Characterisation of the conserved hypothetical proteins SdhE and YgfX in Serratia 39006

Serratia sp. ATCC 39006 (Serratia 39006) is a Gram-negative bacterium from the Enterobacteriaceae family. Serratia 39006 is able to produce a number of secondary metabolites including the red tripyrrole antibiotic prodigiosin (pig). Although the physiological role of pig remains unknown it is of considerable clinical interest due to its immunosuppressive and anti-cancer properties. Furthermore, a complex hierarchy involving a network of regulatory proteins allows pig biosynthesis to be regulated by multiple environmental inputs. Conserved hypothetical proteins are of unknown function and account for approximately 30% of all proteins in eukaryotic and bacterial genomes. Two conserved hypothetical proteins, termed SdhE and YgfX, were previously identified as positive regulators of pig biosynthesis. In an attempt to further understand how SdhE and YgfX regulate pig biosynthesis this investigation has utilised a combination of bioinformatic, genetic and biochemical techniques. SdhE is a highly conserved protein present in both eukaryotes and bacteria. Investigations revealed that it was required for the activity of succinate dehydrogenase, an important component of the electron transport chain and tricarboxylic acid cycle. SdhE interacted with the flavoprotein subunit SdhA, directly bound the flavin adenine dinucleotide (FAD) cofactor and was required for the flavinylation of SdhA. This is the first demonstration of a protein required for FAD incorporation in bacteria. YgfX is a membrane bound protein that is able to multimerise. YgfX also directly interacted with SdhE suggesting that it may regulate the activity of SdhE at a post-transcriptional level or alternatively that SdhE may regulate YgfX. In conclusion, the characterisation of SdhE in this study has identified a novel biochemical function important for bacterial metabolism. Furthermore, the functional linkage between SdhE and YgfX provides a new regulatory pathway linking cellular metabolism and pig biosynthesis