No abstract

A high-throughput approach was used to assess the effect of mono- and disaccharides on MOX, FMD, MPP1 and MAL1 promoters in Hansenula polymorpha. Site-specifically designed strains deficient for (1) hexokinase, (2) hexokinase and glucokinase, (3) maltose permease or (4) maltase were used as hosts for reporter plasmids in which β-glucuronidase (Gus) expression was controlled by these promoters. The reporter strains were grown on agar plates containing varied carbon sources and Gus activity was measured in permeabilized cells on microtitre plates. We report that monosaccharides (glucose, fructose) repress studied promoters only if phosphorylated in the cell. Glucose-6-phosphate was proposed as a sugar repression signalling metabolite for H. polymorpha. Intriguingly, glucose and fructose strongly activated expression from these promoters in strains lacking both hexokinase and glucokinase, indicating that unphosphorylated monosaccharides have promoter-derepressing effect. We also show that maltose and sucrose must be internalized and split into monosaccharides to exert repression on MOX promoter. We demonstrate that at yeast growth on glucose-containing agar medium, glucose-limitation is rapidly created that promotes derepression of methanol-specific promoters and that derepression is specifically enhanced in hexokinase-negative strain. We recommend double kinase-negative and hexokinase-negative mutants as hosts for heterologous protein production from MOX and FMD promoters.

Avoiding food-borne diseases by competitive exclusion agents is a proactive strategy. In the current paper, we report the use of Bacillus smithii TBMI12 spores as potential competitive exclusion agents. One group of mice was predosed for three successive days with 10(8) colony forming units of B. smithii TBMI12 spores followed by inoculation with 10(6) colony forming units of wild-type Salmonella enterica serotype Enteritidis cells. Microbial plate counts of the animals' livers and spleens showed that only 40% of the mice were infected with S. enterica serotype Enteritidis, while the control group was 100% infected. These results suggest that B. smithii TBMI12 spores may protect against infection by S. enterica serotype Enteritidis.