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.     

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.     

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.     

Characterization of a new sporulation factor in Bacillus subtilis.

We report the existence and partial purification of sporulation factor, which stimulates sporulation of Bacillus subtilis at low cell density. Proline or arginine is required for stimulation under the conditions of our assay. Sporulation factor is a small heat-stable substance produced by the cells during exponential growth phase. It is required in small amounts and is resistant to various proteolytic agents. Several spo mutants were tested for the ability to produce functional sporulation factor. All of these mutants produce factor and do not sporulate in the presence of factor from wild-type cells. Sporulation factor is not involved in the induction of alpha-amylase synthesis at the initiation of sporulation.     

Rapid isolation and sequencing of purified plasmid DNA from Bacillus subtilis.

We report two methods for isolation of plasmid DNA from the gram-positive bacterium Bacillus subtilis. The protoplast alkaline lysis procedure was developed for general use, and the protoplast alkaline lysis magic procedure was developed for isolation of DNA for sequencing. Both procedures yielded large amounts of high-quality DNA in less than 1 h, while current protocols require 4 to 7 h to perform and give lower yields and quality. Plasmid DNA was obtained from strains containing either high- or low-copy-number plasmids. In addition, the procedures were easily adapted to yield large amounts of plasmid DNA suitable for sequencing from another gram-positive organism, Staphylococcus aureus. Further, we demonstrated that neither chloramphenicol, used for plasmid selection, nor the mutation recE4 reduced plasmid DNA yield from the strains we examined.     

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.     

Molecular basis of cell-specific endothelial nitric-oxide synthase expression in airway epithelium

Nitric oxide (NO) plays an important role in airway function, and endothelial NO synthase (eNOS) is expressed in airway epithelium. To determine the basis of cell-specific eNOS expression in airway epithelium, studies were performed in NCI-H441 human bronchiolar epithelial cells transfected with the human eNOS promoter fused to luciferase. Transfection with 1624 base pairs of sequence 5' to the initiation ATG (position -1624) yielded a 19-fold increase in promoter activity versus vector alone. No activity was found in lung fibroblasts, which do not express eNOS. 5' deletions from -1624 to -279 had modest effects on promoter activity in H441 cells. Further deletion to -248 reduced activity by 65%, and activity was lost with deletion to -79. Point mutations revealed that the GATA binding motif at -254 is mandatory for promoter activity and that the positive regulatory element between -248 and -79 is the Sp1 binding motif at -125. Electrophoretic mobility shift assays yielded two complexes with the GATA site and three with the Sp1 site. Immunodepletion with antiserum to GATA-2 prevented formation of the slowest migrating GATA complex, and antiserum to Sp1 supershifted the slowest migrating Sp1 complex. An electrophoretic mobility shift assay with H441 versus fibroblast nuclei revealed that the slowest migrating GATA complex is unique to airway epithelium. Thus, cell-specific eNOS expression in airway epithelium is dependent on the interaction of GATA-2 with the core eNOS promoter, and the proximal Sp1 binding site is also an important positive regulatory element.