Regulation of acetohydroxyacid synthetase in Bacillus subtilis

Summary In Bacillus subtilis, the activity of aceto hydroxyacid synthetase is inhibited by L-valine. The valine effect is antagonized by the simultaneous addition of L-isoleucine and L-leucine. Repression of enzyme formation required an excess of leucine and valine in the growth medium.     

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.     

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.     

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.     

Genetic mapping of cadmium resistance mutations inBacillus subtilis

Bacillus subtilis, which accumulates cadnium via the manganese transport system, may acquire cadmium resistance by chromosomal mutations that reduce Cd²⁺ uptake without affecting Mn²⁺ transport. A cadmium resistance mutation,cdr-1, maps at about 40° on theB. subtilis chromosome. The deduced map order wasarol-narB-mtlB-cdr-dal-purB. Thecdr mutations in four other, independently isolated Cd²⁺-resistant mutants demonstrating reduced Cd²⁺ uptake also mapped betweenaroI anddal.     

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.     

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.     

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.     

Variation of antimetabolite sensitivity with different carbon sources inBacillus subtilis

The susceptibility ofBacillus subtilis to amino acid analogues was found to be markedly influenced by the carbon source used in the test media. Thialysine inhibited the bacterium with a greater number of carbon sources than the other two analogues tested. 5-Hydroxylysine was inhibitory with glycerol, lactose,D-xylose,L-arabinose and soluble starch while ethionine showed toxicity with lactose,D-xylose andL-arabinose. None of these analogues were toxic at the levels tested whenD-galactose was used as carbon source. The bacterium was not susceptible to thialysine with glycerol, to 5-hydroxylysine withL-arabinose and to ethionine with lactose.     

Effect of growth rate on the maintenance of a recombinant plasmid inBacillus subtilis

Summary The rate of fall in the proportion of plasmid-containing cells in a population ofBacillus subtilis 1A297[pVC102] grown in continuous culture was independent of growth rate. Plasmid loss could not be ascribed to faulty partitioning during cell division. At a low dilution rate, the specific rate of plasmid loss exceeded the specific growth rate of the plasmid-containing cells.     

Membrane fluidity inBacillus subtilis. Physical change and biological adaptation

The thermotropic behaviour of membrane phospholipids was estimated in intact cells of Bacillus subtilis. Membrane fluidity (microviscosity) of intact cells depended markedly on the ambient temperature - increase in cultivation temperature led to an increase in membrane fluidity. Estimated as anisotropy of 1,6-diphenyl-1,3,5-hexatriene fluorescence, a 30% difference was observed when cells cultivated at 20 and 40 degrees C were compared. This lack of rigorous homeostatic control of bulk-phase lipid fluidity prompted the reevaluation of the physiological significance of the "homeoviscous adaptation" in B. subtilis.