Phylogeny and cross-regulation of the YjjM and LeuO transcription factors translated as multiple protein forms from one gene in Escherichia coli

Until recently, no examples of the in-frame translation of several proteins from one gene in bacteria were known. The first one was the VirF transcription factor controlling pathogenicity development in Shigella flexneri and CobB sirtuin in Salmonella enterica. Recently, we observed synthesis of shortened protein products for YjjM (LgoR) and LeuO functioning as transcription factors in Escherichia coli. To determine the evolutionary factors that could lead to the appearance of alternative start codons, we performed phylogenetic analysis and showed that each protein had a unique phylogenetic history, and additional starting methionines appeared only in Enterobacteria. Using the Western-blot analysis of proteins synthesized from the Escherichia coli K-12 MG1655 chromosome with the his-tagged leuO gene two shortened variants of LeuO, corresponding to translation starting from Met34 and Met48 were detected. Synthesis of all three LeuO forms was inhibited in the absence of the yjjM gene, suggesting interplay of these transcription factors. The YjjM recognition motif revealed from the ChIP-seq data appeared to be very similar to that of LeuO, known previously. Taking this into account, we compared ChIP and SELEX data for LeuO and YjjM and found six common targets. At least five of them were confirmed to be under control of these regulators by qRT-PCR. Interestingly, the effects were more prominent during anaerobic growth at 37$^\circ$C simulating conditions inside a host organism. Two genes, coding for the enterobactin transporter FepA, and a repressor of genes responsible for flagellar biosynthesis and virulence YjjQ, were repressed, mainly by YjjM, only in these conditions, while tsr coding for the chemotaxis receptor protein was more repressed under lower temperature and higher aeration.