Localization of 3-phosphoglyceric acid synthesis in the mother cell compartments and forespores ofBacillus megaterium and the effects of manganous ions on its metabolism

Rapidly metabolizable compounds such as glucose or glycerol were not utilized byBacillus megaterium in the absence of manganese when grown in the supplemented nutrient broth medium. Under these conditions, growth ceased at low cell titre, 3-phosphoglyceric acid accumulated inside the cells and normal sporulation process was arrested. Addition of manganese to the medium caused disappearance of 3-phosphoglyceric acid, growth resumed and normal sporulation was observed. Synthesis of 3-phosphoglyceric acid occurred only in the mother cell compartments and it was transported for accumulation inside the forespores ofBacillus megaterium when grown in supplemented nutrient broth medium. Incubation of forespores in the presence of glucose or glycerol had no effect on 3-phosphoglyceric acid synthesis/accumulation, but it was completely utilized when forespores were incubated with manganese plus ionophore (X 537A). No other metal(s) could substitute for manganese suggesting that manganese plays crucial role in 3-phosphoglyceric acid metabolism     

Role of the Bacillus subtilis gsiA gene in regulation of early sporulation gene expression.

The Bacillus subtilis gsiA operon was induced rapidly, but transiently, as cells entered the stationary phase in nutrient broth medium. A mutation at the gsiC locus caused sporulation to be defective and expression of gsiA to be elevated and prolonged. The sporulation defect in this strain was apparently due to persistent expression of gsiA, since a gsiA null mutation restored sporulation to wild-type levels. Detailed mapping experiments revealed that the gsiC82 mutation lies within the kinA gene, which encodes the histidine protein kinase member of a two-component regulatory system. Since mutations in this gene caused a substantial blockage in expression of spoIIA, spoIIG, and spoIID genes, it seems that accumulation of a product of the gsiA operon interferes with sporulation by blocking the completion of stage II. It apparently does so by inhibiting or counteracting the activity of KinA.     

Regulation of lysine and dipicolinic acid biosynthesis in Bacillus brevis ATCC 10068: significance of derepression of the enzymes during the change from vegetative growth to sporulation

Lysine biosynthetic pathway enzymes of Bacillus brevis ATCC 1068 were studied as a function of stage of development (growth and sporulation). The synthesis of aspartic-2-eemialdehyde dehydrogenase (ASA-dehydrogenase), dihydrodipicolinate synthase (DHDPA-synthase), DHPA-reductase and diaminopimelate decarboxylase (DAP-decarboxylase) was found not to be co-regulated, since lysine was not a co-repressor for these enzymes. Unlike the aspartokinase isoenzymes, the other enzymes of the lysine pathway were not derepressed in thiosine-resistant, lysine-excreting mutants. Thus, the aspartokinase isoenzymes were the key enzymes during growth and regulation of lysine biosynthesis through restriction of l-ASA synthesis via feedback control by lysine on the aspartokinases was therefore suggested.In contrast to other Bacillus species, the levels of the lysine biosynthetic pathway enzymes of strain ATCC 10068 were not derepressed during the change from vegetative growth to sporulation. Two control mechanisms, enabling the observed preferential channelling of carbon for the synthesis of spore-specific diaminopimelic acid (DAP) and dipicolinic acid (DPA) were a) loss of DAP-decarboxylase, b) inhibition of DHDPA-reductase by DPA. Increase in the level of the DAP pool during sporulation, as a consequence of the loss of DAP-decarboxylase, and its relevance to the non-enzymatic formation of DPA has been discussed.Abbreviations l-ASA l-aspartic-2-semialdehyde - DAP diaminopimelic acid - DPA dipicolinic acid - DHDPA dihydrodipicolinate - AGM aspargine-glycerol medium - PY peptone-yeast extract - NB+NSM nutrient broth plus nutrient sporulation medium     

Requirement for Acetate and Glycine (or Serine) for Sporulation Without Growth of Bacillus subtilis

Cells of Bacillus subtilis sporulate when they are transferred, at any time of growth in nutrient sporulation medium, to a potassium-phosphate buffer containing slowly utilizable carbon sources such as l-aspartate, citrate, l-glutamate, or lactate. Transfer to buffer containing more rapidly utilizable carbon sources such as malate or glucose leads to sporulation only when the cells either had reached the end of growth or when the transfer medium also contains glycine. Acetate, which as a sole carbon source does not allow growth, also does not alone permit sporulation; however, the presence of both acetate (0.05 m) and glycine or l-serine (0.01 m) in the buffer medium allows sporulation if the cells are transferred to this medium after they have grown in the nutrient sporulation medium beyond the end of the exponential growth phase (T(0)). The development, required before transfer, does not seem to involve the end of a round of deoxyribonucleic acid duplication, as experiments with tryptophan-starved cells have indicated. Glycine or serine cannot be replaced by any of the known metabolites, which are partially derived from them. Amino acid analysis of nutrient sporulation medium showed that glycine (but not serine) is present at a concentration of 0.3 mm at the beginning of the developmental period, thus allowing, in combination with an acetyl-coenzyme A (CoA) precursor, sporulation but not growth. Acetyl-CoA is required not only for adenosine-triphosphate synthesis but also for some other reactions.     

Activation of intracellular serine proteinase in Bacillus subtilis cells during sporulation.

Cells of Bacillus subtilis 168 (trpC2) growing and sporulating in a single chemically defined medium carried out intracellular protein degradation and increased their levels of intracellular serine protease-1 in a manner very similar to what had previously been reported for cells sporulating in nutrient broth. The results were interpreted to mean that these processes are intrinsic to sporulation rather than medium dependent. To determine the cause of these increases in specific activity of proteinases, we purified the protease, prepared rabbit immunoglobulins directed against it, and monitored changes in protease antigen levels by performing rocket immunoelectrophoresis. In cells sporulating in nutrient broth, the protease antigen levels increased about 7-fold, whereas the specific activity increased about 150-fold, for an activation of about 20-fold. In cells sporulating in the single chemically defined sporulation medium, the protease antigen increased about 10-fold, whereas the specific activity increased at least 400-fold, for an activation of about 40-fold. These results were interpreted to mean that a posttranslational event activated the protease in vivo; a previously described endogenous proteinase inhibitor was confirmed to be present in the strain used. Chloramphenicol added to the cultures inhibited both the increases in antigen levels and in the specific activity of the proteinase.     

Further studies of swarmer cell differentiation of Proteus mirabilis PM23: a requirement for iron and zinc

Proteus mirabilis PM23, unlike other motile strains of the species, differentiates in rich fluid media to form nonseptate filaments resembling the swarmer cells formed on solid media. The swarming activity of PM23 is greater than that of the other strains on solid media and it grows faster than another strain, IM47, in differentiation-supporting broth. This faster growth is not exhibited in broth that does not support differentiation. The differentiation of PM23 in brain-heart infusion broth occurs over a wide range of pH and temperature. Inhibitors of swarming on agar plates (p-nitrophenylglycerol and boric acid) and three chelating agents (EDTA, sodium cyanide, and sodium diethyldithiocarbamate) stop differentiation both on plates and in brain-heart infusion broth; however, EGTA is not effective even at 10 mM (10 times the minimum inhibitory concentration of EDTA). The inhibitory mechanisms of p-nitrophenylglycerol and boric acid are different from that of the chelating agents. The timing of EDTA inhibition suggests generation of a "signal" to differentiate after about 2 h growth. Prevention of differentiation by addition of Fe2+ and Zn2+ up to near the time that differentiation should appear suggests that these cations have a crucial involvement in the process of initiation. However, they are not effective as additives in allowing differentiation to occur in defined media or even nutrient broth; the further addition of nucleotides or cAMP was equally ineffective.     

On the formation of crystal proteins during sporulation in Bacillus thuringiensis var. thuringiensis

The sporulation potential of Bacillus subtilis as a function of position in the cell cycle was determined by transferring cells from growth medium to sporulation medium at various times during growth. Growth was induced by incubating heat-activated spores in rich medium or by diluting stationary phase vegetative cultures with fresh growth medium. The results supported earlier observations that sporulation potential is cell cycle dependent. The rise in sporulation potential was studied by exposing cultures to the inhibitors of cell wall and protein synthesis, vancomycin and chloramphenicol. The delay in the appearance of the peak of sporulation potential caused by these inhibitors compared with the reported lack of effect of nalidixic acid, indicates that the appearance of sporulation potential requires synthesis of a macromolecular component other than deoxyribonucleic acid. The effect of nalidixic acid in preventing the decline of the sporulation potential was compared with the effect of high temperature on a mutant temperature sensitive for the initiation of DNA replication. It was found that prevention of chromosome completion with nalidixic acid maintained a high sporulation potential, whereas prevention of chromosome re-initiation in the temperature sensitive mutant did not affect the decline in sporulation potential as the cells enter stationary phase.Abbreviations NAL Nalidixic acid - HPUra 6-(p-hydroxyphenylazo)-uracil - VAN Vancomycin - CAM Chloramphenicol - BHI Brain heart infusion broth - c.f.u. Colony forming units     

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.     

Single, chemically defined sporulation medium for Bacillus subtilis: growth, sporulation, and extracellular protease production.

The composition and application of a single, chemically defined medium or growth and sporulation of Bacillus subtilis is described. At 37 degrees C cells grew with a doubling time of about 40 min; cultures attained near-maximal spore formation (70 to 80% by 12 h after the end of exponential growth and produced 1 X 10(9) to 2 X 10(9) heat-resistant free spores at 24 h. Dipicolinic acid production was completed between 7 and 11 h. Cells grown in the single, chemically defined medium excreted levels of serine and neutral proteases comparable to those excreted in nutrient broth medium.     

Phosphate limitation induces sporulation in the chytridiomycete Blastocladiella emersonii

The cell cycle is controlled by numerous mechanisms that ensure correct cell division. If growth is not possible, cells may eventually promote autophagy, differentiation, or apoptosis. Microorganisms interrupt their growth and differentiate under general nutrient limitation. We analyzed the effects of phosphate limitation on growth and sporulation in the chytridiomycete Blastocladiella emersonii using kinetic data, phase-contrast, and laser confocal microscopy. Under phosphate limitation, zoospores germinated and subsequently formed 2-4 spores, regardless of the nutritional content of the medium. The removal of phosphate at any time during growth induced sporulation of vegetative cells. If phosphate was later added to the same cultures, growth was restored if the cells were not yet committed to sporulation. The cycles of addition and withdrawal of phosphate from growth medium resulted in cycles of germination-growth, germination-sporulation, or germination-growth-sporulation. These results show that phosphate limitation is sufficient to interrupt cell growth and to induce complete sporulation in B.?emersonii. We concluded that the determination of growth or sporulation in this microorganism is linked to phosphate availability when other nutrients are not limiting. This result provides a new tool for the dissection of nutrient-energy and signal pathways in cell growth and differentiation.     

Non-variable sources of pure water and the germination and outgrowth of Bacillus subtilis spores

Variable germination and outgrowth occurred when Bacillus subtilis NCTC 8236 spores were inoculated into nutrient broth prepared with distilled water. More reproducible findings were achieved when the medium was prepared with Elgastat water and the greatest reproducibility occurred with Elgastat water as vehicle combined with a rigorous acid-washing of all glassware. This combined procedure also produced optimum and reproducible results for the synchronous growth of two B. subtilis 168 strains in casein medium supplemented with appropriate amino acids, a technique of value in monitoring the development of resistance to antibacterial agents during sporulation. The levels of aluminium in distilled water were higher than those of other elements; however, the incorporation of aluminium sulphate into broth prepared with Elgastat water had no effect on germination, and outgrowth was reduced (but not eliminated) only at high concentrations of this salt.     

Expression analysis of the spi gene in the pock-forming plasmid pSA1.1 from Streptomyces azureus and localization of its product during differentiation

The sporulation inhibitory gene spi in the pock-forming conjugative plasmid pSA1.1 of Streptomyces azureus was introduced into cells via a high or low copy number vector to examine the effect of gene dosage on the growth of Streptomyces lividans TK24 as a host. In transformants carrying a high spi copy number, nutrient mycelial growth was inhibited, as was morphological differentiation from substrate mycelium to aerial mycelium on solid media. The degree of inhibition depended on the spi gene dosage, but the presence of pSA1.1 imp genes, which encode negative repressor proteins for spi, relieved the inhibition. Confocal images of Spi tagged with enhanced green fluorescent protein in cells on solid media revealed that spi expression was initiated at the time of elongation of substrate mycelium, that its expression increased dramatically at septation in aerial hyphae, and that the expression was maximal during prespore formation. Expression of spi covered the whole of the hyphae, and the level of expression at the tip of the hyphae during prespore formation was about sixfold greater than during substrate mycelial growth and threefold greater than during aerial mycelial growth. Thus, localized expression of spi at particular times may inhibit sporulation until triggering imp expression to repress its inhibitory effects.     

Chromosome replication in Caulobacter crescentus growing in a nutrient broth.

The pattern of chromosome replication in the Caulobacter crescentus cell cycle was studied by examining the rate of deoxyribonucleic acid (DNA) synthesis during synchronous growth in a fast-growth nutrient broth. As reported previously for the cell cycle in a slow-growth minimal medium (Degnen and Newton, 1972), the Caulobacter cell cycle (at the fastest available growth rate) in nutrient broth consisted of three distinct periods in terms of DNA synthetic activity. The swarmer-cell cycle consisted of a presynthetic period (G1), synthetic period (S), and postsynthetic period (G2) of 30, 50, and 35 min, respectively, whereas the stalked-cell cycle consisted of S and G2 periods of 50 and 35 min, respectively. Synchronously growing cells in the nutrient broth were stained to visualize nuclear bodies. Two nuclear bodies could be discerned in both swarmer and stalked cells, and four could be discerned in predivisional cells. DNA content per cell was determined chemically and found to be about the same in swarmer and stalked cells; it was equivalent to roughly twice the value expected from the kinetic complexity reported previously (Wood et al., 1976) for Caulobacter DNA.     

Irradiation of Escherichia coli in the Visible Spectrum with a Tunable Organic-Dye Laser Energy Source

Pulsed laser energy was shown to be effective in inhibiting the growth of Escherichia coli. The irradiation source was derived from a tunable organic-dye laser utilizing rhodamine 6G (590 ± 5 nm) solutions as lasing media. The organisms, suspended in nutrient broth, were irradiated both with and without an exogenous photosensitizer. One photosensitizer (toluidine blue) did not appreciably alter the inhibitory effect observed. In the presence of acridine orange, however, some additional growth occurred.     

Construction and properties of an intracellular serine protease mutant of Bacillus subtilis.

An intracellular serine protease (ISP-1) mutant of Bacillus subtilis was created by introducing a frameshift into the coding region of the cloned gene. Intracellular protease activity in the mutant was very low, yet sporulation in both nutrient broth and minimal medium was normal. The rate of bulk protein turnover in the mutant was slightly slower than that in the wild-type strain. These results suggest that the gene for ISP-1 is not essential and that ISP-1 is not the major enzyme involved in protein turnover during sporulation.     

Production of a Sporulation Pigment by Streptomyces venezuelae

Streptomyces venezuelae S13 produced a pH-indicating sporulation pigment on a glucose-salts-agar medium consisting of glucose, KNO(3), MgSO(4), and Na(2)HPO(4), pH 7. Pigmentation on this medium appeared to be closely associated with sporulation, which normally required 5 to 7 days at 30 C. The pigment was soluble in water as well as in a number of organic solvents. Butanol-extracted pigment exhibited absorption maxima at 430 and 520 nm at pH 3 and 12, respectively. Although many salts of organic acids and amino acids could replace glucose as the sole carbon source in basal salts-agar medium for growth and pigmentation, most sugars that were tested supported good growth but negligible pigmentation. Among the nitrogenous substances tested, KNO(3) was most desirable for pigmentation. The organism did not exhibit any specific requirements for divalent cations with respect to growth and pigmentation. In the absence of MgSO(4), however, glucose-salts-agar prepared by autoclaving all components together failed to support growth. The production of the sporulation pigment on glucose-salts-agar was comparable to that obtained on tomato paste-oatmeal-agar medium. Incorporation of partially purified pigment material into broth medium that did not normally support sporulation induced sporulation, and amino acid-salts-agar medium could induce vegetative mycelia to pigment when transferred from medium that did not support either pigmentation or sporulation.     

Evaluation of methods to solubilize and analyze cell-associated ectoenzymes

A protocol for production, storage, and use of Shock 1 (Shk1) bioreporter cells for toxicity monitoring in wastewater treatment facilities was developed. Shk1 is a bioluminescent toxicity bioreporter for activated sludge previously constructed by the incorporation of lux genes into an activated sludge microorganism.

A number of factors affecting Shk1 growth and bioluminescence were examined including the growth medium, tetracycline concentration, storage conditions, and test media. Based on the results of these experiments, a toxicity testing protocol was developed that involved growth of cultures in nutrient broth with tetracycline, storage of cultures at 4 °C, cell activation by reinoculation into nutrient broth, and toxicity testing by cell injection into the test media. Effective use of this approach required standardized time intervals for cell growth, storage, activation and exposure in the test media.

Bioluminescence from Shk1 cells was measured in nutrient broth and influent wastewater and activated sludge mixed liquor from a municipal wastewater treatment plant. Using the Shk1 toxicity testing protocol, Zn EC₅₀ values for bioluminescence in nutrient broth, influent wastewater, and activated sludge mixed liquor were approximately 42, 7, and 32 mg/l, respectively. Zn concentrations as low as 1 mg/l could be detected in influent wastewater. The detection limit in influent wastewater is below the Zn concentrations typically reported to affect the activated sludge process.     

Effect of Nitrofurans and Chlortetracycline on Microorganisms Associated with Shrimp

Nitrofuran AF-2 displayed greater inhibitory effect than did nitrofuran Z when a mixed bacterial culture, including several proteolytic bacteria, isolated from shrimp was subjected to these compounds in vitro. Nitrofuran Z exhibited greater bactericidal properties than did chlortetracycline in all cultures used. Only 10 mug of nitrofuran AF-2 per ml was sufficient to inhibit the growth of mixed bacteria in nutrient broth, whereas 50 mug of nitrofuran Z per ml was necessary to accomplish the same inhibition. A 50-mug amount of chlortetracycline per ml displayed about the same inhibitory effect as either 10 mug of AF-2 per ml or 20 mug of Z per ml. The isolated proteolytic bacteria showed greater suppression of growth when subjected to AF-2 than when subjected to Z; however, both nitrofurans were effective in preventing growth. The addition of either 1 mug of AF-2 per ml or 5 mug of Z per ml to nutrient broth inhibited the growth of Achromobacter aquarmarinus, whereas chlortetracycline was less effective, requiring about 20 mug to suppress growth to the same degree.