William T. Doerrler

An Escherichia coli Mutant Defective in Lipid Export

Escherichia coli phospholipids and lipopolysaccharide, made on the inner surface of the inner membrane, are rapidly transported to the outer membrane by mechanisms that are not well characterized. We now report a temperature-sensitive mutant (WD2) with an A270T substitution in a trans-membrane region of the ABC transporter MsbA. As shown by32Pi and 14C-acetate labeling, export of all major lipids to the outer membrane is inhibited by ∼90% in WD2 after 30 min at 44 °C. Transport of newly synthesized proteins is not impaired. Electron microscopy shows reduplicated inner membranes in WD2 at 44 °C, consistent with a key role for MsbA in lipid trafficking.

Loss of Outer Membrane Proteins without Inhibition of Lipid Export in an Escherichia coli YaeT Mutant

Escherichia coli yaeT encodes an essential, conserved outer membrane (OM) protein that is an ortholog of Neisseria meningitidis Omp85. Conflicting data with N. meningitidis indicate that Omp85 functions either in assembly of OM proteins or in export of OM lipids. The role of YaeT in E. coli was investigated with a new temperature-sensitive mutant harboring nine amino acid substitutions. The mutant stops growing after 60 min at 44 °C. After 30 min at 44 °C, incorporation of [35S]methionine into newly synthesized OM proteins is selectively inhibited. Synthesis and export of OM phospholipids and lipopolysaccharide are not impaired. OM protein levels are low, even at 30 °C, and the buoyant density of the OM is correspondingly lower. By Western blotting, we show that levels of the major OM protein OmpA are lower in the mutant in whole cells, membranes, and the growth medium. SecA functions as a multicopy suppressor of the temperature-sensitive phenotype and partially restores OM proteins. Our data are consistent with a critical role for YaeT in OM protein assembly in E. coli.

Accumulation of a polyisoprene-linked amino sugar in polymyxin-resistant Salmonella typhimurium and Escherichia coli: structural characterization and transfer to lipid A in the periplasm.

Polymyxin-resistant mutants of Escherichia coli and Salmonella typhimurium accumulate a novel minor lipid that can donate 4-amino-4-deoxy-l-arabinose units (l-Ara4N) to lipid A. We now report the purification of this lipid from a pss−pmrA C mutant of E. coli and assign its structure as undecaprenyl phosphate-α-l-Ara4N. Approximately 0.2 mg of homogeneous material was isolated from an 8-liter culture by solvent extraction, followed by chromatography on DEAE-cellulose, C18 reverse phase resin, and silicic acid. Matrix-assisted laser desorption ionization/time of flight mass spectrometry in the negative mode yielded a single species [M − H]− at m/z 977.5, consistent with undecaprenyl phosphate-α-l-Ara4N (M r = 978.41). 31P NMR spectroscopy showed a single phosphorus atom at −0.44 ppm characteristic of a phosphodiester linkage. Selective inverse decoupling difference spectroscopy demonstrated that the undecaprenyl phosphate group is attached to the anomeric carbon of the l-Ara4N unit. One- and two-dimensional 1H NMR studies confirmed the presence of a polyisoprene chain and a sugar moiety with chemical shifts and coupling constants expected for an equatorially substituted arabinopyranoside. Heteronuclear multiple-quantum coherence spectroscopy analysis demonstrated that a nitrogen atom is attached to C-4 of the sugar residue. The purified donor supports in vitro conversion of lipid IVA to lipid IIA, which is substituted with a single l-Ara4N moiety. The identification of undecaprenyl phosphate-α-l-Ara4N implies that l-Ara4N transfer to lipid A occurs in the periplasm of polymyxin-resistant strains, and establishes a new enzymatic pathway by which Gram-negative bacteria acquire antibiotic resistance.

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

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.

New Functions for the Ancient DedA Membrane Protein Family

The DedA protein family is a highly conserved and ancient family of membrane proteins with representatives in most sequenced genomes, including those of bacteria, archaea, and eukarya. The functions of the DedA family proteins remain obscure. However, recent genetic approaches have revealed important roles for certain bacterial DedA family members in membrane homeostasis. Bacterial DedA family mutants display such intriguing phenotypes as cell division defects, temperature sensitivity, altered membrane lipid composition, elevated envelope-related stress responses, and loss of proton motive force. The DedA family is also essential in at least two species of bacteria: Borrelia burgdorferi and Escherichia coli. Here, we describe the phylogenetic distribution of the family and summarize recent progress toward understanding the functions of the DedA membrane protein family.

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

The DedA family genes are found in most bacterial genomes. Two of these proteins are Escherichia coli YqjA and YghB, predicted inner membrane proteins of unknown function sharing 61% amino acid identity. The E. coli single deletion mutants are largely without phenotype, but the double mutant (BC202; Delta yqjA::Tet(r) Delta yghB::Kan(r)) is characterized by incomplete cell division, temperature sensitivity, and altered phospholipid levels (K. Thompkins et al., J. Bacteriol. 190:4489-4500, 2008). In this report, we have better characterized the cell division chaining defect of BC202. Fluorescence recovery after photobleaching indicates that 58% of the cells in chains are compartmentalized by at least a cytoplasmic membrane. Green fluorescent protein fusions to the cell division proteins FtsZ, ZipA, FtsI, FtsL, and FtsQ are correctly localized to new septation sites in BC202. Periplasmic amidases AmiC and AmiA, secreted by the twin arginine 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.

Multiple Envelope Stress Response Pathways Are Activated in an Escherichia coli Strain with Mutations in Two Members of the DedA Membrane Protein Family

We have reported that simultaneous deletion of two Escherichia coli genes, yqjA and yghB, encoding related and conserved inner membrane proteins belonging to the DedA protein family results in a number of intriguing phenotypes, including temperature sensitivity at 42°C, altered membrane lipid composition, and cell division defects. We sought to characterize these and other phenotypes in an effort to establish a function for this protein family in E. coli. Here, using reporter assays, we show that the major envelope stress response pathways Cpx, Psp, Bae, and Rcs are activated in strain BC202 (W3110; ΔyqjA ΔyghB) at the permissive growth temperature of 30°C. We previously demonstrated that 10 mM Mg(2+), 400 mM NaCl, and overexpression of tatABC are capable of restoring normal growth to BC202 at elevated growth temperatures. Deletion of the cpxR gene from BC202 results in the loss of the ability of these supplements to restore growth at 42°C. Additionally, we report that the membrane potential of BC202 is significantly reduced and that cell division and growth can be restored either by expression of the multidrug transporter MdfA from a multicopy plasmid or by growth at pH 6.0. Together, these results suggest that the DedA family proteins YqjA and YghB are required for general envelope maintenance and homeostasis of the proton motive force under a variety of growth conditions.

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

The gene bb0250 of Borrelia burgdorferi is a homolog of the dedA family, encoding integral inner membrane proteins that are present in nearly all species of bacteria. To date, no precise function has been attributed to any dedA gene. Unlike many bacterial species, such as Escherichia coli, which has eight dedA genes, B. burgdorferi possesses only one, annotated bb0250, providing a unique opportunity to investigate the functions of the dedA family. Here, we show that bb0250 is able to restore normal growth and cell division to a temperature-sensitive E. coli mutant with simultaneous deletions of two dedA genes, yqjA and yghB, and encodes a protein that localizes to the inner membrane of E. coli. The bb0250 gene could be deleted from B. burgdorferi only after introduction of a promoterless bb0250 under the control of an inducible lac promoter, indicating that it is an essential gene in this organism. Growth of the mutant in the absence of isopropyl-β-d-thiogalactopyranoside resulted in cell death, preceded by cell division defects characterized by elongated cells and membrane bulges, demonstrating that bb0250 is required for proper cell division and envelope integrity. Finally, we show that BB0250 depletion leads to imbalanced membrane phospholipid composition in borrelia. These results demonstrate a strong conservation of function of the dedA gene family across diverse species of Gram-negative bacteria and a requirement for this protein family for normal membrane lipid composition and cell division.

The Sinorhizobium meliloti MsbA2 protein is essential for the legume symbiosis

Sinorhizobium meliloti is a beneficial legume symbiont, closely related to Brucella species, which are chronic mammalian pathogens. We discovered that the S. meliloti MsbA2 protein is essential to ensure the symbiotic interaction with the host plant, alfalfa. S. meliloti invades plant cells via plant-derived structures known as infection threads. However, in the absence of MsbA2, S. meliloti remains trapped within abnormally thickened infection threads and induces a heightened plant defence response, characterized by a substantial thickening of the nodule endodermis layer and the accumulation of polyphenolic compounds. The S. meliloti MsbA2 protein is homologous to the Escherichia coli lipopolysaccharide/phospholipid trafficking protein MsbA. However, MsbA2 was not essential for the membrane transport of either lipopolysaccharide or phospholipids in S. meliloti. We determined that the msbA2 gene is transcribed in free-living S. meliloti and that in the absence of MsbA2 the polysaccharide content of S. meliloti is altered. Consequently, we propose a model whereby the altered polysaccharide content of the S. meliloti msbA2 mutant could be responsible for its symbiotic defect by inducing an inappropriate host response.

Multiple deletions reveal the essentiality of the DedA membrane protein family in Escherichia coli

The DedA family is a highly conserved, ancient family of membrane proteins with representatives in most sequenced genomes. A characteristic of prokaryotic DedA family genes is extensive gene duplication, with most bacterial genomes carrying two or more homologues. The Escherichia coli genome carries eight DedA genes, each individually nonessential. We previously described an E. coli mutant (BC202; ΔyghB : : kan(R), ΔyqjA : : tet(R)) with in-frame deletions of two DedA genes encoding proteins with 61 % amino acid identity. BC202 fails to complete cell division or grow at elevated temperatures. Here, we report that restoration of normal growth and cell division of BC202 is possible by overexpression of a subset of the eight E. coli DedA genes (yabI, yohD, yqjA and yghB) but not others (dedA, ydjX, ydjZ and yqaA), suggesting the existence of two functional groups within the family. We have constructed individual E. coli strains in which all eight DedA genes are deleted in a nonpolar manner, and growth is supported by a single DedA family gene under control of an inducible promoter. Strain BAL801 (with growth supported by cloned dedA) and BAL802 (with growth supported by cloned yqjA) exhibit slow growth that is absolutely dependent upon the presence of the arabinose inducer. Growth in the presence of glucose results in cell death. These results indicate that while not individually essential, the E. coli DedA family proteins are collectively essential. These observations suggest important functions for the E. coli DedA protein family.