Outer Membrane Fraction Research Articles

The Malonyl-CoA-sensitive Form of Carnitine Palmitoyltransferase Is Not Localized Exclusively in the Outer Membrane of Rat Liver Mitochondria

The data used to support the idea that malonyl-coenzyme A (CoA)-sensitive carnitine palmitoyltransferase (CPT-I) is localized on the outer mitochondrial membrane are based on harsh techniques that disrupt mitochondrial physiology. We have turned to the use of the French press, which produces a shearing force that denudes mitochondria of their outer membrane without the physiologically disruptive effects characteristic of phosphate swelling. Our results indicate that the mitoplasts contain just 15-19% of the outer membrane marker enzyme activity while retaining 85% of the total CPT activity and 50% of both CPT-I, as well as long-chain acyl-CoA synthase activity, the latter two supposed outer membrane enzymes. These mitoplasts were shown by electron microscopy to have the configuration of mitochondria that merely have been divested of their outer membranes. Carnitine-dependent fatty acid oxidation was retained in the mitoplasts, showing that they were physiologically intact. Moreover, protein immunoblotting analysis showed that CPT-I, as well as the inner CPT-II, was localized in the mitoplast fraction. The outer membrane fraction, which consisted of membrane "ghosts," contained most (50-60%) of marker enzyme activity, monoamine oxidase-B and porin proteins, but only about 27-29% CPT-I activity. Because CPT-I and long-chain acyl-CoA synthetase appear to be associated with both inner and outer membranes, we postulate that these enzymes reside in contact sites, which represent a melding of both limiting membranes.

Identification and characterization of a newly isolated shiga toxin 2-converting phage from shiga toxin-producing Escherichia coli.

Shiga toxins 1 (Stx1) and 2 (Stx2) are encoded by toxin-converting bacteriophages of Stx-producing Escherichia coli (STEC), and so far two Stx1- and one Stx2-converting phages have been isolated from two STEC strains (A. D. O'Brien, J. W. Newlands, S. F. Miller, R. K. Holmes, H. W. Smith, and S. B. Formal, Science 226:694-696, 1984). In this study, we isolated two Stx2-converting phages, designated Stx2Phi-I and Stx2Phi-II, from two clinical strains of STEC associated with the outbreaks in Japan in 1996 and found that Stx2Phi-I resembled 933W, the previously reported Stx2-converting phage, in its infective properties for E. coli K-12 strain C600 while Stx2Phi-II was distinct from them. The sizes of the plaques of Stx2Phi-I and Stx2Phi-II in C600 were different; the former was larger than the latter. The restriction maps of Stx2Phi-I and Stx2Phi-II were not identical; rather, Stx2Phi-II DNA was approximately 3 kb larger than Stx2Phi-I DNA. Furthermore, Stx2Phi-I and Stx2Phi-II showed different phage immunity, with Stx2Phi-I and 933W belonging to the same group. Infection of C600 by Stx2Phi-I or 933W was affected by environmental osmolarity differently from that by Stx2Phi-II. When C600 was grown under conditions of high osmolarity, the infectivity of Stx2Phi-I and 933W was greatly decreased compared with that of Stx2Phi-II. Examination of the plating efficiency of the three phages for the defined mutations in C600 revealed that the efficiency of Stx2Phi-I and 933W for the fadL mutant decreased to less than 10(-7) compared with that for C600 whereas the efficiency of Stx2Phi-II decreased to 0.1% of that for C600. In contrast, while the plating efficiency of Stx2Phi-II for the lamB mutant decreased to a low level (0.05% of that for C600), the efficiencies of Stx2Phi-I and 933W were not changed. This was confirmed by the phage neutralization experiments with isolated outer membrane fractions from C600, fadL mutant, or lamB mutant or the purified His6-tagged FadL and LamB proteins. Based on the data, we concluded that FadL acts as the receptor for Stx2Phi-I and Stx2Phi-II whereas LamB acts as the receptor only for Stx2Phi-II.

Outer membrane proteins ofBartonella henselaeand their interaction with human endothelial cells

Members of the genusBartonellaare unique in that they are bacteria which cause proliferation of microvascular endothelial cells and neovascularization (angiogenesis). The mechanisms by whichBartonella henselaecauses these processes are unknown. Given the importance of surface-exposed determinants in the pathogenesis of many organisms, outer membrane proteins (OMPs) ofB. henselaewere identified. Enrichment of the outer membrane fraction ofB. henselaeby sarkosyl treatment of total membranes, together with radioiodination and biotinylation of intact organisms, suggest that at least nine proteins, with molecular weights of 28, 30, 35, 43, 58, 61, 79, 92 and 171 kDa, are located in the outer membrane. Triton X-100-extracted biotinylated human umbilical vein endothelial cell (HUVEC) surface proteins bound to the 43 kDaB. henselaeOMP afterB. henselaewhole-cell lysates and sarkosyl-fractionated OMPs were separated by SDS-PAGE and transferred onto nylon. BiotinylatedB. henselaesurface proteins of 28, 32, 43, 52 and 58 kDa were shown to bind intact HUVEC, with the 43 kDa protein being the major adhesin. Preincubation of HUVEC with an increasing concentration (20 μg/ml to 4 mg/ml) of sarkosyl-fractionated unlabelledB. henselaeouter membrane proteins inhibited the attachment of all identified HUVEC binding proteins. The identification ofB. henselaeOMPs, as well as adhesins, should provide a basis for further investigation of the role of adherence in the pathogenesis ofB. henselae.

The spontaneous gating activity of OmpC porin is affected by mutations of a putative hydrogen bond network or of a salt bridge between the L3 loop and the barrel.

Porins are trimeric channel-forming proteins of the outer membrane of Escherichia coli. Each subunit contains 16 beta-strands forming a transmembrane beta-barrel whose pore is constricted by the third extracellular loop (L3). We investigated the effects of site-directed mutations at two critical regions of the OmpC porin: (i) the D315A mutation targets a key component of a putative hydrogen bond network linking the L3 loop to the adjacent barrel wall and (ii) the D118Q, R174Q and R92Q mutations target putative salt bridges at the root of the L3 loop. We purified the outer membrane fractions obtained from each mutant and reconstituted them in liposomes suitable for electrophysiology. Patch clamp experiments showed that the frequency of spontaneous transitions between open and closed states is increased in the D315A, D118Q and R92Q mutants but unchanged in the R174Q mutant. These transitions are not driven by transmembrane voltage changes and represent the thermal oscillations between functionally distinct conformations. The asymmetric voltage-dependent inactivation of the channels is not affected by the mutations, however, suggesting different molecular mechanisms for the spontaneous and voltage-dependent gating processes. We propose that the positioning or flexibility of the L3 loop across the pore, as governed by the putative hydrogen-bond network and a salt bridge, play a role in determining the frequency of spontaneous channel gating.

Mitochondrial NADH–Quinone Oxidoreductase of the Outer Membrane Is Responsible for Paraquat Cytotoxicity in Rat Livers

We investigated the existence of an NADH-dependent paraquat (PQ) reduction system in rat liver mitochondria (Mt) in respect to the cytotoxic mechanisms of PQ. The outer membrane fractions, free from the contamination of inner membranes but with a few microsomes, catalyzed rotenone-insensitive NADH, but not NADPH, oxidation by menadione or PQ. Anti-NADH–cytochrome b5reductase antibody and its inhibitorp-hydroxymercuribenzonate did not inhibit the NADH–PQ reduction activity. Therefore, the respiratory systems of the inner membranes and microsomal cytochrome P450 systems could not have been responsible for the reaction. Dicoumarol, an inhibitor of NAD(P)H–quinone oxidoreductase (NQO), dose dependently suppressed the NADH oxidation in the outer membrane via PQ as well as menadione, withI50values of 190 (for menadione) and 150 μM (for PQ). Because of a lower sensitivity to NADPH and the higher doses of dicoumarol required for its inhibition, the activity in the outer membrane may be an “NADH–quinone oxidoreductase” which partly differs from the NQO previously reported. This outer membrane enzyme produced superoxide anions in the presence of both NADH and PQ and was too tightly membrane-bound to be extracted by Triton X-100 and deoxycholate. From these results, we concluded that the free radical-producing mitochondrial NADH–quinone oxidoreductase is a novel oxidation–reduction system participating in PQ toxicity. This is in good agreement with our previous results showing that PQ selectively damaged Mtin vivoandin vitro, resulting in cell death (K.-I. Hiraiet al., 1992,Toxicology72, 1–16).

Features ofFibrobacter intestinalisDR7 Mutant Which is Impaired With its Ability to Adhere to Cellulose

A spontaneous adhesion-defective mutant (DR7-M) ofFibrobacter intestinalisDR7 was isolated which was capable of growing on glucose and cellobiose, but impaired in its capacity to degrade cellulose.Levels of enzyme activities were determined in solubilized fractions of DR7 and DR7-M. Total endoglucanases and xylanase activity values of parent DR7 fractions were 2.84 and 1.85 folds higher than those of the mutant, and were distributed mainly in the bacterial envelope fractions, with some activity also found in the extracellular fluid. In a separate assay, measurement of the enzymatic activity bound to cellulose showed that a portion of the endoglucanase activity bound to cellulose while most xylanase activity did not bind. Notwithstanding, the wild type DR7 cells had 26-fold higher total activities of cellulose-degrading enzymes than the mutant, and 96% of its activity was exclusively located in outer membrane and periplasm fractions. In the mutant, the lower cellulose degrading enzymes activity was located only in the extracellular fluid. Most of the cellulose degrading enzymes activity of DR7 had the capability to bind to cellulose.SDS-page electrophoresis of outer membrane and periplasm cell fractions showed that DR7 and DR7-M possess similar molecular weight (MW) profiles but different quantities of 16 cellulose-binding-proteins (CBPs) in the MW range of 36 up to 225 kDa. Zymogram analysis with soluble substrates, either carboxymethylcellulose or soluble xylan, following SDS-page of DR7 and DR7-M fractions, suggested that CBPs of approximate MW 120, 110, 100, 90, 70 and 40 kDa have endoglucanase activity, and that CBPs of all fractions lack any xylanase activity.

CD14-Dependent Activation of Human Endothelial Cells byBacteroides fragilisOuter Membrane

We studied the capacity of isolatedBacteriodes fragilisouter membrane,B. fragilisNCTC9343 lipopolysaccharide (LPS; endotoxin), andB. fragilisNCTC9343 capsular polysaccharides to activate human umbilical vein endothelial cell (HUVEC) monolayers. To assess HUVEC activation, E-selectin expression was measured by enzyme-linked immunosorbent assay (ELISA), Northern blot analysis for E-selectin-specific mRNA, and electrophoretic gel mobility shift assay (EMSA) for NF-κB, a transcription factor necessary for E-selectin gene activation. Exposure of HUVECs toB. fragilisouter membrane fractions, separated from other components of theB. fragiliscell wall by isopycnic, sucrose gradient centrifugation, significantly increased surface expression of E-selectin and induced functional endothelial cell-dependent leukocyte adhesion.B. fragilisouter membranes induced translocation of NF-κB to HUVEC nuclei and accumulation of E-selectin mRNA in HUVEC cytoplasm. E-selectin expression induced byB. fragilisouter membranes was not blocked by polymixin B. In contrast, E-selectin expression induced by outer membrane fractions purified fromE. coliwas competitively inhibited by polymixin B. Neither purifiedB. fragilisLPS, a prominent constituent of the outer membrane, nor purifiedB. fragiliscapsular polysaccharides induced HUVEC activation. Two different monoclonal antibodies directed against human CD14 completely inhibitedB. fragilisouter membrane-induced NF-κB activation, E-selectin transcription, and E-selectin surface expression. We conclude that the outer membrane component of theB. fragiliscell wall contains a proinflammatory factor(s), that is not LPS, which induces human endothelial cell activation by a soluble CD14-dependent mechanism.

Adherence ofBilophila wadsworthiato Cultured Human Embryonic Intestinal Cells

Adherence ofBilophila wadsworthiato the cultured human embryonic intestinal cell line, Intestine 407 (Int 407), varied among the strains tested from strongly adherent (76–100% cells positive for one or more adherent bacteria) to non- or weakly adherent (0–25% positive cells). Although negative staining revealed that infrequent cells of an adherent strain, WAL 9077, the adherent type-strain, WAL 7959, and a non-adherent strain, WAL 8448, expressed loosely associated fimbrial structures, a role for these structures in adhesion could not be confirmed with either scanning or thin-section electron micrography. Ruthenium red staining of thin-section preparations and subsequent electron microscopy failed to reveal an extensive extracellular polysaccharide layer. SDS-PAGE analysis of crude outer membrane fractions of WAL 9077 and WAL 8448 demonstrated clear differences in their major and minor outer membrane protein components. Thus, we postulate that the adherence ofB. wadsworthiato Int 407 cells is mediated by an outer membrane or cell wall component.

E.coli PhoE porin has an opposite voltage-dependence to the homologous OmpF.

We used patch clamp analysis to compare the electrophysiological behavior of two related porins from Escherichia coli, the anion-specific PhoE and the cation-selective OmpF. Outer membrane fractions were obtained from strains expressing just one of these porin types, and the channels were reconstituted into liposomes without prior purification. We show that the orientation of the reconstituted channels is not random and is the same for both PhoE and OmpF. Like cation-selective porins, PhoE shows fast and slow gating to closed levels of various amplitudes, testifying that the channels visit multiple functional states and behave as cooperative entities. The voltage-dependence of PhoE closure is asymmetric, but strikingly, occurs at voltages of inverse polarity from those promoting closures of OmpC and OmpF. Both slow kinetics and inverse voltage-dependence are removed when 70 amino acids from the N-terminal of OmpF are introduced into the homologous region of PhoE. This novel observation regarding the voltage-dependence of the two channel types, along with published results on PhoE and OmpF mutants, allows us to propose a molecular mechanism for voltage sensing and sensor charge movements in bacterial porins. It also offers new cues on the possible physiological relevance in bacteria of this common form of channel modulation.

Immunodominant major outer membrane proteins of Ehrlichia chaffeensis are encoded by a polymorphic multigene family.

Several immunodominant major proteins ranging from 23 to 30 kDa were identified in the outer membrane fractions of Ehrlichia chaffeensis and Ehrlichia canis. The N-terminal amino acid sequence of a 28-kDa protein of E. chaffeensis (one of the major proteins) was determined. The gene (p28), almost full length, encoding the 28-kDa protein was cloned by PCR with primers designed based on the N-terminal sequence of the E. chaffeensis 28-kDa protein and the consensus sequence between the C termini of the Cowdria ruminantium MAP-1 and Anaplasma marginale MSP-4 proteins. The p28 gene was overexpressed, and antibody to the recombinant protein was raised in a rabbit. The antibody and serum from a patient infected with E. chaffeensis reacted with the recombinant protein, three proteins (29, 28, and 25 kDa) of E. chaffeensis, and a 30-kDa protein of E. canis. Immunoelectron microscopy with the rabbit antibody revealed that the antigenic epitope of the 28-kDa protein was exposed on the surface of E. chaffeensis. Southern blot analysis with a 32P-labeled p28 gene probe revealed multiple copies of genes homologous to p28 in the E. chaffeensis genome. Six copies of the p28 gene were cloned and sequenced from the genomic DNA by using the same probe. The open reading frames of these gene copies were tandemly arranged with intergenic spaces. They were nonidentical genes and contained a semivariable region and three hypervariable regions in the predicted protein molecules. One of the gene copies encoded a protein with an internal amino acid sequence identical to the chemically determined N-terminal amino acid sequence of a 23-kDa protein of E. chaffeensis. Immunization with the recombinant P28 protein protected mice from infection with E. chaffeensis. These findings suggest that the 30-kDa-range proteins of E. chaffeensis represent a family of antigenically related homologous proteins encoded by a single gene family.

Purified outer membranes of Serpulina hyodysenteriae contain cholesterol.

We have isolated outer and inner membranes of Serpulina hyodysenteriae by using discontinuous sucrose density gradients. The outer and inner membrane fractions contained less than 1 and 2%, respectively, of the total NADH oxidase activity (soluble marker) in the cell lysate. Various membrane markers including lipooligosaccharide (LOS), the 16-kDa outer membrane lipoprotein (SmpA), and the C subunit of the F1F0 ATPase indicated that the lowest-density membrane fraction contained outer membranes while the high-density membrane fraction contained inner membranes and that both are essentially free of contamination by the periplasmic flagella, a major contaminant of membranes isolated by other techniques. The outer membrane fractions (rho = 1.10 g/cm3) contained 0.25 mg of protein/mg (dry weight), while the inner membrane samples (rho = 1.16 g/cm3) contained significantly more protein (0.55 mg of protein/mg [dry weight]). Lipid analysis revealed that the purified outer membranes contained cholesterol as a major component of the membrane lipids. Treatment of intact S. hyodysenteriae with different concentrations of digitonin, a steroid glycoside that interacts with cholesterol, indicated that the outer membrane could be selectively removed at concentrations as low as 0.125%.

Identification and characterization of a metalloprotease activity from Helicobacter pylori.

Helicobacter pylori produces a metalloprotease with a native molecular size of approximately 200 kDa, as determined by size-exclusion chromatography. Subcellular distribution studies demonstrated that the activity was associated with the outer membrane fraction of the bacterium. In addition, the protease was secreted by the bacterium when grown in liquid culture. The enzyme activity was measured by hydrolysis of azocasein and biotinylated casein and exhibited optimal caseinolytic activity at pH 8.0 (37 degrees C). The activity was inhibited by EDTA, 1,10-phenanthroline, phosphoramidon, pyridine-2,6-dicarboxylic acid, and 8-hydroxyquinoline-5-sulfonic acid (HQSA). Inhibition by HQSA was reversed by zinc, whereas inhibition due to EDTA was reversed by excess calcium, thus indicating that the enzyme was a zinc-dependent, calcium-stabilized endoproteinase. Furthermore, titration with Zn2+ of a desalted, active-site zinc-chelated preparation of the protease demonstrated that Zn2+ was essential for activity. Leupeptin, phenylmethylsulfonyl fluoride, E-64, pepstatin A, dithiothreitol, and 2-mercaptoethanol had no effect on enzymatic activity. Addition of Ca2+ or Mg2+ to the incubation medium resulted in approximately a twofold stimulation of the azocaseinolytic activity of the enzyme. The protease was stably expressed since it was active even after repeated subculture of the bacterium. Bovine serum albumin, hide powder azure, and elastin-Congo red remained intact even after prolonged exposure to the enzyme. The surface expression of this metalloprotease activity raises the possibility that this enzyme may be involved in the proteolysis of a variety of host proteins in vivo and thereby contributes to gastric pathology.

Induction and characterization of heat shock proteins of Salmonella typhi and their reactivity with sera from patients with typhoid fever.

The heat shock protein (HSP) response of Salmonella typhi following exposure to elevated growth temperatures was studied. Three major proteins with molecular sizes of 58, 68, and 88 kDa were abundantly expressed when S. typhi cells were shifted from 37 to 45 degrees C and to 55 degrees C. These proteins were also constitutively expressed at 37 degrees C. Western blotting and immunoprecipitation studies with anti-HSP monoclonal antibodies revealed that the 58- and 68-kDa proteins were analogous to the GroEL and DnaK proteins, respectively, of Escherichia coli. These HSPs are also abundantly present in the outer membrane fraction of disrupted cells and, to a lesser extent, in the cytosol. Immunoblotting experiments with sera from patients with a culture-positive diagnosis of typhoid fever showed the presence of antibodies to these HSPs. Nine of twelve sera reacted with the 58-, 68-, and 88-kDa proteins, while three sera reacted only with the 68- and 88-kDa proteins. All 10 sera from healthy individuals showed no binding to these HSPs. In light of the well-documented roles of HSPs in the pathogenesis of microbial infections and as immunodominant antigens, these findings may be relevant for a better understanding of disease processes and for the future development of diagnostic and preventive strategies.

Specific interaction between OutD, an Erwinia chrysanthemi outer membrane protein of the general secretory pathway, and secreted proteins.

OutD is an outer membrane component of the main terminal branch of the general secretory pathway (GSP) in Erwinia chrysanthemi. We analyzed the interactions of OutD with other components of the GSP (Out proteins) and with secreted proteins (PelB, EGZ and PemA). OutD is stabilized by its interaction with another GSP component, OutS. The 62 C-terminal amino acids of OutD are necessary for this interaction. In vivo formation of OutD multimers, up to tetramers, was proved after the dissociation in mild conditions of the OutD aggregates formed in the outer membrane. Thus, OutD could form a channel-like structure in the outer membrane. We showed that OutD is stabilized in vivo when co-expressed with Out-secreted proteins. This stabilization results from the formation of complexes that were detected in experiments of co-immunoprecipitation and co-sedimentation in sucrose density gradients. The presence of the N-terminal part of OutD is required for this interaction. The interaction between OutD and the secreted protein PelB was confirmed in vitro, suggesting that no other component of the GSP is required for this recognition. No interaction was observed between the E. carotovora PelC and the E. chrysanthemi OutD. Thus, the interaction between GspD and the secreted proteins present in the periplasm could be the key to the specificity of the secretion machinery and a trigger for that process.

Disruption of polyamine modulation by a single amino acid substitution on the L3 loop of the OmpC porin channel

Structural studies have demonstrated that the extracellular L3 loop of porin constricts the channel and suggest that this loop might be involved in channel selectivity and gating. We previously showed that positively charged polyamines can induce changes in porin gating kinetics by stabilization of closed states. Here we report the effects of the mutation of two different aspartate residues of Escherichia coli OmpC porin on the polyamine sensitivity of the channel. Aspartate 105 or aspartate 118 on the L3 loop was replaced by glutamine by site-directed mutagenesis. The gating activity of the wild-type and mutant channels were studied by patch-clamp of liposomes containing reconstituted outer membrane fractions, in the absence or the presence of either polyamine spermine or cadaverine. Porin channels with a D118Q mutation, at the root of L3, still showed some, albeit milder, sensitivity to polyamine modulation. On the other hand, the D105Q mutation, at the tip of L3, abolished the increase in closing frequency which is typically observed in the presence of polyamines. We conclude that aspartate 105 primarily, but not aspartate 118, plays an important role in mediating the polyamine-induced changes in gating kinetics that result in the inhibition of the OmpC channel.

Mucosal and systemic immune responses after oral immunization of BALB/c mice with an outer membrane fraction of klebsiella pneumoniae

Mucosal and systemic immune responses after oral immunization of BALB/c mice with an outer membrane fraction of klebsiella pneumoniae

Mucosal and systemic immune responses after oral immunization of BALB/c mice with an outer membrane fraction of klebsiella pneumoniae

Mucosal and systemic immune responses after oral immunization of BALB/c mice with an outer membrane fraction of klebsiella pneumoniae

TonB protein appears to transduce energy by shuttling between the cytoplasmic membrane and the outer membrane in Escherichia coli

The energy source for active transport of iron-siderophore complexes and vitamin B12 across the outer membrane in Gram-negative bacteria is the cytoplasmic membrane proton-motive force (pmf). TonB protein is required in this process to transduce cytoplasmic membrane energy to the outer membrane. In this study, Escherichia coli TonB was found to be distributed in sucrose density gradients approximately equally between the cytoplasmic membrane and the outer membrane fractions, while two proteins with which it is known to interact, ExbB and ExbD, as well as the NADH oxidase activity characteristic of the cytoplasmic membrane, were localized in the cytoplasmic membrane fraction. Neither the N-terminus of TonB nor the cytoplasmic membrane pmf, both of which are essential for TonB activity, were required for TonB to associate with the outer membrane. When the TonB C-terminus was absent, TonB was found associated with the cytoplasmic membrane, suggesting that the C-terminus was required for outer membrane association. When ExbB and ExbD, as well as their cross-talk-competent homologues ToIQ and ToIR, were absent, TonB was found associated with the outer membrane. TetA-TonB protein, which cannot interact with ExbB/D, was likewise found associated with the outer membrane. These results indicated that the role of ExbB/D in energy transduction is to bring TonB that has reached the outer membrane back to associate with the cytoplasmic membrane. Two possible explanations exist for the observations presented in this study. One possibility is that TonB transduces energy by shuttling between membranes, and, at some stages in the energy-transduction cycle, is associated with either the cytoplasmic membrane or the outer membrane, but not with both at the same time. This hypothesis, together with the alternative interpretation that TonB remains localized in the cytoplasmic membrane and changes its affinity for the outer and cytoplasmic membrane during energy transduction, are incorporated with previous observations into two new models, consistent with the novel aspects of this system, that describe a mechanism for TonB-dependent energy transduction.

Recombinant Treponema pallidum rare outer membrane protein 1 (Tromp1) expressed in Escherichia coli has porin activity and surface antigenic exposure.

We recently reported the cloning and sequencing of the gene encoding a 31-kDa Treponema pallidum subsp. pallidum rare outer membrane porin protein, designated Tromp1 (D. R. Blanco, C. I. Champion, M. M. Exner, H. Erdjument-Bromage, R. E. W. Hancock, P. Tempst, J. N. Miller, and M. A. Lovett, J. Bacteriol. 177:3556-3562, 1995). Here, we report the stable expression of recombinant Tromp1 (rTromp1) in Escherichia coli. rTromp1 expressed without its signal peptide and containing a 22-residue N-terminal fusion resulted in high-level accumulation of a nonexported soluble protein that was purified to homogeneity by fast protein liquid chromatography (FPLC). Specific antiserum generated to the FPLC-purified rTromp1 fusion identified on immunoblots of T. pallidum the native 31-kDa Tromp1 protein and two higher-molecular-mass oligomeric forms of Tromp1 at 55 and 80 kDa. rTromp1 was also expressed with its native signal peptide by using an inducible T7 promoter. Under these conditions, rTromp1 fractionated predominantly with the E. coli soluble and outer membrane fractions, but not with the inner membrane fraction. rTromp1 isolated from the E. coli outer membrane and reconstituted into planar lipid bilayers showed porin activity based on average single-channel conductances of 0.4 and 0.8 nS in 1 M KCl. Whole-mount immunoelectron microscopy using infection-derived immune serum against T. pallidum indicated that rTromp1 was surface exposed when expressed in E. coli. These findings demonstrate that rTromp1 can be targeted to the E. coli outer membrane, where it has both porin activity and surface antigenic exposure.

Genetic and immunological analyses of a 38 kDa surface-exposed lipoprotein of Pasteurella haemolytica A1

Pasteurella haemolytica serotype A1 is the bacterial pathogen most frequently isolated from the lungs of cattle with bovine respiratory disease. As part of a study to characterize P. haemolytica antigens the gene encoding a 38 kDa lipoprotein, Lpp38. The deduced amino acid sequence of Lpp38 is similar to those of the Escherichia coli polyamine transport proteins PotD (70%) and PotF (33%). P. haemolytica Lpp38 is present in both inner membrane and outer membrane fractions of the cell envelope. Susceptibility of Lpp38 to cleavage by extracellular proteases indicates that portions of the protein are surface-exposed. A protein of similar molecular mass in P. haemolytica strains from all 12 serotypes of biotype A and in an untypeable strain was detected by an anti-Lpp38 monoclonal antibody. Lpp38 is recognized by sera from calves resistant to infection after natural exposure to P. haemolytica and by sera from calves protected against infection by vaccination with P. haemolytica A1 outer membranes or with live bacteria. These data suggest a role for this protein in the development of immunity to P. haemolytica infection.