Identification of a gamma-glutamyl methyl ester in bacterial membrane protein involved in chemotaxis.

Evidence is presented that a methyltransferase enzyme, previously shown to be necessary for chemotaxis and identified as the cheR gene product, catalyzes the formation of a gamma-glutamyl methyl ester in one or more membrane proteins of Salmonella typhimurium. The rates of release of methyl label from the methylated protein in acid, base, and hydroxylamine are consistent with a methyl ester and not with a methylated imidazole, guanidino, or amino group. A gamma-glutamyl methyl ester was isolated from a proteolytic digest of the modified protein.     

Crystal structure of the dipeptide binding protein from Escherichia coli involved in active transport and chemotaxis.

The Escherichia coli periplasmic dipeptide binding protein functions in both peptide transport and taxis toward peptides. The structure of the dipeptide binding protein in complex with Gly-Leu (glycyl-L-leucine) has been determined at 3.2 A resolution. The binding site for dipeptides is designed to recognize the ligand's backbone while providing space to accommodate a variety of side chains. Some repositioning of protein side chains lining the binding site must occur when the dipeptide's second residue is larger than leucine. The protein's fold is very similar to that of the Salmonella typhimurium oligopeptide binding protein, and a comparison of the structures reveals the structural basis for the dipeptide binding protein's preference for shorter peptides.     

A novel membrane protein involved in protein translocation across the cytoplasmic membrane of Escherichia coli.

A novel factor, which is a membrane component of the protein translocation machinery of Escherichia coli, was discovered. This factor was found in the trichloracetic acid-soluble fraction of solubilized cytoplasmic membrane. The factor was purified to homogeneity by ion exchange column chromatographies and found to be a hydrophobic protein with a molecular mass of approximately 12 kDa. The factor caused > 20-fold stimulation of the protein translocation when it was reconstituted into proteoliposomes together with SecE and SecY. SecE, SecY, SecA and ATP were essential for the factor-dependent stimulation of the activity. The factor stimulated the translocation of all three precursor proteins examined, including authentic proOmpA. Stimulation of the translocation of proOmpF-Lpp, a model presecretory protein, was especially remarkable, since no translocation was observed unless proteoliposomes were reconstituted with the factor. Partial amino acid sequence of the purified factor was determined. An antibody raised against a synthetic peptide of this sequence inhibited the protein translocation into everted membrane vesicles, indicating that the factor is playing an important role in protein translocation into membrane vesicles. The partial amino acid sequence was found to coincide with that deduced from the reported DNA sequence of the upstream region of the leuU gene. Cloning and sequencing of the upstream region revealed the presence of a new open reading frame, which encodes a hydrophobic protein of 11.4 kDa. We propose that the factor is a general component of the protein translocation machinery of E. coli.     

Topology analysis of the SecY protein, an integral membrane protein involved in protein export in Escherichia coli.

The secY (prlA) gene product is an essential component of the Escherichia coli cytoplasmic membrane, and its function is required for the translocation of exocytoplasmic proteins across the membrane. We have analyzed the orientation of the SecY protein in the membrane by examining the hydropathic character of its amino acid sequence, by testing its susceptibility to proteases added to each side of the membrane, and by characterizing SecY-PhoA (alkaline phosphatase) hybrid proteins constructed by TnphoA transpositions. The orientation of the PhoA portion of the hybrid protein with respect to the membrane was inferred from its enzymatic activity as well as sensitivity to external proteases. The results suggest that SecY contains 10 transmembrane segments, five periplasmically exposed parts, and six cytoplasmic regions including the amino- and carboxyterminal regions.     

Effect of outer membrane permeability on chemotaxis in Escherichia coli.

The relationship between outer membrane permeability and chemotaxis in Escherichia coli was studied on mutants in the major porin genes ompF and ompC. Both porins allowed passage of amino acids across the outer membrane sufficiently to be sensed by the methyl-accepting chemotaxis proteins, although OmpF was more effective than OmpC. A mutant deleted for both ompF and ompC, AW740, was almost completely nonchemotactic to amino acids in spatial assays. AW740 required greater stimulation with L-aspartate than did the wild type to achieve full methylation of methyl-accepting chemotaxis protein II. Induction of LamB protein allowed taxis to maltose but not to L-aspartate, which indicates that the maltoporin cannot rapidly pass aspartate. Salt taxis was less severely inhibited by the loss of porins than was amino acid taxis, which implies an additional mechanism of outer membrane permeability. These results show that chemotaxis can be used as a sensitive in vivo assay for outer membrane permeability to a range of compounds and imply that E. coli can regulate chemotactic sensitivity by altering the porin composition of the outer membrane.     

Refolding of an integral membrane protein. OmpA of Escherichia coli

OmpA is an integral membrane protein from the outer membrane of Escherichia coli. Purified, lipopolysaccharide-free OmpA was denatured by boiling in sodium dodecyl sulfate (SDS). Refolding was then induced by replacement of SDS with the nonionic detergent octylglucoside. The structure of both the denatured and refolded protein were investigated by SDS-gel electrophoresis, protease digestion, Raman and fluorescence spectroscopy. Refolded OmpA could be reconstituted into membranes of the synthetic lipid dimyristoylphosphatidylcholine. Thus, lipopolysaccharide is neither necessary for proper folding of OmpA nor for its insertion into lipid membranes. Based on this result, models for sorting of OmpA into the outer membrane of E. coli are discussed.     

Stoichiometry of maltodextrin-binding sites in LamB, an outer membrane protein from Escherichia coli.

We have directly measured the stoichiometry of maltodextrin-binding sites in LamB. Scatchard plots and computer fitting of flow dialysis (rate-of-dialysis) experiments clearly establish three independent binding sites per LamB trimer, with a dissociation constant of approximately 60 microM for maltoheptaose. The current model for LamB's function as a specific pore is discussed with respect to the symmetry in LamB's kinetic properties and the implications of our results.     

Isolation and behavior of Escherichia coli deletion mutants lacking chemotaxis functions.

The major cotransduction gap of the Escherichia coli chromosome extends from mini 31 to 34. We have inserted transposons through this gap which, by sequential transduction, link sbcA (min 29.8) with manA (min 35.7) and thus eliminate the gap. These results indicate that the length of DNA in the region, as measured by transduction, is not significantly different from the length obtained by conjugational time of entry. Since this segment of the E. coli chromosome has few known genes, these transposon insertions will be useful for genetic manipulations in the region of the gap. We describe the usefulness of these markers for rapidly mapping mutations which may be isolated in the region from min 27 to 37.     

Insertion of an outer membrane protein in Escherichia coli requires a chaperone-like protein.

Only one of the characterized components of the main terminal branch of the general secretory pathway (GSP) in Gram-negative bacteria, GspD, is an integral outer membrane protein that could conceivably form a channel to permit protein transport across this membrane. PulD, a member of the GspD protein family required for pullulanase secretion by Klebsiella oxytoca, is shown here to form outer membrane-associated complexes which are not readily dissociated by SDS treatment. The outer membrane association of PulD is absolutely dependent on another component of the GSP, the outer membrane-anchored lipoprotein PulS. Furthermore, the absence of PulS resulted in limited proteolysis of PulD and caused induction of the so-called phage shock response, as measured by increased expression of the pspA gene. We propose that PulS may be the first member of a new family of periplasmic chaperones that are specifically required for the insertion of a group of outer membrane proteins into this membrane. PulS is only the second component of the main terminal branch of the GSP for which a precise function can be proposed.     

Escherichia coli membrane proteins with an affinity for deoxyribonucleic acid.

From the membrane fraction of Escherichia coli K-12 strain, four protein fractions (peaks I, IIa, IIb, and III) which have affinity for deoxyribonucleic acid (DNA) have been isolated. The molecular weights of these proteins are between 12,000 and 8,000. Only the peak III fraction contains a protein that binds preferentially to single-stranded DNA, whereas the others contain proteins that bind also to double-stranded DNA. The binding activity of the peak IIb protein is inhibited in the presence of polyuridylic acid. Peak I and peak IIa protein fractions behave like hydrophobic proteins.     

Metabolism of glutamic acid in a mutant of Escherichia coli

Vender, Joyce (Indiana University, Bloomington), Kunthala Jayaraman, and H. V. Rickenberg. Metabolism of glutamic acid in a mutant of Escherichia coli. J. Bacteriol. 90:1304-1307. 1965.-A mutant strain of Escherichia coli W1485 was selected for its ability to utilize glutamic acid as the sole source of carbon. Growth of the mutant on glutamic acid led to the repression of glutamic acid dehydrogenase formation. The mutant differed from the wild-type strain in that glutamic decarboxylase activity was absent from the mutant under conditions of growth which supported the formation of this enzyme in the parent strain. Evidence is presented which suggests that loss of the decarboxylase activity results in the acquisition of the ability to utilize glutamic acid as sole source of carbon; a pathway of glutamate utilization via transamination is proposed.     

Amino acid residues of Escherichia coli acyl carrier protein involved in heterologous protein interactions

Acyl carrier protein (ACP) is a small, highly conserved protein with an essential role in a myriad of reactions throughout lipid metabolism in plants and bacteria where it interacts with a remarkable diversity of proteins. The nature of the proper recognition and precise alignment between the protein moieties of ACP and its many interactive proteins is not understood. Residues conserved among ACPs from numerous plants and bacteria were considered as possibly being crucial to ACP's function, including protein-protein interaction, and a method of identifying amino acid residue clusters of high hydrophobicity on ACP's surface was used to estimate residues possibly involved in specific ACP-protein interactions. On the basis of this information, single-site mutation analysis of multiple residues, one at a time, of ACP was used to probe the identities of potential contact residues of ACPSH or acyl-ACP involved in specific interactions with selected enzymes. The roles of particular ACP residues were more precisely defined by site-directed fluorescence analyses of various myristoyl-mutant-ACPs upon specific interaction with the Escherichia coli hemolysin-activating acyltransferase, HlyC. This was done by selectively labeling each mutated site, one at a time, with an environmentally sensitive fluoroprobe and observing its fluorescence behavior in the absence and presence of HlyC. Consequently, a picture of the portion of ACP involved in selected macromolecular interaction has emerged.     

Solution structure of the bacterial chemotaxis adaptor protein CheW from Escherichia coli

The bacterial chemotaxis adaptor protein CheW physically links the chemoreceptors (MCPs) and the histidine kinase CheA. Extensive investigations using bacterium Escherichia coli have established the central role of CheW in the MCP-modulated activation of CheA. Here we report the solution structure of CheW from E. coli determined by NMR spectroscopy. The results show that E. coli CheW shares an overall fold with previously reported structure of CheW from Thermotoga maritima, whereas local conformational deviations are observed. In particular, the C-terminal alpha-helix is considerably longer in E. coli CheW and appears to shrink the active binding pocket with CheA. Our study provides the structural basis for further investigations in E. coli chemotaxis.     

Purification and characterization of an outer membrane-bound protein involved in long-chain fatty acid transport in Escherichia coli

We report the purification and localization of the fadL gene product (FLP), an essential component of the long-chain fatty acid transport machinery in Escherichia coli. FLP was extracted from total membranes by differential extraction with the nonionic detergents Tween 20 and Triton X-100. This protein was further purified from a Tween 20-insoluble-Triton X-100-soluble extract by salt fractionation, gel filtration chromatography, and hydrophobic interaction chromatography. This regime results in a 95-fold purification of FLP from total membranes. The purified protein preparation was homogeneous based on silver staining and gave the characteristic behavior established for the fadL gene product in the presence of sodium dodecyl sulfate at different temperatures prior to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Mr of 33,000 when heated at 25 degrees C and Mr of 43,000 when heated at 100 degrees C) and on two-dimensional polyacrylamide gels (pI of 4.6 and a Mr of 33,000). Purified FLP was rich in hydrophobic residues accounting for approximately 45% of the total amino acid composition. To localize FLP, antisera were raised against the purified protein and were used to probe differentially fractionated membranes by Western immunoblotting. This procedure demonstrated the presence of this protein only in the outer membrane fraction of fadL+ strains. We confirmed the outer membrane localization of FLP by measuring long-chain fatty acid transport in fadL+ and fadL strains treated with EDTA to alter outer membrane permeability and in spheroplasts generated from fadL+ and fadL strains. Both EDTA-treated cells and spheroplasts transported long-chain fatty acids at essentially the same rate regardless of whether they contained a wild-type or mutant fadL gene. These data imply that FLP is a protein in the outer membrane which is specifically involved in long-chain fatty acid transport.