Escherichia coli K-12 tolF mutants: alterations in protein composition of the outer membrane.

Outer membrane materials prepared from three independently isolated spontaneous Escherichia coli tolF mutants contained no detectable protein Ia. The loss of this protein was nearly completely compensated for by an increase in other major outer membrane proteins, Ib and II. Thus, the major outer membrane proteins accounted for 40% of the total cell envelope protein in both tol+ and tolF strains. No changes were found in the levels of inner membrane proteins prepared from tolF strains when compared with similar preparations from the tol+ strain. Phage-resistant mutants were selected starting with a tolF strain by using either phage TuIb or phage PA2. These phage-resistant tolF strains contained neither protein Ia nor protein Ib. The mutation leading to the loss of protein Ib in these strains is independent of the tolF mutation and is located near malP on the E. coli genetic map.     

A new mechanism of antibiotic resistance in Enterobacteriaceae induced by a structural modification of the major porin

In Enterobacter aerogenes, multidrug resistance involves a decrease in outer membrane permeability associated with changes in an as yet uncharacterized porin. We purified the major porin from the wild-type strain and a resistant strain. We characterized this porin, which was found to be an OmpC/OmpF-like protein and analysed its pore-forming properties in lipid bilayers. The porin from the resistant strain was compared with the wild-type protein and we observed (i) that its single-channel conductance was 70% lower than that of the wild type; (ii) that it was three times more selective for cations; (iii) a lack of voltage sensitivity. These results indicate that the clinical strain is able to synthesize a modified porin that decreases the permeability of the outer membrane. Mass spectrometry experiments identified a G to D mutation in the putative loop 3 of the porin. Given the known importance of this loop in determining the pore properties of porins, we suggest that this mutation is responsible for the novel resistance mechanism developed by this clinical strain, with changes in porin channel function acting as a new bacterial strategy for controlling beta-lactam diffusion via porins.     

Outer membrane proteins of Escherichia coli. VI. Protein alteration in bacteriophage-resistant mutants.

Protein 1 was shown to be the receptor for phage PA-2 by the observations that the purified protein inactivates the phage, mutants lacking the protein are resistant to the phage, and mutants selected for PA-2 resistance have altered protein. Protein 1 appears as two bands (1a and 1b) on high-resolution polyacrylamide gels. The most abundant classes of mutants (ParI and ParII) selected for PA-2 resistance were found to lack band 1b. The mutations responsible for the ParI and ParII phenotypes were mapped at a locus termed par, which is near nalA on the Escherichia coli chromosome. The cyanogen bromide peptides of proteins 1a and 1b are similar, suggesting that these bands represent modified forms of the same polypeptide. Strains carrying the tolF mutation produce only band 1b. When a par tolF double mutant was constructed, this strain produced only band 1a. These results suggest that genes at the par and tolF loci are involved in modification of protein 1, or regulation of such modification, and are not structural genes for protein 1.     

Outer Membrane Proteins of Escherichia coli VII. Evidence That Bacteriophage-Directed Protein 2 Functions as a Pore

Protein 1, a major protein of the outer membrane of Escherichia coli, has been shown to be the pore allowing the passage of small hydrophilic solutes across the outer membrane. In E. coli K-12 protein 1 consists of two subspecies, 1a and 1b, whereas in E. coli B it consists of a single species which has an electrophoretic mobility similar to that of 1a. K-12 strains mutant at the ompB locus lack both proteins 1a and 1b and exhibit multiple transport defects, resistance to toxic metal ions, and tolerance to a number of colicins. Mutation at the tolF locus results in the loss of 1a, in less severe transport defects, and more limited colicin tolerance. Mutation at the par locus causes the loss of protein 1b, but no transport defects or colicin tolerance. Lysogeny of E. coli by phage PA-2 results in the production of a new major protein, protein 2. Lysogeny of K-12 ompB mutants resulted in dramatic reversal of the transport defects and restoration of the sensitivity to colicins E2 and E3 but not to other colicins. This was shown to be due to the production of protein 2, since lysogeny by phage mutants lacking the ability to elicit protein 2 production did not show this effect. Thus, protein 2 can function as an effective pore. ompB mutations in E. coli B also resulted in loss of protein 1 and similar multiple transport defects, but these were only partially reversed by phage lysogeny and the resulting production of protein 2. When the ompB region from E. coli B was moved by transduction into an E. coli K-12 background, only small amounts of proteins 1a and 1b were found in the outer membrane. These results indicate that genes governing the synthesis of outer membrane proteins may not function interchangeably between K-12 and B strains, indicating differences in regulation or biosynthesis of these proteins between these strains.     

Identification of three genes controlling production of new outer membrane pore proteins in Escherichia coli K-12.

Escherichia coli K-12 strains carrying mutations in the ompB gene or double mutations in the tolF and par genes lack the major outer membrane proteins 1a and 1b. These strains are deficient in the transport of small hydrophylic compounds and are multiply colicin resistant. When revertants of these strains were sought, a number of extragenic pseudorevertants were obtained which produced new outer membrane proteins. These new proteins could be divided into three classes by differences in electrophoretic mobility on polyacrylamide gels, by differing specificities for transport of small molecules, and by the identification of three different genetic loci for genes controlling their production. These genetic loci are designated as nmpA (at approximately 82.5 min on the E. coli K-12 genetic map), nmpB (8.6 min), and nmpC (12 min). The new proteins produced in strains carrying nmpA, nmpB, or nmpC mutations did not cross-react with antiserum against a mixture of proteins 1a and 1b, or with antiserum against phage-directed protein 2. Production of the new membrane proteins restored sensitivity to some of the colicins.     

Chromosomal location and expression of the structural gene for major outer membrane protein Ia of Escherichia coli K-12 and of the homologous gene of Salmonella typhimurium.

The gene determining the structure of a major outer membrane protein of Escherichia coli, protein Ia, has been located between serC and pyrD, at the min 21 region of the linkage map. This is based on the isolation and characterization of E. coli-Salmonella typhimurium intergeneric hybrids as well as analyses of a mutation (ompF2) affecting the formation of protein Ia. When the serC region of the S. typhimurium chromosome was transduced by phage P1 into E. coli, two classes of transductants were obtained; one produced protein Ia like the parental strain of E. coli, whereas the other produced not protein Ia but a pair of outer membrane proteins structurally related to 35K protein, one of the major outer membrane proteins of S. typhimurium. Furthermore, a strain of S. typhimurium harboring an F' plasmid which carries the ompF region of the E. coli chromosome was found to produce a protein indistinguishable from protein Ia, beside the outer membrane proteins characteristic to the parental Salmonella strain. These results suggest that the structural genes for protein Ia (E. coli) and for 35K protein (S. typhimurium) are homologous to each other and are located at the ompF region of the respective chromosome. The bearing of these findings on the genetic control of protein Ia formation is discussed.     

Characterization of a Vibrio cholerae virulence factor homologous to the family of TonB-dependent proteins

IrgA is an iron-regulated virulence factor for infection in an animal model with classical Vibrio cholerae strain 0395. We detected gene sequences hybridizing to irgA at high stringency in clinical isolates in addition to 0395, including another classical strain of V. cholerae, three V. cholerae strains of the El Tor biotype, three non-O1 isolates of V. cholerae, and individual isolates of Vibrio parahaemolyticus, Vibrio fluvialis, and Vibrio alginolyticus. No hybridization to irgA was seen with chromosomal DNA from Vibrio vulnificus or Aeromonas hydrophila. To verify that irgA is the structural gene for the major iron-regulated outer membrane protein of V. cholerae, we determined the amino-terminal sequence of this protein recovered after gel electrophoresis and demonstrated that it corresponds to the amino acid sequence of IrgA deduced from the nucleotide sequence. Gel electrophoresis showed that two El Tor strains of V. cholerae had a major iron-regulated outer membrane protein identical in size and appearance to IrgA in strain 0395, consistent with the findings of DNA hybridization. We have previously suggested that IrgA might be the outer membrane receptor for the V. cholerae siderophore, vibriobactin. Biological data presented here, however, show that a mutation in irgA had no effect on the transport of vibriobactin and produced no defect in the utilization of iron from ferrichrome, ferric citrate, haemin or haemoglobin. The complete deduced amino acid sequence of IrgA demonstrated homology to the entire class of Escherichia coli TonB-dependent proteins, particularly Cir. Unlike the situation with Cir, however, we were unable to demonstrate a role for IrgA as a receptor for catechol-substituted cephalosporins. The role of IrgA in the pathogenesis of V. cholerae infection, its function as an outer membrane receptor, and its potential interaction with a TonB-like protein in V. cholerae remain to be determined.     

The surface properties of Neisseria gonorrhoeae: topographical distribution of the outer membrane protein antigens.

Gonococci were labelled with 125I using the lactoperoxidase system. The amount of label incorporated was similar with all strains including those which appeared capsulated. Electrophoresis on sodium dodecyl sulphate-polyacrylamide gels revealed that the major proteins labelled were those found in outer membrane preparations. Comparison of variants of one strain showed that the major outer membrane protein (protein I) was always present and heavily labelled. The second major protein (protein II) was present in variable amounts but labelling was proportional to the amount present. A third protein (III) was only present in outer membranes from a freshly isolated variant but was present in whole cells of each strain. Protein III was not labelled in whole cells but was labelled in outer membrane preparations suggesting that many membranes have their inner surface exposed. The labelling of a strain adapted to growth in guinea-pig chambers failed to reveal any new major surface proteins. The results demonstrate the variation in surface topography possible with variants of one strain of gonococcus but show that one major protein antigen is always expressed on the surface.     

Two bacteriophages which utilize a new Escherichia coli major outer membrane protein as part of their receptor.

Escherichia coli strain JF694 contains a new major outer membrane protein which we have called protein E (J. Foulds, and T. Chai, J. Bacteriol. 133:1478-1483). Two new bacteriophages, TC45 and TC23, were isolated that require the presence of protein E in the outer membrane of host cells for growth. Both of these bacteriophages have a morphology similar to T-even bacteriophages but are distinct in properties such as plaque morphology, buoyant density, and burst size. Although strain JF694, containing protein E, adsorbs bacteriophage TC45 efficiently, cells killed with heat or chloroform are unable to inactivate this bacteriophage. Purified protein E either in the presence or absence of additional probable cofactors such as lipopolysaccharide was also unable to inactivate bacteriophage TC45. Both bacteriophages probably use protein E as at least part of their receptor but require, in addition, other outer membrane components or a specific orientation or organization of this protein in the outer membrane.     

Genetic interaction between the Escherichia coli AcpT phosphopantetheinyl transferase and the YejM inner membrane protein

Strain LH530, a mutant of Escherichia coli K-12, was reported by others to show increased outer membrane permeability, temperature-sensitive growth, and reduced synthesis of lipid A. The unmapped mutant gene was found to be suppressed by high-copy-number plasmids carrying the wild-type acpT gene, which encodes a protein that catalyzes a post-translational protein modification, the attachment of 4'-phosphopantetheine. We mapped the strain LH530 mutation to a gene of unknown function, yejM, known to encode an inner membrane protein. The mutation is a yejM nonsense mutation that produces a truncated protein lacking the predicted periplasmic domain. Reconstruction of the mutation gave a strain having the same phenotypes as LH530. In contrast to the nonsense mutants, deletion of the entire yejM gene was lethal. Suppression by AcpT overexpression of the yejM nonsense mutants encoding the truncated proteins was specific to AcpT. Moreover, AcpT overexpression also suppressed the lethality due to deletion of the entire yejM gene and this suppression also did not require that AcpT be enzymatically active. The mechanism whereby overexpression of a specific cytosolic protein bypasses the essentiality of an inner membrane protein is unknown.     

Composition of major outer membrane proteins of Vibrio vulnificus isolates: effect of different growth media and iron deficiency

The outer membrane proteins of Vibrio vulnificus including isolates from humans, seawater and an asari clam were examined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. A major outer membrane protein with an apparent molecular weight of 48,000 (48K protein) was common to all the strains grown in 3% NaCl-nutrient broth; however this 48K protein was not produced in any of the strains grown in chemically defined medium. Other major outer membrane proteins with molecular weights ranging from 33,000 to 40,000 varied in number, relative amount and molecular weight depending on the strain. One to three new outer membrane proteins with molecular weights ranging from 74,000 to 85,000 were produced in the cells grown in iron-deficient medium. The 48K protein and one or two major proteins with molecular weights ranging from 35,000 to 37,000 in the cells grown in 3% NaCl-nutrient broth were not solubilized by 2% SDS at 60 C for 30 min and were resistant to trypsin, indicating that they are porins. On the other hand, in cells grown in chemically defined medium, one or two major outer membrane proteins with molecular weights ranging from 33,000 to 40,000 might be porins.     

Pal Lipoprotein of Escherichia coli Plays a Major Role in Outer Membrane Integrity

The Tol-Pal system of gram-negative bacteria is composed of five proteins. TolA, TolQ, and TolR are inner membrane proteins, TolB is a periplasmic protein, and Pal, the peptidoglycan-associated lipoprotein, is anchored to the outer membrane. In this study, the roles of Pal and major lipoprotein Lpp were compared in Escherichia coli. lpp and tol-pal mutations have previously been found to perturb the outer membrane permeability barrier and to cause the release of periplasmic proteins and the formation of outer membrane vesicles. In this study, we showed that the overproduction of Pal is able to restore the outer membrane integrity of an lpp strain but that overproduced Lpp has no effect in a pal strain. Together with the previously reported observation that overproduced TolA complements an lpp but not a pal strain, these results indicate that the cell envelope integrity is efficiently stabilized by an epistatic Tol-Pal system linking inner and outer membranes. The density of Pal was measured and found to be lower than that of Lpp. However, Pal was present in larger amounts compared to TolA and TolR proteins. The oligomeric state of Pal was determined and a new interaction between Pal and Lpp was demonstrated.     

New locus (ttr) in Escherichia coli K-12 affecting sensitivity to bacteriophage T2 and growth on oleate as the sole carbon source.

The nature of resistance to phage T2 in Escherichia coli K-12 was investigated by analyzing a known phage T2-resistant mutant and by isolating new T2-resistant mutants. It was found that mutational alterations at two loci, ompF (encoding the outer membrane protein OmpF) and ttr (T-two resistance), are needed to give full resistance to phage T2. A ttr::Tn10 mutation was isolated and was mapped between aroC and dsdA, where the fadL gene (required for long-chain fatty acid transport) is located. The receptor affected by ttr was the major receptor used by phage T2 and was located in the outer membrane. Phage T2 was thus able to use two outer membrane proteins as receptors. All strains having a ttr::Tn10 allele and most of the independently isolated phage T2-resistant mutants were unable to grow on oleate as the sole carbon and energy source, i.e., they had the phenotype of fadL mutants. The gene fadL is known to encode an inner membrane protein. The most likely explanation is that fadL and ttr are in an operon and that ttr encodes an outer membrane protein which functions in translocating long-chain fatty acids across the outer membrane and also as a receptor for phage T2.     

Purification of protein A, an outer membrane component missing in Escherichia coli K-12 ompA mutants.

Outer membrane materials prepared from an Escherichia coli ompA (tolG) strain do not contain one of the major outer membrane proteins found in ompA+ strains. This protein has been purified in high yield from detergent-solubilized cell envelope material prepared from an ompA+ strain by preparative electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate. The purified protein is homogeneous in three electrophoretic systems, contains 2 mol of reducing sugar/mol of peptide and has alanine as the N-terminal amino acid. The amino acid composition is nearly identical to outer membrane protein II or B purified by others from incompletely solubilized cell envelope material. Thus, the fraction of outer membrane protein II or B that is difficult to solubilize is identical with the more readily solubilized fraction.     

Differential analysis of Saccharomyces cerevisiae mitochondria by free flow electrophoresis

One major problem concerning the electrophoresis of mitochondria is the heterogeneity of mitochondrial appearance especially under pathological conditions. We show here the use of zone electrophoresis in a free flow electrophoresis device (ZE-FFE) as an analytical sensor to discriminate between different yeast mitochondrial populations. Impairment of the structural properties of the organelles by hyperosmotic stress resulted in broad separation profiles. Conversely untreated mitochondria gave rise to homogeneous populations reflected by sharp separation profiles. Yeast mitochondria with altered respiratory activity accompanied by a different outer membrane proteome composition could be discriminated based on electrophoretic deflection. Proteolysis of the mitochondrial surface proteome and the deletion of a single major protein species of the mitochondrial outer membrane altered the ZE-FFE deflection of these organelles. To demonstrate the usefulness of ZE-FFE for the analysis of mitochondria associated with pathological processes, we analyzed mitochondrial fractions from an apoptotic yeast strain. The cdc48(S565G) strain carries a mutation in the CDC48 gene that is an essential participant in the endoplasmic reticulum-associated protein degradation pathway. Mutant cells accumulate polyubiquitinated proteins in microsomal and mitochondrial extracts. Subsequent ZE-FFE characterization could distinguish a mitochondrial subfraction specifically enriched with polyubiquitinated proteins from the majority of non-affected mitochondria. This result demonstrates that ZE-FFE may give important information on the specific properties of subpopulations of a mitochondrial preparation allowing a further detailed functional analysis.     

Expression of Escherichia coli PhoE protein in avirulent Salmonella typhimurium aroA and galE strains

Abstract Escherichia coli K-12 PhoE protein is found to be normally expressed and incorporated into the outer membrane of two avirulent Salmonella typhimurium strains, G30 and SH aroA . A hybrid protein which contains an insertion of an antigenic epitope of VP1 protein of foot-and-mouth disease virus into the PhoE protein, was also normally assembled into the Salmonella outer membranes. In the case of the G30 stain, which carries a galE mutation, the inserted epitope is accessible to antibodies in intact cells. In contrast, the epitope is less accessible in the case of the SH aroA strain, probably due to the shielding effect of the O-antigen in this strain.     

Isolation and characterization of mutants of a marine Vibrio strain that are defective in production of extracellular proteins

A marine Vibrio strain, Vibrio sp. strain 60, produces several extracellular proteins, including protease, amylase, DNase, and hemagglutinin. Mutants of Vibrio sp. strain 60 (epr mutants) pleiotropically defective in production of these extracellular proteins were isolated. They fell into two classes, A and B. In class A, no protease activity was detected in the cells either, whereas in class B, considerable protease activity was detected in the cells. Gel electrophoretic analysis revealed that the protease detected in class B mutant cells was similar to the protease excreted by the parent strain. In addition, the protease in class B mutant cells was found to be localized in the periplasmic space. These results suggest that the passage of the protease through the outer membrane is blocked in class B mutants. Comparison of membrane protein profiles by polyacrylamide gel electrophoresis revealed that all the epr mutants contained an increased amount of a 94,000-Mr protein that may be an outer membrane protein. Four epr mutations were mapped in two different regions of the Vibrio chromosome by transduction; two class A mutations and one class B mutation were located close to each other, whereas another class B mutation was located in a different region of the chromosome.     

Correlation Between the Expression of an Escherichia coli Cell Surface Protein and the Ability of the Protein to Bind to Lipopolysaccharide

The ompA gene of Escherichia coli codes for a major protein of the outer membrane. When this gene was moved between various unrelated strains (E. coli K-12 and two clinical isolates of E. coli) by transduction, the gene was expressed very poorly. Recombinants carrying “foreign” genes produced no OmpA protein which could be detected on polyacrylamide gels and became resistant to bacteriophage K3, which uses this protein as receptor. The recombinants were sensitive to host-range mutants of K3, indicating a very low level of OmpA protein was produced. When an E. coli K-12 recombinant carrying an unexpressed foreign ompA allele was subjected to two cycles of selection for an OmpA⁺ phenotype, a mutant strain was obtained which was sensitive to K3 and which expressed nearly normal levels of OmpA protein in the outer membrane. This strain carried mutations in the foreign ompA gene, as indicated both by genetic mapping and the alteration of a peptide in the mutant OmpA protein. The ability of the OmpA protein to bind to lipopolysaccharide (LPS) showed similar strain specificity, and the mutant OmpA protein which was expressed in an unrelated host showed enhanced ability to bind LPS from its new host. Thus, cell surface expression of the ompA gene appears to depend upon the ability of the gene product to bind LPS, suggesting that an interaction between the protein and LPS plays an essential role in biosynthesis of this outer membrane protein.