A new relaxed mutant of Bacillus subtilis.

A new relaxed mutant of Bacillus subtilis was isolated by screening Rifr clones for alterations in stringent control. The Rifr relaxed mutant which is described was found to contain a second-site mutation conferring a relaxed response to an energy source downshift and was partially relaxed after amino acid starvation. The new rel locus, called relG, was distinct from the two other known rel loci in B. subtilis, relA, and relC.     

A mutant Bacillus subtilis gamma-glutamyltranspeptidase specialized in hydrolysis activity

gamma-Glutamyltranspeptidase (GGT) catalyzes the hydrolysis of gamma-glutamyl compounds and the transfer of their gamma-glutamyl moieties to amino acids and peptides. The transpeptidation activity of Bacillus subtilis GGT is about 10-fold higher than its hydrolysis activity. In B. subtilis GGT, substitution of Asp-445 with Ala abolished its transpeptidation activity. The specific activity for hydrolysis of D445A GGT was 40.2% of that of the wild-type GGT. The K(m) value for L-glutamine was 15.3 mM. D445A GGT was salt tolerant like the wild-type GGT. These results indicate that D445A GGT will be highly useful as a 'glutaminase' in food industry.     

Streptomycin-resistant, asporogenous mutant of Bacillus subtilis.

Summary Mutants of Saccharomyces cerevisiae lacking pyruvate kinase (EC 2.7.1.40) are described. These have less than 0.5% of the pyruvate kinase activity of the wild type. All the other glycolytic enymes are present in normal amounts in these mutants. The mutation is recessive and segregates in diploids as a single gene. Five alleles examined fail to complement one another. Tetrad analysis and mitotic recombination data place the mutation on the left arm of chromosome I distal to cys 1. The majority of single-step spontaneous revertants on glucose regain the enzyme activity fully and this activity appears, by a number of criteria, to be due to the same enzyme present in the wild type. Some of these revertants become nuclear petites. The mutants do neither grow on nor ferment sugars but do grow on ethyl alcohol or pyruvate. Glucose addition to cultures growing on alcohol arrests growth until glucose is exhausted. The steady state rate of glucose utilization is slower than in the wild type. This is associated with the accumulation of as much as 5 moles P-enolpyruvate per g wet weight of cells and proportional amounts of 2-P-glyceric and 3-P glyceric acids.The mutation is believed to involve some regulatory element in the synthesis of pyruvate kinase.     

Membrane characteristics of the hemA mutant of Bacillus subtilis

The membrane of the delta-aminolaevulinic acid synthase (EC. 2.3.1.37) deficient mutant of Bacillus subtilis growing in the presence of delta-aminolaevulinic acid differs only to a limited extent from the wild type. In haemin-containing medium, however, significant differences are observed as regards the osmotic stability of the protoplasts and the membrane protein composition.     

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.     

Genetic analysis of a mutant of Bacillus subtilis producingltraviolet-sensitive spores

Summary A mutant ofBacillus subtilis, uvssp-42-1, producing UV-sensitive spores was studied genetically. By treatment of the cells with DNA prepared from auvr strain two types,uvs-42 (Hcr⁻) andssp-1 (Hcr⁺), of transformants producing UV-resistant spores were obtained. Only strains having both types of mutations together produced UV-sensitive spores.     

Characterization of a pleiotropic succinate dehydrogenase-negative mutant of Bacillus subtilis

A succinate dehydrogenase-negative mutant of Bacillus subtilis is described which lacks all three subunits of the membrane-bound succinate dehydrogenase complex: flavoprotein, iron protein, and cytochrome b558. The corresponding mutation is revertible and it maps at one extreme of the sdh region. The results presented suggest that the structural genes for the subunits of the succinate dehydrogenase complex are part of one operon.     

Bacillus subtilis mutant with temperature-sensitive net synthesis of phosphatidylethanolamine.

Bacillus subtilis mutants with temperature-sensitive growth on complex media were screened for defects in phospholipid metabolism. One mutant was isolated that showed temperature-sensitive net synthesis of phosphatidylethanolamine. The mutant did not accumulate phosphatidylserine at the nonpermissive temperature. In the presence of hydroxylamine, wild-type B. subtilis accumulated phosphatidylserine at both 32 and 45 degrees C, whereas the mutant did only at 32 degrees C. In vitro phosphatidylethanolamine synthesis with bacterial membranes is no more temperature sensitive with mutant membranes than with wild-type membranes. The mutation probably affects the synthesis indirectly, possibly by altering a membrane protein. The mutant bacteria grew at the nonpermissive temperature, 45 degrees C, in a phosphate buffer-based minimal medium, although net synthesis of phosphatidylethanolamine was also temperature sensitive in this medium. One mutation caused both temperature-sensitive growth on complex media and temperature-sensitive net synthesis of phosphatidylethanolamine. The mutation is linked to aroD by transformation.     

A defined medium to investigate sliding motility in a Bacillus subtilis flagella-less mutant

Background  

We have recently shown that undomesticated strains of Bacillus subtilis can extensively colonize the surfaces of rich, semi-solid media, by a flagellum-independent mechanism and suggested that sliding motility is responsible for surface migration. Here we have used a flagella-less hag null mutant to examine and confirm sliding motility.     

A temperature sensitive mutant in Bacillus subtilis with an altered elongation factor G

Summary A temperature sensitive mutant of Bacillus subtilis with an altered elongation factor G is described. The mutation is highly co-transformable with resistance to fusidic acid.This work is part of a Ph. D. Thesis to be submitted to Tel Aviv University.     

Bacillus subtilis mutant with altered ability for heterologous transformation]

Mutant strain of Bacillus subtilis, which produced in certain conditions significantly reduced quantity of trnasformants during transformation by homologous DNA, as compared with transformation by heterologous DNA from Bac. aterrimus, is isolated. The ability to transfection by phage SPO1 DNA and the efficiency of infection of the mutant by this phage are also decreased. The causes of such alterated properties are discussed.     

Pyrimidine dimer excision in a Bacillus subtilis Uvr- mutant.

A technique which allows the measurement of small numbers of pyrimidine dimers in the deoxyribonucleic acid (DNA) of cells of Bacillus subtilis irradiated with ultraviolet light has been used to show that a strain mutant at the uvr-1 locus is able to excise pyrimidine dimers. Excision repair in this strain was slow, but incision may not be rate limiting because single-strand breaks in DNA accumulate under some conditions. Excision repair probably accounted for a liquid-holding recovery previously reported to occur in this strain. Recombinational exchange of pyrimidine dimers into newly replicated DNA was readily detected in uvr-1 cells, but this exchange did not account for more than a minor fraction of the dimers removed from parental DNA. Excision repair in the uvr-1 strain was inhibited by a drug which complexes DNA polymerase III with DNA gaps. This inhibition may be limited to a number of sites equal to the number of DNA polymerase III molecules, and it is inferred that large gaps are produced by excision of dimers. Because the uvr-1 mutation specifically interferes with excision of dimers at incision sites, it is concluded that the uvr-1 gene product may be an exonuclease which is essential for efficient dimer excision.