SmeA, a small membrane protein with multiple functions in Streptomyces sporulation including targeting of a SpoIIIE/FtsK-like protein to cell division septa

Sporulation in aerial hyphae of Streptomyces coelicolor involves profound changes in regulation of fundamental morphogenetic and cell cycle processes to convert the filamentous and multinucleoid cells to small unigenomic spores. Here, a novel sporulation locus consisting of smeA (encoding a small putative membrane protein) and sffA (encoding a SpoIIIE/FtsK-family protein) is characterized. Deletion of smeA-sffA gave rise to pleiotropic effects on spore maturation, and influenced the segregation of chromosomes and placement of septa during sporulation. Both smeA and sffA were expressed specifically in apical cells of sporogenic aerial hyphae simultaneously with or slightly after Z-ring assembly. The presence of smeA-like genes in streptomycete chromosomes, plasmids and transposons, often paired with a gene for a SpoIIIE/FtsK- or Tra-like protein, indicates that SmeA and SffA functions might be related to DNA transfer. During spore development SffA accumulated specifically at sporulation septa where it colocalized with FtsK. However, sffA did not show redundancy with ftsK, and SffA function appeared distinct from the DNA translocase activity displayed by FtsK during closure of sporulation septa. The septal localization of SffA was dependent on SmeA, suggesting that SmeA may act as an assembly factor for SffA and possibly other proteins required during spore maturation.     

Motility of Bacillus subtilis during growth and sporulation.

The change of motility and the presence of flagella were followed throughout growth and sporulation in a standard sporulating strain and in 19 cacogenic sporulation mutants of Bacillus subtilis. For the standard strain, the fraction of motile cells decreased during the developmental period to less than 10% at T4. Motility was lost well before the cells lose their flagella. Conditions reducing the decrease of motility also reduced sporulation: motile cells never contained spores. The decrease of motility was not coupled with a decrease in the cellular concentration of adenosine 5'-triphosphate or a decline in oxygen consumption, but an uncoupling agent immediately destroyed motility at any time. Apparently, motility decreased during development because it became increasingly uncoupled from the energy generating systems of the cell. The motility of sporulation mutants decreased after the end of growth at the same time as or earlier than the motility of the standard strain; the early decrease of motility in an aconitase mutant, but not that in an alpha-ketoglurate dehydrogenase mutant, could be avoided by addition of L-glutamate. Sporulation or related events such as extracellular antibiotic or protease production were not needed for the motility decline.     

Adenosine 5′-Triphosphate Release and Membrane Collapse in Glycerol-Requiring Mutants of Bacillus subtilis

Glycerol-requiring mutants of Bacillus subtilis could not sporulate in nutrient sporulation medium even when additional glycerol was added from the beginning of growth. Sporulation could be partially restored either by the frequent addition of small amounts of glycerol during the developmental period or by the single addition of both 10 mM glycerol and 10 mM malate. But sporulation could be completely restored by the addition of 50 mM glycerol-phosphate from the beginning. At the end of growth of the glycerol mutants in nutrient sporulation medium, the cell membrane collapsed and separated from the cell wall, and much of the cellular adenosine 5'-triphosphate was released into the medium. These observations were made in two glycerol mutants, one derived from strain 168 containing glycerol-teichoic acid in the cell wall and the other derived from strain W23 containing ribitol-teichoic acid.     

Analysis of Sporulation Mutants II. Mutants Blocked in the Citric Acid Cycle

Sporulation mutants that were unable to incorporate uracil during the developmental period recovered this capacity with the addition of ribose and in most cases with the addition of glutamate. Of the mutants that responded to both ribose and glumate, all but three also responded to citrate, and all but five responded to acetate. One of the exceptional strains was deficient in aconitase and another one in aconitase and isocitrate dehydrogenase; both required glutamate for growth. For the mutants which did not respond to glutamate, the products made from (14)C-glutamate were determined by thin-layer chromatography. Significant differences were found which enabled the identification of mutant blocks. The deficiency of the corresponding enzyme activity was verified. Several mutants were deficient in alpha-ketoglutarate dehydrogenase, and one lacked succinic dehydrogenase. These mutants could still grow on glucose as sole carbon source, but not on glutamate. The intact Krebs cycle is therefore not required for vegetative growth of aerobic Bacillis subtilis, but it is indispensable for sporulation.     

ClpC-Mediated Sporulation Regulation at Engulfment Stage in Bacillus anthracis

Bacterial sporulation is a conserved process utilized by members of Bacillus genus and Clostridium in response to stress such as nutrient or temperature. Sporulation initiation is triggered by stress signals perceived by bacterial cell that leads to shutdown of metabolic pathways of bacterial cells. The mechanism of sporulation involves a complex network that is regulated at various checkpoints to form the viable bacterial spore. Engulfment is one such check point that drives the required cellular rearrangement necessary for the spore assembly and is mediated by bacterial proteolytic machinery that involves association of various Clp ATPases and ClpP protease. The present study highlights the importance of degradation of an anti-sigma factor F, SpoIIAB by ClpCP proteolytic machinery playing a crucial role in culmination of engulfment process during the sporulation in Bacillus anthracis.     

Order of expression of genes affecting septum location during sporulation of Bacillus subtilis.

The mean volumes of stationary-phase cells of wild-type and asporogenous mutants of Bacillus subtilis have been measured. Mutants blocked at stage 0 of sporulation either produced cells that had the same volume as the developing sporangium or they divided to produce cells of one-half this volume. The order of expression of the genes affected by the mutations in these strains was determined by biochemical characterization and by construction of double sporulation mutants. Mutants that produced small cells were blocked at an earlier stage of sporulation than those that produced large cells. It is suggested that the following dependent sequence must occur before the formation of the prespore spetum: (i) the initiation of sporulation, (ii) a signal to block the final central division site, and (iii) a signal to activate a polar septum site.     

Partial purine deprivation causes sporulation of Bacillus subtilis in the presence of excess ammonia, glucose and phosphate.

In strains of Bacillus subtilis able to synthesize purines de novo, massive sporulation is suppressed by the combination of excess ammonia, glucose and phosphate. Purine auxotrophs, blocked in the general or the guanine-specific portion of the branched purine pathway, sporulated in such a medium when the purine required for normal growth was removed from the medium. The resulting spore titre and the sporulation frequency increased with the residual growth rate in the purine-free medium, i.e. with the leakiness of the purine mutation. Sporulation was further increased by allowing residual growth in growth-limiting amounts of guanosine. None-leaky purine mutants blocked before 5'-phosphoribosyl-5-amino-4-imidazole carboxamide also sporulated well when supplied with 5-amino-4-imidazole carboxamide at concentrations (2 mM) that supported growth at a suboptimal rate.     

Alcohol-resistant sporulation mutants of Bacillus subtilis.

About 80% of Bacillus subtilis cells form spores when grown in nutrient broth. In medium containing various short-chain aliphatic alcohols, the frequency of sporulation was reduced to 0.5%. Mutants sporulated in the presence of alcohols at a frequency of 30 to 40%. Sporulation in the wild-type cells was sensitive to alcohol at the beginning of sporulation (stage zero). Sensitivity to alcohol in the mutants was also at stage zero, even though the sensitivity was considerably reduced. This sensitivity of sporulation to alcohol is the phenotypic expression of a genetic locus designated ssa. Mutations at this locus lead to a decreased sensitivity of sporulation to alcohol without modifying the sensitivity of growth. Genetic analysis by transduction was bacteriophage PBS1 revealed that ssa mutations are near the previously described spo0A locus. ssa mutants also differ from wild-type cells in the composition of membrane phospholipids. The relative amount of phosphatidylglycerol increased, whereas the relative amount of phosphatidylethanolamine and lysylphosphatidylglycerol decreased relative to the proportions in the wild type. The distribution of fatty acids in membrane lipids is the same as in the wild type. No differential sensitivity of phospholipid metabolism to alcohol could be detected in the mutant. This work therefore reveals that the extensive, pleiotropic changes in the membranes of ssa mutants are the phenotypic reflection of alterations at a specific gene locus.     

Cell-density-dependent lysis and sporulation of Myxococcus xanthus in agarose microbeads.

Vegetative cells of Myxococcus xanthus were immobilized in 25-microns-diameter agarose microbeads and incubated in either growth medium or sporulation buffer. In growth medium, the cells multiplied, glided to the periphery, and then filled the beads. In sporulation buffer, up to 90% of the cells lysed and ca. 50% of the surviving cells formed resistant spores. A strong correlation between sporulation and cell lysis was observed; both phenomena were cell density dependent. Sporulation proficiency was a function of the average number of cells within the bead at the time that sporulation conditions were imposed. A minimum of ca. 4 cells per microbead was necessary for efficient lysis and sporulation to proceed. Increasing this number accelerated the lysis and sporulation process. No lysis occurred when an average of 0.4 cell was entrapped per bead. Entrapping an average of 1.7 cells per bead resulted in 46% lysis and 3% sporulation of survivors, whereas entrapping an average of 4.2 cells per bead yielded 82% lysis and 44% sporulation of the surviving cells. Sporulation and lysis also depended upon the cell density in the culture as a whole. The existence of these two independent cell density parameters (cells per bead and cells per milliliter) suggests that at least two separate cell density signals play a role in controlling sporulation in M. xanthus.     

The asgE locus is required for cell-cell signalling during Myxococcus xanthus development

In response to starvation, Myxococcus xanthus undergoes a multicellular developmental process that produces a dome-shaped fruiting body structure filled with differentiated cells called myxospores. Two insertion mutants that block the final stages of fruiting body morphogenesis and reduce sporulation efficiency were isolated and characterized. DNA sequence analysis revealed that the chromosomal insertions are located in open reading frames ORF2 and asgE, which are separated by 68 bp. The sporulation defect of cells carrying the asgE insertion can be rescued phenotypically when co-developed with wild-type cells, whereas the sporulation efficiency of cells carrying the ORF2 insertion was not improved when mixed with wild-type cells. Thus, the asgE insertion mutant appears to belong to a class of developmental mutants that are unable to produce cell-cell signals required for M. xanthus development, but they retain the ability to respond to them when they are provided by wild-type cells. Several lines of evidence indicate that asgE cells fail to produce normal levels of A-factor, a cell density signal. A-factor consists of a mixture of heat-stable amino acids and peptides, and at least two heat-labile extracellular proteases. The asgE mutant yielded about 10-fold less heat-labile A-factor and about twofold less heat-stable A-factor than wild-type cells, suggesting that the primary defect of asgE cells is in the production or release of heat-labile A-factor.     

Sporulation of Bacillus subtilis in Continuous Culture

Sporulation of Bacillus subtilis 168 was studied in chemostat cultures. Sporulation occurred at high frequency under limitation of growth by glucose or the nitrogen source in minimal medium, whereas rates of sporulation were low for Mg(2+), phosphate, citrate, or tryptophan limitation. Sporulation was found at all growth rates tested, and the incidence of spores increased with decrease in growth rate of the culture. Within the range of growth rates up to the maximum obtainable with the defined medium, no threshold effect of growth rate on sporulation was observed. By studying transient states, it was possible to determine the time taken for the appearance of a refractile spore after initiation of a cell to sporulation. Under conditions of glucose limitation, cells were found to be committed to sporulation as soon as they were initiated. In nitrogen-limited cultures, however, a partial relief of nitrogen limitation prevented the development of spores during the first hour after initiation. The results of experiments with multistep changes in dilution rate of a chemostat culture indicate that initiation to sporulation is probably restricted to a particular point in the cell division cycle.     

Effects of virulence,sporulation, and temperature on Metarhizium anisopliae and Beauveria bassiana laboratory transmission in Coptotermes formosanus

Sporulation characteristics and virulence of Metarhizium anisopliae and Beauveria bassiana were examined in relation to laboratory transmission in Coptotermes formosanus. Fungal isolates significantly affected disease prevalence in termite populations. Sporulation of M. anisopliae played a more important role than virulence in producing epizootics within small groups of termites, but this was not the case for B. bassiana. Isolates characterized by quick sporulation (day 2 after death) did not exhibit better transmission in termites than those with high total sporulation (day 11 after death) in either fungal species. An isolate of M. anisopliae ranking highly in all three categories (virulence, quick sporulation, and total sporulation) produced better epizootics than an isolate that was inferior in all three characteristics. High temperatures (35 degrees C) significantly reduced fungal germination rates, leading to significant reduction of epizootics. M. anisopliae was better than B. bassiana in producing epizootics at 27 degrees C. Thus, fungal characteristics other than virulence should be considered for the seasonal colonization approach to termite microbial control.     

SPORULATION OF THE YEAST, HANSENULA SATURNUS: I. EFFECT OF MAGNESIUM AND CALCIUM IONS

Sporulation in a strain of the wild yeast, Hansenula saturnus,was investigated. The yeast was found to form spores even indistilled water. The sporulation rate (percentage of ascus-bearingindividuals) in this case was found to be markedly affectedby the cell concentration adopted in the test. The addition of inorganic nutrients to the sporulation mediumstimulates sporulation. The yeast requires either magnesiumor calcium for growth and sporulation. Higher concentrationsof these ions are required for sporulation than for growth.In both cases magnesium is effective at more dilute concentrationsthan calcium. Under the conditions of the experiments, in which the yeastforms a pellicle, the sporulation rate in the pellicle far exceedsthat in the sediment. The effects of environmental factors on the sporulation wasconsidered in relation to growth. It was found that, under theconditions of poor growth in the sporulation culture, no exogenousmagnesium and calcium are required for sporulation. In suchcases, the yeast cells are inferred to have an endogenous stockof magnesium and calcium enough for the sporulation. ¹ Present address: Laboratory of Microbiology, Department ofAgriculture, Tôhoku University, Sendai. (Received May 4, 1961; )     

The development of sporulation competence in Physarum polycephalum.

Plasmodial cells of the slime mold Physarum polycephalum become “competent” for sporulation following a prolonged period of dark starvation in the presence of nicotinamide. Sporulation can then be induced by illumination. Plasmodia are found to release into the medium during starvation one or more cellular products that promote sporulation. These products exert their effect specifically during the dark starvation period, rather than during the final phase of fruiting body construction. The sporulation control factor(s) (SCF) is nondialyzable and can stimulate the development of sporulation competence in the absence of nicotinamide.     

Growth, sporulation, and enzyme defects of glucosamine mutants of Bacillus subtilis

Two glucosamine (GCA)-requiring mutants have been isolated which grow on glucose minimal or nutrient sporulation medium only in the presence of either GCA or acetyl-GCA. They lack the l-glutamine-d-fructose-6-phosphate aminotransferase (EC 2.6.1.13), which is repressible by GCA and whose activity in the standard strain decreases after cessation of growth. But the mutants can grow on GCA as sole carbon and ammonia source, because GCA induces the synthesis of 2-amino-2-deoxy-d-glucose-6-phosphate ketol-isomerase (deaminating) (EC 5.3.1.10). With respect to sporulation, the GCA-requiring mutants are in a serious dilemma, as GCA represses the onset of massive sporulation and yet a small amount of GCA-6-phosphate derivatives is necessary to allow sporulation. When GCA is continuously provided in small quantities, sporelike particles are produced which contain little or no spore cortex but a normal spore coat. Apparently, GCA derivatives are needed especially for cortex formation. Many of the sporelike particles can produce colonies after octanol, but not after heat treatment. When they are purified by treatment with lysozyme and sodium dodecylsulfate, they do not show the decrease in optical density at 600 nm typical of germination nor do they produce offspring.     

Are Mitotic Functions Required in Meiosis?

Sporulation of diploid yeasts (Saccharomyces cerevisiae), homozygous or heterozygous for temperature-sensitive mitotic cell-cycle mutations, was examined at the restrictive and permissive temperatures. Twenty genes, represented by 32 heterozygotes and 60 homozygotes, were divided into three groups, showing (i) normal sporulation, (ii) no sporulation at the restrictive temperature but normal sporulation at the permissive temperature, (iii) no sporulation at both temperatures. Group (i) as well as several other strains were tested for their meiotic behavior with regard to intragenic recombination and haploidization. The conclusion reached was that all the mitotic nuclear-division and DNA-synthesis functions were required in meiosis. The only cell-division mutations not to affect meiosis were in three cytokinesis loci and in one budemergence locus.     

Extracellular manganese-stimulated deoxyribonuclease as a marker event in sporulation of Bacillus subtilis.

A considerable amount of Mn2+-stimulated DNAase (deoxyribonuclease) activity is released by Bacillus subtilis 168 during sporulation in a glucose-deficient medium; much smaller amounts are released during starvation for phosphate or nitrogen. Protein synthesis is required. Two forms of evidence are presented that production of the DNAase is associated with events late in stage II of sporulation. 19 Thymidine starvation, which inhibits the biochemical events associated with sporulation, also inhibits release of the DNAase. 2. Several asporogenous mutants blocked at stage II or earlier and unable to produce alkaline phosphatase (a stage-II event) do not produce the enzyme. Mutants blocked towards the end of stage II or later produce both enzymes. During sporulation of the wild-type strain, the DNAase appears about 1 h after alkaline phosphatase. The results suggest that production of the DNAase is controlled by a still-undiscovered stage-II genetic locus.