and chemotaxis in B . subtilis strains in which sigma D is missing or is present at reduced levels . Previous studies showed that a null mutant, which contains a disrupted copy of the sigma D structural gene (sigD), fails to synthesize flagellin and grows as long filaments . We now show that these defects are accompanied by the lack of synthesis of the methyl-accepting chemotaxis proteins and a substantial decrease in two autolysin activities implicated in cell separation . A strain containing an insertion upstream of the sigD gene that reduces the level of sigma D protein grew as short chains and was flagellated but was impaired in chemotaxis and/or motility . This reduced level of sigma D expression suggests that the sigD gene may be part of an operon . A strain containing an insertion downstream of the sigD gene expressed nearly wild-type levels of sigma D protein but was also impaired in chemotaxis and/or motility, suggesting that genes downstream of sigD may also be involved in these functions . Genetic experiments demonstrate that sigD is allelic to the flaB locus, which was initially isolated as a locus affecting flagellin expression (G . F . Grant and M . I . Simon, J . Bacteriol . 99:116-124, 1969).

J Bacteriol, 1990 Jun, 172(6), 3257 - 63
Overproduction, purification, and characterization of Bacillus subtilis RNA polymerase sigma A factor; Chang BY et al.; By use of a T7 expression system, large amounts of active Bacillus subtilis RNA polymerase sigma A factor were produced in Escherichia coli cells . This overproduced protein was found in the form of inclusion bodies and constituted 40% of the total cellular protein . Because of the ease of isolation of the inclusion bodies and the acidic properties of sigma A, the protein was purified to more than 99% purity and the yield was about 90 mg/liter of culture . Gel mobility, antigenicity, specificity of promoter recognition, and N-terminal amino acid sequence of the overproduced sigma were found to be the same as those of native sigma A . Partial proteolysis analysis of sigma A protein suggested the presence of a protease-sensitive surface region in the C-terminal part of the sigma A protein . The promoter -10 binding region of sigma A was less sensitive to proteases and was probably involved in a hydrophobic, tightly folded domain of sigma A protein.

Gene, 1990 May 31, 90(1), 153 - 5
Nucleotide sequence of the sacS locus of Bacillus subtilis reveals the presence of two regulatory genes; Zukowski MM et al.; The nucleotide sequence of 3 kb of Bacillus subtilis chromosomal DNA, including sacS, reveals that the regulatory locus for levansucrase synthesis consists of two genes, sacX and sacY . The sacX gene product is remarkably similar to sucrose-specific enzyme II of the B . subtilis phosphoenolpyruvate-dependent phosphotransferase system . The product of sacY is similar, both in amino acid sequence and most probably in its function, to BglG, an Escherichia coli transcriptional antitermination factor.

Biochem Biophys Res Commun, 1990 May 31, 169(1), 297 - 301
Bacillus amyloliquefaciens alpha-amylase signal sequence fused in frame with human proinsulin is properly processed by Bacillus subtilis cells; Novikov AA et al.; The plasmid pBINS1, containing the promoter, SD and leader peptide sequences of Bacillus amyloliquefaciens alpha-amylase gene and 267 bp long sequence coding for human proinsulin directs the efficient synthesis of hybrid preproinsulin, as well as quantitative secretion of proinsulin outside of protease-deficient Bacillus subtilis AJ73 cells . The recombinant proinsulin has been isolated from the culture medium and its N-terminal sequence shown to be identical with that of natural human prohormone.

Nucleic Acids Res, 1990 May 25, 18(10), 2881 - 6
Functional analysis of the Bacillus subtilis bacteriophage SPP1 pac site; Bravo A et al.; Encapsidation of the DNA of the virulent Bacillus subtilis phage SPP1 follows a processive unidirectional headful-mechanism and initiates at a unique genomic location (pac) . We have cloned a fragment of SPP1 DNA containing the pac site flanked by reporter genes into the chromosome of B . subtilis . Infection of such cells with SPP1 led to highly efficient packaging, initiated at the inserted pac site, of chromosomal DNA . The directionality in the packaging of this DNA was the same as observed with vegetative phage DNA . Mutagenizing the chromosomal pac insert defined an 83 base pair segment containing the pac cleavage site which is sufficient to direct phage specific DNA encapsidation . The packaging recognition signal as defined can also be utilized by the SPP1 related phages 41c, SF6 and rho 15.

Science, 1990 May 25, 248(4958), 1012 - 6
A novel nucleoprotein complex at a replication origin; Serrano M et al.; The viral protein p6, required for the protein-primed initiation of replication of Bacillus subtilis phage phi 29, forms a nucleoprotein complex at the viral replication origins that shows novel features . Deoxyribonuclease I and hydroxyl radical footprinting data, as well as the induction of positive supercoiling, support a model in which a DNA right-handed superhelix tightly wraps around a multimeric p6 core . The interaction occurs through the DNA minor groove . The activity of p6 not only requires the formation of the complex but also its correct positioning, indicating that the other proteins involved in the initiation of replication recognize, at a precise position, either the p6 core or the DNA conformational change induced by p6.

J Mol Biol, 1990 May 20, 213(2), 227 - 8
Crystallization of the arginine-dependent repressor/activator AhrC from Bacillus subtilis; Boys CW et al.; The arginine-dependent repressor/activator AhrC from Bacillus subtilis has been crystallized in space group C222(1), with unit cell dimensions a = 229.8 A, b = 72.8 A, c = 137.7 A and one aporepressor hexamer per asymmetric unit . Preliminary X-ray photographs show measurable intensities beyond 3.0 A.

J Biol Chem, 1990 May 5, 265(13), 7100 - 3
Control of the position of RNase P-mediated transfer RNA precursor processing; Carter BJ et al.; Two Bacillus subtilis tRNA(His) precursors (Green, C . J., and Vold, B . S . (1988) J . Biol . Chem . 263, 652-657) were processed by Escherichia coli RNase P in the presence of varying {Mg2+} . The wild type precursor was processed under all conditions to afford a single tRNA product containing 8 base pairs in the acceptor stem . In contrast, the position of processing of a mutant tRNA(His) precursor (containing a G27----A27 alteration) was shown to be condition-dependent . Processing occurred at A27 under conditions consistent with formation of an A27-C100 base pair in the acceptor stem but at G28 under conditions that disfavored base pair formation . The ability to control the site of RNase P-mediated tRNA precursor processing is unprecedented and permits analysis of the chemical factors that promote processing.

Mol Microbiol, 1990 May, 4(5), 855 - 60
Wolinella succinogenes fumarate reductase contains a dihaem cytochrome b; Kortner C et al.; The fumarate reductase operon of Wolinella succinogenes is made up of three structural genes (frd-CAB) . The frdC gene was located next to the promoter region and identified as the cytochrome b structural gene encoding 256 amino acid residues . The N-terminal amino acid sequences of seven fragments derived from the cytochrome b moiety of the enzyme all mapped within the frdC gene . This suggested that the enzyme contained only one species of cytochrome b . Re-evaluation of earlier measurements of subunit composition, haem B content and molecular weight led to the conclusion that the enzyme contained one molecule of cytochrome b with two haem B groups . The hydropathy plot of the amino acid sequence predicted five membrane-spanning hydrophobic segments, the first four of which contained a single histidine residue each . These residues could form the axial ligands to the two haem B groups . FrdC was found to be homologous with the cytochrome b (SdhC) of the Bacillus subtilis succinate dehydrogenase, but not with the hydrophobic subunits of the fumarate reductase or succinate dehydrogenase of Escherichia coli.

Mol Gen Genet, 1990 May, 221(3), 427 - 34
Functional analysis of the 5' regulatory region and the UUG translation initiation codon of the Arthrobacter oxidans 6-hydroxy-D-nicotine oxidase gene; Mauch L et al.; A functional analysis of the Arthrobacter oxidans 6-hydroxy-D-nicotine oxidase (6-HDNO) gene promoter (-35 region TTGACA and -10 region TATCAAT) and the UUG translation start codon was performed using site-directed mutagenesis . Deletion of the C residue from the -10 promoter region or mutations introduced upstream of the -10 region resulted in an increased 6-HDNO expression in Escherichia coli cells in vivo and in both E . coli and A . oxidans coupled transcription-translation systems in vitro . From the identical behaviour of 6-HDNO promoter mutants in the heterologous and homologous systems, it is concluded that A . oxidans harbours an RNA polymerase functionally homologous to the E . coli sigma 70 and Bacillus subtilis sigma 43 polymerases . Replacement of the TTG codon (UUG translation initiation codon) with ATG led to a 3.7-fold increase in 6-HDNO expression in E . coli . This effect was less pronounced at higher promoter strengths, from 3.7 in the case of the 6-HDNO wild-type promoter, to 2.5 in the case of the consensus -10 region and to 1.7 in the case of the tac promoter . A double point mutation introduced close to the ribosome binding site resulted in almost the same increase in 6-HDNO expression (3.1-fold) as the TTG-to-ATG exchange . The failure of cAMP to stimulate 6-HDNO expression in the A . oxidans system indicated that expression of this gene in stationary phase cells is not regulated by cAMP-catabolite repressore protein-mediated mechanism of catabolite repression.(ABSTRACT TRUNCATED AT 250 WORDS)

J Biochem (Tokyo), 1990 May, 107(5), 732 - 9
Purification and characterization of an initiation protein for chromosomal replication, DnaA, in Bacillus subtilis; Fukuoka T et al.; Bacillus subtilis DnaA protein was overproduced by a recombinant plasmid containing B . subtilis dnaA gene in a mutant Escherichia coli strain which is deficient in its own DnaA and RNaseH . The protein was purified to near homogeneity as judged by SDS-PAGE analysis . The purified protein binds preferentially to DNA fragments which are derived from flanking regions of the B . subtilis dnaA gene and contain various numbers of the repeat of 9 nucleotides, TTATCCACA, and closely related sequences . The purified protein binds ATP with high affinity (Kd = 0.02 microM) and ADP with less affinity, but does not bind cAMP . ATP stimulates the binding of the DnaA protein to the repeated sequences . DNaseI footprinting experiments demonstrated that the DnaA bound first to the consensus 9-mer and then to sequences differing by one base from the consensus . Sequences differing by two bases from the consensus were bound by the DnaA only when they were located contiguous to the strong DnaA-boxes . The three DnaA-box clusters, incA, incB, and incC, derived from the replication origin region of the B . subtilis chromosome showed different levels of growth inhibition when they were introduced into B . subtilis . We demonstrated by assaying competition for DnaA-binding among the DnaA-box clusters that there is a good correlation between the degree of growth inhibition by DnaA-box clusters in vivo and their strength of binding to the DNaA protein in vitro.

Appl Environ Microbiol, 1990 May, 56(5), 1429 - 34
Secretion of the alpha-galactosidase from Cyamopsis tetragonoloba (guar) by Bacillus subtilis; Overbeeke N et al.; A fusion of DNA sequences encoding the SPO2 promoter, the alpha-amylase signal sequence from Bacillus amyloliquefaciens, and the mature part of the alpha-galactosidase from Cyamopsis tetragonoloba (guar) was constructed on a Bacillus subtilis multicopy vector . Bacillus cells of the protease-deficient strain DB104 harboring this vector produced and secreted the plant enzyme alpha-galactosidase up to levels of 1,700 U/liter . A growth medium suppressing the residual proteolytic activity of strain DB104 was used to reach these levels in a fermentor . Purification of the secreted product followed by NH2-terminal amino acid sequencing showed that the alpha-amylase signal sequence had been processed correctly . The molecular mass of the product estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was slightly lower than that of the plant purified enzyme, which is most likely due to glycosylation of the latter . The alpha-galactosidase product was active both on the artificial substrate para-nitrophenyl-alpha-D-galactopyranoside and on the galactomannan substrate, guar gum . The activity of this Bacillus sp.-produced enzyme was similar to that of the glycosylated enzyme purified from guar seeds, indicating that glycosylation has no essential function for enzyme activity.

Mikrobiologiia, 1990 May-Jun, 59(3), 387 - 93
{Change in the level of SH-compounds in Escherichia coli and Bacillus subtilis cultures during transition to the stationary phase}; Smirnova GV et al.; The redox potential "jump" recorded earlier for aerobic Escherichia coli and Bacillus subtilis cultures passing to the stationary phase was shown to result from a rise in the content of SH-compounds in the medium and on the cell surface . The effect was absent from anaerobic cultures as well as aerobic E . coli cells treated with the protonophore CICCP . Apparently, the elevated content of SH-compounds outside the cell upon starvation is part of the process which leads to a shift in the ratio between low-molecular-mass thiols and disulfides (towards disulfides inside the cell and towards thiols outside the cell) and is associated with a drop in the intracellular pH . Therefore, the entire metabolism of the cell can change as a result of reactions with the SH-groups of functionally significant compounds when the cell enters the stationary phase upon starvation.

Genetika, 1990 May, 26(5), 826 - 32
{Absence of various manifestations of adaptive response in Bacillus subtilis transformants}; Zherebtsov SV; Competent cells of Bacillus subtilis are more sensitive to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) than total population, exhibit higher level of spontaneous mutations to kanamycin resistance . However, the absolute number of mutated transformants doesn't rise with MNNG treatment in the range of 5 to 50 micrograms per ml . The adaptation to low concentrations of MNNG affects neither spontaneous nor MNNG-induced mutagenesis in the competent (transformed) cells, in contrast to their resistance which is stronger for the adapted transformants . The transformation by MNNG-treated plasmid pUB 110 doesn't reveal any difference between adapted and non-adapted cultures in transformation efficiency decline.

Int J Food Microbiol, 1990 May, 10(3-4), 291 - 301
Effect of redox potential on Bacillus subtilis and Bacillus licheniformis in broth and in pasteurized sausage mixtures; Rodel W et al.; Bacillus licheniformis (a facultatively anaerobe) and B . subtilis (aerobic) may cause spoilage in certain meat products pasteurized in hermetically sealed containers if these are stored with inadequate refrigeration . After having optimized a method for determination of the redox potential in sausage mixtures, we investigated the contribution of low redox potential (Eh) and anaerobiosis to the inhibition of these bacilli . With the Eh values encountered in pasteurized meat products there was no effect of the redox potential on growth of B . licheniformis, either in model broths or in mildly heated bologna-type sausage mixtures . When cultured in a model broth with head space, B . subtilis grew to about 10(7)/ml and was little affected by the initial Eh value (range between +250 and -150 mV) and the initial dissolved oxygen concentration . During exponential growth of this bacterium in the absence of oxygen, the Eh value increased by about 180 mV . In sausage mixture with air inclusions, B . subtilis attained slightly higher cell densities than in mixtures protected against air uptake but it was subsequently overgrown by facultative anaerobic bacilli . We conclude that knowledge of the Eh value of a meat product does not permit a prediction of the growth potential of bacilli in pasteurized meat products, and that a sufficient heat treatment and proper refrigeration are essential to control these bacteria.

Mol Gen Genet, 1990 May, 221(3), 486 - 90
Multiple active forms of a novel serine protease from Bacillus subtilis; Bruckner R et al.; We have cloned and sequenced a gene (epr) encoding a novel serine protease from Bacillus subtilis . Several active forms of the enzyme with molecular masses between 40 and 34 kDa were found in the medium of B . subtilis cultures containing the epr gene cloned on a plasmid . Deletions at the 3' end of the gene, removing up to 240 amino acids of the reading frame, abolished the expression of the larger species but did not affect the expression of the 34 kDa enzyme . The C-terminal third of the protein is therefore not required for protease activity . The size variation of the active forms expressed by the complete epr gene appears to be the result of partial removal of the C-terminus either by processing or degradation . Thus, the epr gene consists of two domains, one encoding a serine protease homologous to subtilisin and the other a C-terminus of unknown function.

Genes Dev, 1990 May, 4(5), 860 - 72
A Bacillus subtilis regulatory gene product for genetic competence and sporulation resembles sensor protein members of the bacterial two-component signal-transduction systems; Weinrauch Y et al.; A Bacillus subtilis gene, required for genetic competence, was identified immediately upstream from the previously characterized gene comA . The comA gene product has been found to exhibit amino acid sequence similarity to the so-called effector class of signal-transduction proteins . DNA sequencing of the new determinant, named comP, revealed that the carboxy-terminal domain of the predicted ComP protein is similar in amino acid sequence to that of several sensor members of the bacterial two-component signal-transduction systems . The predicted amino-terminal domain contains several hydrophobic segments, postulated to be membrane-spanning . In vitro-derived comP disruptions are epistatic on the expression of all late competence genes tested, including comG, comC, comD, and comE, but not on expression of the early gene comB . Although comA has its own promoter, some transcription of comA, especially later in growth, occurs via readthrough from comP sequences . A roughly twofold epistatic effect of a comP disruption was noted on the downstream comA determinant, possibly due to interruption of readthrough transcription from comP to comA . Overexpression of comA fully restored competence to a comP mutant, providing evidence that ComA acts after ComP, and consistent with a role for the latter protein in activation of the former, possibly by phosphorylation . ComP probably is involved in transmitting information concerning the nutritional status of the medium, particularly the presence of nitrogen- and carbon-containing nutrients . ComP was also shown to play a role in sporulation, at least partly interchangeable with that of SpoIIJ, another putative sensor protein.

Antimicrob Agents Chemother, 1990 May, 34(5), 796 - 802
Bacitracin-induced proteins in Bacillus subtilis and Bacillus thuringiensis and their relationship with resistance; Garcia-Patrone M; Bacitracin induced one protein (bacitracin-induced protein {BIP}) in Bacillus thuringiensis and two proteins (BIP1 and BIP2) in Bacillus subtilis that were localized in the membrane . Divalent cations acted as cofactors for induction in all three cases . Growth was initially inhibited by the antibiotic, but following induction of proteins growth resumed . B . subtilis cells possessing BIPs were able to duplicate at a normal rate in the presence of bacitracin . The amount of B . subtilis BIPs diminished markedly after a few divisions in the absence of the antibiotic and the organism simultaneously reverted to the susceptible state . Induction of the proteins did not take place after the fourth or fifth hour of the stationary phase . The B . thuringiensis BIP was also induced by vancomycin . Bacitracin did not induce the synthesis of specific proteins in susceptible (Micrococcus lysodeikticus) or outer membrane-possessing resistant bacteria (Escherichia coli).

Antimicrob Agents Chemother, 1990 May, 34(5), 781 - 5
Lysis and aberrant morphology of Bacillus subtilis cells caused by surfactants and their relation to autolysin activity; Tsuchido T et al.; The surfactants tested in this study lysed Bacillus subtilis 168 cells at the logarithmic growth phase . Results obtained with inhibitors and a mutant that had defective autolytic enzymes suggested that cell lysis resulted from the deregulation of autolysin activity . The addition of surfactants at sublytic concentrations produced twisted cells, filamented cells, or both . Autolysins extracted with 5 M LiCl from the cell wall fraction and lysozyme added to cells that were treated with surfactants restored the apparently normal cell rod morphology, suggesting that surfactants interfere with the role of autolysins in normal construction of the cell envelope . The rates of cellular autolysis and autolysin activity remaining in growing cells after exposure to a surfactant at a sublytic concentration decreased, although the rate of turnover of cell wall peptidoglycan was the same as that of control cells . Surfactants were suggested to interact with the regulatory system of autolysins and, thus, to affect the activities of autolysins in B . subtilis cells and to cause either morphological changes or cell autolysis, depending on the concentration of surfactants.

J Clin Microbiol, 1990 May, 28(5), 1068 - 70
Modified method for testing the quality of albumin-containing enrichments used in growth media for mycobacteria; Butler WR et al.; Many commercially available media for cultivation of mycobacteria have failed to support the growth of these organisms . This is especially true of media prepared with albumin-containing enrichments . Earlier, we developed a method for rapid identification of good albumin enrichments for agar-based media used to test the susceptibility of tubercle bacilli to pyrazinamide . The method was modified to make testing of the acceptability of albumin enrichments for primary isolation media for mycobacteria possible . We describe here a simple turbidimetric test using a specific Bacillus subtilis strain to assay quickly (24 h) different lots of albumin-containing enrichments that may be used in the preparation of growth media for mycobacteria.

J Bacteriol, 1990 May, 172(5), 2755 - 61
Highly repetitive DNA sequences in cyanobacterial genomes; Mazel D et al.; We characterized three distinct families of repeated sequences in the genome of the cyanobacterium Calothrix sp . strain PCC 7601 . These repeated sequences were present at a level of about 100 copies per Calothrix genome and consisted of tandemly amplified heptanucleotides . These elements were named short tandemly repeated repetitive (STRR) sequences . We used the three different Calothrix STRR sequences as probes to perform Southern hybridization experiments with DNAs extracted from various cyanobacterial strains, Bacillus subtilis, and Escherichia coli . The three different STRR sequences were found as repetitive genomic DNA components specific to the heterocystous strains tested . The role of the STRR sequences, as well as their possible use in taxonomic studies, is discussed.

J Bacteriol, 1990 May, 172(5), 2667 - 74
Characterization of PBSX, a defective prophage of Bacillus subtilis; Wood HE et al.; PBSX, a defective Bacillus subtilis prophage, maps to the metA-metC region of the chromosome . DNA (33 kilobases) from this region of the chromosome was cloned and analyzed by insertional mutagenesis with the integrating plasmid pWD3 . This plasmid had a promoterless alpha-amylase gene (amyL) that provided information on the direction and level of transcription at the site of integration . Transcription under the control of the PBSX repressor proceeded in the direction metA to metC over a distance of at least 18 kilobases . Electrophoretic analysis of proteins produced by different integrant strains upon PBSX induction and by fragments subcloned in Escherichia coli allowed the identification of early and late regions of the prophage . A set of contiguous fragments directing mutagenic integration suggested that the minimum size of an operon that encodes phage structural proteins is 19 kilobases . The adaptation of PBSX transcriptional and replicational functions to a chromosomally based, thermoinducible expression system is discussed.

J Bacteriol, 1990 May, 172(5), 2250 - 8
Cloning and characterization of the hemA region of the Bacillus subtilis chromosome; Petricek M et al.; A 3.8-kilobase DNA fragment from Bacillus subtilis containing the hemA gene has been cloned and sequenced . Four open reading frames were identified . The first is hemA, encoding a protein of 50.8 kilodaltons . The primary defect of a B . subtilis 5-aminolevulinic acid-requiring mutant was identified as a cysteine-to-tyrosine substitution in the HemA protein . The predicted amino acid sequence of the B . subtilis HemA protein showed 34% identity with the Escherichia coli HemA protein, which is known to code for the NAD(P)H:glutamyl-tRNA reductase of the C5 pathway for 5-aminolevulinic acid synthesis . The B . subtilis HemA protein also complements the defect of an E . coli hemA mutant . The second open reading frame in the cloned fragment, called ORF2, codes for a protein of about 30 kilodaltons with unknown function . It is not the proposed hemB gene product porphobilinogen synthase . The third open reading frame is hemC, coding for porphobilinogen deaminase . The fourth open reading frame extends past the sequenced fragment and may be identical to hemD, coding for uroporphyrinogen III cosynthase . Analysis of deletion mutants of the hemA region suggests that (at least) hemA, ORF2, and hemC may be part of an operon.

J Bacteriol, 1990 May, 172(5), 2217 - 23
The 42- and 51-kilodalton mosquitocidal proteins of Bacillus sphaericus 2362: construction of recombinants with enhanced expression and in vivo studies of processing and toxicity; Broadwell AH et al.; After site-directed mutagenesis, the genes coding for the 42- and 51-kilodalton (kDa) mosquitocidal proteins of Bacillus sphaericus 2362 were placed under the regulation of the aprE (subtilisin) promoter of the Bacillus subtilis vector pUE (a derivative of pUB18) . The levels of expression of the gene products in B . subtilis DB104 and B . sphaericus 718 were assessed by bioassays with larvae of Culex pipiens and by Western immunoblots . The results indicated that a higher amount of protein was produced in B . subtilis DB104 . Electron microscopic examination of B . subtilis DB104 and B . sphaericus 718 containing the 42- and 51-kDa proteins indicated that amorphous inclusions accumulated in the former species and that crystals identical in appearance to that found in B . sphaericus 2362 were produced in the latter . Strains producing only the 42- or the 51-kDa protein were not toxic to larvae of C . pipiens . A mixture of both strains, a single strain producing both proteins, or a fusion of the 51- and the 42-kDa proteins was toxic . The amount of B . subtilis DB104 containing the 42- and the 51-kDa proteins necessary to kill 50% of the larvae of C . pipiens was 5.6 ng (dry weight) of cells per ml . This value was significantly lower than that for B . sphaericus 2362 (14 ng {dry weight} per ml) . Larvae consuming purified amorphous inclusions containing the 42-kDa protein degraded this protein this protein to primarily 39- and 24-kDa peptides, whereas inclusions with the 51-kDa protein were primarily degraded to a protein of 44 kDa . Past studies involving purified proteins from B . sphaericus 2362 indicate an associate of toxicity with the 39-kDa peptide . The results presented here suggest that the 44-kDa degradation product of the 51-kDa protein may also be required for toxicity.

Enzyme Microb Technol, 1990 May, 12(5), 330 - 6
Effect of medium composition on the maintenance of a recombinant plasmid in Bacillus subtilis; Shoham Y et al.; Recombinant plasmid pCED3 {confers beta-galactosidase production (LacZ+) and kanamycin resistance (Kmr)} in Bacillus subtilis was found to be both segregationally and structurally unstable . Since many solutions to segregational instability are already available, the problem of structural instability was specifically addressed by inclusion of kanamycin in the growth media . Culture instability was found to be highest in complex and defined media supporting high growth rates . Stabilization over the duration of the experiment (40 generations) was achieved by use of a recently developed chemically defined medium supporting a lower growth rate . Slowing down growth by decreasing temperature was much less effective . A major effect of the growth medium appears to be that of decreasing the growth rate advantage held by cells with plasmid deletions over parental cells containing the intact plasmid.

Gene, 1990 Apr 30, 89(1), 77 - 84
Nucleotide sequence of the Synechococcus sp . PCC7942 branching enzyme gene (glgB): expression in Bacillus subtilis; Kiel JA et al.; The nucleotide sequence of the Synechococcus sp . PCC7942 glgB gene has been determined . The gene contains a single open reading frame (ORF) of 2322 bp encoding a polypeptide of 774 amino acids (aa) with an Mr of 89,206 . Extensive sequence similarity exists between the deduced aa sequence of the Synechococcus sp . glgB gene product and that of the Escherichia coli branching enzyme in the middle portions of the proteins (62% identical aa) . In contrast, the N-terminal portions shared little homology . The sequenced region which follows glgB contains an ORF encoding 79 aa of the N terminus of a polypeptide that shares extensive sequence similarity (41% identical aa) with human and rat uroporphyrinogen decarboxylase . This suggests that the region downstream from glgB contains the hemE gene and, therefore, that the organization of genes involved in glycogen biosynthesis in Synechococcus sp . is different from that described for E . coli . A fusion gene was constructed between the 5' end of the Bacillus licheniformis penP gene and the Synechococcus sp . glgB gene . The fusion gene was efficiently expressed in the Gram+ micro-organism Bacillus subtilis and specified a branching enzyme with an optimal temperature for activity similar to the wild-type enzyme.

Eur J Biochem, 1990 Apr 30, 189(2), 221 - 7
Structural features of neutral protease from Bacillus subtilis deduced from model-building and limited proteolysis experiments; Signor G et al.; The overall folding of neutral protease from Bacillus subtilis has been predicted by computer-aided modelling, taking as a basis the known three-dimensional structure of thermolysin . As expected from the 50% similarity of sequence between the two proteins, the structure of B . subtilis protease is similar to that of thermolysin, including the two-domain topology and location of elements of regular secondary structure (helices and strands), whereas specific differences were predicted in loop regions . A protruding and loose loop predicted in B . subtilis has been detected also experimentally by a limited proteolysis approach . Incubation of B . subtilis protease at pH 9.0 for 24 h at room temperature with trypsin at 20:1 ratio (by mass) leads to a specific and almost quantitative fission of the Arg214-Asn215 peptide bond located in a highly exposed, and thus probably flexible, loop of the protease . On the other hand, thermolysin was completely resistant to tryptic hydrolysis when reacted under identical conditions . The 'nicked' B . subtilis protease can be isolated by gel filtration chromatography at neutral pH, whereas the two constituting fragments 1-214 and 215-300 are separated under protein-denaturing conditions . Overall, these results indicate that the limited proteolysis approach can pinpoint a peculiar difference in surface structure between the two similar protein molecules of B . subtilis neutral protease and thermolysin and emphasize the potential use of proteolytic enzymes as structural probes of globular proteins.

Biochem Biophys Res Commun, 1990 Apr 30, 168(2), 443 - 50
Stretch-activated composite ion channels in Bacillus subtilis; Zoratti M et al.; The presence of ion-conducting pores in the membrane of Bacillus subtilis giant protoplasts was discovered using the patch-clamp technique . Membrane stretch caused the activation of several conductances with values in the nS range . The observations indicate the presence of substate levels and of aggregates of channels behaving in a cooperative manner . Following repeated stretch cycles, the channels exhibited spontaneous activity . The characteristics of the electrical phenomena afterwards changed in time in a manner suggesting the decay of the giant channels into lower-conductance species, presumably corresponding to building blocks of the giant stretch-activated channels.

J Biol Chem, 1990 Apr 25, 265(12), 6845 - 50
Cloning, genetic organization, and characterization of a structural gene encoding bacillopeptidase F from Bacillus subtilis; Wu XC et al.; Bacillopeptidase F is an extracellular serine protease that is expressed at the beginning of the stationary phase . To study its structure, regulation of expression, and physiological roles, we have cloned and characterized the structural gene (bpf) encoding this protease from Bacillus subtilis . DNA sequence analysis suggests this protease is synthesized as a preproenzyme (Mr = 92,000) . Through processing at both the NH2 and COOH termini, it is gradually converted into various forms with molecular mass ranging from 80 to 48 kDa . Shortening the 3' end of bpf demonstrates that at least 290 amino acid residues from the COOH-terminus of bacillopeptidase F are not required for either catalytic activity or secretion . Bacillopeptidase F exhibits sequence similarity with several serine proteases . Its gene is found immediately downstream from the fts operon which was mapped at 135 degrees on the B . subtilis genetic linkage map . Inactivation of the chromosomal copy of bpf shows no effect on cell growth and sporulation . A triple protease-deficient strain (WB300 with the structural genes for bacillopeptidase F and two other major proteases inactivated) was constructed to serve as a better expression host for the production and secretion of foreign proteins.

J Mol Biol, 1990 Apr 20, 212(4), 661 - 8
Chloramphenicol-induced translational activation of cat messenger RNA in vitro; Dick T et al.; The expression of the chloramphenicol-inducible chloramphenicol acetyltransferase gene (cat) of the staphylococcal plasmid pUB112 is regulated at the post-transcriptional level . Previous in vivo analyses suggested that the antibiotic stalls ribosomes that are translating a regulatory leader peptide, and that a stalled ribosome activates the ribosome binding site of the acetyltransferase encoding sequence by opening an attenuating leader mRNA hairpin structure . To test this model, we used a Bacillus subtilis S-30 extract for an in vitro translation system and in vitro synthesized cat in RNAs . We showed that the leader portion of the cat transcript acts as a translational attenuator of cat gene expression in absence of chloramphenicol . The drug stimulates acetyltransferase synthesis by a leader mRNA-dependent activation of translation of the cat message . By using 5' end-labeled transcripts and employing the endogenous RNase activity of the S-30 extract we demonstrated that this activation is due to an antibiotic-induced stalling of a ribosome on cat leader mRNA.

J Mol Biol, 1990 Apr 20, 212(4), 645 - 60
Cascade regulation of spore coat gene expression in Bacillus subtilis; Zheng LB et al.; Endospores of the Gram-positive bacterium Bacillus subtilis are encased in a tough protein shell known as the coat . The coat is composed of a dozen or more different structural proteins . We report the identification of and studies on the regulation of promoters governing the expression of coat protein (cot) genes designated B to E encoding polypeptides of 59, 12, 11 and 24 kDa, respectively . We show that transcription of genes B, C and D is governed by single promoters and that transcription of gene E is governed by tandem promoters designated P1 and P2 . In extension of recent work on the transcription of cot gene A and the mother-cell regulatory genes gerE, sigK and spoIIID, we show that genes involved in coat formation are turned on in a regulatory cascade of at least four co-ordinately controlled gene sets . The cascade consists of: cotE as transcribed from its P1 promoter and spoIIID, which are turned on during hours three to four of sporulation; cotE as transcribed from its P2 promoter and sigK, which are turned on during hour five by the appearance of the product (a small DNA-binding protein) of spoIIID; cotA, cotD and gerE, which are turned on during hours five to six by the appearance of the product (sigma factor sigma K) of sigK; and cotB and cotC, which are turned on during hour seven by the appearance of the product (an inferred DNA-binding protein) of gerE . The cascade is hierarchical in that the first three gene sets each contain the regulatory gene that turns on the expression of the next gene set in the pathway . We also show that the level of expression of a member (cotC) of the terminal class of gene expression is strongly influenced by medium and that this effect directly or indirectly depends on the product of sporulation gene spoIV A.

Planta Med, 1990 Apr, 56(2), 187 - 9
A novel antibacterial diterpene from Premna schimperi; Habtemariam S et al.; A novel diterpene, (5R, 8R, 9S, 10R)-12-oxo-ent-3,13(16)-clerodien-15-oic acid, has been obtained from the leaves of Premna schimperi (Verbenaceae) using an anti-microbial bioassay-guided isolation procedure . The diterpene was identified on the basis of spectroscopic data and is active against the Gram-positive bacteria Staphylococcus aureus and Bacillus subtilis in the concentration range 20-25 micrograms ml-1.

Genes Dev, 1990 Apr, 4(4), 525 - 35
The Bacillus subtilis gene for the development transcription factor sigma K is generated by excision of a dispensable DNA element containing a sporulation recombinase gene; Kunkel B et al.; The structural gene (sigK) for the mother-cell RNA polymerase sigma-factor sigma K in Bacillus subtilis is a composite of two truncated genes, named spoIVCB and spoIIIC, which are brought together by site-specific recombination during sporulation . We now show that the recombination event is compartmentalized in that the mother cell, but not the forespore chromosome, undergoes rearrangement . We also show that spoIIIC (encoding the carboxy-terminal portion of sigma K) lies approximately 42 kb downstream of spoIVCB (encoding the amino-terminal portion) and that the joining of the truncated coding sequences is a reciprocal recombination event in which intervening DNA is deleted from the chromosome as a circle . The rearrangement is governed by the product of a gene named spoIVCA located in the excised DNA, as demonstrated by the observations (1) that the product of spoIVCA, but not the product of any other stage-IV sporulation gene tested, is required for the rearrangement, and (2) that the presence of a cloned copy of the rearranged sigK gene in the chromosome bypasses the requirement for the spoIVCA gene product in sporulation . Because cells engineered to contain an intact copy of sigK sporulate normally, we conclude that the sigK rearrangement is not essential for the control of gene expression during sporulation, and we infer the existence of an additional mechanism for restricting sigma K-directed transcription to the mother-cell chamber of the sporangium . Finally, the construction of a strain deleted for the entire sigK intervening sequence shows that the 42-kb element contains no genes essential for viability.

Farmaco, 1990 Apr, 45(4), 439 - 46
Biological studies on 1,2-benzisothiazole derivatives . III . Evaluation of antibacterial, antifungal and DNA-damaging activities of 1,2-benzisothiazolin-3-ones; Massimo G et al.; The in vitro evaluation of the antibacterial, antifungal and DNA-damaging properties of some 1,2-benzisothiazolin-3-one derivatives is described . Antibacterial activity against Gram+ microorganisms (Bacillus subtilis and Staphylococcus aureus) was demonstrated in some N-alkoxybenzyl, N-aryl and N-alkoxyphenyl derivatives . Antifungal activity turned out to be strongly affected by the substituents considered . Some derivatives exhibited selective activity against the fungi used . Poor antimicrobial activity was observed in the N-chlorocarbonylphenyl and N-carboxyamidophenyl derivatives . None of the compounds tested turned out to have any genotoxic properties.

Mol Gen Genet, 1990 Apr, 221(2), 267 - 72
Translational coupling in a penP-lacZ gene fusion in Bacillus subtilis and Escherichia coli: use of AUA as a restart codon; Peijnenburg AA et al.; An out-of-frame fusion between the penicillinase gene (penP) of Bacillus licheniformis and the beta-galactosidase gene (lacZ) of Escherichia coli was shown to direct the synthesis of an active beta-galactosidase with the same electrophoretic mobility as the wild-type protein, both in B . subtilis and E . coli . This synthesis was dependent on translation of the truncated penP gene and appeared to result from translational coupling . The fusion point between penP and lacZ contained the sequence AUAG, in which the UAG and AUA codons were in-frame with the penP and lacZ reading units, respectively . N-terminal amino acid sequence analysis of the beta-galactosidase protein suggested that, both in B . subtilis and E . coli, reinitiation of translation occurred at the AUA codon present at the gene fusion point.

Wei Sheng Wu Xue Bao, 1990 Apr, 30(2), 117 - 21
{Properties of two Bacillus subtilis bacteriophages}; Na SM et al.; Two lytic bacteriophages of Bacillus subtilis BF7658 were isolated . Their morphology, biological properties and physiochemical characteristics of their DNAs were compared . Electromicroscopic observation indicated that BS31 has a hexagonal head and contracted tail sheath, BS32 is a complex short-tailed phage and similar to phi 29 but differs from phi 29 in size and other properties . Two phages have a narrow spectrum of host range . The molecular weight of BS31 and BS32 DNAs are 62kb and 17kb respectively by EcoR1 endonuclease analysis . The G + C content of the DNAs are 45.7%(BS31) and 40.7%(BS32) . Structural proteins of BS31 gave two major bands and at least ten minor ones; BS32 gave three major bands and six minor ones by SDS-polyacralamide gel electrophoresis.

J Biochem (Tokyo), 1990 Apr, 107(4), 603 - 7
Cloning and characterization of a Bacillus subtilis gene homologous to E . coli secY; Nakamura K et al.; A 3.5-kb HindIII DNA fragment containing the secY gene of Bacillus subtilis has been cloned into plasmid pUC13 using the Escherichia coli secY gene as a probe . The complete nucleotide sequence of the cloned DNA indicated that it contained five open reading frames, and their order in the region, given by the gene product, was suggested to be L30-L15-SecY-Adk-Map by their similarity to the products of the E . coli genes . The region was similar to a part of the spc operon of the E . coli chromosome, although the genes for Adk and Map were not included . The gene product of the B . subtilis secY homologue was composed of 423 amino acids and its molecular weight was calculated to be 46,300 . The distribution of hydrophobic amino acids in the gene product suggested that the protein is a membrane integrated protein with ten transmembrane segments . The total deduced amino acid sequence of the B . subtilis SecY homologue shows 41.3% homology with that of E . coli SecY, but remarkably higher homologous regions (more than 80% identity) are present in the four cytoplasmic domains.

Mol Microbiol, 1990 Apr, 4(4), 543 - 51
Differential gene expression during sporulation in Bacillus subtilis: structure and regulation of the spoIIID gene; Stevens CM et al.; The gene spoIIID, which is essential for spore formation in Bacillus subtilis, was cloned and sequenced . It consists of one open reading frame which would encode a 93-amino-acid protein with a classic helix-turn-helix motif, characteristic of sequence-specific DNA-binding proteins . SpoIIID protein is a previously identified transcription factor, capable of altering the specificity of RNA polymerase containing sigma K in vitro (Kroos et al., 1989) . The spoIIID83 mutation (by which the locus was originally identified), was sequenced and found to be a single base substitution in the ribosome binding site upstream of the spoIIID open reading frame . A transcriptional fusion to lacZ was constructed and used to examine the regulation of spoIIID . Expression of spoIIID occurred only during sporulation, beginning 1.5 to 2 hours after the initiation of sporulation . The dependence of spoIIID expression on other spo loci suggests that it is mother-cell-specific, and that it is transcribed by sigma E-containing RNA polymerase.

EMBO J, 1990 Apr, 9(4), 1007 - 13
The amino acid sequence of the CCGG recognizing DNA methyltransferase M.BsuFI: implications for the analysis of sequence recognition by cytosine DNA methyltransferases; Walter J et al.; The Bacillus subtilis FI DNA methyltransferase (M.BsuFI) modifies the outer cytosine of the DNA sequence CCGG, causing resistance against R.BsuFI and R.MspI restriction . The M.BsuFI gene was cloned and expressed in B.subtilis and Escherichia coli . As derived from the nucleotide sequence, the M.BsuFI protein has 409 amino acids, corresponding to a molecular mass of 46,918 daltons . Including these data we have compared the nucleotide and amino acid sequences of different CCGG recognizing enzymes . These analyses showed that M.BsuFI is highly related to two other CCGG specific methyltransferases, M.MspI and M.HpaII, which were isolated from Gram-negative bacteria . Between M.BsuFI and M.MspI the sequence similarity is particularly significant in a region, which has been postulated to contain the target recognition domains (TRDs) of cytosine-specific DNA methyltransferases . Apparently M.BsuFI and M.MspI, derived from phylogenetic distant organisms, use highly conserved structural elements for the recognition of the CCGG target sequence . In contrast the very same region of M.HpaII is quite different from those of M.BsuFI and M.MspI . We attribute this difference to the different targeting of methylation within the sequence CCGG, where M.HpaII methylates the inner, M.BsuFI/M.MspI the outer cytosine . Also the CCGG recognizing TRD of the multispecific B.subtilis phage SPR Mtase is distinct from that of the host enzyme, possibly indicating different requirements for TRDs operative in mono- and multispecific enzymes.

J Bacteriol, 1990 Apr, 172(4), 2175 - 7
A mutation in P23, the first gene in the RNA polymerase sigma A (sigma 43) operon, affects sporulation in Bacillus subtilis; Zuberi AR et al.; Mutations within P23, the first gene of the Bacillus subtilis sigma A operon, were not detrimental to vegetative growth or sporulation . One deletion of P23 resulted in a strain that sporulated earlier than the wild type . This aberrant phenotype may be due to the simultaneous deletion of a sigma H promoter from the sigma A operon.

J Bacteriol, 1990 Apr, 172(4), 1948 - 53
Bacteriophage-enhanced sporulation: comparison of spore-converting bacteriophages PMB12 and SP10; Silver-Mysliwiec TH et al.; The previously characterized bacteriophage SP10 enhanced the frequency of wild-type sporulation by Bacillus subtilis W23 and 3-13 . Comparison of SP10 with the spore-converting bacteriophage PMB12 indicated that both bacteriophages significantly increased the sporulation frequency of an oligosporogenic mutant that contained spo0J::Tn917 omega HU261 . SP10 and PMB12 caused wild-type bacteria to sporulate in a liquid medium that initially contained enough glucose to inhibit the sporulation and expression of alpha-amylase by uninfected bacteria . SP10 also induced the expression of alpha-amylase in the presence of glucose, whereas PMB12 had no detectable effect . These observations were consistent with the conclusion that SP10 is a spore-converting bacteriophage and that SP10 and PMB12 relieve glucose-mediated catabolite repression of sporulation by different mechanisms.

J Bacteriol, 1990 Apr, 172(4), 1939 - 47
Complex character of senS, a novel gene regulating expression of extracellular-protein genes of Bacillus subtilis; Wang LF et al.; The senS gene of Bacillus subtilis, which in high copy number stimulates the expression of several extracellular-protein genes, has been cloned, genetically mapped, and sequenced . The gene codes for a highly charged basic protein containing 65 amino acid residues . The gene is characterized by the presence of a transcription terminator (attenuator) located between the promoter and open reading frame, a strong ribosome-binding site, and a strong transcription terminator at the 3' end of this monocistronic gene . The amino acid sequence of SenS showed partial homology with the N-terminal core binding domain region of bacterial RNA polymerase sigma factors and a helix-turn-helix motif found in DNA-binding proteins . The gene can be deleted without any effect on growth or sporulation.

J Bacteriol, 1990 Apr, 172(4), 1870 - 6
Transcriptional organization of a cloned chemotaxis locus of Bacillus subtilis; Zuberi AR et al.; A cloned chemotaxis operon has been characterized . Thirteen representative che mutations from different complementation groups were localized on the physical map by recombination experiments . The use of integration plasmids established that at least 10 of these complementation groups within this locus are cotranscribed . An additional three complementation groups may form part of the same transcript . The direction of transcription and the time of expression were determined from chromosomal che-lacZ gene fusions . The promoter was cloned and localized to a 3-kilobase fragment . Expression of beta-galactosidase from this promoter was observed primarily during the logarithmic phase of growth . Three-factor PBS1 cotransduction experiments were performed to order the che locus with respect to adjacent markers . The cheF141 mutation is 70 to 80% linked to pyrD1 . This linkage is different from that reported previously (G . W . Ordal, D . O . Nettleton, and J . A . Hoch, J . Bacteriol . 154:1088-1097, 1983) . The cheM127 mutation is 57% linked by transformation to spcB3 . The gene order determined from all crosses is pyrD-cheF-cheM-spcB.

J Bacteriol, 1990 Apr, 172(4), 1783 - 90
A novel Bacillus subtilis gene involved in negative control of sporulation and degradative-enzyme production; Honjo M et al.; We have cloned a 2.5-kilobase fragment of the Bacillus subtilis genomic DNA which caused the reduction of extracellular and cell-associated protease levels when present in high copy number . This fragment, in multicopy, was also responsible for reduced levels of alpha-amylase, levansucrase, alkaline phosphatase, and sporulation inhibition . The gene relevant to this pleiotropic phenotype is referred to as pai . By DNA sequencing, two open reading frames--ORF1 and ORF2, encoding polypeptides of 172 and 207 amino acid residues, respectively--were found . These open reading frames seemed to form an operon . Deletion analysis revealed that an entire region for ORF1 and ORF2 was necessary for the pai phenotype . In addition, it was observed that the presence of the pai gene, in multicopy, caused overproduction of two proteins (molecular masses, 21 and 24 kilodaltons {kDa}) . Analyses of the N-terminal amino acid sequences of these two proteins suggested that they were products of ORF1 and ORF2 . Disruption of the pai gene at ORF1 in the genomic DNA resulted in the release of repression on protease synthesis and sporulation in glucose-enriched (2%) medium . The mutant carrying insertional disruption at ORF2 could not be constructed, suggesting that the ORF2 product, the 24-kDa protein, is essential for growth . The 21-kDa protein contains a helix-turn-helix domain observed in other DNA-binding proteins . Chromosomal mapping of pai indicated that this gene is located close to thr-5 . These results suggest that the pai gene is a novel transcriptional-regulation gene involved in glucose repression.

Mikrobiyol Bul, 1990 Apr, 24(2), 140 - 3
{Application of microcarriers to Bacillus subtilis culture}; Sarikaya E et al.; Microcarriers, first applied to cell cultures . It was reported that microcarriers affected cell growth positively . In this study Cytodex I microcarriers were used . Because of their surface characteristics mammalian cells can easily attach to Cytodex I microcarriers and grow well . B . subtilis cells also attached to microcarriers and grew well . In this study Cytodex I microcarriers were used for productive bacterial growth.

Gene, 1990 Mar 30, 88(1), 101 - 5
The apparent specificity of NotI (5'-GCGGCCGC-3') is enhanced by M.FnuDII or M.BepI methyltransferases (5'-mCGCG-3'): cutting bacterial chromosomes into a few large pieces; Qiang BQ et al.; The restriction endonuclease (ENase) NotI is blocked by methylation within its recognition sequence at 5'-GCGGCmCGC-3' . This sensitivity to methylation can be used to enhance the specificity of NotI in vivo and in vitro . Modification by M.FnuDII or M.BepI methyltransferases (MTase) (5'-mCGCG-3') will block NotI (5'-GCGGCCGC-3') cleavage at overlapping MTase/ENase sites 5'-CGCGGCCGC-3' (equivalent to 5'-GCGGCCGCG-3'), and increase the apparent cleavage specificity of NotI about twofold . This 'cross-protection' procedure reduces the number of NotI fragments in the genomes of Escherichia coli and Bacillus subtilis, as resolved by pulsed field electrophoresis . Application of this method to large DNAs in vitro requires the preparation of highly purified DNA MTases.

J Biol Chem, 1990 Mar 15, 265(8), 4204 - 9
Riboflavin synthases of Bacillus subtilis . Purification and amino acid sequence of the alpha subunit; Schott K et al.; Bacillus subtilis has two different riboflavin synthases characterized by the subunit structures alpha3 (light enzyme) and alpha3beta60 (heavy enzyme) . The light enzyme was purified by a novel procedure with increased yield and excellent reproducibility . The proposed trimer structure was confirmed by cross-linking experiments with dimethyl suberimidate . Fragments of alpha subunits were prepared by cleavage with cyanogen bromide, trypsin, protease Lys-C, and Staphylococcus aureus protease V8, respectively . Sequences were determined by automated liquid or gas phase Edman degradation . The complete sequence (202 amino acids) was established by direct sequencing of the N terminus and sequencing of overlapping peptides . The sequence shows marked internal homology between the NH2-terminal and COOH-terminal half encompassing 26 identical positions and 23 conservative replacements . This suggests that the protomer forms two structurally similar domains . Since it is known that the enzyme has two binding sites per subunit for the substrate 6,7-dimethyl-8-ribityllumazine, it appears likely that each of the homologous protein domains provides one binding site . The stereochemical features of the enzyme mechanism and the structural relation of the alpha trimer to the beta60 capsid of heavy riboflavin synthase suggest that the six domains corresponding to the alpha subunit trimer are related by pseudo 32 symmetry.

FEBS Lett, 1990 Mar 12, 262(1), 33 - 5
Effect of polyelectrolytes on serine proteinase secretion by Bacillus subtilis; Artemov AV et al.; Addition of polycations with molecular masses of 5-40 kDa as well as Na+, stimulated serine proteinase secretion by Bacillus subtilis cells . Polyanions and higher-molecular-mass polycations (100-200 kDa) were inefficient . The enzyme yields in the presence of polycations or Na+ were equal in magnitude . The results indicate that the cations, apparently counteracting the negative surface charge of the bacterial plasma membrane, cause the desorption of the serine (alkaline) proteinase . The synthesis of the proteinase is inferred to be stopped as the enzyme is bound to the outer surface of the plasma membrane . The desorption of the enzyme thus induces the synthesis of the new portions of proteinase.

J Biol Chem, 1990 Mar 5, 265(7), 4054 - 7
Purification and characterization of Chlamydomonas reinhardtii chloroplast glutamyl-tRNA synthetase, a natural misacylating enzyme; Chen MW et al.; Glutamyl-tRNA synthetase from Chlamydomonas reinhardtii was purified by sequential column chromatography on DEAE-cellulose, phosphocellulose, Mono Q, and Mono S . The apparent molecular mass of the protein when analyzed under both denaturing conditions (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and nondenaturing conditions (rate zonal sedimentation on glycerol gradients) was 62,000 Da; this indicates that the active enzyme is a monomer . The purified glutamyl-tRNA synthetase was identified as the chloroplast enzyme by its tRNA charging specificity . Reversed-phase chromatography of unfractionated C . reinhardtii tRNA resolved four peaks of glutamate acceptor RNA when assayed with the purified enzyme . The enzyme can also glutamylate Escherichia coli tRNA(2Glu), but not cytoplasmic tRNA(Glu) from yeast or barley . In addition, the enzyme misacylates chloroplast tRNA(Gln) with glutamate . A similar mischarging phenomenon has been demonstrated for the barley chloroplast enzyme (Schon, A., Kannangara, C.G., Gough, S., and Soll, D . (1988) Nature 331, 187-190) and for Bacillus subtilis glutamyl-tRNA synthetase (Proulx, M., Duplain, L., Lacoste, L., Yaguchi, M., and Lapointe, J . (1983) J . Biol . Chem . 258, 753-759).

J Mol Biol, 1990 Mar 5, 212(1), 1 - 2
Crystallization of the Bacillus subtilis histidine-containing phosphocarrier protein HPr and of some of its site-directed mutants; Kapadia G et al.; The histidine-containing phosphocarrier protein (HPr) from Bacillus subtilis has been crystallized . Two of the site-directed mutants aimed at probing function produce crystals suitable for X-ray studies . The mutant in which His15 is substituted by an alanyl residue crystallizes from ammonium sulfate solution in space group P3(1)21 or P3(2)21, with unit cell dimensions: a = b = 47.3 A; c = 61.5 A . These crystals diffract to at least 1.8 A resolution . The mutant in which Ser46 is substituted by an aspartyl residue crystallizes from polyethylene glycol 4000 solution in space group P2(1), with unit cell dimensions: a = 49.4 A; b = 25.6 A; c = 60.3 A; beta = 109 degrees . These crystals diffract to at least 2.0 A resolution.

Mikrobiol Zh, 1990 Mar-Apr, 52(2), 9 - 14
{The enzyme activity of bacilli showing promise for incorporation into biopreparations}; Slabospitskaia AT et al.; The enzymic activity (amalyse, protease, lipase, pectolytic and cellulase) has been studied in 5 strains of aerobic spore-forming bacteria (Bacillus subtilis, B . licheniformis, B . coagulans, B . pumilis, B . badius) being of interest for creation of medical and prophylactic biopreparations . The above-mentioned enzymes were found in some studied strains . This may provide participation of bacilli in the degradation processes of a number of substrates in the digestive tract of a human being and animals and is an advantage of preparations from the genus Bacillus bacteria as compared with the available biopreparations of other microbial cultures for prophylaxis and treatment of gastrointestinal diseases.

Mol Biol (Mosk), 1990 Mar-Apr, 24(2), 560 - 5
{Genome polymorphism in dissociative variants of Bacillus subtilis (mesentericus) 76 . Identification of dissociants using genome fingerprinting}; Ivanov PL et al.; DNA fingerprinting procedure with M13 repeat probe as we have shown earlier makes it possible to apply a new approach in theoretical and applied fields of microbiology and bacteriology . In this work, using the method described we have revealed genomic polymorphism of dissociative variants of Bac . subtilis (mesentericus) 76 . The data obtained may be referred as strong evidence that bacterial dissociation do has genetic nature.

Mol Microbiol, 1990 Mar, 4(3), 513 - 7
The life-cycle proteins RodA of Escherichia coli and SpoVE of Bacillus subtilis have very similar primary structures; Joris B et al.; Comparison of the predicted amino acid sequence of the cell-cycle RodA protein with the National Research Foundation protein sequence database shows that the 370-amino-acid RodA, a protein that is essential for wall elongation in Escherichia coli and maintenance of the rod shape of the cell, is highly analogous, in terms of primary structure, with the Bacillus subtilis SpoVE protein involved in stage V of sporulation.

J Appl Bacteriol, 1990 Mar, 68(3), 253 - 61
Hemicellulases of Bacillus species: preliminary comparative studies on production and properties of mannanases and galactanases; Araujo A et al.; A range of Bacillus subtilis strains and other Bacillus species were screened for mannanase, beta-mannosidase and galactanase activities . Maximum mannanase activity, 106.2 units/ml, was produced by B . subtilis NRRL 356 . beta-Mannosidase and galactanase activities from all strains were relatively low . The effect of carbon and nitrogen source on mannanase and galactanase production by B . brevis ATCC 8186, B . licheniformis ATCC 27811, B . polymyxa NRRL 842 and B . subtilis NRRL 356 was investigated . Highest mannanase production was observed in the four strains tested when the mannan substrate, locust bean gum, was used as carbon source . Induction was most dramatic in the case of B . subtilis NRRL 356 where only basal enzyme levels were produced in the presence of other carbon sources . beta-Mannosidase was induced in the four Bacillus cultures by locust bean gum . Results indicated that galactose acted as an inducer for production of galactanase . Organic and inorganic nitrogen sources resulted in induction of high mannanase titres in B . subtilis . Highest galactanase activity was produced by each organism in media containing sodium nitrate as nitrogen source . Mannanases from B . brevis, B . licheniformis, B . polymyxa and B . subtilis retained 100% residual activity after a 3 h incubation at 65 degrees C, 65 degrees C, 60 degrees C and 55 degrees C respectively . Galactanases retained more than 95% activity at 55 degrees C after 3 h . The pH optima of mannanases ranged from 6.5-6.8 whereas galactanases ranged from 5.1 in the case of B . brevis to 7.0 for B . polymyxa.

Can J Microbiol, 1990 Mar, 36(3), 164 - 8
Specific inhibition of iturin biosynthesis by cerulenin; Hourdou ML et al.; Cerulenin, an inhibitor of fatty acid synthesis, inhibits the biosynthesis of iturin by Bacillus subtilis . With a cerulenin concentration of 2 micrograms/mL, 50% inhibition was achieved . At this concentration, cerulenin does not affect growth or total protein synthesis but does inhibit the incorporation of sodium {14C}acetate, {14C}myristic acid, and {14C}asparagine into iturin . Since cerulenin is known to block the condensation of malonyl-CoA subunits in the formation of fatty acids, the inhibition of iturin and beta-amino acid syntheses by cerulenin is discussed in relation with lipid synthesis.

Gene, 1990 Mar 1, 87(1), 71 - 8
The structure of the trpE, trpD and 5' trpC genes of Bacillus pumilus; Rivas MV et al.; The nucleotide (nt) sequences of the Bacillus pumilus trpE, trpD and 5' portions of trpC genes have been determined . Genetic analysis suggested the presence of an internal promoter upstream from the trpC gene, yet no typical consensus sequences were found . The nt and amino acid sequence homologies between the B . pumilus, Bacillus subtilis and Escherichia coli trp genes are presented.

Gene, 1990 Mar 1, 87(1), 53 - 61
Structurally stable Bacillus subtilis cloning vectors; Janniere L et al.; Cloning of long DNA segments (greater than 5 kb) in Bacillus subtilis is often unsuccessful when naturally occurring small (less than 10 kb) plasmids are used as vectors . In this work we show that vectors derived from the large (26.5 kb) plasmids pAM beta 1 and pTB19 allow efficient cloning and stable maintenance of long DNA segments (up to 33 kb) . The two large plasmids differ from the small ones in several ways . First, replication of the large plasmids does not lead to accumulation of detectable amounts of ss DNA, whereas the rolling-circle replication typical for small plasmids does . In addition, the replication regions of the two large plasmids share no sequence homology with the corresponding regions of the known small plasmids, which are highly conserved . Taken together, these observations suggest that the mode of replication of the large plasmids is different from that of small plasmids . Second, short repeated sequences recombine much less frequently when carried on large than on small plasmids . This indicates that large plasmids are structurally much more stable than small ones . We suggest that the high structural stability of large plasmids is a consequence of their mode of replication and that plasmids which do not replicate as rolling circles should be used whenever it is necessary to clone and maintain long DNA segments in any organism.

Gene, 1990 Mar 1, 87(1), 23 - 9
The 'effective number of codons' used in a gene; Wright F; A simple measure is presented that quantifies how far the codon usage of a gene departs from equal usage of synonymous codons . This measure of synonymous codon usage bias, the 'effective number of codons used in a gene', Nc, can be easily calculated from codon usage data alone, and is independent of gene length and amino acid (aa) composition . Nc can take values from 20, in the case of extreme bias where one codon is exclusively used for each aa, to 61 when the use of alternative synonymous codons is equally likely . Nc thus provides an intuitively meaningful measure of the extent of codon preference in a gene . Codon usage patterns across genes can be investigated by the Nc-plot: a plot of Nc vs . G + C content at synonymous sites . Nc-plots are produced for Homo sapiens, Saccharomyces cerevisiae, Escherichia coli, Bacillus subtilis, Dictyostelium discoideum, and Drosophila melanogaster . A FORTRAN77 program written to calculate Nc is available on request.

FEMS Microbiol Lett, 1990 Mar 1, 56(1-2), 143 - 8
Production and secretion of pertussis toxin subunits in Bacillus subtilis; Saris P et al.; Pertussis toxin (PT) is a major component of today's acellular whooping cough vaccines . The use of acellular vaccines is predicted to increase sharply in the near future . There is therefore a need to produce PT in a way that makes its purification as easy as possible . Our approach was to express all five PT subunits individually in Bacillus subtilis . We have used vectors containing the promoter and signal sequences of the alpha-amylase gene of Bacillus amyloliquefaciens followed by an insert encoding the appropriate PT-subunit . All PT-subunits were secreted and found in the culture supernatant . The level of expression varied considerably: S1 and S5 were produced in large quantities whereas much smaller amounts of S2, S3 and S4 were found . The subunits were also present in the membrane fraction of the respective strains.

Proc Natl Acad Sci U S A, 1990 Mar, 87(5), 1801 - 5
The SpoOA protein of Bacillus subtilis is a repressor of the abrB gene; Strauch M et al.; The spoOA gene of Bacillus subtilis is critical for the initial stages in the developmental cycle leading to the formation of an endospore . We show that one function of the SpoOA protein is to negatively regulate another regulatory locus, abrB, which controls the expression of many genes associated with the onset of sporulation . Purified SpoOA protein binds to a specific region of the abrB promoter and functions as a repressor of transcription in an in vitro assay . The binding of the SpoOA protein is independent of the binding of the AbrB protein, which is known to autoregulate its expression . This independence mirrors the temporal sequence of events in abrB control.

J Bacteriol, 1990 Mar, 172(3), 1470 - 7
Bacillopeptidase F of Bacillus subtilis: purification of the protein and cloning of the gene; Sloma A et al.; We have purified a minor extracellular serine protease from Bacillus subtilis . Characterization of this enzyme indicated that it was most likely the previously reported enzyme bacillopeptidase F . The amino-terminal sequence of the purified protein was determined, and a "guess-mer" oligonucleotide hybridization probe was constructed on the basis of that sequence . This probe was used to identify and clone the structural gene (bpr) for bacillopeptidase F . The deduced amino acid sequence for the mature protein (496 amino acids) was preceded by a putative signal sequence of 30 residues and a putative propeptide region of 164 amino acids . The bpr gene mapped near pyrD on the chromosome and was not required for growth or sporulation.

J Bacteriol, 1990 Mar, 172(3), 1306 - 11
Characterization of the spoIVB and recN loci of Bacillus subtilis; Van Hoy BE et al.; Two independent genes, recN and spoIVB, along with their respective promoter and termination regions, were discovered and sequenced in the 3.4-kilobase region between the ahrC and spoOA genes at map position 216 in the Bacillus subtilis chromosome map . The gene encoding a 576-amino-acid protein, which maintains a high homology with the Escherichia coli recN gene product, was adjacent to ahrC . The sequence revealed a 64,472-dalton polypeptide which contained a conserved ATP-binding site and possible lexA-type regulatory binding sequences in its promoter region . A second open reading frame identified as the spoIVB gene was directly downstream of recN . It consisted of 1,275 nucleotides which coded for a 425-amino-acid polypeptide with a molecular weight of 45,976 . Phenotypic, genetic, and transcriptional analyses confirmed that this gene was spoIVB . Although no chloroform-resistant spores were produced by spoIVB-inactivated strains, under microscopic examination, phase-gray forespores were visible . The spoIVB165 mutation was localized to a 200-base-pair region in the amino-terminal portion of the polypeptide, spoIVB was not transcribed until hour 2 of sporulation in wild-type B . subtilis cells, as determined by beta-galactosidase activity assays from lacZ transcriptional fusion constructions . We found no amino acid sequence homology between the spoIVB gene product and other known bacterial proteins.

J Bacteriol, 1990 Mar, 172(3), 1499 - 508
A membrane protein with similarity to N-methylphenylalanine pilins is essential for DNA binding by competent Bacillus subtilis; Breitling R et al.; In a cloned copy of comG open reading frame 3 (ORF3), an in-frame deletion was generated by site-directed in vitro mutagenesis, removing the coding sequence for 15 amino acids from the central portion of this pilin-related protein . The mutagenized ORF3 was incorporated into the Bacillus subtilis chromosome, replacing the wild-type ORF3 . The presence of the deleted ORF3 in the chromosome, as confirmed by Southern analysis, was associated with the complete loss of competence by the mutant strain . The ability of the mutant cells to bind exogenous radiolabeled DNA was reduced to the level of nonspecific binding of DNA by noncompetent cells . The chromosomal ORF3 mutation was partially complemented in trans by a plasmid-encoded wild-type ORF3 copy under PSPAC control upon induction of the PSPAC promoter . Using antiserum raised against a synthetic 14-mer oligopeptide deduced from the ORF3 sequence, an immunoreactive band of approximately the expected molecular size was obtained in Western blot (immunoblot) experiments with extracts of cells containing the plasmid-encoded inducible gene . A signal was also detected when cells harboring the chromosomal wild-type or mutant ORF3 in single copy were grown in competence medium . This signal was detected only in the light-buoyant-density (competent) cell fraction and only after the transition from the exponential to the stationary growth phase . In cell fractionation experiments with competent cell extracts, the immunoreactive protein was found in both the NaOH-insoluble and -soluble membrane fractions and was sensitive to proteinase K treatment of either protoplasts or whole cells.

Appl Microbiol Biotechnol, 1990 Mar, 32(6), 651 - 7
Subtilisin-catalysed peptide synthesis and transesterification in organic solvents; Ferjancic A et al.; Subtilisin from Bacillus subtilis was modified with polyethylene glycol (PEG), or adsorbed either on celite or porous glass, or directly used as a suspended powder to catalyse peptide synthesis and transesterification reactions in organic solvents . The rather low yield of peptide synthesis probably resulted from the enzyme tendency to catalyse hydrolysis and transesterification side reactions . The kinetics of transesterification catalysed by PEG-subtilisin was consistent with a ping-pong mechanism modified by a hydrolytic branch . Initial rates of transesterification were found to be dependent on alcohol and organic base concentrations in the reaction mixture . The high affinity of benzyloxycarbonyl-L-serine-methyl ester as compared to benzyloxycarbonyl-L-phenylalanine-methyl ester for the enzyme indicated that a change in substrate specificity of subtilisin occurred in organic phase . The 50-fold increase in the rate of synthesis of benzyloxycarbonyl-L-serine-L-phenylalanine amide which was observed when PEG-subtilisin was used instead of immobilized or powdered enzyme, suggested that a higher flexibility of the polypeptide chain modified by the covalent attachment of a number of soluble PEG moieties occurred in organic solvents . This also resulted in a lower stability of PEG-subtilisin at high temperature.

Biotechnology (N Y), 1990 Mar, 8(3), 241 - 2
The alpha-amylase gene as a marker for gene cloning: direct screening of recombinant clones; Ikuta N et al.; We report the construction and use of a new system for the direct screening of recombinant clones after transformation . The system uses a Bacillus subtilis-Escherichia coli shuttle vector that carries the B . subtilis structural gene for alpha-amylase . Insertion of foreign DNA into this gene results in a loss of amylolytic activity in the host cells that can be assayed using a simple and inexpensive staining procedure.

Nucleic Acids Res, 1990 Feb 25, 18(4), 739 - 44
Heat-inducible translational coupling in Bacillus subtilis; Fujiwara S et al.; Bacillus subtilis plasmid pGR71 is a promoter-probe shuttle vector derived from pUB110 . The expression of the cat gene on pGR71 in B . subtilis requires the insertion of a Bacillus promoter and a ribosomal binding site (RBS) into the HindIII cloning site immediately upstream from the cat gene . A recombinant plasmid of pGR71, named pGR71-369, was obtained by a spontaneous deletion of a fragment containing most of the inserted HindIII fragment and the replication origin necessary for multiplication in Escherichia coli . The expression of the cat gene in B . subtilis cells carrying this plasmid was inducible by heat . Nucleotide sequence analysis of the upstream region of the cat gene, deletion analysis, and dot blot hybridization analysis of mRNA in various conditions revealed that the cat gene was expressed by heat-inducible translational coupling and that the regulatory region of heat inducibility was present in the upstream region of the cat gene.

J Mol Biol, 1990 Feb 20, 211(4), 713 - 25
Bend induced by the phage phi 29 transcriptional activator in the viral late promoter is required for activation; Rojo F et al.; Transcription initiation from the Bacillus subtilis phage phi 29 late A3 promoter requires the viral protein p4, a transcriptional activator . Protein p4 binds to a region of the A3 promoter, located between nucleotides -50 and -100 relative to the transcription start site, that presents a sequence-directed curvature . This curvature is enhanced when protein p4 binds to the promoter . A number of deletion mutants at the carboxyl end of protein p4 have been constructed and their behavior as transcriptional activators of the late A3 promoter has been investigated . The binding of these deletion mutants to the late A3 promoter has been analyzed by gel retardation, DNase I footprinting, methylation interference and circular permutation assays . The results suggest that the last 12 amino acid residues of protein p4, six of which are positively charged, although not involved in the specific recognition of the promoter are responsible for part of the bend induced by protein p4 in its binding site . Evidence is presented which suggests that full induction of this curvature is needed for the transcription activation process . A model is proposed for protein p4 interaction with the A3 promoter, in which the bend is induced in two steps: first, two monomers of protein p4 bind to the inverted recognition sequences, subsequent interaction between them generating a bend between these sequences; second, the highly basic carboxyl terminus of protein p4 establishes non-specific electrostatic interactions with the DNA backbone inducing a bend at both ends of the protein p4 binding region.

J Biol Chem, 1990 Feb 5, 265(4), 1928 - 32
Methyl group turnover on methyl-accepting chemotaxis proteins during chemotaxis by Bacillus subtilis; Thoelke MS et al.; The addition of attractant to Bacillus subtilis briefly exposed to radioactive methionine causes an increase of labeling of the methyl-accepting chemotaxis proteins . The addition of attractant to cells radiolabeled for longer times shows no change in the extent of methylation . Therefore, the increase in labeling for the briefly labeled cells is due to an increased turnover of methyl groups caused by attractant . All amino acids gave enhanced turnover . This turnover lasted for a prolonged time, probably spanning the period of smooth swimming caused by the attractant addition . Repellent did not affect the turnover when added alone or simultaneously with attractant . Thus, for amino acid attractants, the turnover is probably the excitatory signal, which is seen to extend long into or throughout the adaptation period, not just at the start of it.

J Dent Hyg, 1990 Feb, 64(2), 69 - 73
Monitoring dental sterilizers' effectiveness using biological indicators; Nickerson A et al.; The purpose of this study was to evaluate the effectiveness of dental office sterilizers as measured by their ability to kill bacterial spores present on biological indicator strips . The biological indicators used in this study contained two different spores, Bacillus stearothermophilus and Bacillus subtilis (Spordi, AMSCO/Medical Products) . Ten spore test strips were sent to 87 dental offices; 51 sterilizers were tested . Office personnel were instructed to place four strips in the center of a normal sterilization load and process the load . The procedure was repeated on a second day . The processed strips, along with two unprocessed control strips, were returned by mail for laboratory culturing . The results indicated the overall failure rate (positive test) of sterlizers tested for both days was 51% at the culturing temperature of 37 degrees C and 33.3% at 55 degrees C . McNemar's test indicated a significant difference (p less than .03) in sterilization failures associated with the type and number of microorganisms present on the test strips . This study also showed that the more times a sterilizer was tested, the more likely a failure would occur . Overall, an alarming number of sterilizers (64.7%) were not effective in killing all the spores present on the indicator strips . When office personnel were given information for improving sterilizer performance, there was a noticeable reduction in sterilization failures following retesting.

J Bacteriol, 1990 Feb, 172(2), 793 - 801
Genetic map of the Bacillus stearothermophilus NUB36 chromosome; Vallier H et al.; A circular genetic map of Bacillus stearothermophilus NUB36 was constructed by transduction with bacteriophage TP-42C and protoplast fusion . Sixty-four genes were tentatively assigned a cognate Bacillus subtilis gene based on growth response to intermediates or end products of metabolism, cross-feeding, accumulation of intermediates, or their relative order in a linkage group . Although the relative position of many genes on the Bacillus stearothermophilus and Bacillus subtilis genetic map appears to be similar, some differences were detected . The tentative order of the genes in the Bacillus stearothermophilus aro region is aspB-aroBAFEC-tyrA-hisH-(trp), whereas it is aspB-aroE-tyrA-hisH-(trp)-aroHBF in Bacillus subtilis . The aroA, aroC, and aroG genes in Bacillus subtilis are located in another region . The tentative order of genes in the trp operon of Bacillus stearothermophilus is trpFCDABE, whereas it is trpABFCDE in Bacillus subtilis.

J Bacteriol, 1990 Feb, 172(2), 701 - 8
Comparison of the three aspartokinase isozymes in Bacillus subtilis Marburg and 168; Zhang JJ et al.; The levels of two aspartokinase isozymes, a lysine-sensitive enzyme and an aspartokinase that is inhibited synergistically by lysine plus threonine, differ strikingly in different strains of Bacillus subtilis . In derivatives of B . subtilis 168 growing in minimal medium, the predominant isozyme is the lysine-sensitive aspartokinase . In B . subtilis ATCC 6051, the Marburg strain, the level of the lysine-sensitive aspartokinase is much lower during growth in minimal medium, and the major aspartokinase activity is the lysine-plus-threonine-sensitive isozyme . Molecular cloning and nucleotide sequence determination of the genes for the lysine-sensitive isozymes from the two B . subtilis strains and their upstream control regions showed these genes to be identical . Evidence that the lysine-sensitive aspartokinase, referred to as aspartokinase II, is distinct from the threonine-plus-lysine-sensitive aspartokinase comes from the observation that disruption of the aspartokinase II gene by recombinational insertion had no effect on the latter . Mutants were obtained from the aspartokinase II-negative strain that also lacked the threonine-plus-lysine-sensitive aspartokinase, which will be referred to as aspartokinase III . Aspartokinase II could be selectively restored to these mutants by transformation with plasmids carrying the aspartokinase II gene . Study of the growth properties of the various mutant strains showed that the loss of either aspartokinase II or aspartokinase III had no effect on growth in minimal medium but that the loss of both enzymes interfered with growth unless the medium was supplemented with the three major end products of the aspartate pathway . It appears, therefore, that aspartokinase I alone cannot provide adequate supplies of precursors for the synthesis of lysine, threonine, and methionine by exponentially growing cells.

Biochem Cell Biol, 1990 Feb, 68(2), 492 - 5
High-level expression of Bacillus subtilis tryptophanyl-tRNA synthetase in Escherichia coli; Shi W et al.; The trpS gene encoding Bacillus subtilis tryptophanyl-tRNA synthetase (TrpRS) was prepared from the pUC8-derived pTSQ2 plasmid, mutagenized to introduce an EcoRI site immediately in front of the ATG start codon, and inserted into the pKK223-3 vector downstream to the tac promoter to yield the pKSW1 plasmid . Upon induction with isopropyl-beta-D-thiogalactopyranoside, Escherichia coli JM109{pKSW1} cells synthesized TrpRS to a level corresponding to 45% of total cell proteins . This high level of gene expression facilitates large scale preparation of TrpRS for physical studies, detection of in vivo degradation of mutant forms of TrpRS, and comparative assays of TrpRS by {3H}Trp-tRNA formation and by Trp-hydroxamate formation for the purpose of mutant characterization . Finally, since pKSW1 could complement the temperature-sensitive TrpRS mutation on E . coli trpS 10343 cells, defective mutations of the trpS gene on pKSW1 would be deductible on the basis of complementation testing.

Mol Microbiol, 1990 Feb, 4(2), 275 - 82
The regulation of transcription of the gerA spore germination operon of Bacillus subtilis; Feavers IM et al.; The gerA operon of Bacillus subtilis 168 comprises three genes concerned with the triggering of spore germination by L-alanine and its analogues . The expression of this operon has been characterized using chromosomal lacZ fusions to the gerA promoter . The gerA promoter is switched on 2.5-3 hours after the initiation of sporulation, in parallel with glucose dehydrogenase . A high proportion of the gerA-driven beta-galactosidase detected in sporulating cells is found in the mature spore; the gerA promoter is therefore active in the forespore compartment of the sporulating cell . The gerA promoter is not expressed in spoO, spoII or spoIIIA, B, E and G mutant backgrounds, but is expressed in spoIIIC and D and in spoIV and V mutants . The in vivo transcriptional startpoint of the operon has been mapped by primer extension experiments; sequences upstream from this startpoint show significant homology with recognition sequences for RNA polymerase containing sigma G (E sigma G) . The gerA operon was transcribed in vitro by E sigma G with a startpoint identical to that used in vivo, and expression of the gerA operon was rapidly induced in vegetative cells by induction of sigma G synthesis . These data indicate that the gerA operon is an additional member of the sigma G regulon, which includes a number of genes expressed in parallel only in the forespore compartment of sporulating B . subtilis cells.

Appl Environ Microbiol, 1990 Feb, 56(2), 503 - 6
Vapor-phase hydrogen peroxide as a surface decontaminant and sterilant; Klapes NA et al.; The feasibility of utilizing vapor-phase hydrogen peroxide (VPHP) as a surface decontaminant and sterilant was evaluated in a centrifuge application . The prototype VPHP decontamination system, retrofitted into a Beckman L8-M ultracentrifuge, was designed to vaporize a 30% (wt/wt) solution of aqueous hydrogen peroxide continuously injecting and withdrawing VPHP in a deep-vacuum flow-through system . VPHP cycles of 4, 8, 16, and 32 min were examined for cidal activity against spores of Bacillus subtilis subsp . globigii and Bacillus stearothermophilus . Spore inocula (approximately 10(6)/coupon) were dried onto 0.5-in . (1.27-cm)-square stainless-steel coupons, and coupons were suspended in the centrifuge chamber, the space between the refrigeration can and the barrier ring (inner gap), and the space between the barrier ring and the vacuum ring (outer gap) . At a chamber temperature of 4 degrees C, B . subtilis subsp . globigii spores were inactivated within 8 min, while inactivation of spores located in the outer gap at 27 degrees C required 32 min . The elevated temperature and high surface area/volume ratios in the outer gap may serve to decompose the gas more rapidly, thus reducing cidal efficacy . Of the two test spores, B . stearothermophilus was more resistant to VPHP . Nonetheless, VPHP was shown to possess significant sporicidal capability . For practical decontamination applications of the type described, VPHP shows promise as an effective and safer alternative to currently used ethylene oxide or formaldehyde vapors.

J Bacteriol, 1990 Feb, 172(2), 835 - 44
A target for carbon source-dependent negative regulation of the citB promoter of Bacillus subtilis; Fouet A et al.; Expression of the aconitase (citB) gene of Bacillus subtilis is subject to catabolite repression in cells grown in minimal media . In nutrient broth medium, citB expression is low in growing cells but is induced when cells enter sporulation . A 600-base-pair DNA fragment that extends from positions -400 through +200, relative to the transcription start site, was shown to include all of the cis-acting sequences necessary for catabolite repression and sporulation-associated regulation . This was demonstrated by fusing this DNA fragment to the Escherichia coli lacZ gene, integrating the fusion in the amyE locus of the B . subtilis chromosome, and measuring the regulation of expression of beta-galactosidase . By creating a series of deletions from either end of the 600-base-pair fragment, it was possible to define a target for catabolite repression; at least part of this target lies within the sequence between positions -84 and -68 . DNA fragments that included positions -84 through +36, when carried on high-copy plasmids, caused derepression of aconitase synthesis, as if a negative regulator were being titrated . The same plasmids caused derepression of citrate synthase activity as well . Deletion of the sequence between positions -84 and -67 abolished this titration effect for both enzymes . Mutations that altered the target for catabolite repression also affected the inducibility of citB at the onset of sporulation, at least when sporulation was induced by the addition of decoyinine, an inhibitor of guanine nucleotide synthesis . When sporulation was induced by exhaustion of nutrient broth, there was no detectable difference in expression of citB-lacZ fusions whether or not they had the citB sequence from positions -84 to -67, suggesting that the mechanisms of regulation of citB in minimal medium and nutrient broth are different.

J Bacteriol, 1990 Feb, 172(2), 735 - 40
Evidence for two structural genes for alkaline phosphatase in Bacillus subtilis; Hulett FM et al.; Two secreted alkaline phosphatase proteins were purified from cultures of Bacillus subtilis JH646MS . The two proteins showed slight differences in subunit molecular weight, substrate specificity, and charge characteristics . A total of 62% of the first 22 amino-terminal amino acids were identical . Both sequences showed conservation of structural features identified in Escherichia coli and human alkaline phosphatases . One alkaline phosphatase was a monomer and the other was a dimer . Southern analysis of genomic DNA with degenerative oligomers based on the amino acid sequences suggest that there are two structural genes for alkaline phosphatase in the genome of B . subtilis.

J Bacteriol, 1990 Feb, 172(2), 709 - 15
Negative regulator of sigma G-controlled gene expression in stationary-phase Bacillus subtilis; Rather PN et al.; In some media, Bacillus subtilis can maintain a prolonged stationary growth phase; however, in other media, nutrient depletion triggers a complex differentiation that culminates in production of a dormant endospore . This differentiation requires the expression of many genes . We found that during the stationary phase in media in which the cells do not form endospores and do not normally express these sporulation-essential genes, a recessive mutation in spoIIAB caused increased transcription of a set of genes essential for sporulation . Evidently, the wild-type product of spoIIAB acts during the stationary phase to prevent expression of additional sporulation-specific genes.

J Bacteriol, 1990 Feb, 172(2), 1148 - 50
Methyl transfer in chemotaxis toward sugars by Bacillus subtilis; Thoelke MS et al.; Like amino acids, the sugars glucose and the nonmetabolizable 2-deoxyglucose caused a turnover of methyl groups on the methyl-accepting chemotaxis proteins . These sugars also caused methanol formation on addition . Thus, in contrast to chemotaxis in Escherichia coli, taxis to phosphotransferase sugars by Bacillus subtilis utilizes the methyl-accepting chemotaxis proteins.

J Bacteriol, 1990 Feb, 172(2), 1092 - 8
The cisA cistron of Bacillus subtilis sporulation gene spoIVC encodes a protein homologous to a site-specific recombinase; Sato T et al.; The nucleotide sequence of the sporulation gene spoIVC cisA in Bacillus subtilis was determined and found to encode a protein of 500 amino acid residues with a calculated molecular weight of 57,481, which is in good agreement with the size of the gene product estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . The amino acid sequence of the N-terminal region of this protein is homologous to the site-specific DNA recombinases . Hybridization of a 3.6-kilobase EcoRI fragment carrying the spoIVC cisA gene with the EcoRI-restricted chromosomal DNA prepared from cells of various stages showed that DNA rearrangement occurs only in the mother cell in the region adjacent to spoIVC cisA 3 h after the initiation of sporulation . This result coincides with that of Stragier et al . (P . Stragier, B . Kunkel, L . Kroos, and R . Losick, Science 243:507-512, 1989) . The timing of the DNA rearrangement coincides very well with the timing of spoIVC cisA gene expression . The DNA rearrangement was not observed in spoIVC cisA mutants . These results strongly suggest that the spoIVC cisA gene encodes a site-specific DNA recombinase having a very important role in sporulation.

J Bacteriol, 1990 Feb, 172(2), 1043 - 50
Induction of levansucrase in Bacillus subtilis: an antitermination mechanism negatively controlled by the phosphotransferase system; Crutz AM et al.; The target of the induction by sucrose of the levansucrase gene is a transcription terminator (sacRt) located upstream from the coding sequence, sacB . The two-gene locus sacX-sacY (formerly sacS) and the ptsI gene were previously shown to be involved in this induction . ptsI encodes enzyme I of the phosphoenolpyruvate-dependent phosphotransferase system . SacX is strongly homologous to sucrose-specific phosphotransferase system-dependent permeases . SacY is a positive regulator of sacB . Here we show that SacY is probably an antiterminator interacting directly with sacRt, since in Escherichia coli the presence of the sacY gene stimulates the expression of a reporter gene fused downstream from sacRt . Missense mutations affecting sacY were sequenced, and the sacB regulation was studied in isogenic strains carrying these mutations or in vitro-generated mutations affecting sacX, sacY, or ptsI . The phenotype of double mutants suggests a model in which SacX might be a sucrose sensor that would be phosphorylated by the phosphotransferase system and, in this state, could inhibit the SacY antiterminator . Exogenous sucrose, or a mutation inactivating the phosphotransferase system, would dephosphorylate SacX and allow antitermination at sacRt.

J Bacteriol, 1990 Feb, 172(2), 1024 - 9
Gene encoding a novel extracellular metalloprotease in Bacillus subtilis; Sloma A et al.; The gene for a novel extracellular metalloprotease was cloned, and its nucleotide sequence was determined . The gene (mpr) encodes a primary product of 313 amino acids that has little similarity to other known Bacillus proteases . The amino acid sequence of the mature protease was preceded by a signal sequence of approximately 34 amino acids and a pro sequence of 58 amino acids . Four cysteine residues were found in the deduced amino acid sequence of the mature protein, indicating the possible presence of disulfide bonds . The mpr gene mapped in the cysA-aroI region of the chromosome and was not required for growth or sporulation.

J Bacteriol, 1990 Feb, 172(2), 1019 - 23
Isolation and characterization of a novel extracellular metalloprotease from Bacillus subtilis; Rufo GA Jr et al.; We have isolated and characterized two minor extracellular proteases from culture supernatants of a strain of Bacillus subtilis containing deletion mutations of the genes for the extracellular proteases subtilisin (apr) and neutral protease (npr) and a minor extracellular protease (epr) as well as intracellular serine protease-I (isp-1) . Characterization studies have revealed that one of these enzymes is the previously described protease bacillopeptidase F . The second enzyme, the subject of this report, is a novel metalloprotease, which we designate Mpr . Mpr is a unique metalloprotease that has been purified to apparent homogeneity by using both conventional and high-performance liquid chromatography procedures . Mpr has a molecular mass of approximately 28 kilodaltons on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a basic isoelectric point of 8.7 . The enzyme showed maximal activity against azocoll at pH 7.5 and 50 degrees C . Mpr was inhibited by dithiothreitol and a combination of beta-mercaptoethanol and EDTA . Activity was moderately inhibited by beta-mercaptoethanol and EDTA alone as well as by cysteine and citrate and only marginally by phosphoramidon 1,10-phenanthroline and N-{N-(L-3-trans-carboxyoxiran-2-carbonyl)-L-leucyl}-agmatine . Mpr was not inhibited by phenylmethylsulfonyl fluoride . In addition, Mpr showed esterolytic but not collagenolytic activities . Our studies suggest that Mpr is a secreted metalloprotease containing cysteine residues that are required for maximal activity.

J Bacteriol, 1990 Feb, 172(2), 824 - 34
Signal transduction pathway controlling synthesis of a class of degradative enzymes in Bacillus subtilis: expression of the regulatory genes and analysis of mutations in degS and degU; Msadek T et al.; The rates of synthesis of a class of both secreted and intracellular degradative enzymes in Bacillus subtilis are controlled by a signal transduction pathway defined by at least four regulatory genes: degS, degU, degQ (formerly sacQ), and degR (formerly prtR) . The DegS-DegU proteins show amino acid similarities with two-component procaryotic modulator-effector pairs such as NtrB-NtrC, CheA-CheY, and EnvZ-OmpR . By analogy with these systems, it is possible that DegS is a protein kinase which could catalyze the transfer of a phosphoryl moiety to DegU, which acts as a positive regulator . DegR and DegQ correspond to polypeptides of 60 and 46 amino acids, respectively, which also activate the synthesis of degradative enzymes . We show that the degS and degU genes are organized in an operon . The putative sigma A promoter of the operon was mapped upstream from degS . Mutations in degS and degU were characterized at the molecular level, and their effects on transformability and cell motility were studied . The expression of degQ was shown to be subject both to catabolite repression and DegS-DegU-mediated control, allowing an increase in the rate of synthesis of degQ under conditions of nitrogen starvation . These results are consistent with the hypothesis that this control system responds to an environmental signal such as limitations of nitrogen, carbon, or phosphate sources.

Gene, 1990 Jan 31, 86(1), 1 - 9
Nucleotide sequence of Streptomyces fradiae transposable element Tn4556: a class-II transposon related to Tn3; Siemieniak DR et al.; The first transposable element to be isolated from Streptomyces fradiae, Tn4556, was completely sequenced; the total of 6625 bp have an overall G + C composition of 68% . Computer-aided analysis of this sequence reveals the location of nine open reading frames (ORFs) . Several of these ORFs, numbers 1, 2, and 7, contain ribosome-binding sites (RBS) near their putative translation-initiation sites, which share identity with the consensus RBS sequences of Escherichia coli and Bacillus subtilis . ORF1 potentially encodes an 892-amino acid (aa) protein and this deduced aa sequence shares 61% identity with that of the transposase encoded by the tnpA gene of Tn3 . Three other ORFs, 2, 3 and 5, potentially encode proteins which are similar in size to the resolvase protein encoded by the Tn3 gene tnpR; however, none of the protein products deduced from these ORF share extensive aa sequence identity with other resolvase proteins.

Gene, 1990 Jan 31, 86(1), 63 - 9
Development of a beta-galactosidase alpha-complementation system for molecular cloning in Bacillus subtilis; Haima P et al.; A versatile beta-galactosidase alpha-complementation system for Bacillus subtilis was developed, which can be used for molecular cloning in this Gram+ organism . The cloning system, which is based on the highly efficient host-vector system 6GM-pHP13, offers several advantages over previously described systems: (1) convenient direct selection of recombinants; (2) the cloning of large heterologous DNA fragments with high efficiency; and (3) the availability of six unique target sites: SphI, NdeI, NheI, BamHI, SmaI and EcoRI.

Biochemistry, 1990 Jan 30, 29(4), 959 - 65
Reduced DNA flexibility in complexes with a type II DNA binding protein; Hard T et al.; We studied internal molecular motions in Bacillus subtilis phage SPO1 DNA using the time-resolved fluorescence polarization anisotropy (FPA) of intercalated ethidium . The torsional flexibility of this (hydroxymethyl)uracil-containing DNA is very similar to that of naturally occurring thymine-containing DNAs, as judged from fits of the time-resolved FPA decay to an elastic DNA model . Binding of transcription factor 1 (TF1), a type II procaryotic DNA binding protein encoded by the phage SPO1, enhances the FPA, indicating a substantial decrease in the average DNA torsional flexibility in the DNA-TF1 complex . The FPA increase is correlated with a reduced ethidium binding affinity . The effects can be noticed at TF1 binding ratios less than 1 TF1 dimer/500 DNA base pairs, and the measured torsional rigidity at high TF1 binding ratios (1 TF1 dimer/15-20 DNA base pairs) is about 7 times greater than in the absence of TF1 . On the basis of a discussion of various mechanisms for the observed effect we argue that it is due to protein-induced DNA bending at low binding densities although other explanations are also possible . This interpretation might have implications for understanding the biological function of TF1.

Biochemistry, 1990 Jan 16, 29(2), 527 - 34
Primary structure of a zinc protease from Bacillus mesentericus strain 76; Stoeva S et al.; The amino acid sequence of the neutral zinc protease from Bacillus mesentericus strain 76 (MCP 76) has been determined by using peptides derived from digests with trypsin, chymotrypsin, and cyanogen bromide and from cleavage with o-iodosobenzoic acid . The peptides were purified by means of gel filtration and reversed-phase high-performance liquid chromatography and analyzed by automatic sequencing . The protein contains 300 amino acid residues . It proved to be identical with the neutral protease deduced from the DNA precursor sequence of Bacillus subtilis . The residues for zinc and substrate binding are conserved, whereas the number of calcium binding sites is reduced compared to thermolysin . A classification of the neutral zinc protease is discussed.

Biochemistry, 1990 Jan 16, 29(2), 376 - 83
Monofunctional chorismate mutase from Bacillus subtilis: purification of the protein, molecular cloning of the gene, and overexpression of the gene product in Escherichia coli; Gray JV et al.; The monofunctional chorismate mutase from Bacillus subtilis has been purified 2200-fold to homogeneity . The enzyme is a homodimer of subunit Mr = 14,500 and is the smallest natural chorismate mutase that has been characterized . The purified enzyme follows Michaelis-Menten kinetics with a Km of 100 microM and a kcat of 50 s-1, carries no other associated enzymic activities, and is unaffected by any of the aromatic amino acids . The N-terminal amino acid sequence of the protein has been determined, and this information has been used to construct a precise oligonucleotide probe for the gene by means of in vitro DNA amplification from total chromosomal DNA by the polymerase chain reaction . The cloned aroH gene encodes a protein of 127 amino acid residues and is expressed in Escherichia coli . The cloned gene product is indistinguishable from that purified from Bacillus . The aroH coding region was directly subcloned into a phagemid expression vector by means of the polymerase chain reaction . The resulting construct, with the aroH gene positioned behind efficient transcription and translation initiation sequences of E . coli, results in the production of the monofunctional mutase at levels of 30-35% of the soluble cell protein in E . coli transformants . Chorismate mutases comprise a set of functionally related proteins that show little sequence similarity to each other . This diversity stands in contrast to other chorismate-utilizing enzymes.

FEMS Microbiol Lett, 1990 Jan 15, 55(1-2), 227 - 9
Plasmid transformation in Bacillus subtilis NB22, an antifungal-antibiotic iturin producer; Matsuno Y et al.; A transformation system with plasmids was developed for Bacillus subtilis NB22, an antibiotic iturin producing strain . Treatment of B . subtilis NB22 with 4 M KCl was effective for the induction of competence, followed by uptake of plasmid DNA in the presence of polyethylene glycol . The efficiency of transformation of this bacterium with pC194 and pUB110 was 4.1 X 10(3) and 1.5 X 10(3) transformants per micrograms DNA, respectively and the transformation frequency was 3.3 X 10(-3) and 7.2 X 10(-4), transformants per viable cell, respectively . This method was much faster and three orders of magnitude more efficient in transformation efficiency than protoplast transformation methods.

FEMS Microbiol Lett, 1990 Jan 15, 55(1-2), 135 - 8
Genetic transformation of intact cells of Bacillus subtilis by electroporation; Brigidi P et al.; Plasmid DNAs were introduced by electroporation into Bacillus subtilis PB1424 as an alternative to competent-cell or protoplast transformation . The maximum electroporation efficiency was 10(4) transformants/microgram DNA . Parameters including growth phase of cells, ionic strength of the suspending medium, concentration and size of plasmid DNAs, amplitude and duration of the pulse, were evaluated in order to determine conditions that improved transformation efficiency.

Biochem J, 1990 Jan 15, 265(2), 375 - 82
Secretion of Bacillus subtilis levansucrase . Fe(III) could act as a cofactor in an efficient coupling of the folding and translocation processes; Chambert R et al.; The refolding of levansucrase denatured by urea was studied as a possible model for the second step of the secretion pathway of this protein . The folding-unfolding transition was monitored by measuring intrinsic fluorescence and resistance to proteolysis . Both methods provided the same estimation for the unfolding free energy of levansucrase, delta GD, which was 30.1 +/- 1.7 kJ.mol-1 (7.2 +/- 0.4 kcal.mol-1) at pH 7 in 0.1 M-potassium phosphate buffer . The rate of refolding was greatly enhanced by Fe3+, whereas the Fe3+ chelator EDTA prevented correct refolding . Fe3+ allowed the protein to reach its folded form in medium in which the dielectric constant had been lowered by ethanol . The efficiency in vivo of the export of levansucrase bearing an amino acid modification which blocks the second step of the translocation pathway was greatly increased by high concentrations of Fe3+ in the culture medium . Assuming that the protein folding governs the second step of the secretion process of levansucrase, we discuss from an irreversible thermodynamic point of view the possible role of Fe3+ in the efficient coupling of the two events.

Proc Natl Acad Sci U S A, 1990 Jan, 87(2), 618 - 22
Identification of essential histidine residues in the active site of Escherichia coli xylose (glucose) isomerase; Batt CA et al.; Two conserved histidine residues (His-101 and His-271) appear to be essential components in the active site of the enzyme xylose (glucose) isomerase (EC 5.3.1.5) . These amino acid residues were targeted for mutagenesis on the basis of sequence homology among xylose isomerases isolated from Escherichia coli, Bacillus subtilis, Ampullariella sp . strain 3876, and Streptomyces violaceus-niger . Each residue was selectively replaced by site-directed mutagenesis and shown to be essential for activity . No measurable activity was observed for any mutations replacing either His-101 or His-271 . Circular dichroism measurements revealed no significant change in the overall conformation of the mutant enzymes, and all formed dimers similar to the wild-type enzyme . Mutations at His-271 could be distinguished from those at His-101, since the former resulted in a thermolabile protein whereas no significant change in heat stability was observed for the latter . Based upon these results and structural data recently reported, we speculate that His-101 is the catalytic base mediating the reaction . Replacement of His-271 may render the enzyme thermolabile, since this residue appears to be a ligand for one of the metal ions in the active site of the enzyme.

Arch Microbiol, 1990, 154(1), 82 - 4
Detection and determination of factor C--a regulatory protein--in Streptomyces strains by antiserum and monoclonal antibody; Szeszak F et al.; Rabbit antisera and monoclonal antibodies were raised against factor C, a regulatory protein of Streptomyces griseus . ELISA and immunoblotting techniques suitable to determine and characterize factor C antigen was detected in all the 23 Streptomyces strains and variants examined thus far and in one Bacillus subtilis too . Depending on the strain analysed it has a molecular mass of 34,000 or 70,000 in mycelial homogenates . Most of factor C was found excreted into the cultivation medium . The quantity of factor C antigen in different Streptomyces strains showed great variation . Amy+ strains were usually good producers of factor C while Amy- were not . This was consistent with our assumption that factor C was an inducer of reproductive phase in Streptomyces.

J Bacteriol, 1990 Jan, 172(1), 110 - 5
Four codons in the cat-86 leader define a chloramphenicol-sensitive ribosome stall sequence; Rogers EJ et al.; Genes encoding chloramphenicol acetyltransferase in gram-positive bacteria are induced by chloramphenicol . Induction reflects an ability of the drug to stall a ribosome at a specific site in cat leader mRNA . Ribosome stalling at this site alters downstream RNA secondary structure, thereby unmasking the ribosome-binding site for the cat coding sequence . Here, we show that ribosome stalling in the cat-86 leader is a function of leader codons 2 through 5 and that stalling requires these codons to be presented in the correct reading frame . Codons 2 through 5 specify Val-Lys-Thr-Asp . Insertion of a second copy of the stall sequence 5' to the authentic stall sequence diminished cat-86 induction fivefold . Thus, the stall sequence can function in ribosome stalling when the stall sequence is displaced from the downstream RNA secondary structure . We suggest that the stall sequence may function in cat induction at two levels . First, the tetrapeptide specified by the stall sequence likely plays an active role in the induction strategy, on the basis of previously reported genetic suppression studies (W . W . Mulbry, N . P . Ambulos, Jr., and P.S . Lovett, J . Bacteriol . 171:5322-5324, 1989) . Second, we show that embedded within the stall sequence of cat leaders is a region which is complementary to a sequence internal in 16S rRNA of Bacillus subtilis . This complementarity may guide a ribosome to the proper position on leader mRNA or potentiate the stalling event, or both . The region of complementarity is absent from Escherichia coli 16S rRNA, and cat genes induce poorly, or not at all, in E . coli.

Arch Microbiol, 1990, 154(4), 380 - 5
Natural genetic transformation of Pseudomonas stutzeri by sand-adsorbed DNA; Lorenz MG et al.; In a soil/sediment model system we have shown recently that a gram-positive bacterium with natural competence (Bacillus subtilis) can take up transforming DNA adsorbed to sand minerals . Here we examined whether also a naturally transformable soil bacterium of the gram-negative pseudomonad (Pseudomonas stutzeri) can be transformed by mineral-associated DNA . For these studies the transformation protocol of this species was further improved and characterized . The peak of competence during growth of P . stutzeri was determined to occur at the beginning of the stationary phase . The competence state was conserved during shock freezing and thawing of cells in 10% glycerol . Kinetic experiments showed that transformant formation after addition of DNA to competent cells proceeded for more than 2 h with DNA adsorption to cells being the rate limiting step . By means of the defined protocol P . stutzeri was shown to be transformed by sand-adsorbed DNA . Transformation by adsorbed or dissolved DNA occurred between 16 degrees and 44 degrees C . Efficiency and DNaseI-sensitivity of transformation by DNA adsorbed to sand or in liquid were comparable . It is concluded that uptake of particle-bound DNA by P . stutzeri in soil is possible . This finding adds evidence to the view that transformation occurs in natural environments where DNA is assumed to be significantly associated with mineral/particulate material and thereby is protected against enzymatic degradation.

Yi Chuan Xue Bao, 1990, 17(4), 313 - 20
{Construction of promoter probe vector pFDC4 and gene expression vector pFDC11 with high transformation efficiency in Bacillus stearothermophilus}; He X et al.; A 0.5kb fragment from Sau3A-digested total DNA of Bacillus stearothermophilus CU21 was cloned with the vector pPGV5, a derivative of pPL703 . The insertion of this fragment can activate the expression of the promoteless cat-86 gene on the cloning vector in both B . stearothermophilus and Bacillus subtilis hosts . When the 0.54 kb fragment is present in pPGV5 in either orientation, the transformation efficiency of the plasmid is increased about 10(3) to 10(4) fold in CU21 protoplasts . Southern hybridization showed this 0.54 kb fragment was homologous with a 1.6kb fragment, which was shown by Imanaka et al . (1984, J . Gen . Microbiol . 130, 1399-1408) to originate in a cryptic plasmid resident in CU21 and to enhance the transformat on efficiency of another plasmid . With this 0.54kb fragment a new promoter probe vector pFDC4 and a gene expression vector pFDC11 were constructed . Both can transform the CU21 recipient with high efficiency.

Chemotherapy, 1990, 36(5), 325 - 31
Penetration of cefonicid into human lung tissue and lymph nodes; Cazzola M et al.; The present study was undertaken in order to investigate the penetration of cefonicid, a long-acting parenteral cephalosporin, with enhanced activity against most gram-positive and gram-negative pathogens, into human lung tissue and lymph nodes in patients undergoing open thoracotomy . Samples of lung tissue, lymph nodes and serum were obtained at various times after a single intramuscular dose of 1 g . The concentration of cefonicid was assayed by an agar diffusion method with Bacillus subtilis used as the test organism . The mean concentrations of cefonicid in serum at 2, 4, 8, 12 and 24 h after the injection were 91.5, 66.1, 35.7, 21.8 and 2.9 micrograms/ml, respectively . The mean levels of cefonicid into the hilar lymph nodes at the same times were 22.3, 18.7, 12.0, 6.9 and 1.5 micrograms/ml, respectively, while its concentrations in lung tissue were lower than those in lung lymph nodes up to the 12th hour (12.1, 14.6, 7.8, 5.4 and 1.9 micrograms/ml, respectively) . Our results show that cefonicid was well distributed in interstitial fluid from which pulmonary lymph is formed and that its concentrations in lung tissue and lymph nodes were sufficient to inhibit most pathogens involved in respiratory tract infections . This finding was considered important, because it demonstrated that the high binding by plasma protein of cefonicid did not prevent it from entering lung tissue and fluids in useful quantities.

J Basic Microbiol, 1990, 30(6), 387 - 92
Excision of transposon Tn917 in Bacillus subtilis; Adler B et al.; Excision of Tn917 from chromosomal sites in B . subtilis is characterized by reversion studies . We propose different modes of excision depending on the site of transposon insertion . Excision takes place as precise excision in one step which results in reversion of the mutant phenotype, or by a two-step process where nearly precise excision of the transposon moiety is followed by precise excision of the nearly precise excision remnants . For this type of transposons a minor pathway or nonreplicative transposition is proposed to be connected with precise excision.

Mol Microbiol, 1990 Jan, 4(1), 137 - 41
Pulling the trigger: the mechanism of bacterial spore germination; Foster SJ et al.; In spite of displaying the most extreme dormancy and resistance properties known among living systems, bacterial endospores retain an alert environment-sensing mechanism that can respond within seconds to the presence of specific germinants . This germination response is triggered in the absence of both germinant and germinant-stimulated metabolism . Genes coding for components of the sensing mechanism in spores of Bacillus subtilis have been cloned and sequenced . However, the molecular mechanism whereby these receptors interact with germinants to initiate the germination response is unknown . Recent evidence has suggested that in spores of Bacillus megaterium KM, proteolytic activation of an autolytic enzyme constitutes part of the germination trigger reaction.

J Bacteriol, 1990 Jan, 172(1), 218 - 23
Aspartokinase III, a new isozyme in Bacillus subtilis 168; Graves LM et al.; A previously undetected Bacillus subtilis aspartokinase isozyme, which we have called aspartokinase III, has been characterized . The new isozyme was most readily detected in extracts of cells grown with lysine, which repressed aspartokinase II and induced aspartokinase III, or in extracts of strain VS11, a mutant lacking aspartokinase II . Antibodies against aspartokinase II did not cross-react with aspartokinase III . Aspartokinases II and III coeluted on gel filtration chromatography at Mr 120,000, which accounts for the previous inability to detect it . Aspartokinase III was induced by lysine and repressed by threonine . It was synergistically inhibited by lysine and threonine . Aspartokinase III activity, like aspartokinase II activity, declined rapidly in B . subtilis cells that were starved for glucose . In contrast, the specific activity of aspartokinase I, the diaminopimelic acid-inhibitable isozyme, was constant under all growth conditions examined.

Ukr Biokhim Zh, 1990 Jan-Feb, 62(1), 76 - 82
{The role of membrane processes in Au(III) and Au(0) accumulation by bacteria}; Karamushka VI et al.; The role of structural and functional factors in the processes of the bacterial cell interaction with colloid Au (0) and ionic Au (III) states has been investigated . It is shown that the bacterial walls of Bacillus sp . 4368 aggregating with colloid gold contain glycoprotein with isoelectric point 11 . Glycoprotein from cell walls indifferent to colloid gold strain (Bacillus subtilis 168) has pHiso = 5 . At the same time the cells of both strains accumulate Au (III) introduced into a medium in the form of tetrachloroaurate . The process is energy-dependent because it is suppressed by azide, uncouplers of oxidative phosphorylation and dicyclohexyl carbodiimide (DCCD) . The role of ATPase of Au (III) accumulation has been studied on Bacillus sp . 4368 plasma membrane vesicles . The ATPase activity is inhibited by 70, 50 and 35-50% by vanadate, DCCD and Au (III), respectively, but it does not change in the presence of dinitrophenol and NaN3 . ATP but not ADP and AMP stimulated the Au (III) accumulation by membrane vesicles and prevents the inhibitory action of azide but neither of DNP or DCCD . In the energized state membrane vesicles link gold sol particles . It has been assumed that the Au (III) accumulation is associated with the functioning of transmembrane potential generators, the metal being localized on the membrane surface.

Int J Oral Maxillofac Implants, 1990 Summer, 5(2), 117 - 25
Dynamic ultraviolet sterilization of different implant types; Delgado AA et al.; This paper investigates the use of the dynamic ultraviolet sterilization process with various dental implants, stainless steel orthopedic cortical bone screws, and polysulfone polymer healing caps . These biomaterials were inoculated with the spores of Bacillus subtilis and Bacillus stearothermophilus . They were then exposed to dynamic ultraviolet radiation in the chamber of a BUD Ultraviolet Device . Samples were incubated in trypticase soy broth at 37 degrees C and 56 degrees C, and they were subcultured onto an enriched agar medium . Results indicate that 16 seconds of dynamic ultraviolet radiation is effective in sterilizing these materials . This is significantly less time than other sterilization techniques presently used.

Microbiol Immunol, 1990, 34(12), 1013 - 23
Permeability of gentamicin and polymyxin B into the inside of Bacillus subtilis spores; Fujita Y et al.; The penetration of gentamicin and polymyxin B into the inside of Bacillus subtilis spores was examined by an immunoelectron microscopy method with colloidal gold--immunoglobulin G (IgG) complex . The colloidal gold particles were located predominantly in the coat region of both gentamicin-treated and polymyxin B-treated spores and were hardly observed in the other regions, i.e., the cortex and core regions . When these antibiotic-treated spores were subsequently treated with CaCl2, the number of gold particles bound to the coat region was greatly decreased . These results suggest that these two antibiotics are able to penetrate into the spore coat but not into the cortex or core, that is, the primary permeability barrier to them exists between the coat and the cortex regions.

Acta Microbiol Bulg, 1990, 26, 73 - 6
{Production of Bacillus subtilis mutants by transformation crosses}; Aleksieva Z; A method for obtaining auxotrophic mutants for amino acids coding by regulatory region of cat 86 has been reported . A selection of Bacillus subtilis BR 151 mutants for studying the translational regulation of cat 86 has been carried out . The procedure described is very convenient for genetic analysis.

Acta Microbiol Bulg, 1990, 26, 67 - 72
{Genetic studies of a Bacillus subtilis producer of a milk-coagulating enzyme complex}; Aleksieva Z et al.; Dissociation is a phenomenon when from an initial bacterial population different forms in morphological, physiological aspects have been produced . For bacterial producers of biologically active substances the investigation of this process is important for industrial microbiology . Bacillus subtilis 76 is a producer of proteolytic enzyme complex with milk-coagulating effect . It dissociates in several different forms . Some of them (M, R, S) have been studied . They are plasmidless, prototrophs and tiostrepton-resistant . The results obtained are of a great importance for the future genetic experiments.

J Basic Microbiol, 1990, 30(9), 655 - 62
Intracellular expression of hIFN alpha genes in Escherichia coli and Bacillus subtilis directed by staphylokinase signals; Breitling R et al.; Portable expression units for intracellular formation of heterologous proteins in Escherichia coli and Bacillus subtilis were constructed by inserting the transcription and translation initiation signals of the staphylokinase sak42D gene into the polylinker of plasmid pUC18 . Fusions with ATG-gene cassettes coding for mature human interferons (hIFN) alpha 1 and alpha 2 resulted in intracellular expression of both proteins in E . coli . The 20 fold lower yield of hIFN alpha 2 was not due to unfavorable mRNA secondary structure formation as ruled out by constructing a hybrid hIFN alpha 1/alpha 2 gene . Intracellular expression of IFN alpha 1 in B . subtilis reached 6 x 10(4) IU/ml . Nuclease S1 mapping of transcriptional start sites revealed differential promoter usage in E . coli and B . subtilis . In E . coli transcription from the sak42D promoter was drastically reduced by by transcription initiating from upstream lac and tet promoters . In contrast, in B . subtilis transcription proceeded exclusively from the sak42D promoter.

Arch Microbiol, 1990, 155(1), 62 - 7
Menaquinone is an obligatory component of the chain catalyzing succinate respiration in Bacillus subtilis; Lemma E et al.; The question was investigated as to whether the bacterial menaquinone (MK) is a component of the electron transport chain catalyzing succinate respiration in Bacillus subtilis . Three different methods were applied, and the following consistent results were obtained . (i) Solvent extraction of MK from the bacterial membrane caused total inhibition of the respiratory activities with succinate and NADH, while the activity of succinate dehydrogenase remained unaffected . The respiratory activities were restored on incorporation of vitamin K1 into the membrane preparation . (ii) The membrane fraction of a B . subtilis mutant containing 15% of the wild-type amount of MK, respired succinate and NADH at reduced activities . Wild-type activities were restored on fusion of the preparation to liposomes containing vitamin K1 . (iii) The membrane fraction of B . subtilis catalyzed succinate oxidation by various water-soluble naphtho- or benzoquinones at specific activities exceeding to that of succinate respiration . The results suggest that MK is involved in succinate respiration, although its redox potential is unfavorable.

Drugs Exp Clin Res, 1990, 16(4), 149 - 55
An automated pulse labelling method for structure-activity relationship studies with antibacterial oxazolidinones; Eustice DC et al.; The 3-aryl-2-oxooxazolidinones are a new class of synthetic antibacterial agents that potently inhibit protein synthesis . An automated pulse labelling method with {3H}-lysine was developed with Bacillus subtilis to obtain additional quantitative activity data for structure-activity relationship studies with the oxazolidinones . Inhibition constants were calculated after a Logit fit of the data into the formula: % of control = 100/(1 + e{-B(X - A)}), where B is the slope of the model, X is the natural log of the inhibitor concentration and A is the natural log of the inhibitor concentration required to inhibit protein synthesis by 50% (ln IC50) . When substituents at the 5-methyl position of the heterocyclic ring (B-substituent) were NHCOCH3, OH or Cl, the correlation coefficient was 0.87 between the MIC and IC50 values (for all compounds with MICs less than or equal to 16 micrograms/ml) . The D-isomers of DuP 721 (A-substituent = CH3CO) and DuP 105 (A-substituent = CH3SO) gave MICs of 128 micrograms/ml and IC50s of greater than or equal to 50 micrograms/ml for protein synthesis, showing that only the L-isomers were active . By MIC testing, oxazolidinones with the B-substituent of NHCOCH3 and the A-substituent of CH3CO, NO2, CH3S, CH3SO2 or (CH3)2CH had comparable antibacterial potency; however, pulse labelling analysis showed that compounds with an A-substituent of CH3CO or NO2 were more potent inhibitors of protein synthesis.

Microbiol Immunol, 1990, 34(8), 709 - 14
Detection of common flagella antigen in Bacillus cereus by monoclonal antibody; Mikami T et al.; Bacillus cereus has been classified into 23 types by immunochemical analyses of flagella antigen, but a common antigenic determinant of flagella had not been determined . When the immunochemical method of classification had changed from "agglutination method" to "enzyme-linked immunosorbent assay (ELISA)," the cross-reactivity between each flagella type was increased . These results indicated that the application of ELISA method would enable detection of the common antigenic determinant of B . cereus . Therefore, we attempted to make monoclonal antibody against common flagella antigen that could be detected by ELISA . Monoclonal antibody provided was specific for B . cereus (H-1 to H-23) and did not show the cross-reactivity with Escherichia coli NIH and Bacillus subtilis.

Rev Argent Microbiol, 1990 Jan-Mar, 22(1), 1 - 6
{Viability of Bacillus subtilis auxotrophs in the absence of their essential metabolites}; Franco MA et al.; The effect of tryptophan and uracil starvation on the viability of the transformant B . subtilis BSA 170 trp- ura- and its parent strains Bacillus subtilis PB 168 trp -C and Bacillus subtilis PB 3308 ura- was examined . These studies were performed at the conditions for competence development, during 16 hours . Our results showed that B . subtilis BSA 170 was resistant to tryptophan-less death during all the assay and was also resistant to uracil-less death during three hours . After this time, viability measurements revealed less colony forming units per milliliter, and decrease of the culture absorbances . The uracil-less death required the presence of tryptophan suggesting that protein synthesis is needed . The parental strains exhibit similar behavior . Bacillus subtilis PB 168 was resistant to tryptophan-less death and B . subtilis PB 3308 showed decrease of the viability after uracil starvation comparable to that of the transformant strain.

Folia Microbiol (Praha), 1990, 35(5), 371 - 83
Rotational relaxation rate of 1,6-diphenyl-1,3,5-hexatriene in cytoplasmic membranes of Bacillus subtilis . A new model of heterogeneous rotations; Konopasek I et al.; The temperature dependence of fluorescence anisotropy, lifetime and differential tangent of 1,6-diphenyl-1,3,5-hexatriene (DPH) and its polar trimethylammonium derivative (TMA-DPH) were investigated in cytoplasmic membranes of Bacillus subtilis . The fluorescence parameters were compared in the two types of membranes prepared from bacteria cultivated at 20 and 40 degrees C . Steady-state anisotropy measurements showed that within a broad range of temperatures, membranes cultivated at 20 degrees C exhibit significantly lower values than those prepared from cells cultivated at 40 degrees C . The temperature dependence of lifetime and differential tangent measurements (differential polarized phase fluorimetry) were fully consistent with steady-state anisotropy data of both DPH and TMA-DPH . The low anisotropy values in the case of TMA-DPH could be explained by a shorter lifetime and higher temperature-induced decrease as compared with DPH . Surprisingly, the temperature dependence of rotational rate R calculated according to the model of hindered rotations (Lakowicz 1983) gave misleading results . When increasing the temperature from 5 to 25 degrees C, a marked drop of rotational relaxation rate was observed . The minimum R values were measured between 25 and 30 degrees C and further increase of temperature (up to 60 degrees C) was reflected as increase of the R values . Therefore, a new model of "heterogeneous rotations" was developed . This model assumes that even at low temperatures (approaching 0 degrees C) where the differential tangent reaches zero, a fraction of fast rotating molecules exists . The ratio between fast and slowly rotating molecules may be expressed by this model, the newly calculated rotational rates are fully consistent with anisotropy, lifetime and differential tangent measurements and represent the monotonically increasing function of temperature.

Yi Chuan Xue Bao, 1990, 17(5), 398 - 404
{Stability and gene expression of foreign plasmid in Bacillus subtilis BF7658}; Geng YQ et al.; The plasmid pAmy411 carrying thermostable alpha-amylase gene has been transduced into B . subtilis BF7658 by bacteriophage PBS1 . The transduction frequency was 10(-9) transductants per PFU . The stability of plasmid pAmy411 in B . subtilis BF7658 was lower than that in B . subtilis AS1.1176, whereas the copy numbers were double than that in B . subtilis AS1.1176 . The expressive level of thermostable alpha-amylase gene was about 6 times higher than that in B . subtilis AS1.1176.

Microbios, 1990, 63(256-257), 173 - 86
Effect of metabisulphite on sporulation and alkaline phosphatase in Bacillus subtilis and Bacillus cereus; Abalaka JA et al.; The effect of metabisulphite on spore formation and alkaline phosphatase activity/production in Bacillus subtilis and Bacillus cereus was investigated both in liquid and semi-solid substrates . While supplementary nutrient broth (SNB) and sporulation medium (SM) were used as the liquid growth media, two brands of powdered milk were used as the food (semi-solid) substrates . Under both aerobic and anaerobic conditions, B . subtilis was more resistant to metabisulphite than B . cereus while the level of enzyme production and spores formed were generally higher under aerobic than anaerobic conditions . The metabisulphite concentrations required to inhibit spore production as well as alkaline phosphatase synthesis/activity were found to be relatively low and well within safety levels for human consumption . It is concluded that metabisulphite is an effective anti-sporulation agent and a recommendation for its general use in semi-solid and liquid foods is proposed.

Arch Virol, 1990, 113(3-4), 177 - 81
Action of 6-(p-hydroxyphenylazo)-uracil on bacteriophage IG 1; Fernandes RM et al.; IG 1, a temperate phage of Bacillus subtilis, is strongly induced from its lysogens by 6-(p-hydroxyphenylazo)-uracil, an azopyrimidine known as selectively inhibiting the B . subtilis DNA polymerase III . IG 1 phages originated either by induction or infection multiply, abundantly, in the presence of that azopyrimidine, in spite of the drastic decline of cell viability . Chloramphenicol completely suppresses the induction effect and also blocks the formation of spontaneously induced phage particles.

Microbios, 1990, 63(254), 37 - 44
Effect of disulphite on protein and pyridine-2,6-dicarboxylic acid synthesis in sporulating cells of bacterial species; Oloyede OB et al.; The effect of disulphite on protein and pyridine-2,6-dicarboxylic acid (dipicolinic acid, DPA) synthesis was investigated in sporulating cells of Bacillus subtilis E52 and B . cereus W18 . Progressive reductions were evident in the protein and DPA concentrations for both sporulating cells with increasing concentrations (100 to 600 micrograms ml-1) of disulphite . A significant (P less than 0.05) reduction in protein synthesis by disulphite was exhibited, culminating in a decrease in protein synthesis ranging from 50% to 1.4%, and 50% to 2.5%, in B . subtilis E52 and B . cereus W18, respectively . The same disulphite concentrations caused a significant (P less than 0.05) decrease in DPA synthesis ranging from 75% to 12.5% and 70% to 5%, for B . subtilis E52 and B . cereus W18, respectively . DPA synthesis was completely prevented at 500 and 600 micrograms ml-1 for B . subtilis E52 and B . cereus W18, respectively . A plausible mechanism for the inhibitory action of disulphite on sporulating cells of the bacteria is proposed.

Chemotherapy, 1990, 36(5), 332 - 6
Roxithromycin penetration into gingiva and alveolar bone of odontoiatric patients; Del Tacca M et al.; The concentrations of the new macrolide antibiotic roxithromycin in plasma, saliva, gingiva, and alveolar bone were studied in 24 odontoiatric patients treated with a first dose of 300 mg p.o . followed by three maintenance doses of 150 mg p.o., 12-hourly . Samples of blood, saliva, gingiva, and bone were collected at various time points up to 24 h after the last dosing, and the roxithromycin concentration was measured microbiologically, using Bacillus subtilis ATCC 6633 as the reference organism . Pharmacokinetic analysis was performed according to a two-compartment open model with first-order absorption . The plasma, gingiva, and alveolar bone peak concentrations were 6.12 +/- 1.94 mg/l, 6.55 +/- 2.54 mg/kg, and 5.09 +/- 1.60 mg/kg, respectively . Low levels of roxithromycin were detected in saliva (0.67 +/- 0.12 mg/l at the 3rd h) . The values of the area under the concentration-time curve for plasma, gingiva, and bone were 59.47 mg/l.h, 51.88 mg/kg.h and 46.80 mg/kg.h, respectively; the half-life values were 7.52 h for plasma and 6.36 and 5.20 h for gingiva and bone, respectively . These results indicate that roxithromycin reaches high levels in periodontal tissues.

Acta Microbiol Bulg, 1990, 25, 35 - 9
{Protoplast reversion in Bacillus subtilis}; Valerianov Ts et al.; The reversion of the protoplasts of two strains Bac . subtilis on 3 nutritive media: DP of Elliott et al., DM3 of Chang and Cohen and MRG of Bourne and Dancer, is studied . Special attention is devoted to the role of the components gelatin, bovine serum albumin and osmotic stabilizer . In relation to the conclusions drawn in the study a new medium for reversion of protoplasts is suggested--the glucitole reversion agar GRA ensuring continuous 20-40% reversion of the sown protoplasts.

Microbios, 1990, 62(251), 93 - 9
Mycosubtilins B and C: minor antibiotics from mycosubtilin-producer Bacillus subtilis; Besson F et al.; Mycosubtilins B and C were isolated from the culture medium of Bacillus subtilis . The acid hydrolysates of these new antifungal antibiotics, like mycosubtilin, contain alpha-amino acids (Asp3, Glu1, Pro1, Ser1 and Tyr1) and a mixture of iso-C16, n-C16, iso-C17 and anteiso-C17 beta-amino acids . Mycosubtilins B and C differ by the presence of a carboxyl group and of a carboxymethyl group, respectively, instead of a carboxamide group in previously described mycosubtilin.

Arch Microbiol, 1990, 153(6), 569 - 73
The relative rotation of the ends of Bacillus subtilis during growth; Koch AL; Observation of long single filaments of Bacillus subtilis 168 in depression slide cultures demonstrated that one end rotated relative to the other during growth . This was observed with suspended filaments, filaments attached to glass surfaces and single stranded filaments folded back on themselves growing as a double stranded helix . This extends Mendelson's 1976 conclusion to cases with no alternative interpretation to the hypothesis that as each cell grows, the structure of the peptidoglycan changes to rotate one end relative to the other.

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