myo-inositol utilization by Salmonella enterica serovar Typhimurium

• Applicant: Professor Dr. Thilo Martin Fuchs
• Subject Area: Parasitology and Biology of Tropical Infectious Disease Pathogens
• Term: from 2010 to 2013
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 182444822

While the number of sequence projects is rapidly increasing, a biological function has not been assigned to large parts of the bacterial genomes. Of special interest are so-called genomic islands (GEIs), chromosomal fragments of approximately 6 to 48 kb length that interrupt the genetic collinearity with respect to closely related species. GEIs are assumed to have been acquired upon horizontal gene transfer, and they often encode novel biological functions. Their characterization is expected to provide further knowledge about the evolution, the strategies to adapt to changing envi ronments, and yet unknown enzymatic and regulatory properties of bacteria. Metabolic properties are of special interest due to their potential use in biotechnology and, in case of pathogenic bacteria, due to their possible role during infection. The research project proposed here will focus on the investigation of myo-inositol (MI) utilization by Salmonella enterica serovar Typhimurium (S. enterica serovar Typhimurium). All iol genes involved in this pathway are localized on a 22.6 kb genomic island (GEI4417/4436), and some of their functions have recently been characterized (Kröger & Fuchs, 2009; Kröger et al., 2010a). An extended lag phase of 60 hours and a bistable phenotype appeared as a unique feature of S. enterica serovar Typhimurium growth in the presence of MI (Kröger et al., 2010b). Further, yet unpublished work in the applicant’s group revealed that myo-inositol (MI) utilization contributes to intracellular replication of S. enterica serovar Typhimurium. Gene STM4423 is specific for S. enterica serovars and encodes a novel positive regulator that acts on the promoter of iolE. Since STM4423 is known to be induced during infection of mice, it is suggested to play a key role in initiating MI degradation. To examine several yet underinvestigated aspects of the MI degradation pathway on the molecular level, the following work packages are proposed: WP1 will focus on the function of STM4423 including its interaction with bicarbonate and its binding activity towards iol gene promoters. Bandshift assays and Dnase footprinting will be performed to characterize the activity of STM4423 in more detail. An analysis of the promoters PSTM4423 and PiolE, and of environmental conditions acting on these promoters will be performed in WP2. This work package comprises the identification of their transcription initiation site and a promoter deletion analysis, the putative binding of SsrB, and the transcriptional response of these promoters to a variety of environmental stimuli. WP3 will address the relevance of MI utilization by S. enterica serovar Typhimurium in tissue cells, in Caenorhabditis elegans, and in mice. To this end, we will first determine the substrate spectrum of the MI utilization enzymes, and the effect of iol gene deletions on intracellular growth and proliferation within C. elegans. The induction of iol promoters in cultured cells, in C. elegans, and in mice will be monitored using a series of luciferase reporter fusions, and an in vivo imaging system.

• DFG Programme: Research Grants