Asporogenous Mutants Research Articles

Ultrastructure and characterization of an asporogenic mutant of Clostridium botulinum type E.

The asporogenic mutant, RSpoIIIa, showed septum formation and a nearly completed forespore about 4 h after onset of sporulation. The cells show defects at a few sites of the forespore membrane, an absence of 'germ cell wall,' and within 8 h lysis of the cytoplasm occurred indicating that the mutant was blocked at stage III. Some aberrant envelopes were seen later. Lysis of the asporogenic mutant was inhibited for up to 36 h by the addition of 2.4% glucose or sucrose to the medium and 80% of the cells showed septum formation. A comparison of the phenotypic characteristics of the asporogenic RSpoIIIa and the sporogenic MSp+ mutants, as well as the wild type, showed the same ultrastructural changes during the development of the forespore with the accumulation of intracellular iodophilic granules. In addition, the mutants showed specific immunofluorescence and precipitin lines of identity with antisera against the wild-type strain, but unlike the toxigenic wild type, the mutants were nontoxigenic by mouse pathogenicity tests.

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.

Effect of A‐factor on the growth of asporogenous mutants of Streptomyces griseus, not producing this factor

AbstractA‐factor C13H22O4 was shown to be not only the inductor of the biosynthesis of streptomycin in inactive mutants of Streptomyces griseus (Actinomyces streptomycini), but also the potent regulator of differentiation of the cells: it induces the formation of spores in asporogenous strains during their superficial and submerged growth. This is accompanied by morphological modifications in the course of growth of the colonies.

Isolation of Two Acetyl Esterases from Extracts of Bacillus subtilis

Acrylamide gel electrophoresis of crude cellular extracts of Bacillus subtilis revealed the presence of two acetyl esterases. Esterase A, the slower migrating enzyme, was found to be present in both vegetative and sporulating cells, whereas esterase B activity was more abundant after exponential growth ceased. Both esterases were present in the supernatant fraction of lysed spheroplasts and in a disrupted spore preparation. Of four pleiotropic asporogenous mutants tested, three exhibited decreased esterase B activity. Esterases A and B were partially purified by differential precipitation and co-chromatographed on diethylaminoethyl (DEAE)-cellulose (pH 7.5) and DEAE-Sephadex (pH 8.5). By employing gel filtration chromatography, the two esterases were separated, and molecular weights of 160,000 and 51,000 were estimated for esterases A and B, respectively. Esterase A was further purified to electrophoretic homogeneity by differential heating and preparative starch block electrophoresis. Sodium dodecyl sulfate-acrylamide gel electrophoresis of purified esterase A yielded a single protein band with a molecular weight of 31,000. The pI values of esterases A and B were determined to be 6.4 and 5.4, respectively.

Effect of A-factor on the growth of asporogenous mutants of Streptomyces griseus, not producing this factor.

A-factor C13H22O4 was shown to be not only the inductor of the biosynthesis of streptomycin in inactive mutants of Streptomyces griseus (Actinomyces streptomycini), but also the potent regulator of differentiation of the cells: it induces the formation of spores in asporogenous strains during their superficial and submerged growth. This is accompanied by morphological modifications in the course of growth of the colonies.

Template specificity of ribonucleic acid polymerase in asporogenous mutants of Bacillus subtilis.

Asporogenous mutants of Bacillus subtilis were examined for the change in template specificity of ribonucleic acid (RNA) polymerase characteristic of wild-type cells undergoing sporulation. Mutants blocked at stages II, III, and IV showed a changed specificity of the enzyme after the end of growth and were in this respect indistinguishable from the wild type. The RNA polymerase of eight stage-zero mutants (out of nine tested) which possess mutations that map at six distinct loci retained the template specificity of vegetative cells.

Sporulation in Bacillus subtilis. Properties and time of synthesis of alkali-soluble protein of the spore coat.

An alkali-extractable protein fraction comprising 6% of the dry weight of the spore can be removed from spores of Bacillus subtilis 168. Three different extraction procedures each yield at least one similar protein. Extracted protein behaved as a single species on ion-exchange chromatography or gel filtration, but two polypeptides were found on electrophoresis. Comparison of molecular weights on electrophoresis and by sucrosegradient analysis suggests that the protein(s) undergo aggregation into multimers. Extracted spores remain viable, but are altered in density and lysozyme sensitivity and they aggregate together. Electron microscopy of extracted spores shows that loss of material seems to occur from the outer coat layers. Extraction therefore probably removes a specific fraction of the spore-coat protein, but without impairment to the spore protoplast. This protein can first be detected immunologically 4h after the initiation of sporulation, and the synthesis of this protein is sensitive to chloramphenicol, actinomycin D and rifamycin. Labelling experiments also show that the protein begins to be synthesized early in sporulation. Examination of the ability of asporogenous mutants to produce cross-reacting material indicates that some event in stage II of sporulation determines its production.

Sporulation in Bacillus subtilis. Characterization of oligosporogenous mutants and comparison of their phenotypes with those of asporogenous mutants.

SUMMARY: An investigation was undertaken to determine to what extent the properties of oligosporogenous (Osp) mutants allow them to be considered as a separate class of sporulation mutant, distinct from asporogenous (Sp-) mutants. Of thirty Osp mutants examined, seventeen at least had a phenotype which had previously been identified with a Sp- mutant. The majority of cells in an Osp culture either reached a particular stage in the sporulation process and then stopped, or in some cases went on to produce aberrant forms. Some of these aberrant forms have their counterparts in Sp- mutants described by other authors, but some present new features. The morphological and biochemical sequences were linked so that if the majority of cells were blocked at a certain stage, then the biochemical sequence stopped accordingly. The general similarity in behaviour between the two types of mutant is consistent with the assumption that at least some of the Osp mutants have leaky mutations in genes where mutation can also give rise to Sp- phenotypes. Evidence is presented to suggest that the ability of a cell of an Osp mutant to overcome its block, and so go on to form a spore, is a chance event when that stage in the process is reached. A mutant has been obtained in which the spores are octanol-resistant yet contain no measurable dipicolinate. In several other mutants the spores contained well-developed coat layers, but the cortex was poorly formed or completely missing.

Acid-soluble nucleotides in an asporogenous mutant of Bacillus subtilis.

An asporogenous mutant of Bacillus subtilis Sp(-)H12-3, which is considered to have a block at stage 0, showed general growth characteristics similar to those of sporulating cultures. However, a sudden increase in the total amount of acid-soluble nucleotides observed at t(2) in sporulating bacteria was completely absent in this mutant. In sporulating cells, a marked increase in two nucleotides which were identified to be uridine diphosphate (UDP)-galactose and UDP-N-acetylglucosamine was noted, whereas UDP-glucose appeared to be accumulated in the mutant cells at t(2). No unusual nucleotides were found in the strains of B. subtilis examined. The possible role of these UDP derivatives in early stages of sporulation in B. subtilis is discussed.

Comparison of procedures for extracting transfer RNA from spores of Bacillus

Methods not involving physical disruption for extracting tRNA from spores of Bacillus using phenol, 8 m urea-mercaptoethanol, 8 m urea-thiogycolic acid, or dithiothreitol were compared. On the basis of these data, an improved extraction procedure is described which is applicable to spores, vegetative cells and asporogenous mutants of Bacillus subtilis and Bacillus megaterium.

Comparative studies of an asporogenic mutant and a wild type strain of Clostridium botulinum type E 1 .

An asporogenic mutant of Clostridium botulinum type E, ATCC 9564, was recovered on liver veal agar, which had been supplemented with MnSO4 (0.13 mM) and mercaptoacetate (0.2%). Growth in TPG measured by optical density increased exponentially for the two strains during the first 11 h. But subsequently, the optical density of the wild type rapidly declined while the sp− mutant showed a more gradual decrease. The pH decreased significantly during growth but remained unaltered at pH 5–6 during sporulation. The DPA synthesis in the sp+ strain paralleled the appearance of refractile spores, while the sp− strain showed no measurable amounts of DPA. Protease and antibiotic activities were detected late in the log growth phase of the wild type. Gas–solid chromatography showed that during growth and sporulation the mercaptoethanol and CO2 content declined significantly in the wild type, but remained unchanged in the sp− mutant, whereas N2O was evolved concurrently with sporulation. This study has provided us with an effective method for the isolation of asporogenic mutants of C. botulinum, as well as a basis for the analysis of the events which occur during the stages of sporulation.

A quantitative survey of conidiation mutants in Aspergillus nidulans.

SUMMARY: quantitative survey has been made of conidiation mutants in Aspergillus nidulans. Conidiation mutants were 6 to 13 times more numerous than auxotrophic mutants, but growth tests showed that the mutant defect in 85 % of the conidiation mutants was not confined to conidiation. The number of loci specifically involved in conidiation was estimated to be 45 to 150. Mutants were classified according to the stage of the developmental block and also into asporogenous and oligosporo- genous types; 64 % were oligosporogenous, but in further tests, 84 % of the asporogenous mutants were found to be temperature sensitive or slightly leaky, suggesting that many of the loci involved act only as modifiers in conidiation. The largest group of mutants was blocked before conidiophore formation, while many others failed at ill-defined stages of conidiation, suggesting that failure was due to the gradual build-up of metabolic deficiencies. A class of conidial maturation mutants has also been identified.

Sporulation in Bacillus subtilis. The appearance of sulpholactic acid as a marker event for sporulation.

1. The synthesis of sulpholactic acid in sporulating cultures of Bacillus subtilis was studied. 2. Sulpholactic acid was first detected about 4h after the initiation of sporulation and 1h before refractility. The rate of synthesis paralleled that of the other events of sporulation examined. 3. Sulpholactic acid accounted for 1.7% of the material of the spore. 4. Because the addition of chloramphenicol in the earlier stages of sporulation inhibited formation of the compound, it is likely that the enzymes concerned are synthesized de novo during sporulation. 5. In asporogenous mutants only those blocked at a late stage and showing partial refractility were able to produce sulpholactic acid. This correlation makes sulpholactic acid a useful marker event in sporulation.

Early blocked asporogenous mutants of Bacillus subtilis 168. I. Isolation and characterization of mutants resistant to antibiotic(s) produced by sporulating Bacillus subtilis 168.

A number of mutants (abs)-resistant to antibiotic(s) produced by sporulating Bacillus subtilis 168 have been isolated from an early blocked asporogenous mutant (spoA12). At least four classes were recognized according to their phenotypic properties. Genetic analysis has shown that these mutants were neither partial revertants nor suppressor mutants of the spoA gene. Both nonsense and missense mutants of the spoA gene are reverted partially by a secondary mutation which is resistant to antibiotic of B. subtilis 168. Another asporogenous mutant, spoB, whose locus is closely linked to pheA, is also affected by the same abs mutation. The nature of abs mutants is discussed.

Electron spin resonance evaluation of pigment accumulation during asexual sporulation in Aspergillus flavus

The association between pigment accumulation and a free radical electron spin resonance (ESR) signal produced during sporulation in Aspergillus flavus was investigated. In wild-type cultures, the onset of the ESR signal coincided with the onset of both pigmentation and sporulation. Mutant colonies, affected in their pigmentation and/or their ability to produce conidia, failed to give a free radical signal. However, pigmentation apparently was not essential for either sporulation or spore germination in A. flavus. Pigmentless conidia were obtained which germinated as well as wild-type conidia. There did appear, though, to be some form of association between pigment accumulation and sporulation, since all the asporogenous mutants which were isolated also were non-pigmented.

Sporulation in Bacillus subtilis. Correlation of biochemical events with morphological changes in asporogenous mutants.

A comparison was made of morphological changes and successive, mainly biochemical, marker events for sporulation in 14 asporogenous mutants. The morphological and biochemical sequences are linked so that arrested development in one is accompanied by corresponding effects in the other. Thus mutants that fail to produce both protease and antibiotic do not progress beyond stage 0, formation of alkaline phosphatase appears to be associated with the transition from stage II to stage III and glucose dehydrogenase with that from stage III to stage IV. Stage II mutants may produce ;pygmy' cells or other bizarre cell-division forms. The biochemical sequence is dependent in the sense that if the occurrence of any one event is blocked that of all the succeeding events is also blocked. This has implications for biochemical models that have been proposed to explain the temporal sequence observed in spore development.

The mapping of genes for spore formation on the chromosome of Bacillus licheniformis.

A total of 59 asporogenous mutants of Bacillus licheniformis were isolated after treatment with nitrosoguanidine or ethyl methanesulfonate. Forty-one of the 59 asporogenous sites were mapped by SP-15 mediated transduction on an abbreviated genetic map of B. licheniformis which contained 12 reference markers. Linkage data revealed that genes for sporulation occupied at least five separate and distinct areas of the chromosome. B. licheniformis was noted to resemble B. subtilis in that most of the spore genes in both species mapped in a cluster to the left of an identical sequence of terminal auxotrophic markers. The terminal spore cluster in B. licheniformis, as in B. subtilis, contained a locus which when defective, blocked the synthesis of a protease believed to regulate an early function of spore formation.

Comparison of Lysyl-Transfer Ribonucleic Acid Species from Vegetative Cells and Spores of Bacillus subtilis by Methylated Albumin-Kieselguhr and Reversed-Phase Chromatography

Lysyl-transfer ribonucleic acid (tRNA) species from a spore-forming strain of Bacillus subtilis (168 trp2(-)) and an early blocked asporogenous mutant (spoA 12) were compared on reversed-phase and methylated albumin-kieselguhr columns. Lysyl-tRNA species from spores and the asporogenous mutant in stationary phase both exhibited altered chromatographic profiles compared to that of log-phase cells. The major peak in spore lysyl-tRNA species eluted later than that characteristic of vegetative cells, whereas the major peak of the lysyl-tRNA species from the asporogenous mutant in stationary phase eluted earlier. Although the early eluting lysyl-tRNA species was observable on methylated albumin columns, the late eluting peak was not detectable by that column technique. By using a shallower gradient on an RPC-2 column, the resolution of all lysyl-tRNA species increased. Several subspecies were revealed. The chromatographic comparisons clearly show that both the spore-forming strain and the asporogenous mutant undergo relative increases in different lysyl-tRNA species when grown to late stationary phase. No new species seem to be involved but rather altered amounts of minor species existing in log-phase cells. The experiments also demonstrate the usefulness of reversed-phase columns for such comparisons.

Profiles of soluble protein during sporulation of Bacillus subtilis.

Summary: Electrophoresis in polyacrylamide gels of soluble proteins from Bacillus subtilis showed changing patterns of bands during sporulation. Somewhat different patterns were obtained from extracts of three asporogenous mutants exposed to a sporulation medium but the differences did not correlate well with the stages in sporulation at which the mutants were believed to be blocked.

STUDIES ON SPORULATION AND PRODUCTION OF MYCOBACILLIN, AN ANTIFUNGAL POLYPEPTIDE, BY BACILLUS SUBTILIS

Both mycobacillin production and sporulation are initiated in the postlog phase of growth of B. subtilis B3, reaching their maximum by about the same time. Mycobacillin is released during enndotropic sporulation of vegetative cells though not during germination of spores in complex growth medium. Antisporogenic chemicals like glucose (in excess), diethylmalonate, acriflavin, fluoroacetic acid, sodium bisulphite, β-phenethyl alcohol, α-picolinic acid and m-tyrosine inhibit mycobacillin production. By acriflavin and actinomycin D treatment, two types of mutants, oligosporous and asporogenous, were obtained. Mycobacillin production is affected adversely in oligosporous mutants whereas asporogenous mutants do not produce the antibiotic at all.