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.     

Properties of Thymineless Strains of Bacillus megaterium

Both Bacillus megaterium KM:T(-)R(1), a strain partially resistant to thymineless death, and strain KM:T(-), the parent strain, can satisfy their thymine requirement with either thymidine, 5-methyldeoxycytidine, or 5-methyluridine. Neither strain can use 5-methylcytosine, 5-hydroxymethylcytosine, 5-hydroxymethyluracil, or 5-aminouracil for this purpose. Strain KM:T(-)R(1) requires as little as 0.01 mM thymine for maximum growth, whereas strain KM:T(-) requires 0.10 to 0.20 mM thymine. Lysogenic KM:T(-)R(1) dies more rapidly in the presence of mitomycin C than the corresponding phage-sensitive strain. Unexpectedly, the lysogenic strain was found to be less sensitive to thymineless death than the phage-sensitive strain. Lysogenic KM:T(-)R(1) is induced by exposure to mitomycin C and by thymineless incubation. It is concluded that thymineless death occurs by a mechanism which is unrelated to phage induction and that a major lethal effect of mitomycin C is probably a consequence of phage induction.     

Isolation and characterization of a unique division mutant of Bacillus megaterium.

A filamentous division mutant, PV302, of Bacillus megaterium QM B1551 was isolated while screening for sporulation-defective mutants after nitrosoguanidine mutagenesis. Both phase-contrast and electron microscopy revealed that the mutant produced small spherical cells as well as filaments. It also accumulated large amounts of poly-beta-hydroxybutyrate. Poly-beta-hydroxybutyrate accounted for 16% of the dry weight of the mutant strain even after 28 h growth. In comparison to the parental strain, the division mutant also showed both an inability to sporulate and a reduced growth rate. All these phenotypes transduced together. Revertants gained the ability to sporulate, divide, and grow normally. Transductional mapping of the mutation, designated div-1, established a new linkage group for B. megaterium consisting of div-1 and the pyrimidine biosynthesis genes pyrD BCF. The spherical cells were separated from filaments by sucrose gradients and were tested for nucleic acid content and viability. The purified spherical cell fraction contained one-fifth the amount of DNA per mg protein as compared with the filamentous cell fraction and was shown to contain both non-viable minicells and some cells capable of growing after a lag of about 4 h. This suggests that the mutation not only causes defects in septum placement and sporulation, but may possibly affect DNA partitioning.     

Peptidoglycan turnover during growth of a Bacillus megaterium Dap- Lys- mutant.

The aim of this study was to ascertain whether or not the absence of cell wall growth zones, deduced from the analysis of autoradiographs of DL-[3H]mesodiaminopimelic acid pulse-labeled cells of a Dap- Lys- mutant of Bacillus megaterium, was due to a high peptidoglycan turnover. Turnover was determined in very precise experimental conditions because two kinds of turnover occurred: a low, acid-soluble turnover and a high, acid-insoluble one. The latter was detected during a chase in the culture medium when bacteria were centrifuged before treatment with trichloroacetic acid. Otherwise the acid-insoluble released material precipitated with the bacteria. In the electron microscope this material presented a globular structure and contained both peptidoglycan and teichoic acid. The acid-insoluble turnover was mainly produced by a lytic acitivity that was released into the culture medium. This thermolabile activity was not due to cell lysis. It was implicated in septum cleavage and in the detachment of wall fragments from the cell surface, but did not seem indispensable for cell elongation. The acid-soluble turnover was much weaker and seemed to be indispensable for cell elongation.     

-aminobutyric acid as a required germinant for mutant spores of Bacillus megaterium

Germinated spores of Bacillus megaterium QM B1551 were irradiated with ultraviolet light, and spore-forming survivors were screened for germination requirements. Spore strains which failed to germinate in a variety of defined solutions germinative for spores of the parent strain were obtained. Mutant spores germinated readily in solutions containing yeast extract or one of numerous complex preparations. gamma-Aminobutyric acid, obtained from yeast extract by column chromatography, was shown to be required for germination by the mutant spores. gamma-Aminobutyric acid and l-alanine at final concentrations of 1 mm each, in solutions of KI (40 mm), equaled the potency of yeast extract (1 mg/ml) in the germination of the mutant spores. One of several other amino acids could be substituted, though less effectively, for l-alanine. alpha-Aminobutyric acid, beta-aminobutyric acid, beta-alanine, and 5-aminovaleric acid were ineffective substitutes for gamma-aminobutyric acid in mutant spore germination.     

Properties of Bacillus megaterium temperature-sensitive germination mutants.

Bacillus megaterium mutants JV-9 and JV-10 are temperature sensitive for initiation of spore germination. At 46 C, they did not lose heat resistance, dipicolinic acid, or absorbance, indicating that the temperature-sensitive blocks are very early in the sequence of initiation reactions. Strain JV-9 was temperature sensitive for initiation by glucose alone, and strain JV-10 was temperature sensitive for initiation by glucose, L-leucine, L-proline, KBr, or calcium dipicolinate. The kinetics of initiation were followed after two kinds of temperature change (shift-up and shift-down) experiments. Mutant spores incubated for different times at 46 C and then shifted down to 30 C showed no significant differences in the rates of absorbance decrease, i.e., no stimulation or inhibition. Conversely, when mutant spores were incubated for different times at 30 C, a fraction of the population initiated germination, and after shift-up to 46 C an additional fraction continued initiation while a third fraction stopped. This latter fraction did initiate germination when the temperature was lowered to 30 C. The kinetics of initiation after shift-up and shift-down in temperature suggest that the early events in initiation reagents, whereas the other four initiated sensitivity for all of the above initiation reagents, whereas the other four initiated very poorly. It was suggested that the lesion in strain JV-10 may result in the formation of one temperature-sensitive protein. Revertants of strain JV-9 could not be isolated.     

Expression of Bacillus megaterium and Bacillus subtilis small acid-soluble spore protein genes during stationary-phase growth of asporogenous B. subtilis mutants

The small acid-soluble spore proteins alpha and beta were not detected during stationary-phase growth of asporogenous Bacillus subtilis mutants blocked in stages 0, II, or III, but mutants blocked in stages IV or V accumulated nearly wild-type levels of these small acid-soluble spore proteins. Similar results were obtained when production of Bacillus megaterium C protein (also a small acid-soluble spore protein), as well as alpha and beta, were monitored in these mutants containing a recombinant plasmid carrying the B. megaterium C protein gene. The only exception was a spo0H mutant which synthesized a small amount of C protein, but no alpha or beta.     

Genetic mapping of sporulation operons in Bacillus subtilis using a thermosensitive sporulation mutant.

A thermosensitive sporulation mutant was used to determine the order of sporulation operonsin the urs region of the Bacillus subtilis chromosome. Data from three-factor transformation crosses and three- and four-factor transduction crosses established the order metC-SPO-96(SpoII)-spo-85(SpoV)-spo-279(SpoII)-furA-ura-cysC-spo-NG1.67(SpoIII). Previously, furA was thought to lie to the right of ura and cysC to the left (Dubnau, 1970; Young and Wilson, 1972).     

Regulation of maltose metabolism in stationary phase cultures of an asporogenous mutant of Bacillus licheniformis

An asporogenous strain of Bacillus licheniformis accumulated maltose by an energy dependent transport mechanism during an extended stationary phase. Maltose transport was sensitive to the effects of the uncoupler tetrachlorosalicylanide (TCS), and was also inhibited by glucose. Maltose stimulated synthesis of a p -nitrophenyl-α- D -glucoside-hydrolysing enzyme ( p NPGase) in log phase and in stationary phase cells. In the presence of glucose this induction was inhibited. Glucose was used preferentially to maltose in stationary phase cells. The uptake of maltose from the medium, and the synthesis of p NPGase, were immediately and completely inhibited in the presence of glucose. These results are consistent with a mechanism of inducer exclusion mediating the repressive effect of glucose upon p NPGase synthesis in stationary phase cells. Catabolite repression of α-amylase synthesis by glucose was also demonstrated in late stationary phase mutant cells.     

Isolation and characterization of outermost layer deficient mutant spores of Bacillus megaterium

Outermost layer deficient mutant spores of Bacillus megaterium ATCC 12872 were isolated by Urografin density gradient centrifugation after mutagenesis with ethyl methanesulfonate. Although the composition of the cortex peptidoglycan was the same as that of the parent spores, three major proteins (48, 36, and 22 K daltons) were missing, suggesting that these proteins are components of the outermost layer. All mutant spores were also found to have very hydrophobic surface by 'salt aggregation test,' which would facilitate selection of such mutants.     

Properties of Hg- and Cd-spores of Bacillus megaterium QM B1551

Hg- and Cd-spores of Bacillus megaterium QM B1551 were produced in Schaeffer's medium containing mercuric chloride and cadmium chloride respectively. Metals were added to the medium at 9 hr of incubation (Stage V) to give a final concentration of 50 microM. It was found by electron microscopic and biochemical studies that the coats of both Hg- and Cd-spores were thinner than those of control spores. Of the total Hg and Cd in the spores, 77% of the Hg and 63% of the Cd were detected in the spore coats. Hg- and Cd-spores were less resistant to heat and more sensitive to germinants than control spores. Other properties of Hg- and Cd-spores were similar to those of control spores. These results suggest that the spore coat has some relationship to the heat resistance and germinability of spores.     

Cytochromes of Bacillus megaterium and Bacillus subtilis

Treatment of Bacillus subtilis and Bacillus megaterium cells by ordinary procedures failed to extract any soluble cytochrome from the cell. Sonoration produced small fragments which contained the cytochrome complement and were sedimentable in a centrifugal field of 144,000 × g. Difference spectra of such particles showed absorption maxima at 599, 557, 552, 530, and 428 mμ. Digestion of the fragments with lipase liberated a rather typical cytochrome c in that it had absorption maxima in the reduced form at 550, 520, and 415 mμ, formed cyanide and pyridine hemochromogens which were spectroscopically similar to those obtained from mammalian cytochrome c, could be purified in the usual manner by ammonium sulfate fractionation, and had an oxidation potential at pH 7.0 of 0.25 v. As isolated, however, the cytochrome c was readily oxidized by oxygen in the air.