Pore-forming activity of the Tsx protein from the outer membrane of Escherichia coli. Demonstration of a nucleoside-specific binding site

The Tsx protein from the outer membrane of Escherichia coli is known to be involved in the permeation of nucleosides across the outer membrane under limiting substrate conditions. We purified Tsx from an E. coli strain that overproduces Tsx. The purified protein was still functional since it could neutralize the Tsx-specific bacteriophage T6 in vitro. When the purified Tsx was reconstituted into a lipid bilayer, there was a large increase of the membrane conductance, indicating pore-forming activity of Tsx in vitro. This increase could be strongly blocked with adenosine and to a much lesser extent with cytidine. Titration of the pore conductance with adenosine or cytidine suggested the presence of a binding site for nucleosides in the Tsx pore, with a Ks of 6 X 10(-4) and 2 X 10(-2) M for adenosine and cytidine, respectively. We propose that the Tsx protein functions in vivo as a pore that specifically facilitates the permeation of nucleosides across the outer membrane due to its binding site for nucleosides.     

Linker mutagenesis in the gene of an outer membrane protein of Escherichia coli, lamB

In order to identify sequences involved in the localization of LamB, an outer membrane protein from E coli K12, mutagenesis by linker insertion has been performed on a lamB gene copy carried on a plasmid devised for this purpose. An analysis of the first set of 16 clones constructed by this technique shows that, in these clones, the lamB protein is altered either by frameshift mutations leading to abnormal COOH terminal (usually premature termination) or by in-phase deletions or small insertions. Except for two in-phase linker insertions, which only slightly changed the behavior of the protein, the modified proteins are either toxic to cell growth or unstable. In all cases examined so far, the modified proteins were in the outer membrane. We suggest that toxicity is due to incorrect folding, which leads to disruption of the outer membrane. The nature of the genetic alterations leads to the hypothesis that the first 183 amino acids of the LamB mature protein contain, together with the signal sequence, all the instructions needed for proper localization.     

Functions related to the receptor protein specified by the tsx gene of Escherichia coli

The receptor protein for the phage T6 and colicin K, coded by the tsx gene, facilitated the diffusion of all nucleosides and deoxynucleosides except cytidine and deoxcytidine through the outer membrane of Escherichia coli K-12 and Escherichia coli B. The tsx protein was coregulated with the nucleoside uptake system. Constitutive cytR and deoR mutants contained higher amounts of this protein than wild type strains. There was a good correlation between the initial rate of nucleoside uptake and the adsorption rate of phage T6. From the observation that nucleosides did not compete with each other in the translocation across the outer membrane and that they did not inhibit T6 adsorption it was concluded that the tsx protein forms a pore to which nucleosides have only little if any binding affinity.A major outer membrane protein specified by the ompA gene influenced the function of the tsx protein. Outer membranes of ompA mutants showed an enhanced binding of colicin K but the strains were colicin K insensitive (tolerant). The T6 phage adsorbed at the same rate and plated with the same efficiency as to ompA ⁺ strains. The uptake rate of thymidine and of adenosine was reduced by 16–33% in ompA mutants.The adsorption rate of phage T6 on mutants with altered lipopolysaccharide was the same or even higher than on wild type strains. However the plating efficiency was reduced ranging from 0–46%. Lipopolysaccharide plays no role in the primary adsorption of phage T6 but it is apparently required in a later step of the infection process.Non Standard Abbreviations LPS lipopolysaccharide - cAMP-CRP complex of cyclic adenosine 3,5-monophosphate (cAMP) and its receptor protein (CRP)     

Nonrandom structure in the urea-unfolded Escherichia coli outer membrane protein X (OmpX)

On the basis of sequence-specific resonance assignments for the complete polypeptide backbone and most of the amino acid side chains by heteronuclear nuclear magnetic resonance (NMR) spectroscopy, the urea-unfolded form of the outer membrane protein X (OmpX) from Escherichia coli has been structurally characterized. (1)H-(1)H nuclear Overhauser effects (NOEs), dispersion of the chemical shifts, amide proton chemical shift temperature coefficients, amide proton exchange rates, and (15)N[(1)H]-NOEs show that OmpX in 8 M urea at pH 6.5 is globally unfolded, but adopts local nonrandom conformations in the polypeptide segments of residues 73-82 and 137-145. For these two regions, numerous medium-range and longer-range NOEs were observed, which were used as the input for structure calculations of these polypeptide segments with the program DYANA. The segment 73-82 forms a quite regular helical structure, with only loosely constrained amino acid side chains. In the segment 137-145, the tryptophan residue 140 forms the core of a small hydrophobic cluster. Both nonrandom structures are present with an abundance of about 25% of the protein molecules. The sequence-specific NMR assignment and the physicochemical characterization of urea-denatured OmpX presented in this paper are currently used as a platform for investigations of the folding mechanism of this integral membrane protein.     

Chromosomal location of a gene (nmpA) involved in expression of a major outer membrane protein in Escherichia coli.

The phenotypic expression of protein E, a recently described major outer membrane protein, is associated with a mutation at a locus on the Escherichia coli chromosome that we call nmpA. nmpA is located between rbsK and uncA at 82.7 min on the E. coli linkage map. The nmpA locus is also the site of the mutations which lead to the formation of major outer membrane proteins Ic or e. It is likely proteins E, Ic, and e are closely related or identical. The mutant nmpA allele is dominant.     

Lethal mutations in the structural gene of an outer membrane protein (OmpA) of Escherichia coli K12

Summary The gene ompA encodes a major outer membrane protein of Escherichia coli. Localized mutagenesis of the part of the gene corresponding to the 21-residue signal sequence and the first 45 residues of the protein resulted in alterations which caused cell lysis when expressed. DNA sequence analyses revealed that in one mutant type the last CO₂H-terminal residue of the signal sequence, alanine, was replaced by valine. The proteolytic removal of the signal peptide was much delayed and most of the unprocessed precursor protein was fractioned with the outer membrane. However, this precursor was completely soluble in sodium lauryl sarcosinate which does not solubilize the OmpA protein or fragments thereof present in the outer membrane. Synthesis of the mutant protein did not inhibit processing of the OmpA or OmpF proteins. In the other mutant type, multiple mutational alterations had occurred leading to four amino acid substitutions in the signal sequence and two affecting the first two residues of the mature protein. A reduced rate of processing could not be clearly demonstrated. Membrane fractionation suggested that small amounts of this precursor were associated with the plasma membrane but synthesis of this mutant protein also did not inhibit processing of the wild-type OmpA or OmpF proteins. Several lines of evidence left no doubt that the mature, mutant protein is stably incorporated into the outer membrane. It is suggested that the presence, in the outer membrane, of the mutant precursor protein in the former case, or of the mutant protein in the latter case perturbs the membrane architecture enough to cause cell death.     

A new heat-shock gene, ppiD, encodes a peptidyl-prolyl isomerase required for folding of outer membrane proteins in Escherichia coli.

We have identified a new folding catalyst, PpiD, in the periplasm of Escherichia coli. The gene encoding PpiD was isolated as a multicopy suppressor of surA, a mutation which severely impairs the folding of outer membrane proteins (OMPs). The ppiD gene was also identified based on its ability to be transcribed by the two-component system CpxR-CpxA. PpiD was purified to homogeneity and shown to have peptidyl-prolyl isomerase (PPIase) activity in vitro. The protein is anchored to the inner membrane via a single transmembrane segment, and its catalytic domain faces the periplasm. In addition, we have identified by site-directed mutagenesis some of the residues essential for its PPIase activity. A null mutation in ppiD leads to an overall reduction in the level and folding of OMPs and to the induction of the periplasmic stress response. The combination of ppiD and surA null mutations is lethal. This is the first time two periplasmic folding catalysts have been shown to be essential. Another unique aspect of PpiD is that its gene is regulated by both the Cpx two-component system and the sigma32 heat shock factor, known to regulate the expression of cytoplasmic chaperones.     

Role of a major outer membrane protein in Escherichia coli.

Mutants of Escherichia coli B/r lacking a major outer membrane protein, protein b, were obtained by selecting for resistance to copper. These mutants showed a decreased ability to utilize a variety of metabolites when the metabolites were present at low concentrations. Also, mutants of E. coli K-12 lacking proteins b and c from the outer membrane were shown to have an identical defect in the uptake of various metabolites. These results are discussed with regard to their implications as to the role of these proteins in permeability of the outer membrane,     

Escherichia coli outer membrane protein K is a porin.

Protein K is an outer membrane protein found in pathogenic encapsulated strains of Escherichia coli. We present evidence here that protein K is structurally and functionally related to the E. coli K-12 porin proteins (OmpF, OmpC, and PhoE). Protein K was found to cross-react with antibody to OmpF protein and to share 8 out of 17 peptides in common with the OmpF protein. Strains that are OmpC porin- and OmpF porin- and contain protein K as their major outer membrane protein have increased rates of uptake of nutrients and a faster growth rate relative to the parental porin- strain. The protein K-containing strains are at least 1,000-fold more sensitive to colicins E2 and E3 than is the porin -deficient strain. These data suggest that protein K is a functional porin in E. coli. The porin function of protein K was also demonstrated in vitro, using black lipid membranes. Protein K increased the conductance in these membranes in discrete, uniform steps characteristic of channels with a size of about 2 nS.     

Restoration of membrane incorporation of an Escherichia coli outer membrane protein (OmpA) defective in membrane insertion

The mechanism of sorting, to the outer membrane, of the 325-residue Escherichia coli protein OmpA has been investigated. It is thought to traverse the membrane eight times in antiparallel beta-strands, forming an amphiphilic beta-barrel which encompasses residues 1 to about 170; the COOH-terminal moiety is periplasmic. A mutant, carrying the substitutions Leu164----Pro and Val166----Asp within the last beta-strand (residues 160-170), has been described which was unable to assemble in the membrane (Klose, M., MacIntyre, S., Schwarz, H., and Henning, U. (1988) J. Biol. Chem. 263, 13297-13302). Linkers were inserted between the codons for residues 164 and 165 of the mutant protein. Of 13 different genes recovered, five encoded proteins which had regained the ability to assemble in the membrane. The properties of the mutant proteins, together with a structure prediction method, indicate the following rules for the final beta-strand to be compatible with, or possibly initiate, membrane insertion: (i) it must be amphiphilic or hydrophobic while its primary structure as such is fairly unimportant, (ii) it must extend over at least 9 residues, and (iii) it must not contain a proline residue around its center. One of the genes recovered coded for OmpA up to residue 164 and then followed by 10 linker-encoded residues. This 174-residue polypeptide was assembled in the membrane but did not, in contrast to all other proteins, expose sites sensitive to trypsin at the inner face of the membrane. This behavior agrees perfectly well with the OmpA model.     

Monoclonal antibodies against the FhuA (TonA) protein of the Escherichia coli outer membrane

Abstract Outer membranes of Escherichia coli K-12 were used to isolate hybridoma cell lines that produce monoclonal antibodies against the FhuA (TonA) protein. Two monoclonal antibodies were obtained from independent immunization and fusion experiments. The antibodies belonged to the subclass IgG₁ and κ, and IgG_2b and κ, respectively. The latter antibody was purified by affinity chromatography on protein A-Sepharose. The culture supernatants of the hybridoma cell lines and the isolated antibody inhibited adsorption of the phages T5 and T1 to E. coli cells while binding of phage ø80, which also uses the FhuA protein as a receptor, remained unaffected. The specificity of the antibodies to the FhuA protein was supported by the prevention of killing of cells by colicin M and by the lack of inhibition of colicin B and of phage BG23. Transport of iron(III) as ferrichrome complex was not inhibited by the isolated antibody. However, partial competition with the adsorption of the phages T2, TuIb and T6 was observed which may indicate an organization of certain functional phage receptors into clusters.     

Aperiplasmic protein (Skp) of Escherichia coli selectively binds a class of outer membrane proteins

A search was performed for a periplasmic molecular chaperone which may assist outer membrane proteins of Escherichia coli on their way from the cytoplasmic to the outer membrane. Proteins of the periplasmic space were fractionated on an affinity column with sepharose-bound outer membrane porin OmpF. A 17kDa polypeptide was the predominant protein retained by this column. The corresponding gene was found in a gene bank; it encodes the periplasmic protein Skp. The protein was isolated and it could be demonstrated that it bound outer membrane proteins, following SDS-PAGE, with high selectivity. Among these were OmpA, OmpC, OmpF and the maltoporin LamB. The chromosomal skp gene was inactivated by a deletion causing removal of most of the signal peptide plus 107 residues of the 141-residue mature protein. The mutant was viable but possessed much-reduced concentrations of outer membrane proteins. This defect was fully restored by a plasmid-borne skp gene which may serve as a periplasmic chaperone.     

Molecular analysis of the Escherichia coli ruvC gene, which encodes a Holliday junction-specific endonuclease.

The Escherichia coli ruvC gene is involved in DNA repair and recombination and encodes an endonuclease that resolves Holliday structure in vitro. The 2.8-kb chromosomal DNA fragment that encompasses the ruvC gene and its flanking regions was cloned and sequenced. Four open reading frames were identified in the order orf17-orf26-ruvC-orf23 immediately upstream of the ruvAB operon, and their orientations are the same as the ruvAB operon, except for orf23. Proteins encoded by orf17, orf26, and ruvC (orf19) were identified by the maxicell method, and their sizes agreed with those predicted from the DNA sequences. Among the open reading frames in this region, only ruvC is involved in the repair of UV-damaged DNA. ruvC appeared to be regulated by at least two promoters, but, in contrast to the ruvAB operon, ruvC is not regulated by the SOS system as demonstrated by operon fusions.     

Stability studies of FhuA, a two-domain outer membrane protein from Escherichia coli

FhuA (MM 78.9 kDa) is an Escherichia coli outer membrane protein that transports iron coupled to ferrichrome and is the receptor for a number of bacteriophages and protein antibiotics. Its three-dimensional structure consists of a 22-stranded beta-barrel lodged in the membrane, extracellular hydrophilic loops, and a globular domain (the "cork") located within the beta-barrel and occluding it. This unexpected structure raises questions about the connectivity of the different domains and their respective roles in the different functions of the protein. To address these questions, we have compared the properties of the wild-type receptor to those of a mutated FhuA (FhuA Delta) missing a large part of the cork. Differential scanning calorimetry experiments on wild-type FhuA indicated that the cork and the beta-barrel behave as autonomous domains that unfold at 65 and 75 degrees C, respectively. Ferrichrome had a strong stabilizing effect on the loops and cork since it shifted the first transition to 71.4 degrees C. Removal of the cork destabilized the protein since a unique transition at 61.6 degrees C was observed even in the presence of ferrichrome. FhuA Delta showed an increased sensitivity to proteolysis and to denaturant agents and an impairment in phage T5 and ferrichrome binding.     

Mutants (ompA) affecting a major outer membrane protein of Escherichia coli K12.

Seventy independent mutants have been analyzed affecting a major protein, polypeptide II, of the outer cell envelope membrane from Escherichia coli K12. They were classified as nonsense mutants of the amber type (20%), mutants most likely of the missense type possessing the protein at normal concentrations (9%), and mutants either missing the protein or harboring it at much reduced concentrations for unknown reasons (71%). Forty of the mutants were analyzed genetically and all were found to map at or near ompA, the structural gene for protein II. Two-dimensional electrophoretic analyses of envelopes from such mutants revealed an unusual heterogeneity of the protein which on such patterns appeared as at least 12 well separated spots, and the majority of these is due to artifacts of the method but apparently specific for this protein. In no case was a polypeptide fragment found in envelopes from the nonsense mutants. The results are discussed regarding two different phages which use the protein as a receptor and concerning the biosynthetic incorporation of the protein into the outer membrane.