Suppression of temperature-sensitive sporulation of a Bacillus subtilis elongation factor G mutant by RNA polymerase mutations.

A class of rifampin-resistant (rfm) mutations of Bacillus subtilis suppresses the temperature-sensitive sporulation of a fusidic acid-resistant mutant. FUS426, which has an altered elongation factor G. The rfm mutation suppressed only the sporulation defect caused by the elongation factor G mutation, but could not suppress other types of induced sporulation defects. Genetic and biochemical analyses showed that the sporulation suppression by the rfm mutation was caused by a single mutation in RNA polymerase. After the early sporulation phase, the apparent rate of RNA synthesis of FUS426, measured by [3H]uracil or [3H]uridine incorporation into RNA, became lower than that of the wild-type strain, and this decrease was reversed by the rfm mutation. However, when the total rate of RNA synthesis of FUS426 was calculated by measuring the specific activity of [3H]UTP and [3H]CTP, it was higher than that of the rfm mutant, RIF122FUS426. The possible mechanism of the functional interaction between elongation factor G and RNA polymerase during sporulation is discussed.     

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 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.     

Characterization of RNA synthesis in an Escherichia coli mutant with a temperature-sensitive lesion in stable RNA synthesis

Previous experiments with Escherichia coli strain 2S142 have shown that the synthesis of stable RNA is preferentially blocked at the restrictive temperature. In this paper, we have examined the capacity of this mutant strain to synthesize RNA in vitro. Growth of the strain for as short a period as 10 min at 42 degrees C resulted in a 40 to 60% loss of RNA synthetic capacity and a fourfold decrease in percent rRNA synthesized in toluenized cell preparations. The time course for the loss and recovery of this RNA synthetic capacity correlated very well with the changes in RNA synthesis observed in vivo. We found no difference in temperature sensitivity of the purified RNA polymerase from the mutant and the parental strains. Moreover, there was no detectable alteration in the amount of enzyme, specific activity of the enzyme, or electrophoretic mobility of the subunits when the mutant strain was grown at 42 degrees C. The capacity for rRNA synthesis was also measured with the Zubay in vitro system (Reiness et al., Proc. Natl. Acad. Sci. 72:2881-2885, 1975). Supernatant fractions (S-30) prepared from cells grown at 30 degrees C were capable of up to 31.2% rRNA synthesis, using phi 80d3 DNA as template. S-30 fractions from cells grown at 42 degrees C synthesized 8.6% rRNA. The bottom one-third of the S-100 fraction and the ribosomal salt wash from 30 degrees C cells contained one or more factors which partially restored preferential rRNA synthesis in S-30 fractions from cells grown at 42 degrees C. Preliminary evidence suggests that the factor(s) is protein in nature.     

Distribution of muropeptides in walls of Bacillus subtilis and a temperature-sensitive mutant

Attempts to correlate differences in cell shape with aspects of peptidoglycan structure were investigated. The parent strain, Bacillus subtilis 168, and its temperature-sensitive tagB mutant were grown in the chemostat under different growth conditions. The composition of the peptidoglycan was similar in all samples, regardless of cellular shape and anionic polymer content. Muropeptides, released by digestion with muramidase, comprised mainly dimers and monomers with only small amounts of trimer and traces of tetramer muropeptide. Overall, cross-linking did not vary greatly and the cross-linking index was less than 38%. Reverse-phase HPLC separation showed a complex fine structure. The principal muropeptides in all samples appeared to be the tetra monomer, tetra-tetra dimer and tetra-tetra-tetra trimer. While the major components looked the same in all samples, two specific components, a monomer and a dimer, were seen exclusively in the samples that had coccal morphology.     

Guanosine polyphosphate concentration and stable RNA synthesis in Bacillus subtilis following suppression of protein synthesis

Amount of guanosine-5'-triphosphate, 3'-diphosphate (pppGpp) and guanosine-5'-diphosphate, 3'-diphosphate (ppGpp) in the cells of b. subtilis increased several times during starvation for lysine or after treatment with serine hydroxamate (analog of serine) or norvaline (analog of leucine), or in the presence of trimethoprim, which induced deficiency of methionine and leucine. In exponentially growing cells the concentration of pppGpp was found to be 10-20 pmol/A600. When serine hydroxamate or trimethoprim were added, concentration of pppGpp increased to 500-800 pmol/A600 and then slowly diminished. Elimination of lysine or addition to the culture medium of norvaline caused slight transitory accumulation of pppGpp (150 pmol/A600). The amount of another nucleotide ppGpp was always 2-3 times lower than one of pppGpp. Accumulation of (p)ppGpp in rel+ cells was accompanied by cessation of stable RNA synthesis. Under conditions described above rel- cells continued RNA synthesis and did not accumulate (p)ppGpp. In the rel+ cells treated with serine hydroxamate synthesis of stable RNA resumed and the amount of (p)ppGpp decreased after addition of serine or tetracycline and chloramphenicol. The half-life period for pppGpp in the presence of chloramphenicol was determined to be 30-40 seconds. Thus, during aminoacyl-tRNA deficiency rel+ cells of B. subtilis accumulate (p)ppGpp, which are believed to participate in negative regulation of RNA synthesis. Slight accumulation of pppGpp without concomitant inhibition of stable RNA synthesis was observed after treatment of growing cells with chloramphenicol.     

Translocation in Bacillus subtilis: characterization of elongation factor G by peptidyl-[3H]puromycin synthesis.

This communication describes the characterization of elongation factor G from Bacillus subtilis by the translocation of "native" peptide donors. Translocation was followed by elongation factor G-dependent increase in the synthesis of peptidyl-[3H]puromycin using "washed" ribosomes carrying in vivo-bound peptidyl-transfer ribonucleic acid ("native" peptidyl-transfer ribonucleic acid) molecules as peptide donors. Such ribosomes were obtained from cell extracts by washing at a high salt concentration. The use of "native" peptide donors facilitated the study of translocation under conditions that are closer to the in vivo state than those in the methods previously employed.     

RNA synthesis during the stationary growth phase in the Bacillus subtilis Cgr4 mutant

RNA synthesis was studied in Bacillus subtilis Cgr4 grown in the mineral sporulation medium enriched with glucose up to 2% and amino acids up to 1%. To study mRNA synthesis, a method of transfer of the 3H-uridine pulse-labeled culture to the supernatant of physiologically identical, not labeled culture, followed by further incubation was used, the amount of 3H-uridine in the supernatant as well as in cells being measured. RNA was also analysed electrophoretically and distribution of the label among the fractions was determined. It is shown that mRNA synthesized in the logarithmic phase degrades up to 12% on the 2nd hour of growth during 10 min; the mRNA in the stationary phase is stable on the 7th hour of growth; no degradation is observed in the course of 2-3 hours. The beginning of degradation coincides in time with secondary induction of the synthesis of serine proteases and with the onset of sharp decrease in incorporation of 3H-uridine in RNA as well as with induction of spore morphogenesis. On the basis of electrophoretical analysis of pulse-labeled RNA, it was demonstrated that, prior to the transfer, labeled uridine was included and preserved in RNA fraction for 2-3 hours after the transfer, this fraction corresponding in mobility with mRNA in polyacrylamide gel. The following conclusion may be drawn: stable mRNAs are synthesized in the stationary phase and may be used for the translation of extracellular serine protease.     

Small stable RNA maturation and turnover in Bacillus subtilis

Stable RNA maturation is a key process in the generation of functional RNAs, and failure to correctly process these RNAs can lead to their elimination through quality control mechanisms. Studies of the maturation pathways of ribosomal RNA and transfer RNA in Bacillus subtilis showed they were radically different from Escherichia coli and led to the identification of new B. subtilis‐specific enzymes. We noticed that, despite their important roles in translation, a number of B. subtilis small stable RNAs still did not have characterised maturation pathways, notably the tmRNA, involved in ribosome rescue, and the RNase P RNA, involved in tRNA maturation. Here, we show that tmRNA is matured by RNase P and RNase Z at its 5′ and 3′ extremities, respectively, whereas the RNase P RNA is matured on its 3′ side by RNase Y. Recent evidence that several RNases are not essential in B. subtilis prompted us to revisit maturation of the scRNA, a component of the signal recognition particle involved in co‐translational insertion of specific proteins into the membrane. We show that RNase Y is also involved in 3′ processing of scRNA. Lastly, we identified some of the enzymes involved in the turnover of these three stable RNAs.     

Characterization of Bacillus subtilis mutants temperature-sensitive in the synthesis of ribonucleic acid

Two mutants of Bacillus subtilis temperature-sensitive in RNA synthesis were isolated. One mutation (rna-20) was demonstrated to be an allele of a previously identified gene (Riva et al., 1976). The other mutation (rna-16) identified a different gene and was mapped near aroI. The rna-16 mutation at the permissive temperature affected the spore outgrowth process. Purified RNA polymerase from rna-16 did not show any temperature sensitivity or structural defect.     

Anucleate cell production and surface extension in a temperature-sensitive chromosome initiation mutant of Bacillus subtilis.

At 45 C, in a temperature-sensitive initiation mutant (TsB134) of Bacillus subtilis 168 Thy- tryp-, growing in a glucose-arginine minimal medium, chromosome completion occurred over a period of 80 to 90 min, after which there was no further nuclear division. Normal symmetrical cell divisions continued for a generation afterwards, so that nuclei were segregated into separate cells. During this period asymmetric divisions started to occur. Septa appeared at 25 to 30% from one end of the cell, giving a small anucleate cell and a larger nucleate cell. During inhibition of deoxyribonucleic acid (DNA) synthesis by thymine starvation under the restrictive conditions, asymmetrical division also occurred until there was approximately one nucleus per cell (about one generation time). Asymmetric division, giving anucleate cells, then occurred. Similar results were obtained when DNA synthesis was inhibited by nalidixic acid. After 3 h at 45 C, the rate of anucleate cell production in the presence and absence of thymine was constant at one division per 85 min per chromosome terminus present when DNA synthesis stopped. In the absence of DNA synthesis (during thymine starvation) at 35 C, growth in cell length was linear (i.e., the rate was constant), but at 45 C during thymine starvation the rate gradually increased by more than twofold. It is suggested that this was due to the establishment of new sites of growth associated with anucleate cell production. In the presence of thymine at 45 C, the rate of length extension increased by more than fourfold, which it is suggested was caused by the appearance of new growth zones as a result of chromosome termination and a contribution associated with anucleate cell production. If the mutant was incubated at 45 C for 90 min, both in the presence and absence of thymine, then anucleate cell formation could continue on restoration to 35 C in the absence of thymine...     

Electron microscope study of the rod-to-coccus shape change in a temperature-sensitive rod- mutant of Bacillus subtilis.

The changes in cell morphology of Bacillus subtilis rodB during a temperature shift from 20 to 42 degrees C, in the absence of added anions, are described. At 20 degrees C the organisms grow as rods but gradually become spherical in shape when placed at 42 degrees C. The shape change is initiated by an increase in diameter at the cell equator, resulting in a bulged morphology, which is further modified to the morphology of a coccus. This change may involve a modification of the pattern of normal cylindrical extension such that incorporation of newly synthesized wall leads only to increase in diameter, perhaps from a growth zone of limited extent. The pattern of surface growth was followed by reconstructing the sequence of cross wall formation and pole construction in rods grown at 20 degrees C and in organisms incubated at 42 degrees C for 75 and 150 min. In thin section, wall forming the septum and nascent poles can be distinguished from the surface distal to the division site by the presence of raised tears, perhaps analogous to the wall bands of streptococci. By using an analog rotation technique involving the three-dimensional reconstruction of cells by mathematical rotation of axial thin sections about their longitudinal axis, it is shown that the proportion of septal wall increases during the shape change. In the coccal forms, all surface growth may arise from septal growth sites.     

Mutant of Bacillus subtilis with a temperature-sensitive lesion in ribonucleic acid synthesis during germination.

We have isolated a mutant of Baccillus subtilis with a temperature-sensitive lesion in the process of spore germination. The temperature-sensitive mutation affects only germination and outgrowth, and the earliest defect observed is an early block of ribonucleic acid synthesis during germination at 46 C. Upon return to 35 C there is a complete repair of the impaired function, even in the absence of protein synthesis. Protein synthesis inhibition during germination of the mutant spores at 46 C has the effect of increasing the amount of ribonucleic acid made. The temperature-sensitive mutation is located near aroI.     

Structural and functional properties of a Bacillus subtilis temperature-sensitive sigma(A) factor

Bacillus subtilis DB1005 is a temperature-sensitive (Ts) sigA mutant containing double-amino-acid substitutions (I198A and I202A) on the hydrophobic face of the promoter -10 binding helix of sigma(A) factor. We have analyzed the structural and functional properties of this mutant sigma(A) factor both in vivo and in vitro. Our data revealed that the Ts sigma(A) factor possessed predominantly a multimeric structure which was prone to aggregation at restrictive temperature. The extensive aggregation of the Ts sigma(A) resulted in a very low core-binding activity of the Ts sigma(A) factor and a markedly reduced sigma(A)-RNA polymerase activity in B. subtilis DB1005, suggesting that extensive aggregation of the Ts sigma(A) is the main trigger for the temperature sensitivity of B. subtilis DB1005. Partial proteolysis, tryptophan fluorescence and 1-anilinonaphthalene-8-sulfonate-binding analyses revealed that the hydrophobic face of the promoter -10 binding helix and also the hydrophobic core region of the Ts sigma(A) factor were readily exposed on the protein surface. This hydrophobic exposure provides an important cue for mutual interaction between molecules of the Ts sigma(A) and allows the formation of multimeric Ts sigma(A). Our results also indicate that Ile-198 and Ile-202 on the hydrophobic face of the promoter -10 binding helix are essential to ensure the correct folding and stabilization of the functional structure of sigma(A) factor.     

Biochemical and genetic characterization of a temperature-sensitive, tryptophanyl-transfer ribonucleic acid synthetase mutant of Bacillus subtilis

A temperature-sensitive, 5-fluorotryptophan (5FT)-resistant mutant of Bacillus subtilis was isolated which forms an altered tryptophanyl transfer ribonucleic acid synthetase [l-tryptophan: sRNA ligase (AMP), EC 6.1.1.2]. The mutant grows well at 30 C but not at 42 C. At the latter temperature, protein and ribonucleic acid (RNA) synthesis are abolished while deoxyribonucleic acid (DNA) synthesis proceeds for a considerable time. Tryptophanyl-transfer RNA (tRNA) synthetase activity is not detectable in the extracts of the mutant grown at 30 C whether this activity is measured by the attachment of l-tryptophan to tRNA or the l-tryptophan-dependent exchange of (32)P-pyrophosphate with adenosine triphosphate. Mixing experiments with extracts from the wild type and the mutant have ruled out the presence of an inhibitor or the absence of an activator as possible causes. Attempts to retrieve enzyme activity in vitro by various means (different conditions for cell disruption, addition of l-tryptophan, and adenosine triphosphate to the extraction buffer containing glycerol) were unsuccessful. The mutation in the locus of the tryptophanyl tRNA synthetase (trpS) was mapped on the bacterial chromosome by transformation and transduction. It is located between argC and metA. All temperature-resistant transformants recover wild-type levels of tryptophanyl tRNA synthetase activity and sensitivity to 5FT. Spontaneous revertants to temperature resistance are 5FT sensitive, but their levels of tryptophanyl tRNA synthetase activity and the thermolability of this enzyme in cell-free extracts varies. These revertants do not support the growth of a presumed nonsense mutant of phase SPO-1. Transduction experiments with phage PBS-1 indicated that reversion must be the result of an event at the site of the original mutation or at a site extremely close to it.