Edinburgh Napier University

  Using bioinformatics we have identified stm3071 as a possible regulator of anaerobically induced genes involved in metal homeostasis (Price-Carter et al., 2001) and the aim of this study is to determine the function of stm3071 and define the conditions that induce its expression. Cobalt is required for incorporation into cobalamin (vitamin B12) which is important during S. Typhimurium infection. Vitamin B12 is synthesised de novo under anaerobic conditions and is required for metabolism of 1,2-propanediol and ethanolamine which act as sources of carbon and nitrogen when Salmonella is in the gut (Raux et al., 1996; Thiennimitr et al., 2011). Therefore, sensing Co2+ from the environment, and maintaining Co2+ homeostasis, to avoid metal-mediated toxicity, is required for vitamin B12 biosynthesis.
Using λ-red based mutagenesis we have constructed a deletion mutant in order to investigate the function of stm3071. We examined the effect of mutation on the utilisation of 1,2-propanediol under anaerobic conditions and ability to produce vitamin B12. We have also tested the effect of mutation on tolerance to cobalt both aerobically and anaerobically. In order to monitor conditions in which Pstm3071 is switched on, a Pstm3071::lacZ transcriptional fusion was constructed in plasmid pRS415. Levels of β-galactosidase activity were measured in the presence of cobalt in both Δstm3071 and SL1344 (wild type strain) under anaerobic conditions.
Anaerobic growth experiments and B12 assays showed that stm3071 is not essential for growth or synthesis of vitamin B12. In addition, cobalt tolerance in both aerobic and anaerobic conditions was unaffected. However, as measured by β-galactosidase assay, our data suggests that Pstm3071 expression is induced in the presence of cobalt in the deletion mutant. In contrast, we observed no difference in expression of Pstm3071 in the presence or absence of cobalt in SL1344.