Inefficient Tat-Dependent Export of Periplasmic Amidases in an Escherichia coli Strain with Mutations in Two DedA Family Genes

Roughly 25 to 30% of the genes in sequenced genomes are predicted to encode integral membrane proteins (12). The functions of many of these genes, even in a well-studied organism such as Escherichia coli, remain unknown. We have reported on the functional redundancy of two highly conserved and related E. coli inner membrane proteins, YqjA and YghB (40). These proteins belong to a large family (commonly called the DedA family) found widespread in most sequenced genomes. yghB and yqjA encode predicted inner membrane proteins with multiple membrane-spanning domains and 61% amino acid identity. In addition, E. coli contains three other genes predicted to encode proteins with significant similarity to YqjA and YghB (YabI, YohD, and DedA; amino acid BLAST E value of 1,000 genes in the NCBI protein database annotated as either belonging to this family or possessing significant amino acid identity to E. coli DedA/YghB/YqjA (protein BLAST E values of <0.02). No member of this family has a known function, nor is it known whether they possess common functions across phylogenetic groups. Individually, yghB and yqjA are nonessential genes, as each single deletion mutant grows normally (2). However, BC202, an E. coli strain with targeted deletions of both yqjA and yghB, does not grow above 42°C and displays a dramatic cell division phenotype by forming chains of cells when grown at the permissive temperature of 30°C. Phase-contrast and scanning electron microscopy analysis of BC202 suggests that mutants can begin septation but are blocked at a later step in constriction (40). The cause of this phenotype is unclear. BC202 also has alterations in membrane phospholipid composition (40). While BC202 is capable of synthesizing all classes of phospholipids at all growth temperatures, it is depleted of phosphatidylethanolamine (PE), with elevated levels of the acidic phospholipids phosphatidylglycerol (PG) and cardiolipin (CL). In some respects, BC202 resembles phosphatidylserine synthase deletion mutants, such as AD90 (pss93::Kanr), which produces no membrane PE (14). Mutants deficient in PE are viable, but they require divalent cations for growth (14) and display cell division abnormalities (28, 33). Likewise, normal growth and cell division are restored to BC202 when LB growth medium is supplemented with millimolar concentrations of divalent cations (40). Unlike many mutants defective in cell wall synthesis, BC202 is not hypersensitive to detergents or antibiotics, indicating the presence of an intact outer membrane when grown at the permissive temperature. BC202, therefore, displays several phenotypes: a block at an apparent late stage of cell division, temperature sensitivity, and an imbalance in membrane phospholipid composition. To better understand the functions of YghB and YqjA, we have hypothesized two roles for these genes that are not mutually exclusive to explain the phenotypes of BC202. First, YqjA/YghB may play direct roles in cell division. The phospholipid phenotype may be a consequence secondary to the primary cell division defect in this scenario. Second, YqjA/YghB may play a direct role in efficient PE synthesis or controlling membrane phospholipid composition. The cell division phenotype may be a secondary consequence of the lipid imbalance. Here, we have better characterized the cell division phenotype of BC202 by using green fluorescent protein (GFP) fusions of cell division proteins and fluorescence recovery after photobleaching (FRAP) analysis. We find that while most of the cell division proteins are correctly localized to new septal rings, the periplasmic amidase AmiC is not localized to the septal ring as was reported previously (5), and this may be responsible for the observed cell division phenotype of BC202. AmiC is found mostly in the cytoplasmic compartment in BC202, as is AmiA, both of which are exported to the periplasm by the twin arginine pathway (5). The cell division defect of BC202 can be corrected by overexpression of periplasmic amidases or the TatABC operon, collectively suggesting that the Tat pathway functions inefficiently in BC202.