Effects on Bacillus subtilis of a conditional lethal mutation in the essential GTP-binding protein Obg.

The obg gene is part of the spo0B sporulation operon and codes for a GTP-binding protein which is essential for growth. A temperature-sensitive mutant in the obg gene was isolated and found to be the result of two closely linked missense mutations in the amino domain of Obg. Temperature shift experiments revealed that the mutant was able to continue cell division for 2 to 3 generations at the nonpermissive temperature. Such experiments carried out during sporulation showed that Obg was necessary for the transition from vegetative growth to stage 0 or stage II of sporulation, but sporulation subsequent to these stages was unaffected at the nonpermissive temperature. Spores of the temperature-sensitive mutant germinated normally at the nonpermissive temperature but failed to outgrow. The primary consequence of the obg mutation may be an alteration in initiation of chromosome replication.     

Ultrastructural Analysis of Sporulation in a Conditional Serine Protease Mutant of Bacillus subtilis

The morphological characteristics of wild-type Bacillus subtilis and a temperature-sensitive serine protease derivative have been observed during vegetative and sporulation time periods. At 30 C wild-type and mutant cells grow and sporulate identically. At 47.5 C wild-type and mutant cells grow identically, but the mutant cells are blocked at stage 0 or I in the sporulation sequence. Wild-type cells sporulate normally at 47.5 C.     

Characterization of arithmetic deoxyribonucleic acid synthesis at restrictive temperature in a dnaE mutant of Escherichia coli K-12.

The dna-293 mutation is shown to be a dnaE allele. The linear deoxyribonucleic acid synthesis previously observed in this mutant has been further characterized. The production of small deoxyribonucleic acid intermediates and their subsequent joining were identical in the mutant and its dnaE+ parent at 42.5 degrees C. Though the mutant cells continued to divide at the nonpermissive temperature, the rate of division was reduced. The data are consistent with a lack of production of replicationally active deoxyribonucleic acid polymerase III at the restrictive temperature.     

Isolation and Properties of a Temperature-Sensitive Sporulation Mutant of Bacillus subtilis

A thermosensitive sporulation mutant (t(s)-4) of Bacillus subtilis was isolated, and its morphological, physiological, and enzymatic properties were investigated. This mutant is able to grow equally well at 30 and 42 C, but is unable to sporulate at the higher temperature. Electron microscope studies have shown that the t(s)-4 mutant is blocked at stage zero of spore development. This was further confirmed by its inability to produce antibiotic when grown at the restrictive temperature and by the relatively low ribonucleic acid (RNA) and protein turnover during the stationary growth phase, characteristic for stage zero asporogenic mutants. At the permissive temperature, however, antibiotic production and RNA and protein turnover took place at the rate normally found in sporogenic strains of B. subtilis. The above properties were not altered in the parent strain when grown at either 30 or 42 C. By shifting cultures of the t(s)-4 mutant from 30 to 42 C and from 42 to 30 C at different stages of growth, we have been further able to show that the event affected at the high temperature takes place at a very early stage of spore development. As a consequence of this early block in the sporulation process, the t(s)-4 mutant grown at 42 C became defective in the late spore-specific enzymes involved in the biosynthesis of dipicolinic acid. This study suggests that the sporulation process is mediated by a regulatory protein which is altered in the thermosensitive mutant when grown at the restrictive temperature. As a result of this alteration, a pleiotropic phenotype is produced which has lost the ability to catalyze the late biochemical reactions required for spore formation.     

Ribonucleic Acid Polymerase in a Thermosensitive Sporulation Mutant (ts-4) of Bacillus subtilis

Partially synchronized cultures of a Bacillus subtilis thermosensitive sporulation mutant (ts-4) and the 168 try⁻try⁻ (168tt) parental strain were infected with the virulent phage e at various times during their growth cycle at 30 and 42 C (permissive and restrictive temperatures, respectively). It was shown that at the restrictive temperature the burst size in the parental strain was two- to threefold lower than in the ts-4 mutant. No such difference was observed at the permissive temperature. However, the time at which this difference was observed excludes a correlation between the burst size and initiation of the sporulation process. It was further found that the capacity to transcribe in vitro phage e deoxyribonucleic acid by partially purified ribonucleic acid (RNA) polymerase from both strains decreased sharply if the source of enzyme was sporulating cells instead of vegetative ones. However, a similar decrease, although to a lesser extent, was observed with the RNA polymerase isolated from stationary-phase cells of the ts-4 mutant grown at the nonpermissive temperature, or with the enzyme derived from several other zero-stage sporulation mutants. At no time was a structural modification in the β subunits of the RNA polymerase observed during growth of the sporulating bacteria. We have also shown that, in addition to the relatively low specific activity of the RNA polymerase, the level of the intracellular protease activity is about 15-fold lower in the ts-4 mutant grown at the restrictive temperature than that of the parental strain grown at the same temperature. At the permissive temperature no such difference was observed between these two strains. However, the present data do not allow us to establish a correlation among the low content of intracellular protease, the weak specific activity of the RNA polymerase, and the loss of the sporulation capacity in the ts-4 mutant grown at the restrictive temperature.     

New types of RNA polymerase mutations causing temperature-sensitive sporulation in bacillus subtilis.

A single site mutant of Bacillus subtilis with a streptovaricin-resistant RNA polymerase has been isolated; this mutation caused temperature-sensitive sporulation, but had no effect on vegetative growth. The mutant (ts710) temperature-sensitive period irreversibly affected the middle and late stages of sporulation. Mutant cells grown at the nonpermissive temperature exhibited abnormal serine protease accumulation, serine esterase accumulation, alkaline phosphatase accumulation, RNA polymerase template specificity changes, and pulse-labeled RNA synthesis profiles. The accumulation of metal protease was not affected at the nonpermissive temperature. Attempts to isolate single site mutants which were streptolydigin-resistant, and temperature-sensitive for sporulation, were unsuccessful.     

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.     

Temperature-sensitive sporulation caused by a mutation in the Bacillus subtilis secY gene.

A thermosensitive sporulation mutant of Bacillus subtilis containing a mutation in the secY gene was isolated and characterized. No asymmetric septum specific to the sporulation was detected by electron microscopy at the nonpermissive temperature, indicating that the block occurred at a very early stage of sporulation. Furthermore, competence development in the mutant cell was affected even at the sporulation-proficient temperature. It is assumed that the SecY protein of B. subtilis has multiple roles both in the regulation of spore formation and in stationary-phase-associated phenomena.     

Isolation and characterization of a temperature-sensitive dnaK mutant of Escherichia coli B.

A temperature-sensitive dnaK mutant (strain MT112) was isolated from Escherichia coli B strain H/r30RT by thymineless death selection at 43 degrees C. By genetic mapping, the mutation [dnaK7(Ts)] was located near the thr gene (approximately 0.2 min on the may). E. coli K-12 transductants of the mutation to temperature sensitivity were assayed for their susceptibility to transducing phage lambda carrying the dnaK and/or the dnaJ gene. All of the transductants were able to propagate phage lambda carrying the dnaK gene. When macromolecular synthesis of the mutant was assayed at 43 degrees C, it was observed that both deoxyribonucleic acid and ribonucleic acid syntheses were severely inhibited. Thus, it was suggested that the conditionally defective dnaK mutation affects both cellular deoxyribonucleic acid and ribonucleic acid syntheses at the nonpermissive temperature in addition to inability to propagate phage lambda at permissive temperature.     

Appearance of a ribonucleic acid polymerase-binding protein in asporogenous mutants of Bacillus subtilis

A 70,000-dalton protein that is found in sporulating Bacillus subtilis and that binds to ribonucleic acid polymerase is present in asporogenous mutants that proceed to or beyond stage II of sporulation, but is absent from mutants blocked at stage zero.     

Replication of plasmids in mutants of Escherichia coli temperature-sensitive for initiation of deoxyribonucleic acid synthesis

Plasmid deoxyribonucleic acid (DNA) replication was studied in Escherichia coli hosts carrying temperature-sensitive (ts) initiation mutations. The replication of the R plasmid NR1 continues at the nonpermissive temperature in a ts dnaA mutant host but at a decreasing rate in proportion to the residual chromosome synthesis. The replication of NR1, as well as of the F plasmid F′lac, ceases immediately at the nonpermissive temperature in a ts dnaC mutant host. The ability to reinitiate R plasmid replication in the absence of protein or ribonucleic acid synthesis is accumulated at the nonpermissive temperature in a dnaC mutant host.     

Ribonucleic Acid Polymerase Mutant of Escherichia coli Defective in Flagella Formation

Escherichia coli K-12 mutants that are resistant to bacteriophage chi, defective in motility, and unable to grow at high temperature (42 degrees C) were isolated from among those selected for rifampin resistance at low temperature (30 degrees C) after mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine. Genetic analysis of one such mutant indicated the presence of two mutations that probably affect the beta subunit of ribonucleic acid (RNA) polymerase: one (rif) causing rifampin resistance and the other (Ts-74) conferring resistance to phage chi (and loss of motility) and temperature sensitivity for growth. Observations with an electron microscope revealed that the number of flagella per mutant cell was significantly reduced, suggesting that the Ts-74 mutation somehow affected flagella formation at the permissive temperature. When a mutant culture was transferred from 30 to 42 degrees C, deoxyribonucleic acid synthesis accelerated normally, but RNA or protein synthesis was enhanced relatively little. The rate of synthesis of beta and beta' subunits of RNA polymerase was low even at 30 degrees C and was further reduced at 42 degrees C, in contrast to the parental wild-type strain. Expression of the lactose and other sugar fermentation operons, as well as lysogenization with phage lambda, occurred normally at 30 degrees C, suggesting that the mutation does not cause general shut-off of gene expression regulated by cyclic adenosine 3',5'-monophosphate.     

Mutants of vesicular stomatitis virus blocked at different stages in maturation of the viral glycoprotein

Maturation of the vesicular stomatitis virus (VSV) glycoprotein (G) to the cell surface is blocked at the nonpermissive temperature in cells infected with temperature-sensitive mutants in the structural gene encoding for G. We show here that these mutants fall into two discrete classes with respect to the stage of post-translational processing at which the block occurs. In all cases the mutant glycoproteins are inserted normally into the endoplasmic reticulum membrane, receive the two-high-mannose oligosaccharides, and apparently lose the NH2-terminal signal sequence of 16 amino acids. In cells infected with one class of mutants, no further processing of the glycoprotein occurs, and we conclude that the mutant protein is blocked at a pre-Golgi stage. In cells infected with ts L511(V), however, addition of the terminal sugars galactose and sialic acid occurs normally. Thus the maturation of G proceeds through several Golgi functions but is blocked before its appearance on the cell surface. The oligosaccharide chain of ts L511(V) G, accumulated at either the permissive (where surface maturation occurs) or the nonpermissive temperature, lacks one saccharide residue, probably fucose. In addition, no fatty acid residues are added to the ts L511(V) G protein at the nonpermissive temperature, although addition does occur under permissive conditions.     

Physiological studies of a temperature-sensitive sporulation mutant of Bacillus cereus T.

Growth of temperature-sensitive mutant Bacillus cereus T JS22-C occurred normally at the restrictive temperature (37 degrees C), but sporulation was blocked at stage 0. The production of extracellular and intracellular proteases and of alkaline phosphatase occurred at 37 degrees C, but the expression of a functional tricarboxylic acid cycle did not. At the permissive temperature (26 degrees C), the mutant sporulated at a slightly lower frequency (60%) and at a lower rate than the parent strain. The oxidation of organic acids, which accumulate in the growth medium began at T0 in cultures of the parent strain but was delayed until about T3 in cultures of the mutant. Later events in sporulation were also delayed in the mutant by about 3 h. Experiments in which the temperature of growth was shifted from 37 to 26 degrees C or from 26 to 37 degrees C at various times showed that the temperature-sensitive event began approximately 1 h after the end of exponential growth and ended when the cells reached the end of stage II (septum formation). The absence of a functional tricarboxylic acid cycle in cells of the mutant grown at 37 degrees C or shifted from 26 to 37 degrees C before T1 did not appear to be due to a lesion in one of the structural genes of the tricarboxylic acid cycle but was more likely due to the inability of the cells to derepress the synthesis of some of the enzymes of that cycle.     

Erythromycin resistant mutations in Bacillus subtilis cause temperature sensitive sporulation.

Summary All of several hundred erythromycin resistant (ery^R) single site mutants ofBacillus subtilis W168 are temperature sensitive for sporulation (spo^ts). The mutants and wild type cells grow vegetatively at essentially the same rates at both permissive (30° C) and nonpermissive (47° C) temperatures. In addition, cellular protein synthesis, cell mass increases and cell viabilities are similar in mutant and wild type strains for several hours after the end of vegetative growth (47° C). In the mutants examined, the temperature sensitive periods begin when the sporulation process is approximately 40% completed, and end when the process is 90% complete. At nonpermissive temperatures, the mutants produce serine and metal proteases at 50% of the wild type rate, accumulate serine esterase at 16% of the wild type rate, and do not demonstrate a sporulation related increase in alkaline phosphatase activity.The ery^R and spo^ts phenotypes cotransform 100%, and cotransduce 100% using phage PBS1. Revertants selected for ability to sporulate normally at 47° C (spo⁺), simultaneously regain parental sensitivity to erythromycin. No second site revertants are found.Ribosomes from ery^R spo^ts strains bind erythromycin at less than 1% of the wild type rate. A single 50S protein (L17) from mutant ribosomes shows an altered electrophoretic mobility. Ribosomes from spo⁺ revertants bind erythromycin like parental ribosomes and their proteins are electrophoretically identical to wild type. These data indicate that the L17 protein of the 50S ribosomal subunit fromBacillus subtilis may participate specifically in the sporulation process.     

Aspartate transcarbamylase synthesis ceases prior to inactivation of the enzyme in Bacillus subtilis.

Aspartate transcarbamylase is synthesized during exponential growth of Bacillus subtilis and is inactivated when the cells enter the stationary phase. This work is a study of the regulation of aspartate transcarbamylase synthesis during growth and the stationary phase. Using specific immunoprecipitation of aspartate transcarbamylase from extracts of cells pulse-labeled with tritiated leucine, we showed that the synthesis of the enzyme decreased very rapidly at the end of exponential growth and was barely detectable during inactivation of the enzyme. Synthesis of most cell proteins continued during this time. When the cells ceased growing because of pyrimidine starvation of a uracil auxotroph, however, synthesis and inactivation occurred simultaneously. Measurement of pools of pyrimidine nucleotides and guanosine tetra- and pentaphosphate demonstrated that failure to synthesize aspartate transcarbamylase in the stationary phase was not explained by simple repression by these compounds. The cessation of aspartate transcarbamylase synthesis may reflect the shutting off of a "vegetative gene" as part of the program of differential gene expression during sporulation. However, aspartate transcarbamylase synthesis decreased normally at the end of exponential growth at the nonpermissive temperature in a mutant strain that is temperature-sensitive in sporulation and RNA polymerase function. Cessation of aspartate transcarbamylase synthesis appeared to be normal in three other temperature-sensitive RNA polymerase mutants and in several classes of spo0 mutants.     

Bacterial Sporulation and Regulation of Dihydrodipicolinate Synthase in Ribonucleic Acid Polymerase Mutants of Bacillus subtilis

The activity of dihydrodipicolinate synthase increased late in sporulation in Bacillus subtilis. Mutants blocked at several stages of sporulation due to having an altered ribonucleic acid polymerase failed to exhibit this increase.