Genetic mapping of regulatory mutations of Bacillus subtilis riboflavin operon

Summary Seven mutations leading to riboflavin overproduction inBacillus subtilis were found to be linked to the markerdnaF133 (145° on theB. subtilis genetic map) by transformation. Cotransfer indexes (42.5%–61.7%) suggest that theribC mutations are alleles of the same locus. Results of transduction and transformation crosses suggest the following order of markers:pyrD26 –ts-6 –dnaF133 –ribC –recA1.     

Intraspecific transformation inBacillus subtilis

The study of a greater number ofBacillus subtilis strains showed the differences in the ability to transform the standard acceptor strain. The results seem to support the possibility that this species is genetically heterogenous. This fact could be of importance even in practical taxonomy.     

Genetic mapping and physiological consequences of metE mutations of Bacillus subtilis.

Three metE mutations of Bacillus subtilis, which cause cells to have a 25- to 200-fold decrease in L-methionine S-adenosyltransferase (EC 2.5.1.6) activity, were mapped between bioB and thr. The corresponding three metE mutants contained three- to fourfold less intracellular S-adenosylmethionine (SAM) but at least sevenfold more methionine than the metE+ strain when grown in synthetic medium. This indicates a strong feedback control of SAM on its synthesis. However, only the metE2 strain, with the lowest SAM concentration, grew at a slightly lower rate than the parent, which showed that an intracellular concentration of about 25 microM SAM was critical for growth at the normal rate. Neither DNA methylation (measured by bacteriophage luminal diameter 105 restriction) nor sporulation was affected at this low SAM concentration. Addition of methionine to the growth medium caused an increase in the pool of SAM in some but not all metE mutants. Coaddition of adenine did not change this result. However, the extent of sporulation (induced by mycophenolic acid) was decreased 50-fold in all mutants by the addition of methionine and adenine. Therefore, the combination of methionine and adenine suppresses sporulation regardless of whether it causes an increase in the level of SAM.     

Genetic mapping of aminoglycoside and fusidic acid resistant mutations in Bacillus subtilis

Summary Mutations governing resistance to neamine, neomycin, kanamycin and fusidic acid have been mapped within the ribosomal region of theBacillus subtilis genome using PBS1-mediated transduction. The probable order of these closely linked markers is: This work was supported by a predoctoral training grant, GM-1290, from the National Institutes of Health, administered by the Department of Microbiology, N.Y.U. School of Medicine, and by a research grant from the National Science Foundation, GB-16782, awarded to I.S.     

Genetic and biochemical characterization of kirromycin resistance mutations in Bacillus subtilis.

Spontaneous mutations causing resistance to the EF-Tu-specific antibiotic kirromycin have been isolated and mapped in Bacillus subtilis. Three-factor transductional and transformational crosses have placed the kir locus proximal to ery-1 and distal to strA (rpsL) and several mutations affecting elongation factors EF-G and EF-Tu, in the order: cysA strA [fus-1/ts-6(EF-G)] [ts-5(EF-Tu)] kir ery-1 spcA. Purified EF-Tu from mutant strains is more resistant to kirromycin as measured by in vitro protein synthesis and also shows a more acidic isoelectric point than wild-type EF-Tu. This indicates that the kir locus is the genetic determinant (tuf) for EF-Tu and that there is a single active gene for this enzyme in B. subtilis.     

Bacilysin production and sporulation inBacillus subtilis

Bacilysin-negative(bac^–) strain NG79 was found to be oligosporogenous. When compared with the parental strain, it was 200–300 times less resistant to heat, chloroform, and lysozyme treatments, and the spores contained considerably less dipicolinate. When NG79 was transduced, the oligosporogenous phenotype was found to be cured in all the transductants tested. External addition of bacilysin to the cultures of this strain markedly improved each measure of spore quality. The time of its addition determined the extent of acquired resistance, the optimum being 4–7 h after inoculation. This suggested that bacilysin might influence sporulation prior to stage I. Bacilysin activity completely disappeared from the extracellular fluid of aged cultures. It was demonstrated that the dipeptide can be cleaved by the alkaline serine protease that is produced byBacillus subtilis.     

Chemotaxis toward amino acids inBacillus subtilis

Quantitative measurement of positive chemotaxis inBacillus subtilis was performed by means of adaption of the procedure used in studies withEscherichia coli. The motility ofB. subtilis was optimal in the presence of an exogeneous energy source and a nonionic detergent,e. g. Tween 80 or Brij-36. B. subtilis is chemotactic toward the commonly occurringL-amino acids except arginine, lysine, aspartate and glutamate. No chemotactic response was observed towardD-amino acids. Threshold, optimal response and peak concentration were determined. Chemotaxis toward glutamine was optimal at pH 6-7 and a temperature of 32°C. The maximum response toward a particular attractant was presumably influenced by the aerotactic behavior ofB. subtilis.     

Utilization ofEscherichia coli tac promoter inBacillus subtilis

Summary The tac promoter originally constructed for the use inEscherichia coli was fused to an endoglucanase structural gene isolated fromBacillus subtilis and the expression of the chimeric gene inB. subtilis was observed. The tac promoter-controlled gene expressed well inB. subtilis and produced endoglucanase during the exponential growth phase.     

Expression ofBordetella pertussis toxin subunits inBacillus subtilis

Summary Pertussis toxin subunits (S1–S5) were expressed inBacillus subtilis using a vector with the promoter, Shine-Dalgarno sequence and the sequence coding for the first 7 amino acids of the signal sequence of the -amylase gene fromB. amyloliquefaciens. The use of this vector resulted in a high level of intracellular expression of each pertussis toxin subunit as aggregates in the cytoplasm ofB. subtilis.     

Stabilization of a plasmid-encoded LacZ phenotype inBacillus subtilis

Recombinant plasmid pCED3 was structurally unstable inBacillus subtilis cultures grown in the presence of kanamycin to eliminate the effects of segregational instability. Analysis of 96 modified plasmids indicated that deletions in the plasmid occur at many different sites. The presence of plasmid pCED3 slowed the growth rate of theB. subtilis host. Cells that contained modified plasmids grew faster than the parental cells and took over the population. Two different methodologies were developed to reduce the cultural instability of the plasmid-directed LacZ⁺ phenotype. By growing the cells in a medium that supports a low growth rate, the growth rate ratio between modified and parental cells was reduced, resulting in a partial stabilization (40 generations) of the LacZ⁺ phenotype in the population [35]. Removal of a 4.77 kbEcoRI fragment (which consists primarily of the pBR322 replicon) from plasmid pCED3 produced a more stable plasmid derivative, designated pYS1. Cells harboring plasmid pYS1 grew faster than pCED3-bearing cells, although the level of activity of -galactosidase was similar in both strains. By combining the two approaches (i.e., growth of pYS1-bearing cells in a medium that supports low growth rate), the LacZ⁺ phenotype was stably maintained in the cell population for over 170 generations. Under these conditions, there was no detectable difference between the growth rates of cells bearing the pYS1 plasmid and further modified plasmids.     

Gluconimycin an inhibitor of pyrimidine synthesis inBacillus subtilis

Pyrimidine synthesis inBacillus subtilis PCI-219 cells was the primary site of action of the antibiotic gluconimycin as indicated by its arresting the activity of aspartate carbamoyltransferase as well as accumulation of aspartic acid in cultures fortified with gluconimycin. Microbial growth and viable counts were suppressed by the antibiotic whereas glycolysis and aerobic respiration were insignificantly affected. Gluconimycin failed to induce a lytic effect on cell protoplasts while considerable amounts of substances absorbing at 260 nm were released from mierobial cells treated with gluconimycin.     

Membrane fluidity inBacillus subtilis. Validity of homeoviscous adaptation

The validity of the principle of homeoviscous adaptation for Bacillus subtilis was tested by comparing fluorescence anisotropy (1,6-diphenyl-1,3,5-hexatriene) and electron-spin resonance (16-doxylstearate) measurements carried out in isolated plasma membranes and in phospholipid fractions. The physical measurements were supplemented by fatty-acid analysis. The results support our previous findings on intact cells. The thermoadaptive mechanism of B. subtilis manifested as an increase in relative proportion of branched anteiso-C15 and anteiso-C17 fatty acids, are not strong enough to compensate for the marked physical change of membrane fluidity induced by temperature decrease.     

Molecular characterization of specific heat shock proteins inBacillus subtilis

Heat shock inBacillus subtilis may induce as many as 66 proteins after temperature upshift from 37° to 48°C. Four induced proteins were analyzed by microsequencing techniques. These were identified as the homologues for GroEL, DnaK, enolase, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which are heat shock proteins in other systems. The identities of GroEL and DnaK were confirmed additionally by Western blot analysis. As a control, a protein whose synthesis was repressed approximately threefold by heat shock was identified by microsequencing as flagellin.