Isolation and characterization of fusidic acid-resistant, sporulation-defective mutants of Bacillus subtilis.

Fusidic acid-resistant, sporulation-defective mutants were isolated from Bacillus subtilis 168 thy trp. About two-thirds of the fusidic acid-resistant (fusr) mutants were defective in sporulation ability and fell into three classes with respect to sporulation character. The representative mutants FUS426 and FUS429 were characterized in detail. FUS426 [fusr spo (Ts)], a temperature-sensitive sporulation mutant, grew well at 30 and 42 degrees C but did not sporulate at 42 degrees C. FUS429 [fusr spo (Con)], conditional sporulation mutant, grew and sporulated normally in the absence of fusidic acid, but its sporulation and growth rates decreased in the presence of fusidic acid, depending on the concentration of the drug. Although electron microscopic observation showed that both mutants were blocked at stage I of sporulation, the physiological analyses indicate that these mutants belong to the SpoOB class. Both mutants formed a thickened cell wall as compared with that of the parental strain. Genetic and in vitro protein synthesis analyses led to the conclusion that the sporulation-defective character of mutants FUS426 and FUS429 resulted from an alteration in elongation factor G caused by a single lesion in the fus locus. The possible role of elongation factor G in sporulation is discussed.     

Sporulation properties of cytochrome a-deficient mutants of Bacillus subtilis

Three classes of cytochrome a-deficient mutants of Bacillus subtilis have been found to be asporogenic or oligosporogenic. All three classes showed declines in adenosine 5'-triphosphate (ATP) concentrations during early sporulation, at a time when ATP levels in wild-type strains are constant. Class III mutants were found to be deficient in aconitase and isocitric dehydrogenase, and showed reduced maximum growth in nutrient sporulation medium. These mutants also suffered the most rapid decline in ATP concentration in early sporulation, and exhibited neither the biphasic oxygen consumption curve nor the increase in pH normally observed at the end of logarithmic growth in nutrient sporulation medium. Nicotinamide adenine dinucleotide oxidase activities of purified membrane preparations were approximately normal for mutants in all classes, except for two of the class II mutants and one class III mutant. Neither cytochrome a nor cytochrome c appears to be an obligatory intermediate in cyanide-sensitive nicotinamide adenine dinucleotide oxidation in B. subtilis.     

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.     

Autocide AMI rescues development in dsg mutants of Myxococcus xanthus

Low concentrations of autocide AMI rescued aggregation and sporulation in the dsg mutant class of Myxococcus xanthus but were incapable of rescuing asg, bsg, or csg mutants. AMI-induced spores of dsg mutants were resistant to heat and sonication and germinated when plated on nutrient-rich agar. AMI accelerated aggregation and sporulation and increased the final spore number in submerged cultures of a wild-type strain of M. xanthus. Development of M. xanthus was accompanied by release of a fluorescent material (emission maximum, 438 nm) into the supernatant fluid. The release of this material began early and continued throughout development. All Spo- mutant strains tested released significantly reduced levels of this material. These levels were increased in the presence of AMI in all Spo- mutant classes, most dramatically in the dsg mutants.     

Bacteriophage PMB12 conversion of the sporulation defect in RNA polymerase mutants of Bacillus subtilis.

The pseudotemperate phage PMB12 was isolated from soil on the basis of its ability to enhance the rate of sporulation of Bacillus subtilis 168. PMB12 was subsequently shown to convert the sporulation defect in two genetically distinct classes of sporulation mutants. One class includes those rifampin-resistant mutants that are also spore-negative (mutated at the rif locus). The other class includes a strain carrying the sporulation mutation spoCM-1. The spoCM-1 mutation is linked to cysA15 by PBS1 transduction but is distinct from the rif locus. Several other sporulation mutants were not converted by PMB12. PMB12 is related to phage PBS1. However, PBS1 did not convert the above sporulation mutants. The replication of PBS2, a clear-plaquing derivative of PBS1, is rifampin insensitive, apparently due to a phage-induced rifampin-insensitive RNA polymerase. PMB12 replication is also rifampin insensitive.     

Bacteriophage-resistant mutants of Escherichia coli K12. Location of receptors within the lipopolysaccharide.

A series of mutants of Escherichia coli K12 resistant to lipopolysaccharide (LPS)-specific bacteriophages were isolated, and examined with regard to their general properties, phage typing, chemical analysis of their LPS, and genetic analysis. Fourteen classes of mutants were distinguished on the basis of phage typing and sensitivity to bile salts. Three of the mutant classes are sensitive to phages to which the parent is resistant. Mutants which are sensitive to bile salts generally lack heptose in their LPS, but two mutant classes are exceptions to this rule. Analyses of the sugars in the purified LPS of all mutant classes indicated that mutants were obtained which are blocked at most stages in core polysaccharide synthesis. On the basis of the chemical analysis, in conjunction with phage typing data and other known properties of the mutants, it is deduced which residue(s) is involved as a receptor for each of the phages used and which residues hinder these receptors. Some of the mutant classes do not seem to be changed in their LPS structure. Many of the mutations map in or near the rfa locus, but some are far removed from this region.     

Developmental defects resulting from arginine auxotrophy in Aspergillus nidulans

A mutant of Aspergillus nidulans, isolated for inability to form asexual spores (conidia) on complete medium, was found to regain the ability to conidiate if the medium was supplemented with arginine. On minimal medium the mutant required arginine for growth but at a much lower concentration than that required for conidiation. This mutant, designated argB12, thus defines a phase-critical gene, i.e. a gene whose function is in greater demand for development than for growth. In addition to its aconidial phenotype, the mutant also exhibited (depending on the medium) aberrant sexual development and a low efficiency of conidial germination. In crosses, each of these developmental phenotypes segregated with arginine auxotrophy. Genetic and biochemical analyses showed the argB12 mutation to be an allele of the previously described argB locus, mutants of which lack ornithine transcarbamylase. Arginine-requiring mutants at at least two other loci were also found to be defective in asexual sporulation, but the germination defect appears to be specific to argB mutants.     

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.     

Emergence and development of chlorhexidine resistance during sporulation of Bacillus subtilis 168

Abstract During sporulation of Bacillus subtilis strain 168 initiated by step-down conditions, resistance to chlorhexidine diacetate (CHA) developed at about t _3.5, before heat but after toluene resistance. Mutants blocked at stage IV of sporulation were sensitive to all three treatments. Stage V mutants were toluene resistant but moderately sensitive to heat and CHA. A stage VI mutant was resistant to all three treatments. Thus, chlorhexidine resistance is likely to be a result of spore coat, rather than of cortex, development.     

Requirement for peptidoglycan synthesis during sporulation of Bacillus subtilis.

Cultures of Bacillus subtilis were treated during sporulation with antibiotics (bacitracin and vancomycin) that affect peptidoglycan synthesis. The cells were resistant to the effects of the antibiotics only when the drugs were added about 2 h after the beginning of sporulation. This was about 1 h later than the escape time of a temperature-sensitive sporulation mutant that is unable to complete prespore septation. Similar experiments were done with a mutant temperature sensitive for peptidoglycan synthesis. This showed an escape curve similar to that shown by the antibiotics. When sporulating cells were treated with antibiotics, they produced alkaline phosphatase earlier than normal. Enzyme production was unaffected by inhibition of deoxyribonucleic acid synthesis but was inhibited by chloramphenicol. Sporulation mutants that are unable to make alkaline phosphatase under normal conditions were able to make it in the presence of bacitracin. The alkaline phosphatase made under these conditions was under "sporulation-type" control since its synthesis was repressible by casein hydrolysate and unaffected by inorganic phosphate. When cells were treated with bacitracin in the growth medium as well as in the sporulation medium, alkaline phosphatase synthesis was at the same level as in an untreated control. A number of other antibiotics and surfactants were tested for the ability to cause premature production of the phosphatase of those tested, only taurodeoxycholate whowed this behavior. Moreover, incubation of cells with taurodeoxycholate in the growth medium as well as in the sporulation medium prevented premature enzyme production.     

Fructose-bisphosphatase-deficient mutants of the yeast Schizosaccharomyces pombe

We showed that in the yeast Schizosaccharomyces pombe, fructose-bisphosphatase is not subject to catabolite inactivation as it was observed in Saccharomyces cerevisiae. However, this enzyme activity is sensitive to catabolite repression in both yeasts. Two mutants lacking completely fructose-bisphosphatase activity were found. They were unable to grow on glycerol medium. They were still respiratory competent and exhibited the ability to derepress partially malate dehydrogenase activity. In glucose exponential phase culture, the parental strain lacks completely the fructosebisphosphatase activity due to catabolite repression. In these conditions, the growth is slowed down only in the mutants eventhough both mutants and their parental strain lack this enzyme activity. Normal sporulation and poor spore germination were observed for one mutant whereas, only in the presence of glucose, normal sporulation and normal spore germination were observed for the second mutant. Mendelian segregation of glycerol growth was found for the well germinating mutant. It is of nuclear heredity. The two mutations appeared to be closely linked.Abbreviations FBPase Fructose-1,6-bisphosphatase - fbp ^- genetic symbol for FBPase deficiency - glr ^- symbol for inability to grow on glycerol A. M. Colson is Research Associate au Fonds National de la Recherche Scientifique     

Isolation and characterization of vanadate-resistant mutants of Saccharomyces cerevisiae

Cellular vanadium metabolism was studied in Saccharomyces cerevisiae by isolating and characterizing vanadate [VO4(3-), V(V)]-resistant mutants. Vanadate growth inhibition was reversed by the removal of the vanadate from the medium, and vanadate resistance was found to be a recessive trait. Vanadate-resistant mutants isolated from glucose-grown cells were divided into five complementation classes containing more than one mutant. Among the vanadate-resistant mutants isolated in maltose medium, the majority of mutants were found in only two complementation groups. Three of the classes of vanadate-resistant mutants were resistant to 2.5 mM vanadate but sensitive to 5.0 mM vanadate in liquid media. Two classes of vanadate-resistant mutants were resistant to growth in media containing up to 5.0 mM vanadate. Electron spin resonance studies showed that representative strains of the vanadate-resistant complementation classes contained more cell-associated vanadyl [VO2+, V(IV)] than the parental strains. 51 Vanadium nuclear magnetic resonance studies showed that one of the vanadate resonances previously associated with cell toxicity (G. R. Willsky, D. A. White, and B. C. McCabe, J. Biol. Chem. 259:13273-132812, 1984) did not accumulate in the resistant strains compared with the sensitive strain. The amount of vanadate remaining in the media after growth was larger for the sensitive strain than for the vanadate-resistant strains. All of the strains were able to accumulate phosphate, vanadate, and vanadyl.     

Isolation, characterization, and mapping of Bacillus subtilis 168 germination mutants.

After mutagenesis with nitrosoguanidine, germination mutants of Bacillus subtilis 168 were selected by killing, with heat, spores that germinated at 42 C and collecting survivors at 30 C. The germination properties of nine mutants variously affected in amino acid biosynthesis and sugar utilization were studied in detail. They were divided into two groups: (i) Ger-ALA mutants, failed to germinate in 10 mM L-alanine but germinated in complex media (some of these mutants were temperature sensitive); (ii) Ger-PAB mutants, germinated poorly, even in complex media, suggesting that they were blocked in important germination functions. All the mutants failed to germinate in L-alpha-amino-n-butyrate or L-valine (including temperature-sensitive mutants only at the restrictive temperature) showing that there is a step necessary for germination affected by all three acids. The mutants had normal growth rates, indicating that the defective gene products were specific for germination functions. These defects were not identified. Eight of the mutants were mapped by transduction with phage PBS-1. The recombinants were scored either by observations, by microscopy of phase darkening of the spores, or by a plate test involving the reduction of tetrazolium by heated colonies of spores. Five of the mutations, of at least three phenotypes, were between thr-5 and cysB3 away from all the sporulation markers that have been previously mapped. A linked ald (alanine dehydrogenase) locus was on the other side of thr-5. The other Ger markers were located in at least two additional positions. Auxotrophic strains that were used for mapping germinated normally, but germination of the Ger mutants differed slightly in different genetic backgrounds.     

Spermine-resistant mutants of Arabidopsis thaliana with developmental abnormalities

Fourteen spermine-resistant mutants of Arabidopsis thaliana (L.) Heynh. have been isolated from EMS-mutagenized M₂ seeds by screening for germination seedlings on a medium containing a germination inhibitory concentration of spermine. Two of these mutants have been studied initially. Genetic analysis indicated that the two mutants are allelic and the spermine resistance is due to recessive nuclear mutations at a locus we have designated SPR1. Mutant spr1-1 exhibits large, longitudinally folded-in cauline leaves whereas mutant spr1-2 is characterized by vigorous growth, large longitudinally folded-in cauline leaves, prominent flowers with 4–8 sepals, 6–8 petals, 6 (rarely 7) stamens of equal lengths, and 2–4 carpellary club-shaped pistil. Both mutants are resistant to exogenous spermine but are as sensitive as the wild-type to spermidine and putrescine.     

Analysis of the germination of spores of Bacillus subtilis with temperature sensitive spo mutations in the spoVA operon

A Bacillus subtilis strain with a base substitution in the ribosome-binding site of spoVAC was temperature sensitive (ts) in sporulation and spores prepared at the permissive temperature were ts in L-alanine-triggered germination, but not in germination with Ca2+-dipicolinic acid (DPA) or dodecylamine. Spores of a ts spo mutant with a missense mutation in the spoVAC coding region were not ts for germination with l-alanine, dodecylamine or Ca2+-DPA. These findings are discussed in light of the proposal that SpoVA proteins are involved not only in DPA uptake during sporulation, but also in DPA release during nutrient-mediated spore germination.     

A single base change in the acceptor stem of tRNA(3Leu) confers resistance upon Escherichia coli to the calmodulin inhibitor, 48/80

We have isolated several classes of spontaneous mutants resistant to the calmodulin inhibitor 48/80 which inhibits cell division in Escherichia coli K12. Several mutants were also temperature sensitive for growth and this property was exploited to clone a DNA fragment from an E. coli gene library restoring growth at 42 degrees C and drug sensitivity at 30 degrees C in one such mutant. Physical and genetic mapping confirmed that both the mutation and the cloned DNA were located at 15.5 min on the E. coli chromosome at a locus designated feeB. By subcloning, complementation analysis and sequencing, the feeB locus was identified as identical to the tRNA(CUALEU) gene. When the mutant locus was isolated and sequenced, the mutation was confirmed as a single base change, C to A, at position 77 in the acceptor stem of this rare Leu tRNA. In other studies we obtained evidence that this mutant tRNA, recognizing the rare Leu codon, CUA, was defective in translation at both permissive and non-permissive temperatures. The feeB1 mutant is defective in division and shows a reduced growth rate at non-permissive temperature. We discuss the possibility that the mutant tRNA(3Leu) is limiting for the synthesis of a polypeptide(s), requiring several CUA codons for translation which in turn regulates in some way the level or activity of the drug target, a putative cell cycle protein.     

Sensitivity ofBacillus subtilis sporulation to methylene blue inhibition

The dye methylene blue was found to inhibit sporulation inBacillus subtilis 168. The compound 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 the inhibitor through T₆, but germination and outgrowth were not affected by the presence of the compound. The inhibition of sporulation may be related to the ability of the compound to inhibit oxidative phosphorylation.     

The GanB Galpha-protein negatively regulates asexual sporulation and plays a positive role in conidial germination in Aspergillus nidulans

We isolated the ganB gene encoding the Galpha-protein homolog from Aspergillus nidulans. To investigate the cellular function of GanB, various mutant strains were isolated. Deletion of constitutively inactive ganB mutants showed conidiation and derepressed brlA expression in a submerged culture. Constitutive activation of GanB caused a reduction in hyphal growth and a severe defect in asexual sporulation. We therefore propose that GanB may negatively regulate asexual sporulation through the BrlA pathway. In addition, deletion or constitutive inactivation of GanB reduced germination rate while constitutive activation led to precocious germination. Furthermore, conidia of a constitutively active mutant could germinate even without carbon source. Taken together, these results indicated that GanB plays a positive role during germination, possibly through carbon source sensing, and negatively regulates asexual conidiation in A. nidulans.     

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.