Translated abstract:
In this study, the genetics, the transport and the regulation of the myo-inositol metabolism of Salmonella enterica serovar Typhimurium (S. Typhimurium) are characterized on a molecular level.
The iol genes, which are involved in the myo-inositol metabolism, are encoded by the genomic island GEI4417/4436. By deletion, the genes iolA, iolB, iolC1, iolC2, iolD2, iolE, iolG1 and STM4423 were identified to be essential for growth with myo-inositol as the sole carbon source. Except for STM4423, the mentioned genes encode the enzymes, which degrade myo-inositol to dihydroxyacetone phosphate, acetyl-CoA and CO2. As investigated by RT-PCR, the genes iolA and iolB, iolC1 and iolC2, iolD1 and iolD2, as well as iolE and iolG1 form transcriptional units. By fusing the iol-promoters with the luciferase, it was shown that the promoters of iolA/B, iolC1/C2, iolD1/D1 and iolE/G1 are highly expressed during growth using myo-inositol as a C-source. In LB medium or in minimal medium containing glucose, all iol promoters except for the promoter of iolE/G1 are repressed by the regulator IolR. Using DNA-protein studies, IolR was shown to bind the remaining and its own promoter indicating its auto-regulation. The induction of the genes iolE/G1 is probably mediated by the regulator STM4423, since its expression suppresses the growth defect caused by the STM4423 deletion.
IolT1 (STM4418) could be identified to act as the main myo-inositol transporter in S. Typhimurium. An optimum of activity of IolT1 at pH 5,5 and a Km value between 0,49 and 0,79 mM was measured in uptake experiments using radio-labelled myo-inositol. The strong reduction of the uptake in the presence of protonophores suggests that IolT1 operates as a myo-inositol/H+ symporter. IolT2 (STM4419) was identified to be a weak myo-inositol transporter. The expression of these two transporters was strongly induced in cells growing with myo-inositol as a carbon source and repressed by IolR binding to their promoters in LB medium or minimal medium containing glucose, as shown by DNA-protein binding assays.
The term biological bistability describes the differential behaviour of isogenic organisms under identical conditions. Such a bistable phenotype could be observed for S. Typhimurium in minimal medium containing myo-inositol as the sole carbon source resulting in a variable transition of each single cell from the lag to the growth phase. On the cellular level, the bistable phenotype was measured as exemplified by the induction of the iolE promoter by fluorescence microscopy and flow cytometry in a qualitative and quantitative manner. The deletion of iolR as well as addition of CO2/bicarbonate to the medium abolishes the bistability. CO2/bicarbonate does not act on IolR, but probably mediates induction of the iolR-independent operon iolE/G1 by binding STM4423. This example shows for the first time an influence of CO2/bicarbonate on the gene expression of S. Typhimurium. The dual regulation of the myo-inositol metabolism by IolR and STM4423 is unique for all organisms investigated so far. A model of the regulation of the myo-inositol metabolism derived from the data presented in this study is postulated.