A two-dimensional protein gel electrophoresis study of the heat stress response of Bacillus subtilis cells during sporulation

The heat resistance of spores of Bacillus subtilis formed at 30 degrees C was enhanced by pretreatment at 48 degrees C for 30 min, 60 min into sporulation, for all four strains examined. High-resolution two-dimensional gel electrophoresis showed the generation and/or overexpression of 60 proteins, 11 of which were specific to heat shock, concurrent to this acquired thermotolerance. The greatest number of new proteins was observed between 30 and 60 min after heat shock, and the longer the time between exponential growth and heat treatment, the fewer differences were observed on corresponding protein profiles. The time at which heating produced the maximum increase in spore resistance and the most new proteins on two-dimensional gels occurred before alkaline phosphatase and dipicolinic acid production and corresponded to stage I or II of sporulation. The stress proteins formed disappeared later in sporulation, suggesting that heat shock proteins increase spore heat resistance by altering spore structure rather than by repairing heat damage during germination and outgrowth.     

The program of protein synthesis during sporulation in Bacillus subtilis

The program of protein synthesis was examined during sporulation in Bacillus subtilis as an index of the control of gene expression. At various stages of growth and spore formation, cells of B. subtilis were pulse-labeled with ³⁵S-methionine. Protein was extracted from the radioactively labeled bacteria and then subjected to high resolution one-dimensional and two-dimensional slab gel electrophoresis. We report that sporulating cells restricted or “turned off” the synthesis of certain polypeptides characteristic of the vegetative phase of growth. In certain cases, this “turn off” was prevented in a mutant (SpoOa-5NA) blocked at the first stage of spore formation. Sporulating bacteria also elaborated new polypeptide species that could not be detected in vegetatively growing cells or in cells of the asporogenous mutant SpoOa-5NA in sporulation medium. The synthesis of these sporulation-specific proteins was “turned on” in a temporally defined sequence throughout the period of spore formation. Spore coat protein, for example, was first synthesized at 4 hr after the onset of sporulation, the time at which refractile prespores appeared. Certain sporulation-specific polypeptides including the coat protein were among the most actively produced polypeptides in sporulating cells.     

Covalent modification of proteins in Bacillus subtilis during the process of sporulation, germination and outgrowth

Cells of Bacillus subtilis 168+ were labeled with 32P-orthophosphate during the process of sporulation, germination and outgrowth. By two-dimensional gel electrophoresis, at least 30 protein species were found to be radioactively labeled; 30% of these were modified by phosphorylation. Significant changes in the protein phosphorylation pattern during growth and cellular differentiation could be demonstrated. Using gamma-32P-ATP evidence for an ATP-dependent protein kinase was also obtained. Under these conditions 4 proteins with a molecular mass of 109,600; 103,100; 73,300 and 32,200 Da were found to be phosphorylated.     

In Vivo Aminoacylation of Transfer Ribonucleic Acid in Bacillus subtilis and Evidence for Differential Utilization of Lysine-Isoaccepting Transfer Ribonucleic Acid Species

The presence or absence of certain amino acids has different effects on the ability of Bacillus subtilis to sporulate, and the intracellular pool size of amino acids has been reported to vary during sporulation. The idea that these variations might exert a regulatory effect through aminoacylation of transfer ribonucleic acid (tRNA) was investigated by studying the levels of aminoacylation in vivo in the logarithmic or stationary phase of growth. Both the periodate oxidation method and the amino acid analyzer were used to evaluate in vivo aminoacylation. The results indicated that in general the level of aminoacylation of tRNA's remained constant through stage III of sporulation, although there were detectable variations for specific amino acid groups. Our studies also showed that periodate oxidation damaged certain tRNA's; therefore, the results obtained by such a method should be interpreted with caution. Because the damage can affect certain isoaccepting species specifically, the periodate oxidation method cannot be used to establish which isoaccepting species are acylated in vivo. We also investigated the possibility of preferential use of particular tRNA species by polyribosomes. These results demonstrated a preferential use of lysyl-tRNA's at different growth stages. Control mechanisms operating during the early stages of sporulation, therefore, do not affect the overall level of aminoacylation. However, there is an effect on the levels of aminoacylation of specific amino acids and on which isoaccepting species are utilized by the polyribosome system.     

Analysis of Isoaccepting Transfer Ribonucleic Acid Species of Bacillus subtilis: Changes in Chromatography of Transfer Ribonucleic Acids Associated with Stage of Development

Changes in chromatographic profiles of tyrosyl-, leucyl-, tryptophanyl-, and lysyl-transfer ribonucleic acids (tRNAs) are presented as a function of the growth stage in Bacillus subtilis. All of the tRNA groups investigated expressed different temporal patterns of change in isoaccepting species. Tyrosyl-tRNAs were the earliest to change and were followed by changes in leucyl- and then tryptophanyl-tRNAs. Lysyl-tRNAs were unique in having two times of change: one early and one very late. As an aid in understanding the temporal aspect of tRNA alterations during sporulation, the chromatographic profiles of aminoacyl tRNAs from an early blocked asporogenous mutant were studied. The asporogenous mutant used was blocked at the axial filament stage, stage 0 of sporulation. Nevertheless, those tRNAs which showed differences between the spore and cells in exponential growth exhibited similar changes in the asporogenous mutant after 24 h of growth. The data suggest that several tRNA changes occur during development in B. subtilis but that the events leading to these changes are either independent of, or occur before, stage 0 of sporulation, except in the case of lysyl-tRNA.     

Properties and developmental roles of the lysyl- and tryptophanyl-transfer ribonucleic acid synthetases of Bacillus subtilis: common genetic origin of the corresponding spore and vegetative enzymes

The lysyl-transfer ribonucleic acid synthetase (LRS) and tryptophanyl-transfer ribonucleic acid synthetases (TRS) (l-lysine:tRNA ligase [AMP], EC 6.1.1.6; and l-tryptophan:tRNA ligase [AMP], EC 6.1.1.2) have been purified 60- and 100-fold, respectively, from vegetative cells and spores of Bacillus subtilis. There are no significant differences between the corresponding spore and vegetative enzymes with respect to their elution characteristics from columns of phosphocellulose or hydroxylapatite, their molecular weight (~130,000 for LRS and ~87,000 for TRS as determined by gel filtration), their kinetic constants for substrates (in the amino acid-dependent adenosine triphosphate-pyrophosphate exchange reaction), and the kinetics of inactivation by heat and by antibody. The Mg(2+) requirement for optimal enzyme activity of the corresponding spore and vegetative enzyme differ slightly. Mutants having defective (temperature sensitive) vegetative LRS or TRS activities produce spores in which these enzymes are also defective. The mutant spores are more heat sensitive than the parental type, but contain normal levels of dipicolinic acid. They germinate normally at the restrictive temperature (43 C), but are blocked at specific developmental stages in outgrowth. No modification in temperature sensitivity phenotype occurs during outgrowth, nor is there a change in molecular weight of the two enzymes. The implication is that the LRS and TRS activities of the vegetative and spore stages are each coded (at least in part) by the same structural gene. The temperature sensitivity of mutant spores is discussed with respect to those factors which are involved in the formation of the heat-resistant state.     

Analysis of the function of a putative 2,3-diphosphoglyceric acid-dependent phosphoglycerate mutase from Bacillus subtilis

A Bacillus subtilis gene termed yhfR encodes the only B. subtilis protein with significant sequence similarity to 2, 3-diphosphoglycerate-dependent phosphoglycerate mutases (dPGM). This gene is expressed at a low level during growth and sporulation, but deletion of yhfR had no effect on growth, sporulation, or spore germination and outgrowth. YhfR was expressed in and partially purified from Escherichia coli but had little if any PGM activity and gave no detectable PGM activity in B. subtilis. These data indicate that B. subtilis does not require YhfR and most likely does not require a dPGM.     

Nature and timing of some sporulation-specific protein changes in Saccharomyces cerevisiae.

During meiosis and spore formulation in Saccharomyces cerevisiae, changes that occur in a/alpha diploids, but not in isogenic nonsporulating a/a diploids, have been detected in cellular polypeptides. These were found by the technique of prelabeling growing cells with 35SO4(2-) and suspending them in sulfur-free sporulation medium. Under the conditions used, about 400 polypeptides were detected by two-dimensional gel electrophoresis, and 45 were altered during sporulation; of these, 21 changes were specific to a/alpha strains. These alterations were mainly due to the appearance of new polypeptides or to marked increases in the concentrations of a few polypeptides produced during vegetative growth. They could have been due either to modifications of existing polypeptides present in growing cells or to de novo synthesis of new gene products. They occurred at characteristic times during sporulation; whereas the majority of changes took place early (within the first 6 h in sporulation conditions), there were several changes characterizing the later stages of sporulation. Ten of the 35SO4(2-)-labeled polypeptides were also labeled with 32P in the presence of [32P]orthophosphate; of these, three were previously found to be sporulation specific. One of these was phosphorylated at all stages of sporulation and was labeled when [32P]orthophosphate was added either during growth of the culture of 1 h after transfer to sporulation medium. Another was labeled in the same way by adding 32P at either time, so that by 7 h in sporulation medium it was phosphorylated, but was dephosphorylated by 24 h. The third sporulation-specific peptide was labeled in extracts prepared at 7 h in sporulation medium (but not at 24 h) when [32P]-orthophosphate was added during presporulation growth, but not when [32P]-orthophosphate was added 1 h after transfer of the culture to sporulation medium. This polypeptide appeared early during sporulation; it is probably phosphorylated as it appears and is dephosphorylated at some time between 7 h and 24 h of sporulation.     

Survival of Microorganisms in a Simulated Martian Environment: II. Moisture and Oxygen Requirements for Germination of Bacillus cereus and Bacillus subtilis var. niger Spores1

The effects of moisture and oxygen concentration on germination of Bacillus cereus and B. subtilis var. niger spores were investigated in a simulated Martian environment. Less moisture was required for germination than for vegetative growth of both organisms. A daily freeze-thaw cycle lowered moisture requirements for spore germination and vegetative growth of both organisms, as compared with a constant 35 C environment. Oxygen had a synergistic effect by lowing the moisture requirements for vegetative growth, and possibly germination, of both organisms. Oxygen was not required for spore germination of either organism, but was required for vegetative growth of B. subtilis and for sporulation of both organisms.     

Protein synthesis and breakdown in the mother-cell and forespore compartments during spore morphogenesis in Bacillus megaterium

Recently developed techniques for isolating forespores from bacilli at all stages of spore morphogenesis have been exploited to investigate the contribution of each of the two compartments of the sporulating cell to the overall pattern of protein synthesis and degradation during sporulation in Bacillus megaterium. These studies have shown: (1) that protein synthesis continues in both compartments throughout spore morphogenesis; (2) that the degradation of proteins made at all times during vegetative growth and sporulation is confined to the mother-cell compartment; (3) that proteins synthesized in the mother-cell compartment during sporulation are subsequently degraded more rapidly than proteins synthesized during vegetative growth. This rate of degradation increases the later the proteins are synthesized in the sporulation sequence. Mature spores were disrupted, and the percentage of the total protein in soluble and particulate fractions was determined. Pulse-labelling experiments were performed to investigate the extent to which the proteins of these two fractions are newly synthesized during sporulation. These data were used to calculate the extent of capture of vegetative cell protein at the time of formation of the forespore septum. The value obtained is consistent with evidence from electron micrographs and supports a model for the origin of spore protein in which there is no protein turnover in the developing forespore.     

Biochemical Studies of Bacterial Sporulation and Germination XIII. Adenylate Kinase of Vegetative Cells and Spores of Bacillus subtilis

The spore and vegetative cell adenylate kinases of Bacillus subtilis, purified about 1,000-fold, proved indistinguishable by several physical and functional tests, including polyacrylamide gel electrophoresis, DEAE cellulose chromatography, and specificity toward substrates. Adenylate kinase activity in cell extracts, followed throughout growth and sporulation, was found to reach a maximum near the end of exponential growth, remain at that level during sporulation, until shortly before the appearance of refractile forms, and then decline, along with total protein, during the subsequent maturation of the spores. The enzyme, stable in extracts of exponential growing cells, was unstable in extracts of sporulating cells, presumably as a result of degradation by protease(s) appearing after the end of exponential growth.     

Conditional dihydrostreptomycin resistance in Bacillus subtilis

Mutants resistant to dihydrostreptomycin were isolated and genetically analyzed in Bacillus subtilis. Two new classes of mutants distinct from the ribosomal strA locus were found. One class, strB, was located between metC3 and ura-1 on the chromosome. The second class, strC, mapped in the spore gene region close to the spoA locus. Both mutant classes were resistant to dihydrostreptomycin during growth but sensitive to the antibiotic during sporulation. Resuspension sporulation experiments with a strB mutant showed that sensitivity to the antibiotic was acquired early in the sporulation process. The germination and outgrowth of strB spores was sensitive to the antibiotic until growth commenced, whereupon the culture was resistant. Thus the mutants are sensitive to dihydrostreptomycin during both sporulation and germination but resistant during the growth phase.     

Membrane protein alterations during the early stages of sporulation of bacillus subtilis

Membrane protein alterations during the early stages of sporuloation were examined by polyacrylamide gel electrophoresis. Solubilized samples of the vegetative cell membrane (VCM), sporulation membrane fraction (SMF), and inner forespore membranes (IFM) were compared with respect to their protein compositions. The VCM contained 39 protein components, distinguishable as separate bands on gel electrophoresis, and these ranged in molecular weight from 16,000 to greater than 100,000. During the first 5 hr of sporulation, 6 of these 39 protein bands disappeared, 8 increased and 12 decreased in concentration, and 13 showed no discernible change. In addition, 15 new protein components were identified in the SMF during the fireist 5 hr. The new components consisted of 7 protein bands that were transiently associated with the SMF, and 8 proteins that persisted in the SMF from their time of appearance until at least T5 of sporulation. Comparison of the protein composition of the IFM with those of the VCM and SMF revealed that membrane protein alterations occur during sporulation. The turnover of H³-tryptophan-labeleld membrane protein was followed during growth and sporulation. During the 30 min of growth following a simple chase with excess unlabeled tryptophan, membrane protein appeared stable, whereas 5–10% of the nonmembrane protein turned over to acid-soluble material. However, manipulation of the cells by dilution ito fresh medium, or centrifugation, as part of the chase procedure, resulted in elution of membrane protein to the cytoplasm. In contrast, proteins labeled during vegetative growth were always eluted to the cytoplasm during the first 2 hr of sporulation, and this was followed by a period of reassociation with the membrane fraction. The results are discussed with respect to membrane differentiation as it relates to spore development.