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