Mutants of Aspergillus nidulans blocked at an early stage of sporulation secrete an unusual metabolite

Mutants of Aspergillus nidulans defective in conidiation (asexual sporulation) can be classified according to whether they are blocked before or after induction of conidiation. Mutants blocked before induction (preinduction mutants) appear to be unable to respond to the inducing stimulus and thus are defective in one of the earliest events in the sporulation process. Three preinduction mutants have been isolated and characterized. Each was found to exhibit the same pleiotropic phenotype: they also were defective in sexual sporulation and secreted a set of phenolic metabolites at a level much higher than did wild type or mutants blocked at later stages of conidiation. One of the metabolites has been identified as the antibiotic diorcinal (3,3'-dihydroxy-5,5'-dimethyldiphenyl ether) which is known to be involved in the synthesis of certain farnesyl phenols of unknown function. These results suggest that preinduction mutants are blocked in a phenolic metabolic pathway, one or more product of which participates in the initiation of sporulation.     

Analysis of Sporulation Mutants I. Response of Uracil Incorporation to Carbon Sources, and Other Mutant Properties

Mutants deficient in sporulation were isolated and characterized with respect to antibiotic and protease activity, transformability, growth, and sporulation. All but two mutants could grow on minimal medium containing glucose. The inability of most mutants to incorporate uracil into trichloroacetic acid-precipitable material (ribonucleic acid) during the developmental period, and their response to a number of carbon sources, were used to characterize their biochemical blocks. Reproducible measurements of these responses were possible when the pH of the culture, which changed during growth and greatly influenced the rate of uracil uptake, was adjusted to 6.5. By their response to ribose and glutamate, the sporulation mutants could then be divided into four groups. All mutants of the first three groups produced antibiotic activity against Staphylococcus aureus, whereas all mutants, except one, of the fourth group produced none or very little of this activity. Mutants which did not respond to glutamate belonged to the first three groups; they also grew slowly or not at all on glutamate as sole carbon source. One of these mutants lacked succinic dehydrogenase activity. The results indicate that most of our sporulation mutants are unable to produce or utilize a natural carbon precursor, which is normally used as a slowly available carbon and energy source via the Krebs cycle when other carbon sources are used up. It enters the Krebs cycle as a precursor of alpha-ketoglutarate, probably via acetylcoenzyme A. All mutants of group four are blocked in this pathway before alpha-ketoglutarate.     

Conditional dihydrostreptomycin resistance in Bacillus subtilis

Mutants resistant to dihydrostreptomycin were isolated and genetically analyzed in Bacillus subtilis. Two new classes of mutants distinct from the ribosomal strA locus were found. One class, strB, was located between metC3 and ura-1 on the chromosome. The second class, strC, mapped in the spore gene region close to the spoA locus. Both mutant classes were resistant to dihydrostreptomycin during growth but sensitive to the antibiotic during sporulation. Resuspension sporulation experiments with a strB mutant showed that sensitivity to the antibiotic was acquired early in the sporulation process. The germination and outgrowth of strB spores was sensitive to the antibiotic until growth commenced, whereupon the culture was resistant. Thus the mutants are sensitive to dihydrostreptomycin during both sporulation and germination but resistant during the growth phase.     

Genetic antagonism and hypermutability in Mycobacterium smegmatis

Multidrug-resistant strains of Mycobacterium tuberculosis are a serious and continuing human health problem. Such strains may contain as many as four or five different mutations, and M. tuberculosis strains that are resistant to both streptomycin and rifampin contain mutations in the rpsL and rpoB genes, respectively. Coexisting mutations of this kind in Escherichia coli have been shown to interact negatively (S. L. Chakrabarti and L. Gorini, Proc. Natl. Acad. Sci. USA 72:2084-2087, 1975; S. L. Chakrabarti and L. Gorini, Proc. Natl. Acad. Sci. USA 74:1157-1161, 1977). We investigated this possibility in Mycobacterium smegmatis by analyzing the frequency and nature of spontaneous mutants that are resistant to either streptomycin or rifampin or to both antibiotics. Mutants resistant to streptomycin were isolated from characterized rifampin-resistant mutants of M. smegmatis under selection either for one or for both antibiotics. Similarly, mutants resistant to rifampin were isolated from streptomycin-resistant strains. The second antibiotic resistance mutation occurred at a lower frequency in both cases. Surprisingly, in both cases a very high rate of reversion of the initial antibiotic resistance allele was detected when single antibiotic selection was used; the majority of strains resistant to only one antibiotic were isolated by this process. Determinations of rates of mutation to antibiotic resistance in M. smegmatis showed that the frequencies were enhanced up to 10(4)-fold during stationary phase. If such behavior is also typical of slow-growing pathogenic mycobacteria, these studies suggest that the generation of multiply drug-resistant strains by successive mutations may be a more complex genetic phenomenon than suspected.     

Sporulation of Streptomyces griseus in submerged culture.

A wild-type strain of Streptomyces griseus forms spores both on solid media (aerial spores) and in liquid culture (submerged spores). Both spore types are highly resistant to sonication, but only aerial spores are resistant to lysozyme digestion. Electron micrographs suggest that lysozyme sensitivity may result from the thinner walls of the submerged spores. Studies of the life cycle indicate that neither streptomycin excretion nor extracellular protease activity is required for sporulation: the analysis of mutants, however, suggests that antibiotic production may be correlated with the ability to sporulate. A method was devised to induce the rapid sporulation of S. griseus in a submerged culture. This method, which depends on nutrient deprivation, was used to determine that either ammonia or phosphate starvation can trigger sporulation and that the enzyme glutamine synthetase may be useful as a sporulation marker after phosphate deprivation.     

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.     

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.     

The A-factor-binding protein of Streptomyces griseus negatively controls streptomycin production and sporulation.

A-factor, 2-(6'-methylheptanoyl)-3R-hydroxymethyl-4-butanolide, is an autoregulator essential for streptomycin production and sporulation in Streptomyces griseus. S. griseus 2247 that requires no A-factor for streptomycin production or sporulation was found to have a defect in the A-factor-binding protein. This observation implied that the A-factor-binding protein in the absence of A-factor repressed the expression of both phenotypes in the wild-type strain. Screening among mutagenized S. griseus colonies for strains producing streptomycin and sporulating in the absence of A-factor yielded three mutants that were also deficient in the A-factor-binding protein. Reversal of the defect in the A-factor-binding protein of these mutants led to the simultaneous loss of streptomycin production and sporulation. These data suggested that the A-factor-binding protein played a role in repressing both streptomycin production and sporulation and that the binding of A-factor to the protein released its repression. Mutants deficient in the A-factor-binding protein began to produce streptomycin and sporulate at an earlier stage of growth than did the wild-type strain. These mutants produced approximately 10 times more streptomycin than did the parental strain. These findings are consistent with the idea that the intracellular concentration of A-factor determines the timing of derepression of the gene(s) whose expression is repressed by the A-factor-binding protein.     

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.     

Morphological stages of Bacillus subtilis sporulation and resistance to fusidic acid.

During spore development of Bacillus subtilis both protein synthesis and sporulation become resistant to the antibiotic fusidic acid. This resistance develops at the time when asymmetric prespore septa are formed. Simultaneously ribosomes lose their ability to bind fusidic acid, as demonstrated by their affinity chromatography with the immobilized drug. Mutants resistant to fusidic acid during growth are oligosporogenous; their sporulation development is blocked before septum formation. These results indicate that normal ribosomes are needed for prespore septation sporulation; only after septation can protein synthesis be maintained, throughout the development period, by fusidate resistant ribosomes.     

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.     

Potency factors in the delta-endotoxin of Bacillus thuringiensis var. aizawi and the significance of plasmids in their control

Mutants defective in delta-endotoxin crystal production from four closely related isolates of Bacillus thuringiensis var. aizawi with aizawi serotype crystals were as vigorous as the parents in terms of growth, extracellular protease production, sporulation and heat resistance of spores. Spores produced by mutants germinated faster than wild type spores possibly due to deficiency of protein, in the form of delta-endotoxin in the spore coat. Acrystalliferous (cry—) mutants were not active in Galleria mellonella or Pieris brassicae larvae. Mutants with small crystals (sm cry) lost activity or gained extra activity against either one or the other host, revealing the presence of different toxicity factors. Solubilized crystals of parent isolates were composed of two major polypeptides with Mr values of 130 000 and 138 000. Sm cry mutants lost either polypeptide irrespective of which insect potency had been lost. Some cry — and some sm cry mutants had the same plasmid pattern as the parent; others lost one plasmid sometimes gaining another of different size. No consistent correlation was found between plasmid loss in mutants and any loss or increase of potency indicated by bioassays. It is concluded that the delta-endotoxins of the isolates under investigation are composed of at least two toxins. The results suggest that genes coding for the production of toxic factors or for their expression may be carried on both the plasmids and the chromosome.     

Macrolide and aminoglycoside antibiotic resistance mutations in the Bacillus subtilis ribosome resulting in temperature-sensitive sporulation

Summary Mutants of Bacillus subtilis resistant to various macrolide antibiotics have been isolated and characterized with respect to their sporulation phenotype and the electrophoretic mobility of their ribosomal proteins (r-proteins). Two types of major alterations of r-protein L17, one probably due to a small deletion, are found among mutants exhibiting high-level macrolide resistance. These mutants are all temperature-sensitive for sporulation (Spo^ts). Low-level resistance to some macrolides is found to be associated with minor alterations in r-protein L17. These mutations do not cause a defective sporulation phenotype. All of the macrolide resistance mutations map at the same locus within the Str-Spc region of the B. subtilis chromosome. Hence, changes in a single ribosomal protein can result in different sporulation phenotypes.Mutants resistant to the aminoglycoside antibiotics neomycin and kanamycin have been isolated. Approximately 5% of these are Spo^ts. Representative mutations, neo 162 and kan25, cause concomitant drug resistance and sporulation temperature-sensitivity and map as single-site lesions in the Str-Spc region of the chromosome. Strains bearing neo162 or kan25 are equally cross-resistant to several aminoglycoside antibiotics but show no resistance to streptomycin or spectinomycin. These mutations define a new B. subtilis drug resistance locus at which mutation can cause defective sporulation.     

Mutations in a new Streptomyces coelicolor locus which globally block antibiotic biosynthesis but not sporulation

Streptomyces coelicolor produces four known antibiotics. To define genetic elements that regulate antibiotic synthesis, we screened for mutations that visibly blocked synthesis of the two pigmented antibiotics and found that the mutant strains which we recovered were of two classes--double mutants and mutants in which all four antibiotics were blocked. The mutations in these multiply blocked strains define a new locus of S. coelicolor which we have named absA. The genetic location of absA, at 10 o'clock, is distinct from the locations of the antibiotic gene clusters and from other known mutations that affect antibiotic synthesis. The phenotype of the absA mutants suggests that all S. coelicolor antibiotic synthesis genes are subject to a common global regulation that is at least in part distinct from sporulation and that absA is a genetic component of the regulatory mechanism.     

Identification and characterization of TnphoA mutants of Salmonella that are unable to pass through a polarized MDCK epithelial cell monolayer

Surface protein mutants of the invasive Salmonella species, S. choleraesuis, were generated using the transposon TnphoA. 626 alkaline phosphatase (PhoA+) fusion mutants were identified and screened for their ability to pass through (transcytose) polarized epithelial monolayers of Madin Darby canine kidney (MDCK) cells grown on membrane filters. Forty two mutants were unable to pass through this barrier. All of these transcytosis mutants were unable to adhere to or invade MDCK monolayers, yet these mutations were not in the genes encoding type 1 pili or mannose-resistant haemagglutination (MRHA). These transcytosis mutants could be grouped into six classes. Class 1 mutants had altered lipopolysaccharide (LPS) O side-chain structures while Class 2 mutants had defects in their LPS core. Mutants belonging to Classes 5 and 6 did not decrease the transepithelial electrical resistance of polarized MDCK cell monolayers, in contrast to the parental strain and the other mutants (Classes 1, 2, 3 and 4). Mutants belonging to Class 1 were less virulent in mice, while Class 2 (defective core) and Classes 4 and 5 (normal LPS) mutant strains were avirulent in mice. Mutants from Classes 3 and 6 were as virulent in mice as S. choleraesuis. These results suggest that the ability to pass through epithelial barriers may be an important virulence characteristic of Salmonella. These data indicate that bacterial adherence, internalization and monolayer transcytosis are closely linked events. It was also demonstrated that a mutant with decreased rates of intracellular replication still passed through the monolayer at rates similar to wild-type S. choleraesuis.     

Deletion and insertion mutants in the structural gene for ribosomal protein S1 from Escherichia coli

Mutants have been constructed by deleting regions of the gene rpsA for ribosomal protein S1, which had been cloned in plasmid pACYC184. The mutant genes were analyzed for their ability to complement an S1 amber mutant containing a temperature-sensitive suppressor. Another series of mutants was constructed using the tac promoter plasmid pKK223-3, and the effect of the mutant proteins was analyzed in a strain wild type for rpsA. The gene products of all mutants were identified by the immunoblotting technique. Plasmids with a mutant rpsA gene which do not or only poorly complement the S1 amber mutation cause drastic growth reduction, whereas the overall protein synthesis is affected to different extents depending on the site of the deletion. Mutants which express S1 fragments comprising at least the NH2-terminal 100 amino acids stimulate or inhibit the synthesis of certain cellular proteins. The amount of chromosomal coded S1 was reduced by each mutant plasmid. Our data suggest that S1 has a general regulatory role during protein biosynthesis.     

Abortive Infection of Sporulating Bacillus subtilis 168 by φ2 Bacteriophage

Bacteriophage phi2 is unable to replicate in Bacillus subtilis 168. Although some phage deoxyribonucleic acid (DNA) synthesis can occur, the DNA made is not biologically active and sedimentation analysis reveals that it is smaller in size than that of mature DNA or DNA isolated from phi2-infected permissive hosts. Messenger ribonucleic acid hybridizable with phi2 DNA is also synthesized in phi2-infected cells of 168. Mutants of 168 which are permissive hosts for phi2 have been isolated. These mutants are defective in sporulation and possess the phenotype of "early sporulation mutants." The majority map in two locations, one near the lys locus opposite the trp locus (spoA locus) and the other tightly linked to a phe locus.     

The pachytene checkpoint in S. cerevisiae depends on Swe1-mediated phosphorylation of the cyclin-dependent kinase Cdc28

Mutants defective in meiotic recombination and synaptonemal complex formation undergo checkpoint-mediated arrest in mid-meiotic prophase. In S. cerevisiae, this checkpoint requires Swe1, which phosphorylates and inactivates the cyclin-dependent kinase Cdc28. A swe1 deletion allows mutants that normally arrest in meiotic prophase to sporulate at wild-type levels, though sporulation is delayed. This delay is eliminated by overproducing Clb1, the major cyclin required for meiosis I. The Swe1 protein accumulates and is hyperphosphorylated in checkpoint-arrested cells. Our results suggest that meiotic arrest is mediated both by increasing Swe1 activity and limiting cyclin production, with Swe1 being the primary downstream target of checkpoint control. The requirement for Swe1 distinguishes the pachytene checkpoint from the DNA damage checkpoints operating in vegetative cells.