Tagging morphogenetic genes by insertional mutagenesis in the yeast Yarrowia lipolytica.

The yeast Yarrowia lipolytica is distantly related to Saccharomyces cerevisiae, can be genetically modified, and can grow in both haploid and diploid states in either yeast, pseudomycelial, or mycelial forms, depending on environmental conditions. Previous results have indicated that the STE and RIM pathways, which mediate cellular switching in other dimorphic yeasts, are not required for Y. lipolytica morphogenesis. To identify the pathways involved in morphogenesis, we mutagenized a wild-type strain of Y. lipolytica with a Tn3 derivative. We isolated eight tagged mutants, entirely defective in hyphal formation, from a total of 40,000 mutants and identified seven genes homologous to S. cerevisiae CDC25, RAS2, BUD6, KEX2, GPI7, SNF5, and PPH21. We analyzed their abilities to invade agar and to form pseudomycelium or hyphae under inducing conditions and their sensitivity to temperature and to Calcofluor white. Chitin staining was used to detect defects in their cell walls. Our results indicate that a functional Ras-cyclic AMP pathway is required for the formation of hyphae in Y. lipolytica and that perturbations in the processing of extracellular, possibly parietal, proteins result in morphogenetic defects.

Disruption of the Paenibacillus polymyxa levansucrase gene impairs its ability to aggregate soil in the wheat rhizosphere.

Inoculation of wheat roots with Paenibacillus (formerly Bacillus) polymyxa CF43 increases the mass of root-adhering soil. We tested the role of levan, a fructosyl polymer produced by strain CF43, in the aggregation of soil adhering to wheat roots. The P. polymyxa gene homologous to the Bacillus subtilis sacB gene encoding levansucrase was cloned and sequenced. The corresponding gene product synthesises high molecular weight levan. A P. polymyxa mutant strain, SB03, whose sacB gene is disrupted, was constructed using heterogramic conjugation. Effects of wheat inoculation with the wild type and the mutant strain were compared using two different cultivated silt loam soils in four independent pot experiments. Roots of wheat plantlets inoculated with CF43 or SB03 were colonized after 7-14 days at the same level, and root and shoot masses were not significantly different from those of the non-inoculated control plants. The ratio of root-adhering soil dry mass to root tissue dry mass was significantly higher for plants inoculated with strain CF43 than for those inoculated with mutant strain SB03: +30% in Orgeval soil and +100% in Dieulouard soil. Thus the levan produced by P. polymyxa is implicated in the aggregation of root-adhering soil on wheat.

Cloning, sequencing, and disruption of a levanase gene of Bacillus polymyxa CF43.

The Bacillus polymyxa CF43 lelA gene, expressing both sucrose and fructan hydrolase activities, was isolated from a genomic library of B. polymyxa screened in Bacillus subtilis. The gene was detected as expressing sucrose hydrolase activity; B. subtilis transformants did not secrete the lelA gene product (LelA) into the extracellular medium. A 1.7-kb DNA fragment sufficient for lelA expression in Escherichia coli was sequenced. It contains a 548-codon open reading frame. The deduced amino acid sequence shows 54% identity with mature B. subtilis levanase and is similar to other fructanases and sucrases (beta-D-fructosyltransferases). Multiple-sequence alignment of 14 of these proteins revealed several previously unreported features. LelA appears to be a 512-amino-acid polypeptide containing no canonical signal peptide. The hydrolytic activities of LelA on sucrose, levan, and inulin were compared with those of B. subtilis levanase and sucrase, confirming that LelA is indeed a fructanase. The lelA gene in the chromosome of B. polymyxa was disrupted with a chloramphenicol resistance gene (cat) by "inter-gramic" conjugation: the lelA::cat insertion on a mobilizable plasmid was transferred from an E. coli transformant to B. polymyxa CF43, and B. polymyxa transconjugants containing the lelA::cat construct replacing the wild-type lelA gene in their chromosomes were selected directly. The growth of the mutant strain on levan, inulin, and sucrose was not affected.