The RIM101 pathway contributes to yeast cell wall assembly and its function becomes essential in the absence of mitogen-activated protein kinase Slt2p.

The Saccharomyces cerevisiae ynl294cDelta (rim21Delta) mutant was identified in our lab owing to its moderate resistance to calcofluor, although it also displayed all of the phenotypic traits associated with its function as the putative sensor (Rim21p) of the RIM101 pathway. rim21Delta also showed moderate hypersensitivity to sodium dodecyl sulfate, caffeine, and zymolyase, and the cell wall compensatory response in this mutant was very poor, as indicated by the almost complete absence of Slt2 phosphorylation and the modest increase in chitin synthesis after calcofluor treatment. However, the cell integrity pathway appeared functional after caffeine treatment or thermal stress. rim21Delta and rim101Delta mutant strains shared all of the cell-wall-associated phenotypes, which were reverted by the expression of Rim101-531p, the constitutively active form of this transcription factor. Therefore, the absence of a functional RIM101 pathway leads to cell wall defects. rim21Delta, as well as rim101Delta, was synthetic lethal with slt2Delta, a synthetic defect alleviated by osmotic stabilization of the media. The double mutants grown in osmotically stabilized media were extremely hypersensitive to zymolyase and showed thicker cell walls, with poorly defined mannoprotein layers. In contrast, rim21Delta rlm1Delta and rim101Delta rlm1Delta double mutants were fully viable. Taken together, these results show that the RIM101 pathway participates directly in cell wall assembly and that it acts in parallel with the protein kinase C pathway (PKC) in this process independently of the transcriptional effect of the compensatory response mediated by this route. In addition, these results provide new experimental evidence of the direct involvement of the PKC signal transduction pathway through the Sltp2 kinase in the construction of yeast cell walls.

Isolation of Salmonella spp. from liquid and solid excreta prior to and following ensilage in ten swine farms located in central Mexico.

A study was carried out to define selected bacteriological characteristics of residues from 10 swine farms, 5 with or without prior clinical enteric disease (PCED) and to determine the effect of ensilage on the bacteria present in the solid fraction. At each farm, samples were taken from the sedimentation basin (SB), the solid fraction (SF), and the liquid fraction (LF). For each sample, CFU/g for enteric bacteria were quantified; Salmonella spp. were isolated and typified. Solid phase samples from each farm were used to prepare the ensilage, with a mixture of solids (80%), sorghum (12%) and molasses (8%). The quantity of enteric bacteria was significantly greater in farms without PCED (P < 0.05). Salmonella enterica were isolated from 8/10 of the farms with and without PCED; in 8 from SB; in 6 from LF; and in 5 from SF. Enteric bacteria were not isolated from silage, therefore, ensilage may be an alternative treatment for excreta that allows the elimination of pathogens such as Salmonella spp.

Saccharomyces cerevisiae Bni4p directs the formation of the chitin ring and also participates in the correct assembly of the septum structure.

In Saccharomyces cerevisiae cytokinesis is efficiently achieved when a concerted series of events take place at the neck region, leading to septum formation. Here it is shown that Bni4p plays a crucial role in this process. Deltabni4 mutants contain normal amounts of chitin and show normal chitin synthase III (CSIII) activity, but are partially resistant to Calcofluor White (CFW), probably due to the striking pattern of chitin distribution. CFW vital staining shows that chitin is synthesized in daughter cells and that it is also asymmetrically deposited at the mother-side of the neck in large-budded cells. This specific pattern coincides with that of Chs4p and Chs3p proteins. Alternatively, staining of unbudded cultures confirmed that Bni4p directs early chitin ring assembly, but is no longer required for the chitin deposition that occurs late in the cell cycle at cytokinesis. Consequently, this work provides a strategy to genetically discriminate between the absence of chitin synthesis (Deltachs3 mutant) and failure in chitin ring assembly (Deltabni4 mutants). The characterization of double mutants affected in chitin synthesis and primary septum (PS) assembly (Deltamyo1 and Deltachs2) provides evidence for the cooperation of Bni4p in PS formation besides its role in chitin ring assembly. In addition, it is shown that the chitin ring, but not the late deposition of chitin, cooperates in the correct assembly of the actomyosin ring and the PS when the biological function of the septins is compromised. We conclude that Bni4p is not only required for the assembly of the chitin ring, but is also involved in septum architecture and the maintenance of neck integrity.

Persistence of Escherichia coli, Salmonella choleraesuis, Aujeszky's Disease virus and Blue Eye Disease virus in ensilages based on the solid fraction of pig faeces.

AIMS: This study was carried out to determine the survival time of Escherichia coli, Salmonella choleraesuis, Aujeszky's Disease virus and Blue Eye Disease virus in ensilages based on the solid fraction of pig faeces. METHODS AND RESULTS: The four micro-organisms were inoculated into microsilos based on the solid fraction of pig faeces, sorghum and molasses. They were left for 0, 7, 14, 28 and 56 days, after which the state of each microsilo was evaluated, and isolation of the inoculated agents was attempted. The four inoculated agents were isolated only on day 0 of ensilage. The viral agents were identified through the cytopathic effect and fluorescence. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: It is concluded that ensilages based on the solid fraction of pig faeces appear to reduce the risk of the transmission of the agents inoculated in this study and help to reduce the environmental impact by using the solid in animal feed.

Improved properties of baker's yeast mutants resistant to 2-deoxy-D-glucose.

We isolated spontaneous mutants from Saccharomyces cerevisiae (baker's yeast V1) that were resistant to 2-deoxy-D-glucose and had improved fermentative capacity on sweet doughs. Three mutants could grow at the same rate as the wild type in minimal SD medium (0.17% Difco yeast nitrogen base without amino acids and ammonium sulfate, 0.5% ammonium sulfate, 2% glucose) and had stable elevated levels of maltase and/or invertase under repression conditions but lower levels in maltose-supplemented media. Two of the mutants also had high levels of phosphatase active on 2-deoxy-D-glucose-6-phosphate. Dough fermentation (CO2 liberation) by two of the mutants was faster and/or produced higher final volumes than that by the wild type, both under laboratory and industrial conditions, when the doughs were supplemented with glucose or sucrose. However, the three mutants were slower when fermenting plain doughs. Fermented sweet bakery products obtained with these mutants were of better quality than those produced by the wild type, with regard to their texture and their organoleptic properties.

Genomic stability of Saccharomyces cerevisiae baker's yeasts.

The objective of this study has been to gather data on genomic stability of baker's yeast strains during long-term mitotic growth under restrictive conditions so that comparisons could be made to other studies indicating genomic instability during meiosis. The work describes the analysis of mitotic stability of the nuclear and mitochondrial genomes in the baker's yeast strain V1 during incubation in continuous culture for 190 generations (300 days). The cells were cultured in complete medium containing 2% glucose and 8 to 12% ethanol, as a mutagenic agent specific for mtDNA. The high concentration of ethanol severely limited the growth rate of the cells. DNA samples were monitored for chromosomal pattern, polymorphisms in selected nuclear genes (SUC2, MALIT, ADH1) and mobile genetic elements (Ty1 and Y'), and for RFLPs in mtDNA. The results show that both the nuclear and mitochondrial genomes of grande cells were very stable. However, the frequency of petite mutants in the population varied dramatically during the course of the experiment, reaching as high as 87% petite during the first 27 days of the experiment and declining to 5.8% petite at the end. This decline can be attributed to selection against petite mutants in media containing high concentrations of ethanol. Moreover, when samples and the parental strain were compared at the end of the experiment, no change could be observed in parameters such as their growth rate in different media, capacity to leave doughs, viability in ethanol or frequency of petite mutants. Results therefore indicated that the majority of the cells in the population were very similar to the parental throughout the experiments, with no apparent molecular or phenotypical changes.