Identification of novel subunits of the TOM complex from Arabidopsis thaliana

The preprotein translocase of the outer mitochondrial membrane (also called TOM complex) from Arabidopsis thaliana was characterized by Blue-native gel electrophoresis (BN-PAGE) and Electrospray Tandem Mass Spectrometry (ESI-MS/MS). BN-PAGE allows to prepare a very stable 390 kDa complex that includes six different protein types: the 34 kDa translocation pore TOM40, the 21/23 kDa preprotein receptor TOM20, the small TOM component TOM7 and three further subunits of 10, 6.3 and 6.0 kDa. Primary structures of all TOM subunits were elucidated. The 10 kDa subunit represents a truncated version of the TOM22 preprotein receptor and the two 6 kDa proteins represent subunits possibly homologous to fungal TOM6 and TOM5, although sequence conservation is at the borderline of significance. TOM40, TOM7 and one or both of the 6 kDa subunits form a subcomplex of about 100 kDa. The six TOM proteins from Arabidopsis are encoded by 12 genes, at least 11 of which are expressed. While the subunit composition of the TOM complex from fungi, animals and plants is remarkably conserved, the domain structure of individual TOM proteins differs, e.g. acidic domains in TOM22 and the 6 kDa TOM subunits from Arabidopsis are absent. The domain structure of the Arabidopsis TOM complex does not support the so-called ‘acid chain hypothesis’, which explains the translocation of proteins across the outer mitochondrial membrane of mitochondria by the binding of preproteins to acidic protein domains within the TOM complex. Functional implications are discussed.     

Location and characterization of genes involved in binding of starch to the surface of Bacteroides thetaiotaomicron.

Previous studies of starch utilization by the gram-negative anaerobe Bacteroides thetaiotaomicron have demonstrated that the starch-degrading enzymes are cell associated rather than extracellular, indicating that the first step in starch utilization is binding of the polysaccharide to the bacterial surface. Five transposon-generated mutants of B. thetaiotaomicron which were defective in starch binding (Ms-1 through Ms-5) had been isolated, but initial attempts to identify membrane proteins missing in these mutants were not successful. We report here the use of an immunological approach to identify four maltose-inducible membrane proteins, which were missing in one or more of the starch-binding mutants of B. thetaiotaomicron. Three of the maltose-inducible proteins were outer membrane proteins (115, 65, and 43 kDa), and one was a cytoplasmic membrane protein (80 kDa). The genes encoding these proteins were shown to be clustered in an 8.5-kbp segment of the B. thetaiotaomicron chromosome. Two other loci defined by transposon insertions, which appeared to contain regulatory genes, were located within 7 kbp of the cluster of membrane protein genes. The 115-kDa outer membrane protein was essential for utilization of maltoheptaose (G7), whereas loss of the other proteins affected growth on starch but not on G7. Not all of the proteins missing in the mutants were maltose regulated. We also detected two constitutively produced proteins (32 and 50 kDa) that were less prominent in all of the mutants than in the wild type. Both of these were outer membrane proteins.     

Structural determinants of processing and secretion of the Haemophilus influenzae Hap protein

Haemophilus influenzae elaborates a surface protein called Hap, which is associated with the capacity for intimate interaction with cultured epithelial cells. Expression of hap results in the production of three protein species: outer membrane proteins of approximately 155 kDa and 45 kDa and an extracellular protein of approximately 110 kDa. The 155 kDa protein corresponds to full-length mature Hap (without the signal sequence), and the 110 kDa extracellular protein represents the N-terminal portion of mature Hap (designated Hap_s). In the present study, we examined the mechanism of processing and secretion of Hap. Site-directed mutagenesis suggested that Hap is a serine protease that undergoes autoproteolytic cleavage to generate the 110 kDa extracellular protein and the 45 kDa outer membrane protein. Biochemical analysis confirmed this conclusion and established that cleavage occurs on the bacterial cell surface. Determination of N-terminal amino acid sequence and mutagenesis studies revealed that the 45 kDa protein corresponds to the C-terminal portion of Hap, starting at N1037. Analysis of the secondary structure of this protein (designated Hap_β) predicted formation of a β-barrel with an N-terminal transmembrane α-helix followed by 14 transmembrane β-strands. Additional analysis revealed that the final β-strand contains an amino acid motif common to other β-barrel outer membrane proteins. Upon deletion of this entire C-terminal consensus motif, Hap could no longer be detected in the outer membrane, and secretion of Hap_s was abolished. Deletion or complete alteration of the final three amino acid residues had a similar but less dramatic effect, suggesting that this terminal tripeptide is particularly important for outer membrane localization and/or stability of the protein. In contrast, isolated point mutations that disrupted the amphipathic nature of the consensus motif or eliminated the C-terminal tryptophan had no effect on outer membrane localization of Hap or secretion of Hap_s. These results provide insight into a growing family of Gram-negative bacterial exoproteins that are secreted by an IgA1 protease-like mechanism; in addition, they contribute to a better understanding of the structural determinants of targeting of β-barrel proteins to the bacterial outer membrane.     

Properties of the Burkholderia pseudomallei insertional mutants deficient in membrane proteins production

Transposon-induced B. pseudomallei mutants deficient in membrane proteins production were obtained for evaluation of the functional role of these cell components. In comparison with the wild type strain B. pseudomallei 57576, mutant clones TTM6, TTM7 and TTM9 carrying Tn5 chromosome insertions were characterized by lost or decreased production of outer membrane proteins 27, 48, 52, 150, 200 kDa. Alterations in outer membrane protein spectra were accompanied by twofold increase in susceptibility of bacteria to fluoroquinolones (pefloxacin, ofloxacin) and cephalosporins (ceftazidime) and noticeable reduction of virulence for white mice and guinea pigs in contrast to the initial strain, the obtained mutants were also less resistant in in vitro phagocyte killing.     

Protein interactions in the outer membrane of Escherichia coli.

Specific protein interactions in Escherichia coli outer membrane were analyzed using chemical cross-linking with truly cleavable reagents and symmetrical two-dimensional sodium dodecyl sulphate/polyacrylamide gel electrophoresis. The major outer membrane proteins were shown to form cross-linked complexes. These include multimers of lambda receptor, protein I, II, III and the free form of lipoprotein. Lipoprotein was also found to be cross-linked to proteins II and III. The identity of many of these complexes was verified using appropriate mutants missing the proteins in question. No new protein interactions were detected in the mutants even when three of the major proteins were missing. Proteins II, III and the free form of lipoprotein could also be cross-linked to the peptidoglycan layer of the cell wall.     

The N-terminal 21 amino acids of a 70 kDa protein of the yeast mitochondrial outer membrane direct E. coli beta-galactosidase into the mitochondrial matrix space in yeast cells

The intracellular location of fusion proteins was investigated in yeast cells. They consisted of the N-terminal 21, 61 or 292 amino acids of the 70 kDa protein of the yeast mitochondrial outer membrane and an enzymatically active E. coli beta-galactosidase. The hybrids containing 61 or 292 residues of the 70 kDa protein, as well as the original 70 kDa protein, were localized on the outer membrane in a tightly membrane-bound form. In contrast, the other hybrid was exclusively localized in the mitochondrial matrix space as a soluble protein.     

Identification, isolation, and characterization of the 42-kilodalton major outer membrane protein (MompA) from Treponema pectinovorum ATCC 33768.

The major protein present in the isolated outer membrane of Treponema pectinovorum ATCC 33768, MompA, was identified, purified, and characterized. Immuno-gold electron microscopy, using anti-MompA serum, and cell fractionation experiments confirmed the localization of MompA to the outer membrane. MompA was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to have a molecular mass of 42 kDa when heat denatured, whereas native MompA formed a number of detergent-stable forms with molecular masses of 71, 76, and 83 kDa. A temperature of 60 degrees C was required to convert the native protein to the 42-kDa form. A number of detergents and chemical agents that are capable of breaking ionic and hydrogen bonds of proteins did not convert native MompA to the 42-kDa species. The native forms of the protein were resistant to the combined action of proteinase K, trypsin, and chymotrypsin, whereas the 42-kDa form of MompA was not. The N-terminal amino acid sequence of MompA was determined to be DVTVNINSRVRPVLYTT, and database searches did not identify any homology with known protein sequences. Amino acid compositional analysis showed the protein to be rich in proline and glycine, with these amino acids accounting for 28 and 13%, respectively, of the total amino acids. Antiserum raised against the major outer membrane protein of T. denticola GM-1 and ATCC 35405 did not cross-react with MompA, and antiserum raised against MompA did not react with any cellular components of Treponema denticola, Treponema vincentii, or Treponema socranskii. A major outer membrane protein similar in molecular mass to MompA was identified in eight clinical isolates of T. pectinovorum. The major outer membrane protein produced by four of the clinical isolates reacted strongly, by Western blotting, with anti-MompA serum, whereas proteins of the other strains did not.     

Purification and proteomic analysis of outer membrane vesicles from a clinical isolate of Leptospira interrogans serovar Copenhageni

The severe pulmonary form of leptospirosis (SPFL) is an especially serious and rapid disease process characterized by alveolar hemorrhage and acute respiratory failure. The outer membrane of Leptospira facilitates direct interactions with the environs and likely contains important constituents involved during infection, transmission, survival, and adaptation to environmental conditions, including putative vaccinogen and diagnostic candidates. Outer membrane vesicles (OMVs) were purified by incubation in low-pH citrate buffer, treatment in a French press, and centrifugation over a continuous sucrose gradient. OMVs characterized by two-dimensional gel electrophoresis (2-DE) contained the previously described outer membrane proteins OmpL1, Qlp42, LipL32, LipL41, LipL36 and Loa22. In addition, unknown, hypothetical and putative outer membrane proteins were identified. High-performance liquid chromatography (HPLC) coupled with mass spectrometry and fraction collection (LC-MS+) measured the intact mass profile of the major outer membrane protein, LipL32, and the putative lipoprotein Qlp42. In contrast to a predicted molecularmass of 27,653.5 Da for LipL32 after cleavage of its signal peptide, intact mass proteomics measured the mass as ranging from 28,468 to 28,583 Da, consistent with lipidation of LipL32. In contrast to a predicted molecular mass of 39.8 kDa for Qlp42, the actual mass was measured as 24,811 and 26,461 Da consistent with a 30 kDa doublet observed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and processing of the N-terminus of the mature protein. These studies indicate that purified OMVs are highly compatible with proteomics technologies including 2-DE and intact mass proteomics using LC-MS+ that facilitates definition of actual molecular masses of intact outer membrane proteins, and heterogeneity associated with them.     

The virulence factor PEB4 (Cj0596) and the periplasmic protein Cj1289 are two structurally related SurA-like chaperones in the human pathogen Campylobacter jejuni

The PEB4 protein is an antigenic virulence factor implicated in host cell adhesion, invasion, and colonization in the food-borne pathogen Campylobacter jejuni. peb4 mutants have defects in outer membrane protein assembly and PEB4 is thought to act as a periplasmic chaperone. The crystallographic structure of PEB4 at 2.2-? resolution reveals a dimer with distinct SurA-like chaperone and peptidyl-prolyl cis/trans isomerase (PPIase) domains encasing a large central cavity. Unlike SurA, the chaperone domain is formed by interlocking helices from each monomer, creating a domain-swapped architecture. PEB4 stimulated the rate of proline isomerization limited refolding of denatured RNase T(1) in a juglone-sensitive manner, consistent with parvulin-like PPIase domains. Refolding and aggregation of denatured rhodanese was significantly retarded in the presence of PEB4 or of an engineered variant specifically lacking the PPIase domain, suggesting the chaperone domain possesses a holdase activity. Using bioinformatics approaches, we identified two other SurA-like proteins (Cj1289 and Cj0694) in C. jejuni. The 2.3-? structure of Cj1289 does not have the domain-swapped architecture of PEB4 and thus more resembles SurA. Purified Cj1289 also enhanced RNase T(1) refolding, although poorly compared with PEB4, but did not retard the refolding of denatured rhodanese. Structurally, Cj1289 is the most similar protein to SurA in C. jejuni, whereas PEB4 has most structural similarity to the Par27 protein of Bordetella pertussis. Our analysis predicts that Cj0694 is equivalent to the membrane-anchored chaperone PpiD. These results provide the first structural insights into the periplasmic assembly of outer membrane proteins in C. jejuni.     

Identification of an OprD homologue in Acinetobacter baumannii

With the increased number of resistant Acinetobacter baumannii strains, it is urgently required to decipher the molecular bases of outer membrane permeability. The analyses of the outer membrane from different A. baumannii strains indicated a modification in the expression of two proteins of 29 and 43 kDa, respectively. By electrophoresis and MALDI-MS analyses, the 43 kDa OMP was identified as a protein belonging to the OprD family, a basic amino acid and imipenem porin.     

Phenylalanine hydroxylase-like antibody in human chorionic villi]

An antigen similar by electrophoretic mobility to liver phenylalanine hydroxylase (PH) and cross-reacting with monoclonal antibody PH8 against liver PH was detected in extracts of soluble proteins in 6 from 23 samples of chorionic villi. An antigen with electrophoretic mobility corresponding to 40-41 kDa was detected in extracts of membrane proteins from these 23 samples by immunoblotting with monoclonal antibody PH8. Its molecular weight was similar to that of major chymotryptic peptide of human liver PH. The content of the antigen varied with samples and was less than 20 ng/mg of the extracted protein. Two-dimensional gel electrophoresis revealed only 1 spot of the antigen. The antigen did not react with monoclonal antibodies PH7 and PH9 epitopes of which were located in N-terminal fragment of liver PH. These data suggest that the antigen of membrane fraction could be a PH protein without N-terminal domain.     

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.     

Membrane proteins PmpG and PmpH are major constituents of Chlamydia trachomatis L2 outer membrane complex

The outer membrane complex of Chlamydia is involved in the initial adherence and ingestion of Chlamydia by the host cell. In order to identify novel proteins in the outer membrane of Chlamydia trachomatis L2, proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. By silver staining of the protein profile, a major protein doublet of 100-110 kDa was detected. In-gel tryptic digestion and matrix-assisted laser desorption/ionization mass spectrometry identified these proteins as the putative outer membrane proteins PmpG and PmpH.     

The chloroplastic protein import machinery contains a Rieske-type iron-sulfur cluster and a mononuclear iron-binding protein.

Transport of precursor proteins across the chloroplastic envelope membranes requires the interaction of protein translocons localized in both the outer and inner envelope membranes. Analysis by blue native gel electrophoresis revealed that the translocon of the inner envelope membranes consisted of at least six proteins with molecular weights of 36, 45, 52, 60, 100 and 110 kDa, respectively. Tic110 and ClpC, identified as components of the protein import apparatus of the inner envelope membrane, were prominent constituents of this complex. The amino acid sequence of the 52 kDa protein, deduced from the cDNA, contains a predicted Rieske-type iron-sulfur cluster and a mononuclear iron-binding site. Diethylpyrocarbonate, a Rieske-type protein-modifying reagent, inhibits the translocation of precursor protein across the inner envelope membrane, whereas binding of the precursor to the outer envelope membrane is still possible. In another independent experimental approach, the 52 kDa protein could be co-purified with a trapped precursor protein in association with the chloroplast protein translocon subunits Toc86, Toc75, Toc34 and Tic110. Together, these results strongly suggest that the 52 kDa protein, named Tic55 due to its calculated molecular weight, is a member of the chloroplastic inner envelope protein translocon.     

Fuctional organization of the outer membrane of escherichia coli: Phage and colicin receptors as components of iron uptake systems

The functional interaction of outer memberane proteins of E. coli can be studied using phage and colicin receptors which are essential components of penetration systems. The uptake of ferric iron in the form of the ferrichrome complex requires the ton A and ton B functions in the outer membrane of E. coli. The ton A gene product is the receptor protein for phage T5 and is required together with the ton B function by the phages T1 anf ?80 to infect cells and by colicin M and the antibiotic albomycin, a structural analogue of ferrichrome, to kill cells. The ton B function is necessary for the uptake of ferric iron complexed by citrate. Iron complexed by enterochelin is only transported in the presence of the ton B and feu functions. Cells which have lost the feu function are resistant to the colicins B, I or V while ton B mutants are resistant to all colicins. The interaction of the ton A, Ton B, and feu functions apparently permits quite different “substrates” to overcome the permeablility barrier of the outer membrane. It was shown for ferrichrome dependent iron uptake that the complexing agent was not altered and could be used repeatedly. Only very low amounts of ³H-labeled ferrichrome were found in the cell. It is possible that the iron is mobilized in the membrane and that desferriferrichrome is released into the medium without having entered the cytoplasm. Growth on ferrichrome as the sole iron source waw used to select revertants of T5 resistant ton A mutants. All revertants exhibited wild-type properties with the exception of partial revertants. In these 4 strains, as in the ton A mutants, the ton A protein was not detectable by SDS polyacrylamide gel electrophoreses of outer membranes. Albomycin resistant mutants were selected and shown to fall into 5 categories: (1) ton A; (2) ton B mutants; (3) mutants with no iron transport defects and normal ton A/ton B functions, which might be target site mutants; (4) mutants which were deficient in ferrichrome-mediated iron uptake but had normal ton A/ton B functions. We tentatively consider that the defect might be located in the active transport system of the cytoplasmic membrane; (5) a variety of mutants with the following general properties: most of them were resistant to colicin M, transported iron poorly, and, like ton B mutants, contained additional proteins in the outer membrane. The outer membrane protein patterns of wild-type and ton B mutant strains were compared by slab gel electrophoresis in an attempt to identify a ton B protein. It was observed that under most growth conditions, ton B mutants overproduced 3 proteins of molecular weights 74,000–83,000. In extracted, iron-deficient medium, both the wild-type and ton B mutant strains had similar large amounts of these proteins in their outer membranes. The appearance of these proteins was suppressed by excess iron in both wild-type and mutant. From this evidence it is apparent that the proteins appear as a response to low intracellular iron rather than being controlled by the ton B gene. The nature of these proteins and their possible role in iron transport is disussed.     

Identification and localization of Myxococcus xanthus porins and lipoproteins

Myxococcus xanthus DK1622 contains inner (IM) and outer membranes (OM) separated by a peptidoglycan layer. Integral membrane, β-barrel proteins are found exclusively in the OM where they form pores allowing the passage of nutrients, waste products and signals. One porin, Oar, is required for intercellular communication of the C-signal. An oar mutant produces CsgA but is unable to ripple or stimulate csgA mutants to develop suggesting that it is the channel for C-signaling. Six prediction programs were evaluated for their ability to identify β-barrel proteins. No program was reliable unless the predicted proteins were first parsed using Signal P, Lipo P and TMHMM, after which TMBETA-SVM and TMBETADISC-RBF identified β-barrel proteins most accurately. 228 β-barrel proteins were predicted from among 7331 protein coding regions, representing 3.1% of total genes. Sucrose density gradients were used to separate vegetative cell IM and OM fractions, and LC-MS/MS of OM proteins identified 54 β-barrel proteins. Another class of membrane proteins, the lipoproteins, are anchored in the membrane via a lipid moiety at the N-terminus. 44 OM proteins identified by LC-MS/MS were predicted lipoproteins. Lipoproteins are distributed between the IM, OM and ECM according to an N-terminal sorting sequence that varies among species. Sequence analysis revealed conservation of alanine at the +7 position of mature ECM lipoproteins, lysine at the +2 position of IM lipoproteins, and no noticable conservation within the OM lipoproteins. Site directed mutagenesis and immuno transmission electron microscopy showed that alanine at the +7 position is essential for sorting of the lipoprotein FibA into the ECM. FibA appears at normal levels in the ECM even when a +2 lysine is added to the signal sequence. These results suggest that ECM proteins have a unique method of secretion. It is now possible to target lipoproteins to specific IM, OM and ECM locations by manipulating the amino acid sequence near the +1 cysteine processing site.     

Isolation and partial characterization of a 39 kDa major outer membrane protein of Actinobacillus actinomycetemcomitans Y4

The outer membrane fractions of Actinobacillus actinomycetemcomitans, which were extracted from whole cells with cetyl trimethyl ammonium bromide and CaCl2, contained four major outer membrane proteins (MOMP) of 39, 37, 36 and 30 kDa. The 39 kDa MOMP of A. actinomycetemcomitans was sequentially purified by extraction with Zwittergent 3-14 detergent, anion-exchange chromatography and gel filtration chromatography. Analysis of amino acid composition and N-terminal amino acid sequence of 20 residues of purified 39 kDa MOMP was performed. Although some of the periodontitis patient sera reacted strongly with 39 kDa and 30 kDa MOMP in crude outer membrane fractions, purified 39 kDa MOMP showed decreased immunoreactivity with the human sera.     

Expression, folding, and characterization of small proteins with increasing disulfide complexity by a pT7-7-derived phagemid

The expression, folding, and characterization of a series of small proteins with increasingly complex disulfide bond patterns were characterized. A phagemid was prepared from the pT7-7 plasmid to facilitate mutagenic studies with these proteins. cDNAs coding for bovine, rat, and human prolactin; human growth hormone; and bovine alpha-lactalbumin were amplified by PCR using primers that inserted restriction sites at the 5' and 3' ends and reduced the coding sequence to the mature methionyl protein with bacterially preferred codons in the 5' region. The expressed proteins were folded and oxidized by methods that allowed disulfide bond formation to occur either during or following folding. The effectiveness of the folding procedures was determined for each protein by electrophoresis, absorption spectroscopy, and functional studies. The redox conditions required for folding functional proteins varied as the number of disulfide bonds per unit molecular weight increased. Human growth hormone, 22 kDa; human prolactin, 23 kDa; and bovine prolactin, 23 kDa, contain two, three, and three disulfides, respectively, and are folded correctly by air oxidation performed during renaturation under alkaline conditions. Proper disulfide bond formation of rat prolactin, 23 kDa, containing three disulfide bonds required the addition of a reducing agent at the initiation of renaturation. Bovine alpha-lactalbumin, 14 kDa with four disulfide bonds, required complete renaturation prior to the removal of a reducing agent. SDS-gel electrophoresis under nonreducing conditions provided information regarding the proper folding of these proteins. The absorption of 250-nm light by disulfide bonds also provided information regarding the proper folding of rat prolactin and bovine alpha-lactalbumin.     

Identification and characterization of a novel outer membrane protein (OMP J) of Moraxella catarrhalis that exists in two major forms

Moraxella catarrhalis is a common commensal of the human respiratory tract that has been associated with a number of disease states, including acute otitis media in children and exacerbations of chronic obstructive pulmonary disease in adults. During studies to investigate the outer membrane proteins of this bacterium, two novel major proteins, of approximately 19 kDa and 16 kDa (named OMP J1 and OMP J2, respectively), were identified. Further analysis indicated that these two proteins possessed almost identical gene sequences, apart from two insertion/deletion events in predicted external loops present within the putative barrel-like structure of the proteins. The development of a PCR screening strategy found a 100% (96/96) incidence for the genes encoding the OMP J1 and OMP J2 proteins within a set of geographically diverse M. catarrhalis isolates, as well as a significant association of OMP J1/OMP J2 with both the genetic lineage and the complement resistance phenotype (Fisher's exact test; P < 0.01). Experiments using two DeltaompJ2 mutants (one complement resistant and the other complement sensitive) indicated that both were less easily cleared from the lungs of mice than were their isogenic wild-type counterparts, with a significant difference in bacterial clearance being observed for the complement-resistant isolate but not for its isogenic DeltaompJ2 mutant (unpaired Student's t test; P < 0.001 and P = 0.32). In this publication, we characterize a novel outer membrane protein of Moraxella catarrhalis which exists in two variant forms associated with particular genetic lineages, and both forms are suggested to contribute to bacterial clearance from the lungs.