Spectinomycin-resistant mutants of Bacillus subtilis with altered sporulation properties.

Summary Specitinomycin-resistant mutants of Bacillus subtilis show three different types of alterations in sporulation ability. Class 1 mutants can both grow and sporulate in the presence of spectinomycin. Class 2 mutants can grow in the presence of spectinomycin, but are unable to sporulate in either the presence or absence of spectinomycin. Class 3 mutants have a conditional phenotype, and are able to sporulate in the absence of spectinomycin, but not in its presence. The ability of these strains to produce alkaline phosphatase, a biochemical marker for early sporulation events, is correlated with the ability to sporulate in the presence or absence of antibiotic. All of the spectinomycin-resistance mutations could be genetically linked to the cysA marker, and a mutational alteration of a protein of the 30S ribosomal subunit has been identified in one of the Class 3 strains (Spc1–11). Fine-structure mapping of the spectinomycin resistance mutation of strain Spc 1–11 confirmed its location in the cluster of genes for ribosomal components on the B. subtilis genetic map. Genetic analysis indicated that the properties of the Class 1 and Class 2 mutants result from more than one mutation. The spectinomycin-resistance and altered sporulation properties of the two Class 3 mutants probably result from a single genetic lesion.     

Cyclic nucleotides and development ofMyxococcus xanthus: Analysis of mutants

Approximately 60 developmental mutants ofMyxococcus xanthus M300 were obtained through nitrosoguanidine mutagenesis and placed into three operationally defined categories. Type-I strains exhibited no aggregation or sporulation. Type-II strains were able to aggregate but did not sporulate. A strain classed as a type-III strain was a low-capacity fruiter. Each category displayed defects in cyclic nucleotide behavior that could be predicted from the current model. Most significantly, several aggregationless (type I) mutants lacking cGMP phosphodiesterase aggregated in the presence of externally applied phosphodiesterase. A requirement for cell-cell contact in sporulation has been confirmed. Evidence is presented that suggests the involvement of cAMP phosphodiesterase in sporulation and that sporulation may be a developmental pathway independent of aggregation. These results support a previously published hypothesis of the role of cyclic nucleotides in the development ofM. xanthus.     

Catabolite repression-resistant mutants of Bacillus subtilis.

Mutants of Bacillus subtilis that are able to sporulate under the condition of catabolite repression were isolated by a simple selection technique. The mutants used in the present study were able to grow normally on minimal medium with ammonium sulphate as the nitrogen source and glucose as the carbon source. Studies carried out with these mutants show that there is no close relation between catabolite repression of an inducible enzyme, acetoin dehydrogenase, and that of sporulation. Certain mutants are able to sporulate in the presence of all the carbon sources tested but some mutants are resistant only to the carbon source used in isolation. It is suggested that several metabolic steps may be affected in catabolite repression of sporulation.     

Mutants of the fission yeast Schizosaccharomyces pombe which alter the shift between cell proliferation and sporulation

Summary Mutants of S. pombe have been isolated which undergo conjugation and sporulation in rich medium, conditions which are normally inhibitory for these processes. Two of these mutants are also able to sporulate from the haploid state in the absence of heterozygosity at the mating type locus. These recessive mutants define a single nuclear gene called ran1 which is unlinked to mating type. It is proposed that the ran1 gene codes for an inhibitor in the control of the initiation of conjugation and sporulation. In wild type cells the inhibitory effect is released by nutritional starvation and heterozygosity at the mating type locus. This allows the cells to proceed to sporulation. The ran1 mutants are unusual in that they attempt to undergo a reductional meiotic division from the haploid state. They are also genetically unstable and generate extragenic suppressors at high frequency.     

Changes in regulation of ribosome synthesis during different stages of the life cycle of Saccharomyces cerevisiae.

Summary Diploid strains of Saccharomyces cerevisiae, each homozygous for one of the temperature sensitive mutations rna2, rna4, rna6 or rna8, are temperature sensitive for ribosome synthesis during vegetative growth, but are not inhibited for ribosomal synthesis at the restrictive temperature under sporulation conditions. The continued ribosome biosynthesis at the restrictive temperature (34° C) during sporulation includes de novo synthesis of both ribosomal RNA and ribosomal proteins. This lack of inhibition of ribosome biosynthesis is found even when cells committed to complete sporulation are returned to vegetative growth medium. The ribosomes synthesized at 34° C are apparently functional, as they are found in polyribosomes. Although the rna mutants do not regulate ribosome synthesis during sporulation, all of these diploid strains fail to complete sporulation at 34° C. The cells are arrested after the second meiotic nuclear division but before ascus formation. The failure to complete sporulation at the restrictive temperature and the inhibition of ribosome biosynthesis during growth are caused by the same mutation, because revertants selected for temperature independent growth were also able to sporulate at 34° C.     

Mannitol is required for asexual sporulation in the wheat pathogen Stagonospora nodorum (glume blotch)

The physiological role of the mannitol cycle in the wheat pathogen Stagonospora nodorum (glume blotch) has been investigated by reverse genetics and metabolite profiling. A putative mannitol 2-dehydrogenase gene (Mdh1) was cloned by degenerate PCR and disrupted. The resulting mutated mdh1 strains lacked all detectable NADPH-dependent mannitol dehydrogenase activity. The mdh1 strains were unaffected for mannitol production but, surprisingly, were still able to utilize mannitol as a sole carbon source, suggesting a hitherto unknown mechanism for mannitol catabolism. The mutant strains were not compromised in their ability to cause disease or sporulate. To further our understanding of mannitol metabolism, a previously developed mannitol-1-phosphate dehydrogenase (gene mpd1) disruption construct [Solomon, Tan and Oliver (2005) Mol. Plant-Microbe Interact. 18, 110-115] was introduced into the mutated mdh1 background, resulting in a strain lacking both enzyme activities. The mpd1mdh1 strains were unable to grow on mannitol and produced only trace levels of mannitol. The double-mutant strains were unable to sporulate in vitro when grown on minimal medium for extended periods. Deficiency in sporulation was correlated with the depletion of intracellular mannitol pools. Significantly sporulation could be restored with the addition of mannitol. Pathogenicity of the double mutant was not compromised, although, like the previously characterized mpd1 mutants, the strains were unable to sporulate in planta. These findings not only question the currently hypothesized pathways of mannitol metabolism, but also identify for the first time that mannitol is required for sporulation of a filamentous fungus.     

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.     

Phenotypic reversion in some early blocked sporulation mutants of Bacillus subtilis. Genetic study of polymyxin resistant partial revertants

Summary A class of suppressor mutations restores, in pleiotropic sporulation mutants of B. subtilis (SPO mutants), the wild type level of resistance to Polymyxin, and, most often, other properties of the wild strain as well, but never the ability to sporulate. These suppressors, extracistronic, are active on mutations occurring in any one of the 5 genes in which SPO mutations have been found. The phenotype of the suppressed strains is dependent on both the suppressed (SPO) and the suppressive mutations. All these suppressors are located in a single locus and some of them are thermosensitive. The evidence suggests that a physiological compensation is at work in the partial revertants, so that the locus at which the suppressors are located was called cps X. Two hypotheses are discussed that might account for these observations.     

Production of Bacteriophage by Temperature-Sensitive Sporulation Mutants of Bacillus cereus T

Five temperature-sensitive sporulation mutants of Bacillus cereus T have been isolated. These mutants are blocked at stage 0 of sporulation at the restrictive temperature (37 C) but are able to sporulate at nearly normal frequencies at the permissive temperature (26 C). A bacteriophage that forms a stable lysogen in the parent strain is induced at increased frequencies in the mutants. This induction is accompanied, in some of the mutants, by a reduction in immunity to the phage. Revertants, selected for their ability to sporulate normally at both temperatures, lose their ability to produce high titers of the phage. In addition to this lytic phage, an apparently defective phage has been found in lysates of the mutants. Strains cured of the plaque-forming phage still carry the defective phage. Comparisons of physical and biological properties of the plaque-forming phage with those of the two Bacillus cereus phages most similar to it have shown that this phage is not identical to either of them. The maximal titer of phage produced in cultures of the parent strain is about 10(3) plaque-forming units (PFU) per ml at both temperatures. The maximal titers of phage produced by the mutant are 4 x 10(9) PFU/ml at 37 C and 7 x 10(8) PFU/ml at 26 C. Both mutant and parent strains release over 90% of the phage they produce after the onset of stationary phase.     

An easy method for screening and isolating rod mutants of<Emphasis Type="Italic"> Bacillus subtilis</Emphasis>

A convenient and rapid method for screening and identifying rod mutants of Bacillus subtilis is described. At the restrictive temperature (45 °C), all rod mutants of B. subtilis screened lost their ability to sporulate. The morphology and colour of mutant colonies grown on sporulation agar plates differed from those of rod⁺ cells, which were able to sporulate even at elevated temperature. These characteristics provide an alternative approach for the identification of rod mutants in B. subtilis culture by streaking the cells onto a minimal glucose agar plate and incubating at the restrictive temperature. After 30 h of incubation at this temperature, rod mutants are easily identified. This method will facilitate the screening and isolation of rod mutants of B. subtilis.     

Mutants of Schizosaccharomyces pombe which sporulate in the haploid state

Summary Suppressor mutants of mei1–102, a mutation in one of the mating type cassette genes (mat2-P) which blocks the progression into meiosis, were isolated and characterized in Schizosaccharomyces pombe. These suppressor mutations conferred either temperature-sensitivity or cold-sensitivity. The growth of these strains is halted and sporulation initiated at the restrictive temperatures, regardless of other conditions usually required for the initiation of meiosis i.e. they sporulate in the presence of a nitrogen source and mating type homozygosity. Their most striking feature is that they can sporulate from the haploid state. The haploidy of these mutants was confirmed by genetical analysis and by measurement of the DNA content of the cells. The mutants are all recessive and define a single gene pat1. The pat1 gene maps very close to the centromere of chromosome II. A meiosis defective mutation in mei5 can suppress the temperature-sensitivity caused by pat1, indicating some interaction between them. Spores produced from a haploid cell have poor viability and appear to contain only 1/2C DNA on average.     

Erythromycin resistant mutations in Bacillus subtilis cause temperature sensitive sporulation.

Summary All of several hundred erythromycin resistant (ery^R) single site mutants ofBacillus subtilis W168 are temperature sensitive for sporulation (spo^ts). The mutants and wild type cells grow vegetatively at essentially the same rates at both permissive (30° C) and nonpermissive (47° C) temperatures. In addition, cellular protein synthesis, cell mass increases and cell viabilities are similar in mutant and wild type strains for several hours after the end of vegetative growth (47° C). In the mutants examined, the temperature sensitive periods begin when the sporulation process is approximately 40% completed, and end when the process is 90% complete. At nonpermissive temperatures, the mutants produce serine and metal proteases at 50% of the wild type rate, accumulate serine esterase at 16% of the wild type rate, and do not demonstrate a sporulation related increase in alkaline phosphatase activity.The ery^R and spo^ts phenotypes cotransform 100%, and cotransduce 100% using phage PBS1. Revertants selected for ability to sporulate normally at 47° C (spo⁺), simultaneously regain parental sensitivity to erythromycin. No second site revertants are found.Ribosomes from ery^R spo^ts strains bind erythromycin at less than 1% of the wild type rate. A single 50S protein (L17) from mutant ribosomes shows an altered electrophoretic mobility. Ribosomes from spo⁺ revertants bind erythromycin like parental ribosomes and their proteins are electrophoretically identical to wild type. These data indicate that the L17 protein of the 50S ribosomal subunit fromBacillus subtilis may participate specifically in the sporulation process.     

Sporulation and Enterotoxin Production by Mutants of Clostridium perfringens

The ability of Clostridium perfringens type A to produce an enterotoxin active in human food poisoning has been shown to be directly related to the ability of the organism to sporulate. Enterotoxin was produced only in a sporulation medium and not in a growth medium in which sporulation was repressed. Mutants with an altered ability to sporulate were isolated from an sp(+) ent(+) strain either as spontaneous mutants or after mutagenesis with acridine orange or nitrosoguanidine. All sp(0) (-) mutants were ent(-). Except for one isolate, these mutants were not disturbed in other toxic functions characteristic of the wild type and unrelated to sporulation. A total of four of seven osp(0) mutants retained the ability to produce detectable levels of enterotoxin. None of the ent(-) mutants produced gene products serologically homologous to enterotoxin. A total of three sp(-) mutants, blocked at intermediate stages of sporulation, produced enterotoxin. Of these mutants, one was blocked at stage III, one probably at late stage IV, and one probably at stage V. A total of three sp(+) revertants isolated from an sp(-) ent(-) mutant regained not only the ability to sporulate but also the ability to produce enterotoxin. The enterotoxin appears to be a sporulation-specific gene product; however, the function of the enterotoxin in sporulation is unknown.     

Mannitol 1-phosphate metabolism is required for sporulation in planta of the wheat pathogen Stagonospora nodorum

An expressed sequence tag encoding a putative mannitol 1-phosphate dehydrogenase (Mpd1) has been characterized from the fungal wheat pathogen Stagonospora nodorum. Mpd1 was disrupted by insertional mutagenesis, and the resulting mpd1 strains lacked all detectable NAD-linked mannitol 1-phosphate dehydrogenase activity (EC 1.1.1.17). The growth rates, sporulation, and spore viability of the mutant strains in vitro were not significantly different from the wild type. The viability of the mpd1 spores when subjected to heat stress was comparable to wild type. Characterization of the sugar alcohol content by nuclear magnetic resonance spectroscopy revealed that, when grown on glucose, the mutant strains contained significantly less mannitol, less arabitol, but more trehalose than the wild-type strains. The mannitol content of fructose-grown cultures was normal. No secreted mannitol could be detected in wild type or mutants. Pathogenicity assays revealed the disruption of Mpd1 did not affect lesion development, however the mutants were unable to sporulate. These results throw new light on the role of mannitol in fungal plant interactions, suggesting a role in metabolic and redox regulation during the critical process of sporulation on senescing leaf material.     

Population changes induced in Candida albicans by nalidixic acid

Cells of Candida albicans plated on media containing nalidixic acid (Nal) either die, adapt physiologically to Nal-tolerance or mutate to Nal-resistance. The fraction of a population exhibiting each response depends on the growth phase of cells when plated and their nitrogen and carbon nutrition and growth temperatures before and after plating. Nal induces Nal-resistant mutants in very high frequency but only at 37 C on plates containing i) glucose as primary carbon source and ii) adenine, a sulfur amino acid or a representative of the glutamic acid family of amino acids. Nal does not affect either forward mutation to caffeine-resistance or reverse mutation from histidine auxotrophy to prototrophy. Nal-resistant mutants produce minute colonies on Nal-free medium, respire oxidatively and are unusually sensitive to inhibitors of oxidative phosphorylation. They revert spontaneously to wild type at very high rates but can be propagated indefinitely in the absence of Nal by serial selection and replating of minute colonies. Cellular inactivation and induction of Nal-resistant mutants are greatly affected by specific inhibitors of mitochondrial macromolecular syntheses. The presence of chloramphenicol or erythromycin during exposure to Nal prevents cell death and mutation but has no effect on adaptation to Nal-tolerance. Growth on acriflavin or ethidium bromide enhances resistance of cells to inactivation when subsequently plated on Nal containing media. It is concluded that Nal-induced cellular inactivation and mutation to Nal-resistance, but not adaptation to Nal-tolerance, result from damages to the mitochondrion which are fixed or promoted by macromolecular syntheses within the mitochondrion. Implications of these findings for the therapeutic use of Nal are discussed.     

Mating Type and Sporulation in Yeast I. Mutations Which Alter Mating-Type Control over Sporulation

In Saccharomyces cerevisiae, meiosis and spore formation as well as mating are controlled by mating-type genes. Diploids heterozygous for mating type (aα) can sporulate but cannot mate; homozygous aa and αα diploids can mate, but cannot sporulate. From an αα diploid parental strain, we have isolated mutants which have gained the ability to sporulate. Those mutants which continue to mate as αα cells have been designated CSP (control of sporulation). Upon sporulation, CSP mutants yield asci containing 4α spores. The mutant gene which allows αα cells to sporulate is unlinked to the mating-type locus and also acts to permit sporulation in aa diploid cells. Segregation data from crosses between mutant αα and wild-type aa diploids and vice versa indicate (for all but one mutant) that the mutation which allows constitutive sporulation (CSP) is dominant over the wild-type allele. Some of the CSP mutants are temperature-sensitive, sporulating at 32°, but not at 23°. In addition to CSP mutants, our mutagenesis and screening procedure led to the isolation of mutants which sporulate by virtue of a change in the mating-type locus itself, resulting in loss of ability to mate.     

Properties of a Bacillus subtilis mutant able to sporulate continually during growth in synthetic medium

Several mutants of Bacillus subtilis were isolated which sporulate continually during exponential growth in glucose medium. The spdA1 mutation, responsible for the continual sporulation of one of the mutants, mapped near thr. When an exponentially growing culture of a strain containing spdA1 was maintained at essentially constant turbidity, 5% of the viable cells contained heat-resistant spores. The continual sporulation depended on the stringent response since it was absent in spdA relA double mutants. Genetic and biochemical analysis indicated that the continual sporulation of spdA1 strains was associated with a lower specific activity of pyruvate carboxylase, which limited the rate of oxaloacetate synthesis from glucose via pyruvate and thereby the supply of compounds depending on the citrate cycle, especially aspartate. Therefore, the mild stringent response caused by the spdA1 mutation seems to result from a partial deficiency of aspartyl-tRNA which may exert its sporulation-initiating effect during a limited time interval in each growth cycle. A mutant blocked in fumarase activity (citG) behaved similarly. It grew only slowly in glucose medium because much of the limiting oxaloacetate was wasted for the excretion of fumarate. The mutant produced little aspartate and sporulated at a high frequency in glucose medium, even in the presence of glutamate; the sporulation was again prevented by aspartate or malate or by introduction of the relA marker into the strain.     

Remarks on the modifications of cell wall during the sporulation of Saccharomyces cerevisiae Hansen (author's transl)]

Evolution of cell wall during sporulation was studied by means of scanning electron microscopy and by immunological techniques. Experiments were done simultaneously with a strain a/alpha able to sporulate and a strain alpha/alpha unable to sporulate. Under such conditions it was possible to clarify whether the changes observed were related to the sporulation or to the culture conditions. Cell wall structure modifications during sporulation were not obvious morphologically but have been revealed by immunological methods. During vegetative growth, antigenic sites of strains a/alpha and alpha/alpha were different. During incubation in the sporulation medium, antigenic structure of the cell wall was modified. Some antigenic sites seem to be specific of sporulation.     

ScoC Mediates Catabolite Repression of Sporulation in <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

Sporulation in Bacillus subtilis can be triggered by carbon catabolite limitation. Conversely, carbon source excess can repress the production of extracellular enzymes, motility, and sporulation. Recent studies have implicated a pH-sensing mechanism, involving AbrB, the TCA cycle, Spo0K, and Ï^H in controlling the catabolite repression of sporulation gene expression. In an accompanying paper, we demonstrate that the AbrB-dependent pH-sensing mechanism may not be the only means by which carbon catabolites affect sporulation. In the studies reported here, we have examined the molecular basis underlying the catabolite repression phenotype of mutations in the hpr (scoC), rpoD (crsA47), and spo0A (rvtA11) loci. Loss of function mutations in hpr (scoC) restored sporulation gene expression and sporulation in the presence of excess catabolite(s), suggesting that Hpr (ScoC) has a pivotal role in mediating catabolite repression. Moreover, hpr gene expression increased substantially in the presence of excess catabolite(s), further supporting the involvement of Hpr (ScoC) in the carbon catabolite response system. We suggest that alterations in the phosphorelay response to catabolites may be one mechanism by which catabolite-resistant mutants such as crsA and rvtA are able to sporulate in the presence of excess glucoseReceived: 12 November 2002 / Accepted: 13 December 2002     

Acid exposure enhances sporulation of certain strains of Clostridium perfringens

A gastroenteritis results when Clostridium perfringens is ingested in high numbers and sporulates releasing enterotoxin in the intestines. Since the organism must pass through the stomach, its ability to form spores may be affected by the acidic environment. Five strains of C. perfringens were exposed to acidic conditions and then assessed for survival and their ability to form spores. An acidic pH environment kills the bacteria over time but surviving cells are able to recover and form spores. Two of the five strains demonstrated enhanced sporulation following a 30-min exposure to a pH 2 environment. For four of the strains tested, enterotoxin concentrations were higher from acid-exposed cells than from untreated cells. Exposure to a pH 3.5 environment did not affect sporulation when compared to an untreated control. Bacteria in the stationary phase of growth were the most able to resist the acid and sporulate. The results indicate that some strains will produce more spores and enterotoxin following exposure to an acidic environment.