KNR4, a suppressor of Saccharomyces cerevisiae cwh mutants, is involved in the transcriptional control of chitin synthase genesHelene M a r t i n, Ad i I ia Dag kessa ma ns ka ia, Ga I i na Sa t c ha ns ka, Nathalie Dallies and Jean Franqois The KNR4 gene, originally isolated by complementation of a K9 killer-toxinresistant mutant displaying reduced levels of both 1,3-/?-glucan and 1,3-pglucan synthase activity, was recloned from a YCp50 genomic library as a suppressor of Saccharomyces cerevisiae calcofluor-white-hypersensitive (cwh) mutants. In these mutants, which were characterized by increased chitin levels, the suppressor effect of KNR4 resulted, for some of them, in a lowering of polymer content to close to wild-type level, with no effect on the contents of P-glucan and mannan. In all cases, this effect was accompanied b y a strong reduction in mRNA levels corresponding to CHS7, CHS2 and CHS3, encoding chitin synthases, without affecting expression of FKS7 and RHO7, two genes encoding the catalytic subunit and a regulatory component of 1,3-P-glucan synthase, respectively. Overexpression of KNR4 also inhibited expression of CHS genes in wild-type strains and in two other cwh mutants, whose sensitivity to calcofluor white was not suppressed by this gene. The physiological relevance of the KNR4 transcriptional effect was addressed in two different ways. In a wild-type strain exposed t o a-factor, overexpression of this gene inhibited CHSl induction and delayed shmoo formation, two events which are triggered in response to the pheromone, whereas it did not affect bud formation and cell growth in a chs1 chs2 double mutant. A chimeric protein made by fusing green fluorescent protein t o the C terminus of Knr4p which fully complemented a knr4A mutation was found to localize in patches a t presumptive bud sites in unbudded cells and a t the incipient bud site during bud emergence. Taken together, these results demonstrate that KNR4 has a regulatory role in chitin deposition and in cell wall assembly. A mechanism by which this gene affects expression of CHS genes is proposed.Keywords : Saccharomyces cerevisiae, cell wall, chitin, KNR4, localization INTRODUCTIONYeasts and fungi are surrounded by a thick cell wall which accounts for 25 O/O of the dry mass (Valentin et al., 1987; Fleet, 1991; Dallies et al., 1998). The cell wall is essential for maintenance of cell shape and offers protection against harmful environmental conditions. It is a dynamic structure which can adapt to different physiological states (conjugation, sporulation, station- ary phase, etc.) and morphological changes (i.e. pseudohyphal and agar-invasive growth; Girneno et al., 1992;Roberts & Fink, 1994). It is composed of P-glucans and mannoproteins, which account for about 80-95 % of wall dry mass, and of smaller amounts of chitin (2%) and lipids (3-5%) (for a review, see .

The aim of this study was to assess the capability of bacterial isolates immobilized on poly(ethylene oxide) (PEO) cryogels to degrade and utilize phenol as a sole source of carbon and energy. Two xenobiotic-degrading bacteria were isolated from industrial areas polluted with heavy metals and aromatics. Sequencing of their 16S rDNA classified them as Pseudomonas rhodesiae (denoted as KCM R 5 ) and Bacillus subtilis (denoted as KCM RG 5 ). The following operation parameters were used: sequencing batch process, 24 h cycle of feeding, increasing phenol concentrations from 300 to 1000 mg¢L ¡1 , volume of inflow À 250 mL, volume of outflow À from 212 to 7 mL and temperature of 28 C. The PEO-KCM R 5 biofilter was found to remove phenol at a concentration of 1000 mg¢L ¡1 , while the PEO-KCM RG 5 system was unable to degrade phenol at a concentration of about 600 mg¢L ¡1 . After four weeks of biodegradation, the PEO biofilms remained compact, porous and elastic, while containing compact microbial biofilm as shown by scanning electron microscopy analysis of the cryogels. Taken together, our results demonstrate that our novel bacterial entrapment system in PEO cryogels is highly effective and sustainable for phenol degradation and can be relevant for application in the detoxification technologies of industrially polluted waters.

The bacterial diversity in two uranium waste piles was studied. Total DNA was recovered from a large number of soil samples collected from different sites and depths in the piles using two procedures for direct lysis. Significant differences in the bacterial composition of the samples were revealed by the use of rep-APD, RISA and 16S ARDREA. The 16S rDNA analyses showed that the uranium wastes were dominated by Acidithiobacillusferrooxidans and by several Pseudomonas species classified in the gamma-subdivision of the Proteobacteria. The three kinds of A. ferrooxidans 16S and IGS rDNA specific fragments that were found corresponded to the three phylogenetic groups recognised in this species. This microdiversity probably reflects the genetic adaptation of the uranium waste strains to different concentrations of heavy metals.