Induction of microcycle sporulation in Bacillus brevis spp. AG4 by amethopterin

Addition of amethopterin to medium before inoculation inhibited DNA synthesis and induced microcycle sporogenesis in Bacillus brevis spp. AG4. Synthesis of RNA and proteins occurred at a considerably reduced rate.Abbreviations TVC total viable counts - HSC heat stable counts - CDGS chemically defined medium for growth and sporulation - TCA trichloro acetic acid     

Repression of sporulation in Bacillus subtilis by L-malate.

L-Malate repressed sporulation in the wild-type strain of Bacillus subtilis. When 75 mM L-malate was added to the growth medium at the time of inoculation, the appearance of heat-resistant spores was delayed 6 to 8 h. The synthesis of extracellular serine protease, alkaline phosphatase, glucose dehydrogenase, and dipicolinic acid was similarly delayed. Sporulation was not repressed when malate was added to the culture at t4 or later. A mutant was selected for ability to sporulate in the presence of malate. This strain could also sporulate in the presence of glucose. The malate-resistant mutant grew poorly with malate as sole carbon source, although it possessed an intact citric acid cycle, and it showed increased levels of malic enzyme. This indicates a defect in the metabolism of malate in the mutant. A mutant lacking malate dehydrogenase activity was also able to sporulate in the presence of malate. A model for the regulation of sporulation by malate is presented and discussed. Citric acid cycle intermediates other than malate did not affect sporulation. In contrast to previous results, sporulation of certain citric acid cycle mutants could be greatly increased or completely restored by the addition of intermediates after the enzymatic block. The results indicate that the failure of citric acid cycle mutants to sporulate can be adequately explained by lack of energy and lack of glutamate.     

Inhibition of Bacillus subtilis growth and sporulation by threonine.

A 1-mg/ml amount of threonine (8.4 mM) inhibited growth and sporulation of Bacillus subtilis 168. Inhibition of sporulation was efficiently reversed by valine and less efficiently by pyruvate, arginine, glutamine, and isoleucine. Inhibition of vegetative growth was reversed by asparate and glutamate as well as by valine, arginine, or glutamine. Cells in minimal growth medium were inhibited only transiently by very high concentrations of threonine, whereas inhibition of sporulation was permanent. Addition of threonine prevented the normal increase in alkaline phosphatase and reduced the production of extracellular protease by about 50%, suggesting that threonine blocked the sporulation process relatively early. 2-Ketobutyrate was able to mimic the effect of threonine on sporulation. Sporulation in a strain selected for resistance to azaleucine was partially resistant. Seventy-five percent of the mutants selected for the ability to grow vegetatively in the presence of high threonine concentrations were found to be simultaneously isoleucine auxotrophs. In at least one of these mutants, the threonine resistance phenotpye could not be dissociated from the isoleucine requirement by transformation. This mutation was closely linked to a known ilvA mutation (recombination index, 0.16). This strain also had reduced intracellular threonine deaminase activity. These results suggest that threonine inhibits B. subtilis by causing valine starvation.     

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.     

Decadent sporulation mutants of Bacillus subtilis.

In decadent sporulation mutants, sporulating populations are heterogeneous: the cells reach successive chemical and physical resistances with progressively decreasing frequencies. Each decadent mutant can be characterized by the shape and slope of the curve describing the frequency of cells resistant to various agents ('the resistance spectrum'). In some mutants the resistance spectrum decreases progressively from xylene resistance to heat resistance; in other mutants it decreases rapidly between octanol resistance and chloroform resistance. Electron microscopy showed that in two mutants the majority of the cells are blocked at stages III and IV; the number of cells that develop further to reach successive morphological stages falls off progressively. In two other mutants most cells reach stage V. Cortexless spores are also frequent. One of the decadent mutations, SpoL1, was localized between aroD and acf. The phenotype of decadent mutants is discussed in terms of sequential gene activation.     

Characterization of the Bacillus subtilis sporulation gene spoVK.

The sporulation gene spoVK of Bacillus subtilis was cloned by use of the insertional mutation spoVK::Tn917 omega HU8. The spoVK gene was shown to be the site of an incorrectly mapped mutation called spoVJ517. Thus, a separate spoVJ gene as defined by the 517 mutation does not exist and is instead identical with spoVK.     

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.     

Selective inhibition of Bacillus subtilis sporulation by acridine orange and promethazine.

Two structurally similar compounds were found to inhibit sporulation in Bacillus subtilis 168. A dye, acridine orange, and an antischizophrenic drug, promethazine, blocked spore formation at concentrations subinhibitory to vegetative growth, while allowing synthesis of serine protease, antibiotic, and certain catabolite-repressed enzymes. The sporulation process was sensitive to promethazine through T2, whereas acridine orange was inhibitory until T4. The drug-treated cells were able to support the replication of phages phie and phi29, although the lytic cycles were altered slightly. The selective inhibition of sporulation by these compounds may be related to the affinity of some sporulation-specific genes to intercalating compounds.     

Antibiotic-resistant mutants of Bacillus subtilis conditional for sporulation.

Among spontaneously occurring antibiotic-resistant mutants of Bacillus subtilis 168 we have identified a sub-class that is conditionally sporulative. Mutants in this sub-class are resistant to antibiotic during vegetative growth but are sensitive during sporulation. Mutants conditionally-resistant to erythromycin, kanamycin, spectinomycin, and streptomycin have been isolated and characterized by phase contrast microscopy and with respect to their ability to synthesize heat-resistant endospores or the sporulation-associated enzyme alkaline phosphatase. The results suggest that several entirely different genetic lesions may result in this single phenotype. This group includes mutants whose properties suggest that both th 30S and 50S ribosomal subunits may be altered concomitant with early spore specific metabolism. The blockage imposed by antibiotic may be at or near Stage 2 of sporulation.     

Cell cycle and sporulation in Bacillus subtilis

Recent work on cell division and chromosome orientation and partitioning in Bacillus subtilis has provided insights into cell cycle regulation during growth and development. The cell cycle is an integral part of development and entrance into sporulation is modulated by signals that transmit the status of DNA integrity, chromosome replication and segregation. In addition, B. subtilis modifies cell division and DNA segregation to establish cell-type-specific gene expression during sporulation.     

Cloning of sporulation gene spoIIG in Bacillus subtilis.

Two specialized transducing phages carrying a sporulation gene, spoIIG , of Bacillus subtilis were constructed from B. subtilis temperate phages p11 and phi 105 by the "prophage transformation" method. Restriction enzyme analysis and transformation experiments showed that the spoIIG gene was present on a 6.2 X 10(6)-dalton (6.2-Md) EcoRI fragment in both transducing phage genomes. Further analysis showed that spoIIG + transforming activity resides on a 2.25-Md EcoRI-BamHI fragment within the 6.2-Md EcoRI fragment. The 2.25-Md fragment was subcloned into the region between the EcoRI and BamHI sites of pUB110, and deletion plasmids lacking PstI or HindIII fragments within the 2.25-Md fragment were constructed. The recombinant plasmid carrying the intact spoIIG gene restored sporulation of strain HU1002 ( spoIIG41 recE4 ) to a frequency of 10(4) spores per ml and inhibited sporulation of strain 4309 ( spo + recE4 ) to a level of 10(3) spores per ml.     

Identification of proteins phosphorylated by ATP during sporulation of Bacillus subtilis.

Protein phosphorylation in Bacillus subtilis was assayed in vitro by using extracts prepared from cells at various times during growth and sporulation. At least six proteins were labeled in vitro by using [gamma-32P]ATP and extracts of vegetative cells. In extracts prepared at the end of exponential growth and during stationary phase, 12 to 13 proteins were labeled. Seven of the phosphoproteins were purified by fast-performance liquid chromatography and polyacrylamide gel electrophoresis, blotted to Immobilon membranes, and subjected to partial protein sequencing. One of the sequences had sequence homology (greater than 45%) to elongation factor G from several bacterial species, and four sequences matched the predicted amino-terminal sequences of the outB, orfY-tsr, orfU, and ptsH genes.     

Septation and chromosome segregation during sporulation in Bacillus subtilis.

The early stages of sporulation in Bacillus subtilis incorporate a modified, highly asymmetric cell division. It is now clear that most, if not all, of the components of the vegetative division machinery are used also for asymmetric division. However, the machinery for chromosome segregation may differ significantly between vegetative growth and sporulation. Several interesting checkpoint mechanisms couple cell cycle events to gene expression early in sporulation. This review summarises important advances in the understanding of chromosome segregation and cell division at the onset of sporulation in B.subtilis in the past three years.     

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.