coligenome contains up to five additional genes encoding proteins with similarity to YqjA and YghB.yabI,yohD(40), anddedA(Doerrler, unpublished) can complement the growth phenotype of BC202 when overexpressed from plasmids. transport (Tat) pathway, are localized to the cytoplasm in BC202. Overexpression of AmiA, AmiC, or AmiB, a periplasmic amidase secreted via the general secretory pathway, restores normal cell division but does not suppress the temperature sensitivity of BC202, indicating that YghB and YqjA may play additional roles in cellular physiology. Strikingly, overexpression of the Tat export machinery (TatABC) results in normal cell division and growth at elevated temperatures. These data collectively suggest that the twin arginine pathway functions inefficiently in BC202, likely due to the altered levels of membrane phospholipids in this mutant. These results underscore the importance of membrane composition in the proper function of the Tat protein export pathway. 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 asEscherichia coli, remain unknown. We have reported on the PKA inhibitor fragment (6-22) amide functional redundancy of two highly conserved and relatedE. coliinner membrane proteins, YqjA and YghB (40). These proteins belong to a large family (commonly called the DedA family) found widespread in most sequenced genomes.yghBandyqjAencode predicted inner membrane proteins with multiple membrane-spanning domains and 61% amino acid identity. In addition,E. colicontains three other genes predicted to encode proteins with significant similarity to YqjA and YghB (YabI, YohD, and DedA; amino acid BLAST E value of <1 106) and two other proteins with lower PKA inhibitor fragment (6-22) amide degrees of similarity (YdjX and YdjZ). Currently, there are >1,000 genes in the NCBI protein database annotated as either belonging to this family or possessing significant amino acid identity toE. coliDedA/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,yghBandyqjAare nonessential genes, as each single deletion mutant grows normally (2). However, BC202, anE. colistrain with targeted deletions of bothyqjAandyghB, does not grow above 42C and displays a dramatic cell division phenotype by forming chains of cells when grown at the permissive temperature of 30C. 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 Rabbit Polyclonal to APOL4 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 PKA inhibitor fragment (6-22) amide permissive temperature. BC202, therefore, displays several phenotypes: a block at an apparent late stage of cell division, temperature sensitivity, and an imbalance PKA inhibitor fragment (6-22) amide 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 PKA inhibitor fragment (6-22) amide 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 definitely AmiA, both of which.
