Cloning of the Bacillus subtilis sulfanilamide resistance gene in Bacillus subtilis

A recombinant plasmid was constructed by ligation of chromosomal DNA from a sulfanilamide-resistant strain of Bacillus subtilis to the plasmid vector pUB110 which specifies neomycin resistance. Recombinant molecules generated in vitro were introduced into a B. subtilis recipient strain which carried the recE4 mutation, and selection was for neomycin-sulfanilamide-resistant transformants. A single colony was isolated containing the recombinant plasmid pKO101. This 6.3-megadalton plasmid simultaneously conferred resistance to neomycin and sulfanilamide when transferred into sensitive Rec+ or Rec- cells by either transduction or transformation.     

Antibiotic-resistant mutants of Bacillus subtilis conditional for sporulation.

Among spontaneously occurring antibiotic-resistant mutants of Bacillus subtilis 168 we have identified a sub-class that is conditionally sporulative. Mutants in this sub-class are resistant to antibiotic during vegetative growth but are sensitive during sporulation. Mutants conditionally-resistant to erythromycin, kanamycin, spectinomycin, and streptomycin have been isolated and characterized by phase contrast microscopy and with respect to their ability to synthesize heat-resistant endospores or the sporulation-associated enzyme alkaline phosphatase. The results suggest that several entirely different genetic lesions may result in this single phenotype. This group includes mutants whose properties suggest that both th 30S and 50S ribosomal subunits may be altered concomitant with early spore specific metabolism. The blockage imposed by antibiotic may be at or near Stage 2 of sporulation.     

Conjugative chromosomal gene transfer in Bacillus subtilis

Conjugative transfer of 20-kb chromosomal fragment carrying genes encoding tetracycline (tet(r)) and lincomycin (lin(r)) resistance in the soil strain Bacillus subtilis 19 is described. Transfer was preceded by this fragment insertion into the large conjugative pl9cat plasmid producing a hybrid plasmid. Insertion frequency was 10(-4)-10(-5). Then genes tet(r) and lin(r) were transferred to the recipient strains. The transfer of chromosomal genes inserted into the plasmid and plasmid gene cat occurred sequentially and resembled sexduction, which represents chromosomal gene transfer by F'- and R' plasmids during conjugation in Escherichia coli and other gram negative bacteria.     

Glutamine synthetase gene of Bacillus subtilis

The glutamine synthetase gene (glnA) of Bacillus subtilis was purified from a library of B. subtilis DNA cloned in phage lambda. By mapping the locations of previously identified mutations in the glnA locus it was possible to correlate the genetic and physical maps. Mutations known to affect expression of the glnA gene and other genes were mapped within the coding region for glutamine synthetase, as determined by measuring the sizes of truncated, immunologically cross-reacting polypeptides coded for by various sub-cloned regions of the glnA gene. When the entire B. subtilis glnA gene was present on a plasmid it was capable of directing synthesis in Escherichia coli of B. subtilis glutamine synthetase as judged by enzymatic activity, antigenicity, and ability to allow growth of a glutamine auxotroph. By use of the cloned B. subtilis glnA gene as a hybridization probe, it was shown that the known variability of glutamine synthetase specific activity during growth in various nitrogen sources is fully accounted for by changes in glnA mRNA levels.     

Electrophoretic mobility of Bacillus subtilis knockout mutants with and without flagella

Mutants of Bacillus subtilis 168 strain were obtained by inactivation of a specific gene by homologous recombination with the plasmid pMutinT3. The cell surface properties of these strains were characterized by measuring the electrophoretic mobility of the cells as a function of pH and ionic strength. The surface properties were different for the strains possessing flagella on their cells and strain FlgB, having no flagellum, due to knockout of the corresponding gene. The cell surface properties of the strains possessing flagella become similar to those of strain FlgB after acid treatment. It was confirmed that the acid treatment degraded the flagella without causing any apparent structural change on the cell surface via observations made using atomic force microscopy, transmission electron microscopy, and scanning electron microscopy. These results indicate that the flagella are a key factor influencing cell surface properties.     

Characterization of a rifampin-resistant, conditional asporogenous mutant of Bacillus subtilis.

A rifampin-resistant, conditionally asporgoenous mutant of Bacillus subtilis was isolated that sporulates poorly in Sterlini-Mandelstam sporulation medium, but that sporulates normally in modified Difco sporulation medium. Rifampin-resistant (Rif-r) and conditional asporogenous (Spo-c) phenotypes co-transformed at 100% frequency. Preliminary genetic studies indicated the Rif-r trait to lie between cysA14 and ery, a locus (rnp) common to Rif-r mutants. Ribonucleic acid polymerase from strains bearing this mutation was found to be rifampin resistant in vitro. The loss of ability to sporulate in Sterlini-Mandelstam medium was found to be corrected, to a large extent, by addition to the medium of arginine, methionine, valine, and isoleucine. Several other amino acids had small effects, whereas others had no effect at all. The restorative effect is approximately additive. Growth studies indicated that Rif-r strains grew more rapidly than the corresponding parent in minimal medium at temperatures higher than 37 C. Addition of certain amino acids to the medium resulted in identical growth rates at these temperatures. Extracellular protease and esterase activities of the Rif-r Spo-c mutant were normal. A slight difference was found in the heat sensitivity of partially purified ribonucleic acid polymerase preparations of this mutant compared to the wild type.     

Cloning of sporulation gene spoIVC in Bacillus subtilis

Summary Sporulation gene spoIVC of Bacillus subtilis was cloned by the prophage transformation method in temperate phage 105. The specialized transducing phage, 105spoIVC-1, restored the sporulation of the asporogenous mutant of B. subtilis strain 1S47 (spoIVC133). Transformation experiments showed that the spoIVC gene resides on a 7.3 kb HindIII restriction fragment. Subsequent analysis of the 7.3 kb HindIII fragment with restriction endonuclease EcoRI showed that the spoIVC gene resides on a 3.6 kb EcoRI fragment within the 7.3 kb fragment. The 3.6 kb fragment was recloned into the unique EcoRI site of plasmid pUB110 and deletion derivatives having a deletion within the 3.6 kb insert were constructed. The plasmid carrying the entire spoIVC gene restored the sporulation of strain HU1214 (spoIVC133, recE4) at a frequency of 10⁷ spores/ml, and reduced the sporulation of strain HU1018 (spo ⁺, recE4) to 10⁷ spores/ml.     

A Bacillus subtilis malate dehydrogenase gene.

A Bacillus subtilis gene for malate dehydrogenase (citH) was found downstream of genes for citrate synthase and isocitrate dehydrogenase. Disruption of citH caused partial auxotrophy for aspartate and a requirement for aspartate during sporulation. In the absence of aspartate, citH mutant cells were blocked at a late stage of spore formation.     

The forespore line of gene expression in Bacillus subtilis

Endospore formation by Bacillus subtilis involves three differentiating cell types, the predivisional cell, the mother cell, and the forespore. Here we report the program of gene expression in the forespore, which is governed by the RNA polymerase sigma factors sigma(F) and sigma(G) and the DNA-binding proteins RsfA and SpoVT. The sigma(F) factor turns on about 48 genes, including the gene for RsfA, which represses a gene in the sigma(F) regulon, and the gene for sigma(G). The sigma(G) factor newly activates 81 genes, including the gene for SpoVT, which turns on (in nine cases) or stimulates (in 11 cases) the expression of 20 genes that had been turned on by sigma(G) and represses the expression of 27 others. The forespore line of gene expression consists of many genes that contribute to morphogenesis and to the resistance and germination properties of the spore but few that have metabolic functions. Comparative genomics reveals a core of genes in the sigma(F) and sigma(G) regulons that are widely conserved among endospore-forming species but are absent from closely related, but non-spore-forming Listeria spp. Two such partially conserved genes (ykoU and ykoV), which are members of the sigma(G) regulon, are shown to confer dry-heat resistance to dormant spores. The ykoV gene product, a homolog of the non-homologous end-joining protein Ku, is shown to associate with the nucleoid during germination. Extending earlier work on gene expression in the predivisional cell and the mother cell, we present an integrated overview of the entire program of sporulation gene expression.     

Deletion of an rRNA gene set in Bacillus subtilis

One of the 10 rRNA gene sets found in a wild-type strain of Bacillus subtilis 168 was deleted, apparently by recombination between two tandemly repeated rRNA gene sets. The deletion strain grew as well as the wild-type strain under a variety of growth conditions and also showed no change in the sporulation efficiency.     

Cloning of sporulation gene spoIIG in Bacillus subtilis.

Two specialized transducing phages carrying a sporulation gene, spoIIG , of Bacillus subtilis were constructed from B. subtilis temperate phages p11 and phi 105 by the "prophage transformation" method. Restriction enzyme analysis and transformation experiments showed that the spoIIG gene was present on a 6.2 X 10(6)-dalton (6.2-Md) EcoRI fragment in both transducing phage genomes. Further analysis showed that spoIIG + transforming activity resides on a 2.25-Md EcoRI-BamHI fragment within the 6.2-Md EcoRI fragment. The 2.25-Md fragment was subcloned into the region between the EcoRI and BamHI sites of pUB110, and deletion plasmids lacking PstI or HindIII fragments within the 2.25-Md fragment were constructed. The recombinant plasmid carrying the intact spoIIG gene restored sporulation of strain HU1002 ( spoIIG41 recE4 ) to a frequency of 10(4) spores per ml and inhibited sporulation of strain 4309 ( spo + recE4 ) to a level of 10(3) spores per ml.     

Patterns of gene expression in Bacillus subtilis colonies.

Bacillus subtilis 5:7, a derivative of macrofiber-producing strain FJ7, carries the lacZ reporter gene within Tn917 at an unknown location in the host genome. Expression of the host gene carrying lacZ within colonies of 5:7 was observed by examining growth under different conditions in the presence of 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal). At a high plating density small colonies arose that expressed the host gene early and throughout the colony, whereas at a low density large colonies were produced that expressed the host gene late in development and only in cells forming a ring pattern close to the colony periphery. A highly regulated spatial and temporal gene expression pattern was observed in growth from cross-streaks, suggesting that gene expression is responsive to concentration gradient fields established by neighboring growth. Colonies cultured on agar blocks revealed that expression was governed by depletion of a medium component and also by the geometry of the substrate upon which the colonies grew. At least three factors influenced the control of expression: (i) the concentration of a diffusible component of the medium exhausted by cell growth, (ii) a spatial-temporal factor related to growth within the colony, and (iii) the geometry of the growth substrate.     

Designed gene amplification on the Bacillus subtilis chromosome

We previously reported the cloning of a 1.6 kb HindIII fragment (containing the junction of the repeating unit) from chromosomal DNA of Bacillus subtilis strain B7 in which tandem amplification of a 16 kb region occurred, and the induction of B7-type gene amplification by competence transformation with this cloned fragment. Based on this result, we designed, on the B. subtilis chromosome, a gene amplification of the 22 kb repeating unit containing the alpha-amylase structural gene (amyE), the tunicamycin-resistance gene (tmrB) and the shikimate kinase structural gene (aroI). We cloned only two short DNA fragments from both termini of the 22 kb region, constructed a junction structure of the designed repeating unit on pBR327 and transformed a B. subtilis wild-type strain by this constructed plasmid. As a result, we succeeded in obtaining tunicamycin-resistant (Tmr) transformants in which the designed gene amplification of 22 kb occurred on the chromosome. The Tmr transformants showed high productivity of alpha-amylase and shikimate kinase. The copy number of the repeating unit was estimated to be 10-20. This system may provide an effective means of amplifying long (greater than 20 kb) DNA regions on the chromosome.     

Complex regulation of the Bacillus subtilis aconitase gene

The roles of the CcpC, CodY, and AbrB proteins in regulation of the Bacillus subtilis aconitase (citB) gene were found to be distinct and to vary with the conditions and phase of growth. CcpC, a citrate-inhibited repressor that is the primary factor regulating citB expression in minimal-glucose-glutamine medium, also contributed to repression of citB during exponential-phase growth in broth medium. A null mutation in codY had no effect on citB expression during growth in minimal medium even when combined with ccpC and abrB mutations. However, a codY mutation slightly relieved repression during exponential growth in broth medium and completely derepressed citB expression when combined with a ccpC mutation. An abrB mutation led to decreased expression of citB during stationary phase in both broth and minimal medium. All three proteins bound in vitro to specific and partially overlapping sites within the citB regulatory region. Interaction of CcpC and CodY with the citB promoter region was partially competitive.