In vivo and in vitro characterisation of bacterial DedA proteins

The DedA protein superfamily is a group of integral membrane proteins distributed throughout eukaryotes, prokaryotes and archaea. The function of some DedA proteins has been elucidated, an example of which is the link of numerous eukaryotic DedA homologues with phospholipid transport. The role of the prokaryotic DedA proteins is more complex and the overarching function of these proteins less understood, but potential roles in antimicrobial resistance, phospholipid transport, PMF maintenance, Na+/H+ transport and heavy metal transport have been indicated. Deletion of DedA genes results in a range of pleiotropic phenotypes in numerous bacterial species e.g. Escherichia coli K-12 (E. coli K-12), Burkholderia thailandensis (B. thailandensis), Klebsiella pneumoniae (K. pneumoniae) and Borrelia burgdorferi (B. burgdorferi).

Phylogenetic analysis of the DedA proteins present in numerous bacterial species demonstrated that the DedA were widespread in pathogenic, non-pathogenic and symbiotic species. Analysis of 272 E. coli genomes displayed that 6 of the 8 E. coli K-12 homologues were completely conserved amongst these genomes, with only ydjX and ydjZ absent in 37. Amino acid sequence analysis of YqjA in pathogenic and non-pathogenic E. coli species highlighted that YqjA is remarkably well conserved for a membrane protein, and that potentially mutations in YqjA are not well tolerated.

Deletion of yqjA in E. coli str. BW25113 results in cells that could not grow in media of pH 9.25 at 37 °C but could at 30 °C, suggestive that the alkaline sensitivity at pH 9.25 previously described for this strain is temperature dependent. BW25113ΔyqjA and BW25113ΔyqjAΔyqhB were also found to exhibit alkalinisation of the cytoplasm when exposed to growth media of alkaline pH of 9.25. Mutational analysis highlighted the potential roles of amino acid residues S52, L53 and L117 in the ability of YqjA to restore growth to BW25113ΔyqjA at pH 9.25. Additionally, in vitro inverted membrane vesicle assays suggest that YqjA is capable of Na+/H+ transport. A significant increase in membrane fluidity was seen in BW25113ΔyqjAΔyqhB, possibly due to roles of YqjA and YghB in phospholipid transport.

Overexpression of several DedA genes was able to restore growth to BW25113ΔyqjAΔyqhB in a non-permissive temperature of 42 °C and low sodium conditions. The DedA genes were overexpressed, DedA proteins isolated from the cell membrane and analysed by SEC. The main finding of this work was that 7 of the 8 the DedA genes tested could be overexpressed using the pBAD and pET vectors and isolated using DDM or DM, but a number of the proteins when analysed by SEC were unstable in solution after membrane extraction.