Search PubMed⌕ Search

Biomedical subjects

G H Chambliss

Publications and source records attributed to G H Chambliss.

At least 37 records · Page 2Linked to original sources
PubMed1987

Effect of decoyinine on the regulation of alpha-amylase synthesis in Bacillus subtilis.

Decoyinine, an inhibitor of GMP synthetase, allows sporulation in Bacillus subtilis to initiate and proceed under otherwise catabolite-repressing conditions. The effect of decoyinine on alpha-amylase synthesis in B. subtilis, an event which exhibits regulatory features resembling sporulation initiation, was examined. Decoyinine did not overcome catabolite repression of alpha-amylase synthesis in a wild-type strain of B. subtilis but did cause premature and enhanced synthesis in a mutant strain specifically blocked in catabolite repression of alpha-amylase synthesis. Decoyinine had no effect on alpha-amylase enzymatic activity. Thus, it appears that the catabolite control mechanisms governing alpha-amylase synthesis and sporulation in B. subtilis differ in their responses to decoyinine and hence must consist at least partially of separate components.

Adenosine↗
PubMed1987

Endo-beta-1,4-glucanase gene of Bacillus subtilis DLG.

The DNA sequence of the Bacillus subtilis DLG endo-beta-1,4-glucanase gene was determined, and the in vivo site of transcription initiation was located. Immediately upstream from the transcription start site were sequences closely resembling those recognized by B. subtilis sigma 43-RNA polymerase. Two possible ribosome-binding sites were observed downstream from the transcription start site. These were followed by a long open reading frame capable of encoding a protein of ca. 55,000 daltons. A signal sequence, typical of those present in gram-positive organisms, was observed at the amino terminus of the open reading frame. Purification of the mature exocellular beta-1,4-glucanase and subsequent amino-terminal protein sequencing defined the site of signal sequence processing to be between two alanine residues following the hydrophobic portion of the signal sequence. The probability of additional carboxy-terminal processing of the beta-1,4-glucanase precursor is discussed. S1 nuclease protection studies showed that the amount of beta-1,4-glucanase mRNA in cells increased significantly as the culture entered the stationary phase. In addition, glucose was found to dramatically stimulate the amount of beta-1,4-glucanase mRNA in vivo. Finally, the specific activities of purified B. subtilis DLG endo-beta-1,4-glucanase and Trichoderma reesei QM9414 endo-beta-1,4-glucanase (EC 3.2.1.4) were compared by using the noncrystalline cellulosic substrate trinitrophenyl-carboxymethyl cellulose.

Amino Acid Sequence↗
PubMed1986

Production of toxin by Clostridium botulinum type A strains cured by plasmids.

Twelve strains of Clostridium botulinum type A and seven strains of Clostridium sporogenes were screened for plasmids by agarose gel electrophoresis of cleared lysates of cells from 5 ml of mid-log-phase culture. Nine type A strains had one or more plasmids of 4.3, 6.8, or 36 megadaltons (MDa); several strains showed a large plasmid of 61 MDa, but it was not consistently recovered. Four C. sporogenes strains had one or more plasmids of 4.3, 5.6 or 36 MDa. Isolates obtained from cultures of plasmid-containing C. botulinum type A strains grown in ionic detergent broth and from spontaneously arising variants were screened both for toxin production and for plasmid content. Toxigenicity of C. botulinum could not be correlated with the presence of any one plasmid.

Botulinum Toxins↗
PubMed1986

Cloning of the Bacillus subtilis DLG beta-1,4-glucanase gene and its expression in Escherichia coli and B. subtilis.

The gene encoding beta-1,4-glucanase in Bacillus subtilis DLG was cloned into both Escherichia coli C600SF8 and B. subtilis PSL1, which does not naturally produce beta-1,4-glucanase, with the shuttle vector pPL1202. This enzyme is capable of degrading both carboxymethyl cellulose and trinitrophenyl carboxymethyl cellulose, but not more crystalline cellulosic substrates (L. M. Robson and G. H. Chambliss, Appl. Environ. Microbiol. 47:1039-1046, 1984). The beta-1,4-glucanase gene was localized to a 2-kilobase (kb) EcoRI-HindIII fragment contained within a 3-kb EcoRI chromosomal DNA fragment of B. subtilis DLG. Recombinant plasmids pLG4000, pLG4001a, pLG4001b, and pLG4002, carrying this 2-kb DNA fragment, were stably maintained in both hosts, and the beta-1,4-glucanase gene was expressed in both. The 3-kb EcoRI fragment apparently contained the beta-1,4-glucanase gene promoter, since transformed strains of B. subtilis PSL1 produced the enzyme in the same temporal fashion as the natural host B. subtilis DLG. B. subtilis DLG produced a 35,200-dalton exocellular beta-1,4-glucanase; intracellular beta-1,4-glucanase was undetectable. E. coli C600SF8 transformants carrying any of the four recombinant plasmids produced two active forms of beta-1,4-glucanase, an intracellular form (51,000 +/- 900 daltons) and a cell-associated form (39,000 +/- 400 daltons). Free exocellular enzyme was negligible. In contrast, B. subtilis PSL1 transformed with recombinant plasmid pLG4001b produced three distinct sizes of active exocellular beta-1,4-glucanase: approximately 36,000, approximately 35,200, and approximately 33,500 daltons. Additionally, B. subtilis PSL1(pLG4001b) transformants contained a small amount (5% or less) of active intracellular beta-1,4-glucanase of three distinct sizes: approximately 50,500, approximately 38,500 and approximately 36,000 daltons. The largest form of beta-1,4-glucanase seen in both transformants may be the primary, unprocessed translation product of the gene.

Bacillus subtilis↗
PubMed1986

Molecular cloning of cis-acting regulatory alleles of the Bacillus subtilis amyR region by using gene conversion transformation.

Three cis-acting alleles (gra-10, gra-5, and amyR2) of the Bacillus subtilis amyR promoter locus each cause catabolite repression-resistance of amyE-encoded alpha-amylase synthesis. The gra-10, gra-5, and amyR2 alleles were transferred from the chromosomes of their respective hosts to a plasmid carrying the amyR1-amyE+ gene by the process of gene conversion which is carried out during transformation of competent B. subtilis by plasmid clones carrying homologous DNA. The cloned amyR promoter regions containing the gra-10 and gra-5 mutations were shown to confer catabolite repression-resistance in cis to the synthesis of chloramphenicol acetyltransferase encoded by the cat-86 indicator gene when subcloned into the promoter-probe plasmid pPL603B. Implications concerning both the regulation of amyR utilization and the process of gene conversion in B. subtilis are discussed.

Alleles↗
PubMed1985

Isolation and expression of a constitutive variant of the chloramphenicol-inducible plasmid gene cat-86 under control of the Bacillus subtilis 168 amylase promoter.

The amyR1 region controls the regulated expression of the Bacillus subtilis 168 amylase gene amyE. When cloned into the B. subtilis promoter-cloning plasmid pPL603, amyR1 has been shown to activate expression of the promoter-indicator gene cat-86. In this chimeric plasmid, p5' alpha B10, cat-86 expression was maximal in stationary phase B. subtilis cells and cat-86 expression was repressible by glucose. Both these properties are similar to the regulated expression of the B. subtilis amyE gene. In addition, cat-86 expression in p5' alpha B10 was inducible with chloramphenicol (Cm). The inducibility phenotype of cat-86 has been shown to be independent of the promoter that is used to activate the gene, and inducibility has been suggested to result from the presence of a pair of inverted-repeat sequences that span the ribosome-binding site (RBS) for cat-86. A spontaneous deletion mutant of p5' alpha B10 was isolated, p5' alpha B10 delta 1, in which cat-86 expression was constitutive with respect to Cm, but the basic pattern of amyR1-directed regulation of cat-86 was intact. The rightward deletion endpoint was within the upstream member of the pair of inverted repeats that immediately precede cat-86. This result is therefore consistent with the role proposed for the inverted repeats in Cm inducibility. The leftward endpoint of the deletion is within the amyR1 region and thus allows a more precise determination of the functional domain of amyR1.

Acetyltransferases↗
PubMed1985

Induction of the chloramphenicol acetyltransferase gene cat-86 through the action of the ribosomal antibiotic amicetin: involvement of a Bacillus subtilis ribosomal component in cat induction.

The plasmid gene cat-86 and the cat gene resident on pC194 each encode chloramphenicol-inducible chloramphenicol acetyltransferase activity in Bacillus subtilis. Chloramphenicol induction has been proposed to result from chloramphenicol binding to ribosomes, which then permits the drug-modified ribosomes to perform events essential to induction. If this proposal were correct, B. subtilis mutants containing chloramphenicol-insensitive ribosomes should not permit chloramphenicol induction of either cat-86 or pC194 cat. However, we and others have been unable to isolate chloramphenicol-resistant ribosomal mutants of B. subtilis 168. We therefore developed a simple procedure for screening other antibiotics for the potential to induce cat-86 expression. One antibiotic, amicetin, was found to be an effective inducer of cat-86 but not of the cat gene on pC194. Amicetin and chloramphenicol each interact with the 50S ribosomal subunit, and the mechanism of cat-86 induction by both drugs may be similar. Amicetin-resistant mutants of B. subtilis were readily isolated, and in none of six mutants tested was cat-86 detectably inducible by amicetin, although the chloramphenicol-inducible phenotype was retained. The ami-1 mutation which is present in one of these amicetin-resistant mutants was mapped by PBS1 transduction to the "ribosomal gene cluster" adjacent to cysA. Additionally, ribosomes from cells harboring the ami-1 mutation contained an altered BL12a protein, as detected in two-dimensional polyacrylamide gel electrophoresis. Lastly, an in vitro protein-synthesizing system that uses ribosomes from an ami-1-containing cell line was more resistant to amicetin than a system that uses ribosomes from an amicetin-sensitive but otherwise isogenic strain. These results indicate that the host mutation, ami-1, which effectively abolished the inducibility of cat-86 by amicetin, altered a ribosomal component.

Acetyltransferases↗
PubMed1985

Isolation and characterization of a cis-acting mutation conferring catabolite repression resistance to alpha-amylase synthesis in Bacillus subtilis.

Bacillus subtilis 168GR10 was shown to contain a mutation, gra-10, which allowed normal temporal activation of alpha-amylase synthesis in the presence of a concentration of glucose that is inhibitory to activation of amylase synthesis in the parent strain, 168. The gra-10 mutation was mapped by phage PBS-1-mediated transduction and by transformation to a site between lin-2 and aroI906, very tightly linked to amyE, the alpha-amylase structural gene. The gra-10 mutation did not pleiotropically affect catabolite repression of sporulation or of the synthesis of extracellular proteases or RNase and was unable to confer glucose-resistance to the synthesis of chloramphenicol acetyltransferase encoded by the cat-86 gene driven by the amyE promoter region (amyR1) inserted into the promoter-probe plasmid pPL603B. It therefore appears that gra-10 defines a cis-regulatory site for catabolite repression, but not for temporal activation, of amyE expression. The evidence shows that temporal activation and glucose-mediated repression of alpha-amylase synthesis in B. subtilis 168 are distinct phenomena that can be separated by mutation.

Bacillus subtilis↗
PubMed1984

Genetic mapping of a mutation causing an alteration in Bacillus subtilis ribosomal protein S4.

A mutation causing an alteration in Bacillus subtilis ribosomal protein S4 was mapped by transformation and PBS-1 transduction to a site between aroG and argA, a region of the B. subtilis chromosome not previously demonstrated to contain ribosomal protein genes. The S4 mutation conferred a spore-plus phenotype in a streptomycin-resistant, spore-minus genetic background. The altered protein was detectable by polyacrylamide gel electrophoresis of ribosomal proteins of recombinants scored for the spore-plus phenotype in genetic crosses.

Bacillus subtilis↗
PubMed1984

Characterization of the cellulolytic activity of a Bacillus isolate.

A group I Bacillus strain, DLG, was isolated and characterized as being most closely related to Bacillus subtilis. When grown on any of a variety of sugars, the culture supernatant of this isolate was found to possess cellulolytic activity, as demonstrated by degradation of trinitrophenyl-carboxymethyl cellulose. Growth in medium containing cellobiose or glucose resulted in the greatest production of cellulolytic activity. The cellulolytic activity was not produced until the stationary phase of growth, and the addition of glucose or cellobiose to a culture in this phase had no apparent effect on enzyme production. Fractionation of the culture supernatant showed that the molecular weight of the enzymatic activity was less than 100,000. Maximum cellulolytic activity in assays was observed at pH 4.8 and at 58C, although maximum thermal stability of the activity. Kinetic experiments suggested that more than one enzyme was acting upon trinitrophenyl-carboxymethyl cellulose. Exocellular protein produced by this Bacillus isolate showed roughly one-fifth the cellulolytic activity displayed by Trichoderma reesei C30 on noncrystalline, cellulosic substrates. In contrast to T. reesei cellulase, the Bacillus enzymatic activity showed no ability to degrade crystalline forms of cellulose, nor was cellobiase activity detectable.

Bacillus↗
PubMed1984

Genetic analysis of a streptomycin-resistant oligosporogenous Bacillus subtilis mutant.

Strain SRB15T+, a streptomycin-resistant, oligosporogenous mutant of Bacillus subtilis, contains two mutations, fun and strR. These mutations were mapped by PBS-1 mediated transduction and by transformation to two different sites in the cysA-linked region of the B. subtilis chromosome. The fun mutation mapped very close to rpsLl, a classic strA mutation, whereas strR mapped to a site distal to rpsE. The effects of these mutations on growth, sporulation, and streptomycin resistance in vivo and in vitro were determined. The fun mutation gave a different phenotype than did the rpsLl mutation and caused altered migration of a ribosomal protein which was identified as S12, the protein encoded by rpsL. It therefore appears that fun is an allele of the rpsL gene.

Alleles↗
PubMed1984

New chloramphenicol resistance locus in Bacillus subtilis.

A spontaneously occurring, noninducible, chloramphenicol-resistant mutant of Bacillus subtilis 168 has a mutation (cam-2) which maps in the ribosomal protein region of the chromosome near dal. Its presence does not confer dependence on chloramphenicol. Ribosomes of the cam-2 strain remained sensitive to chloramphenicol in in vitro protein synthesis. No chloramphenicol acetyltransferase activity could be detected.

Acetyltransferases↗
PubMed1982

Revertants of a streptomycin-resistant, oligosporogenous mutant of Bacillus subtilis.

Revertants of a streptomycin-resistant (Strr), oligosporogenous (Spo-) mutant of Bacillus subtilis were selected form the ability to sporulate. The revertants obtained fell into two phenotypic classes: Strs Spo+ (streptomycin-sensitive, sporeforming), which arose by reversion of the streptomycin resistance mutations of the parent strain; and Strr Spo+, which arose by the acquisition of additional mutations, some of which were shown to affect ribosomal proteins. Alterations of ribosomal proteins S4 and S16 in the 30S subunit and L18 inthe 50S subunit were detected in Strr Spo+ revertants by polyacrylamide gel electrophoresis. Streptomycin resistance of the parental strain and the Strr revertants was demonstrated to reside in the 30S ribosomal subunit. The second site mutations of the revertants depressed the level of streptomycin resistance in vivo and in the in vitro translation of phage SP01 messenger ribonucleic acid (mRNA) relative to the resistance exhibited by the Strr parental strain. The Strr parent grew slowly and sporulated at approximately 1% of the wild type level. The Strr revertants closely resembled the wild type strain with regard to growth and sporulation. The Strr revertants grew at rates intermediate between those of the Strr patent and wild type, and sporulated at wild type levels.

Bacillus subtilis↗
PubMed1982

Synthesis of acid-soluble spore proteins by Bacillus subtilis.

The major acid-soluble spore proteins (ASSPs) of Bacillus subtilis were detected by immunoprecipitation of radioactively labeled in vitro- and in vivo-synthesized proteins. ASSP synthesis in vivo began 2 h after the initiation of sporulation (t2) and reached its maximum rate at t7. This corresponded to the time of synthesis of mRNA that stimulated the maximum rate of ASSP synthesis in vitro. Under the set of conditions used in these experiments, protease synthesis began near t0, alkaline phosphatase synthesis began at about t2, and refractile spores were first observed between t7 and t8. In vivo- and in vitro-synthesized ASSPs comigrated in sodium dodecyl sulfate-polyacrylamide gels. Their molecular weights were 4,600 (alpha and beta) and 11,000 (gamma). The average half-life of the ASSP messages was 11 min when either rifampin (10 micrograms/ml) or actinomycin D (1 microgram/ml) was used to inhibit RNA synthesis.

Alkaline Phosphatase↗
PubMed1979

DNA-directed cell-free protein-synthesizing system of Bacillus subtilis.

A cell-free coupled system of transcription and translation using cell extracts from Bacillus subtilis and DNA from phage SP82 has been developed. Under optimum conditions, it incorporated approx. 300 pmol methionine during a 1 h incubation. The activity of the system increased linearly as the concentration of S-150 supernatant fraction protein increased from 125 to 325 microgram per assay. The optimum Mg2+ concentration was 12.5-15 mM. Ribosomes required treatment with DNAase in order to reduce endogenous activity, but the S-150 fraction was kept DNAase-free to prevent degradation of exogenously added DNA. The coupled system was sensitive to inhibitors of RNA and protein synthesis. Kinetic studies showed that the number of pmol of nucleotides present in newly synthesized RNA increased linearly for the first 20-min reaction and that the rate of amino acid incorporation increased linearly for the first 30 min. Polyacrylamide gel electrophoresis of the in vitro synthesized products yielded a band pattern that closely resembled the pattern of early phage SP82 proteins produced in vivo.

Bacillus subtilis↗