Temperature Sensitivity and Cell Division Defects in an Escherichia coli Strain with Mutations in yghB and yqjA , Encoding Related and Conserved Inner Membrane Proteins

Abstract

Ludox density gradients were used to enrich for Escherichia coli mutants with conditional growth defects and alterations in membrane composition. A temperature-sensitive mutant named Lud135 was isolated with mutations in two related, nonessential genes: yghB and yqjA. yghB harbors a single missense mutation (G203D) and yqjA contains a nonsense mutation (W92TGA) in Lud135. Both mutations are required for the temperature-sensitive phenotype: targeted deletion of both genes in a wild-type background results in a strain with a similar phenotype and expression of either gene from a plasmid restores growth at elevated temperatures. The mutant has altered membrane phospholipid levels, with elevated levels of acidic phospholipids, when grown under permissive conditions. Growth of Lud135 under nonpermissive conditions is restored by the presence of millimolar concentrations of divalent cations Ca 2؉ , Ba 2؉ , Sr 2؉ , or Mg 2؉ or 300 to 500 mM NaCl but not 400 mM sucrose. Microscopic analysis of Lud135 demonstrates a dramatic defect at a late stage of cell division when cells are grown under permissive conditions. yghB and yqjA belong to the conserved and widely distributed dedA gene family, for which no function has been reported. The two open reading frames encode predicted polytopic inner membrane proteins with 61% amino acid identity. It is likely that YghB and YqjA play redundant but critical roles in membrane biology that are essential for completion of cell division in E. coli.

BB0250 of Borrelia burgdorferi Is a Conserved and Essential Inner Membrane Protein Required for Cell Division

“…The membrane and cell division defects of BC202 are present at both the permissive and nonpermissive growth temperatures. However, BC202 is not hypersensitive to antibiotics or detergents, likely signifying an intact outer membrane, under permissive growth conditions (35). We have demonstrated that the periplasmic amidases AmiA and AmiC are not exported to the periplasm in E. coli mutant BC202 (31).…”

“…Each of the dedA homologs in E. coli (yqjA, yghB, yabI, yohD, dedA, ydjX, ydjZ, and yqaA) is individually nonessential as the single gene knockouts have been made and are available in the Keio collection (1). Our group has determined that simultaneous deletion of yghB and yqjA from E. coli results in a strain (named BC202; ⌬yghB::Kan r ⌬yqjA::Tet r ) that has abnormal membrane phospholipid composition, does not complete cell division (forming chains of cells), and fails to grow at 42°C (35). YghB and YqjA are proteins of 219 and 220 amino acids, respectively, displaying 61% amino acid identity.…”

“…The dedA family is a highly conserved bacterial gene family encoding inner membrane proteins of unknown function (35). There are more than 2,000 homologs currently found in the NCBI protein database (protein BLAST score versus Escherichia coli DedA of Ͻ0.02), and many species of bacteria have multiple homologs.…”

Insights into thermoadaptation and the evolution of mesophily from the bacterial phylum Thermotogae

“…Therefore, while the lipid composition of the membrane is crucial for its function, integral membrane proteins may also play a significant role, particularly with respect to the temperature limit of an organism's growth range. For example, mutations of integral membrane proteins of the DedA family cause temperature sensitivity and cell division defects in Escherichia coli (Thompkins et al 2008). Interestingly, proteins from the DedA family have been shown to be essential in at least 2 bacterial species (E. coli and Borrelia burgdorferi), but their homologs are not detected in several thermophilic and hyperthermophilic Thermotogae genomes (Doerrler et al 2013).…”

“…Interestingly, in Bacillus a transmembrane 2-component response regulator, which controls the desaturase that introduces double bonds in preexisting fatty acids, senses changes in membrane fluidity and not the actual temperature changes (de Mendoza 2014). In addition to the lipid structure of cell membranes, integral membrane proteins affect the temperature tolerance of an organism (Thompkins et al 2008). Therefore, while the lipid composition of the membrane is crucial for its function, integral membrane proteins may also play a significant role, particularly with respect to the temperature limit of an organism's growth range.…”