Isolation and characterization of Schizosaccharomyces pombe mutants defective in cell wall (1-3)beta-D-glucan.
Schizosaccharomyces pombe thermosensitive mutants requiring the presence of an osmotic stabilizer to survive and grow at a nonpermissive temperature were isolated. The mutants were genetically and biochemically characterized. In all of them, the phenotype segregated in Mendelian fashion as a single gene which coded for a recessive character. Fourteen loci were defined by complementation analysis. Studies of cell wall composition showed a reduction in the amount of cell wall beta-glucan in three strains (JCR1, JCR5, and JCR10) when growing at 37 degrees C. Galactomannan was diminished in two others. Strains JCR1 and JCR5, with mutant alleles cwg1-1 and cwg2-1, respectively, were further studied. The cwg1 locus was mapped on the right arm of chromosome III, 18.06 centimorgans (cM) to the left of the ade5 marker; cwg2 was located on the left arm of chromosome I, 34.6 cM away from the aro5 marker. (1-3)beta-D-Glucan synthase activities from cwg1-1 and cwg2-1 mutant strains grown at 37 degrees C were diminished, as measured in vitro, compared with the wild-type strain; however, Km values and activation by GTP were similar to the wild-type values. Mutant synthases behaved like the wild-type enzyme in terms of thermostability. Analyses of round shape, lytic behavior, and low (1-3)beta-D-glucan synthase activity in cultures derived from ascospores of the same tetrad showed cosegregation of all these characters. Detergent dissociation of (1-3)beta-D-glucan synthase into soluble and particulate fractions and subsequent reconstitution demonstrated that the cwg1-1 mutant was affected in the particulate fraction of the enzymatic activity while cwg2-1 was affected in the soluble component. The antifungal agents Papulacandin B and Aculeacin A had similar effects on the enzymatic activities of the wild type and the cwg2-1 mutant strain, whereas the cwg1-1 mutant, when growing at 37 degrees C, had a more inhibitor-resistant (1,3)beta-D-glucan synthase. It is concluded that the cwg1+ and cwg2+ genes are related to (1,3)beta-D-glucan biosynthesis.
Ribas
J C
JC
Instituto de Microbiología Bioquímica, Consejo Superior de Investigaciones Científicas, Facultad de Biología, Universidad de Salamanca, Spain.
Diaz
M
M
Duran
A
A
Perez
P
P
D000617:Aminoglycosides; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D000935:Antifungal Agents / Q000494:pharmacology; D000970:Antineoplastic Agents / Q000378:metabolism*; D002455:Cell Division / Q000187:drug effects; D002473:Cell Wall / Q000737:chemistry / Q000378:metabolism*; D002874:Chromosome Mapping; D005816:Genetic Complementation Test; D005838:Genotype; D005936:Glucans / Q000096:biosynthesis*; D005964:Glucosyltransferases / Q000378:metabolism; D006358:Hot Temperature / Q000009:adverse effects; D008565:Membrane Proteins; D008858:Microscopy, Phase-Contrast; D016296:Mutagenesis; D010456:Peptides, Cyclic; D012568:Schizosaccharomyces / Q000235:genetics*; D029702:Schizosaccharomyces pombe Proteins; D013012:Sorbitol / Q000494:pharmacology; D047071:beta-Glucans
D000617:Aminoglycosides; D000900:Anti-Bacterial Agents; D000935:Antifungal Agents; D000970:Antineoplastic Agents; D005936:Glucans; D008565:Membrane Proteins; D010456:Peptides, Cyclic; D029702:Schizosaccharomyces pombe Proteins; D047071:beta-Glucans; D013012:Sorbitol; C011634:aculeacin A; C020789:papulacandin B; C033363:beta-1,3-glucan; D005964:Glucosyltransferases; C020570:1,3-beta-glucan synthase