Isolation and characterization of outer and inner membranes from Pseudomonas aeruginosa and effect of EDTA on the membranes.

The outer and inner cytoplasmic membranes of Pseudomonas aeruginosa were separated as small and large membranes, respectively, from the cell envelope of this organism treated with lysozyme in Tris-chloride buffer containing sucrose and MgCl2 by differential centrifugation. The small membrane fraction contained predominantly 2-keto-3-deoxyoctonate (KDO), and little cytochromes or oxidase activities. The small membrane was composed of only 9 polypeptides and showed homogeneous small vesicles electron-microscopically. On the other hand, the large membrane fraction had high cytochrome contents and oxidase activities, and little KDO. The large membrane was composed of a number of polypeptides and showed large fragments or vesicles electron-microscopically. These results indicate that the small and large membranes are the outer and inner cytoplasmic membranes of P. aeruginosa, respectively. The isolated outer membrane showed a symmetrical protein peak with a density of 1.23 on sucrose density gradient centrifugation and the isolated inner membrane showed an unusually high density, probably due to association with ribosomes and extrinsic or loosely bound proteins. EDTA lowered the density of both membranes and caused lethal damage to the outer membrane, causing disintegration with the release of lipopolysaccharide (LPS), proteins and phospholipid.     

Flagellin inhibits Myoviridae phage φCTX infection of Pseudomonas aeruginosa strain GuA18: purification and mapping of binding site

PhiCTX is a double-stranded DNA phage of the Myoviridae family that converts Pseudomonas aeruginosa into a cytotoxin producer. A 42-kDa phiCTX-inhibiting protein was purified from the outer membrane fraction of P. aeruginosa strain GuA18 by octyl-beta-glucoside extraction, DEAE-chromatography, and mono-Q HPLC. This protein had an isoelectric point of 5.4 and bound specifically [125I]-labeled phiCTX. The N-terminal amino acid sequence of six out of seven Lys-C fragments was highly similar (87%) to that of the entire of type-a flagellin of P. aeruginosa strain PAK. At a concentration of 14 nM, purified flagellin protein caused a 50% decrease in the phage titer after a 20-min incubation at 37 degrees C (PhI50). The presence of ethanol was necessary to reconstitute the inhibitory activity. In contrast, no ethanol treatment was necessary for the inhibitory activity of the sheared flagellin filaments from P. aeruginosa strain GuA18, which consists of the 42-kDa flagellin subunits and the synthesized 17-mer phage-binding-peptide NGSNSDSERTALNGEAK, representing flagellin residues 100-116 of P. aeruginosa strain PAK. The PhI50 was 10 nM and 200 nM, respectively. Antisera against the flagellin filament protein as well as against the 17-mer peptide neutralized phage infection. These results indicated that the amino acid region 100-116 of the flagellin subunit of strain GuA18 is involved in phiCTX binding. This region might play a role in phage attachment.     

Mapping the surface regions of Pseudomonas aeruginosa PAK pilin: the importance of the C-terminal region for adherence to human buccal epithelial cells

The adherence of non-mucoid Pseudomonas aeruginosa strains is believed to be mediated by the pilus, which consists of a single protein subunit of 15,000 Daltons called pilin. Ten antipeptide antisera were raised to map the surface regions of pilin from P. aeruginosa strain K (PAK). Only one of the antipeptide antisera to the eight predicted surface regions failed to react with PAK pili in direct ELISA. Five out of eight synthetic peptides representing the eight predicted surface regions reacted with anti-PAK pilus antiserum, indicating their surface exposure. Combining the antipeptide and antipilus antisera results, all eight predicted surface regions were demonstrated to be surface-exposed. The PAK 128-144-OH peptide produced the best binding antiserum to PAK pili. Only antipeptide Fab fragments directed against the disulphide bridged C-terminal region of PAK pilin blocked the adherence of pili to human buccal epithelial cells, which suggests that this region contains the receptor-binding domain of the PAK pilus.     

Expression of the Pseudomonas aeruginosa PAK pilin gene in Escherichia coli.

Pseudomonas aeruginosa is a piliated opportunistic pathogen. We have recently reported the cloning of the structural gene for the pilus protein, pilin, from P. aeruginosa PAK (B. L. Pasloske, B. B. Finlay, and W. Paranchych, FEBS Lett. 183:408-412, 1985), and in this paper we present evidence that this chimera (pBP001) expresses P. aeruginosa PAK pilin in Escherichia coli independent of a vector promoter. The strength of the promoter for the PAK pilin gene was assayed, and the cellular location of the pilin protein within E. coli was examined. This protein was present mainly in the inner membrane fraction both with and without its six-amino-acid leader sequence, but it was not assembled into pili.     

A Toluene-tolerant Mutant of Pseudomonas aeruginosa Lacking the Outer Membrane Protein F

The outer membrane protein profiles of a toluene-tolerant mutant, Pseudomonas aeruginosa, strain PAK103, were compared with those of its parent strain PAO1161. Protein F (OprF), the most abundant outer membrane protein in the parental strain PAO1161, was missing in the toluene-tolerant strain PAK103. The absence of OprF may lead to the loss of toluene diffusion across in the outer membrane of the mutant cells     

Assembly of mutant pilins in Pseudomonas aeruginosa: formation of pili composed of heterologous subunits.

Recently, we reported the degree of N-terminal processing within the cytoplasmic membranes of three mutant pilins from Pseudomonas aeruginosa PAK with respect to leader peptide removal and the methylation of the N-terminal phenylalanine (B. L. Pasloske and W. Paranchych, Mol. Microbiol. 2:489-495, 1988). The results of those experiments showed that the deletion of 4 or 8 amino acids within the highly conserved N terminus greatly inhibited leader peptide removal. On the other hand, the mutation of the glutamate at position 5 to a lysine permitted leader peptide cleavage but inhibited transmethylase activity. In this report, we have examined the effects of these mutant pilins upon pilus assembly in a P. aeruginosa PAO host with or without the chromosomally encoded pilin gene present. Pilins with deletions of 4 or 8 amino acids in the N-terminal region were not incorporated into pili. Interestingly, pilin subunits containing the glutamate-to-lysine mutation were incorporated into compound pili together with PAO wild-type subunits. However, the mutant pilins were unable to polymerize as a homopolymer. When wild-type PAK and PAO pilin subunits were expressed in the same bacterial strain, the pilin subunits assembled into homopolymeric pili containing one or the other type of subunit.     

Identification of Pseudomonas aeruginosa flagellin as an adhesin for Muc1 mucin

We reported previously that Muc1 mucin on the epithelial cell surface is an adhesion site for Pseudomonas aeruginosa (Lillehoj EP, Hyun SW, Kim BT, Zhang XG, Lee DI, Rowland S, and Kim KC. Am J Physiol Lung Cell Mol Physiol 280: L181-L187, 2001). The present study was designed to identify the adhesin(s) responsible for bacterial binding to Muc1 mucin using genetic and biochemical approaches. Chinese hamster ovary (CHO) cells stably transfected with a Muc1 cDNA (CHO-Muc1) or empty plasmid (CHO-X) were compared for adhesion of P. aeruginosa strain PAK. Our results showed that 1) wild-type PAK and isogenic mutant strains lacking pili (PAK/NP) or flagella cap protein (PAK/fliD) demonstrated significantly increased binding to CHO-Muc1 cells, whereas flagellin-deficient (PAK/fliC) bacteria were no more adherent to CHO-Muc1 than CHO-X cells, and 2) P. aeruginosa adhesion was blocked by pretreatment of bacteria with antibody to flagellin or pretreatment of CHO-Muc1 cells with purified flagellin. We conclude that flagellin is an adhesin of P. aeruginosa responsible for its binding to Muc1 mucin on the epithelial cell surface.     

Identification of an additional member of the secretin superfamily of proteins in Pseudomonas aeruginosa that is able to function in type II protein secretion

Pseudomonas aeruginosa is a prolific exporter of virulence factors and contains three of the four protein secretion systems that have been described in Gram-negative bacteria. The P. aeruginosa type II general secretory pathway (GSP) is used to export the largest number of proteins from this organism, including lipase, phospholipase C, alkaline phosphatase, exotoxin A, elastase and LasA. Although these exoproteins contain no sequence similarity, they are specifically and efficiently transported by the secretion apparatus. Bacterial homologues of XcpQ (GspD), the only outer membrane component of this system, have been proposed to play the role of gatekeeper, by presumably interacting and recognizing the exported substrates to allow their passage through the outer membrane. While determining the phenotype of non-polar deletions in each of the xcp genes, we have shown that a deletion of the P. aeruginosa strain K xcpQ does not completely abolish protein secretion. As the proposed function of XcpQ should be requisite for secretion, we searched for additional factors that could carry out this role. A cosmid DNA library from a PAK strain deleted for xcpP-Z was tested for its ability to increase protein secretion by screening for enhanced growth on lipid agar, a medium that selects for the secretion of lipase. In this manner, we have identified an XcpQ homologue, XqhA, that is solely responsible for the residual export observed in a Δ xcpQ strain, although it is not required for efficient secretion in wild-type P. aeruginosa . We have also demonstrated that this protein is capable of recognizing all of the exoproteins of P. aeruginosa , arguing against the proposed role of members of the secretin family as determinants of specificity.     

Composition and molecular weight of pili purified from Pseudomonas aeruginosa K.

Pseudomonas aeruginosa strain K (PAK) bears polar pili that promote infection by at least six bacteriophages. Moreover, a recently isolated mutant of strain K (PAK/2PfS) is many times more piliated than the wild-type strain and facilitates the preparation of large amounts of pure pili for biochemical studies. The present investigation was carried out to establish the structural relatedness of PAK and PAK/2PfS pili and to determine their biochemical composition. A purfication procedure is described for PAK and PAK/2PfS pili that yields about 8 mg of pure pili per 100 g (wet weight) of PAK/2PfS cells and 0.8 mg of pure pili per 100 g (wet weight) of PAK cells. PAK and PAK/2PfS pili were found to be free from phosphate, carbohydrate, and lipid and to contain a single polypeptide subunit of 17,800 daltons. Isopycnic centrifugation studies revealed that PAK and PAK(2PfS pili have the same buoyant density in sucrose (1.221) and CsC1 (1.295). Both types of pili banded at pH 3.9 when subjected to isoelectric focusing. Amino acid analyses showed that PAK and PAK/2PfS pili have identical amino acid compositions, whereas microimmunodiffusion studies revealed that the two types of pili are immunologically indistinguishable. It was concluded that PAK and PAK/2PfS pili are identical and that the mutation responsible for producing the multipiliated state in PAK/2PfS is probably located outside the structural gene for PAK pili.     

α<Superscript>5</Superscript>β<Subscript>1</Subscript> integrins and fibronectin are involved in adherence of the<Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> ER97314 clinical strain to A549 cells

The role of fibronectin (Fn) and its natural receptors alpha5beta1 integrins in the interaction of P. aeruginosa with A549 epithelial cells was compared in the clinical isolate ER97314 and the reference PAK strain. Both strains expressed functional type IV pili, as shown by the results of the twitching motility assay. The ER97314 strain was highly adherent to immobilized Fn (640 000+/-20 000 CFU per well) while the PAK strain adhered less efficiently (70 000+/-10 000 CFU per well). Both strains adhered to A549 cells (33 400+/-1200 and 1200+/-100 CFU per well, for PAK and ER97314, respectively), only the PAK strain being significantly internalized (9430+/-2020 CFU per well). Cytochalasin D and genistein significantly decreased bacterial adherence of the 2 strains and caused also a significant decrease in PAK internalization. This inhibitory activity was not related to changes in the expression of alpha5beta1 integrins. Antibodies to Fn and alpha5beta1 integrins inhibited the adherence of the ER97314 strain but had no significant effect on PAK interaction with human cells. These findings suggest that only some P. aeruginosa strains can target Fn and their natural receptors alpha5beta1 integrins for adherence to A549 cells.     

Conservation of the gene for outer membrane protein OprF in the family Pseudomonadaceae: sequence of the Pseudomonas syringae oprF gene.

The conservation of the oprF gene for the major outer membrane protein OprF was determined by restriction mapping and Southern blot hybridization with the Pseudomonas aeruginosa oprF gene as a probe. The restriction map was highly conserved among 16 of the 17 serotype strains and 42 clinical isolates of P. aeruginosa. Only the serotype 12 isolate and one clinical isolate showed small differences in restriction pattern. Southern probing of PstI chromosomal digests of 14 species from the family Pseudomonadaceae revealed that only the nine members of rRNA homology group I hybridized with the oprF gene. To reveal the actual extent of homology, the oprF gene and its product were characterized in Pseudomonas syringae. Nine strains of P. syringae from seven different pathovars hybridized with the P. aeruginosa gene to produce five different but related restriction maps. All produced an OprF protein in their outer membranes with the same apparent molecular weight as that of P.aeruginosa OprF. In each case the protein reacted with monoclonal antibody MA4-10 and was similarly heat and 2-mercaptoethanol modifiable. The purified OprF protein of the type strain P. syringae pv. syringae ATCC 19310 reconstituted small channels in lipid bilayer membranes. The oprF gene from this latter strain was cloned and sequenced. Despite the low level of DNA hybridization between P. aeruginosa and P. syringae DNA, the OprF gene was highly conserved between the species with 72% DNA sequence identity and 68% amino acid sequence identity overall. The carboxy terminus-encoding region of P. syringae oprF showed 85 and 33% identity, respectively, with the same regions of the P. aeruginosa oprF and Escherichia coli ompA genes.     

Outer membranes of gram-negative bacteria. XIX. Isolation from Pseudomonas aeruginosa PAO1 and use in reconstitution and definition of the permeability barrier.

A method for separating the outer and inner membranes of Pseudomonas aeruginosa PAO1 in the absence of added ethylenediaminetetraacetic acid was devised. The method yields two outer membrane fractions which show the same protein pattern on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but differ substantially in their relative contents of phospholipids. One of these outer membrane fractions and the inner membrane fraction are less than 4% cross-contaminated, as judged by the content of typical inner and outer membrane markers. The outer membrane contains four major protein bands with apparent molecular weights of 37,000, 35,000, 21,000 and 17,000. Vesicles reconstituted from lipopolysaccharide and phospholipids were impermeable to all saccharides included in the vesicles during vesicle formation. When the vesicles contained outer membrane proteins, they fully retained only those saccharides of greater than 9,000 molecular weight, suggesting that the exclusion limit of the outer membrane of P. aeruginosa for saccharides is substantially larger than the figure (500 to 600 daltons) obtained for certain enteric bacteria. The advantages and potential disadvantages of having an outer membrane with a higher exclusion limit for hydrophilic substances are discussed.     

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.     

Resistance of spheroplasts and whole cells of Pseudomonas aeruginosa to bactericidal activity of various biocides: evidence of the membrane implication

To emphasise the role of outer and inner membranes in the resistance of Pseudomonas aeruginosa to bactericidal activity of various disinfectants, spheroplasts and whole cells were compared. Spheroplasts are more sensitive than whole cells to quaternary ammonium compounds such as didecyl dimethyl ammonium bromide (DDAB) and C16-benzalkonium chloride. The outer membrane acts as a barrier to prevent these disinfectants from entering the cell. It seems to have no influence on activities of smaller molecules such as C12, C14-benzalkonium chlorides and sodium dichloroisocyanurate. For tri-sodium phosphate, the presence of outer membrane emphasized the action of the molecule. Moreover, resistance of DDAB-adapted spheroplasts to bactericidal activity of DDAB is higher than the resistance of non-adapted spheroplasts. This suggests that the inner membrane could also play a role in resistance to DDAB.     

Acquisition of iron from citrate by Pseudomonas aeruginosa

Transport of [14C]citrate, ferric [14C]citrate and [55Fe]ferric citrate into Pseudomonas aeruginosa grown in synthetic media containing citrate, succinate, or succinate and citrate as carbon and energy sources was measured. Cells grown in citrate-containing medium transported radiolabelled citrate and iron, whereas the succinate-grown cells transported iron but not citrate. Binding studies revealed that isolated outer and inner membranes of citrate-grown cells contain a citrate receptor, absent from membranes of succinate-grown cells. [55Fe]Ferric citrate bound to the isolated outer membranes of each cell type. The failure of citrate to compete with this binding suggests the presence of a ferric citrate receptor on the outer membranes of each cell type. Citrate induced the synthesis of two outer-membrane proteins of 41 and 19 kDa. A third protein of 17 kDa was more dominant in citrate-grown cells than in succinate-grown cells.     

Possibility of detecting pyocyanine in Pseudomonas aeruginosa cells

Pyocyanin can be detected in the cells of Pseudomonas aeruginosa using UV and IR spectroscopy of disturbed complete inner reflection (DCIR). Intact cells of the parent strain liberating the pigment into the cultural broth and mutant cells lacking the ability contain pyocyanin within the cells. Occasionally, pyocyanin can be detected in the outer layers of the cells, which is more typical of the parent strain. In the freshly isolated fractions of the parent strain cellular walls, pyocyanin seems to be pesent in the bound state that has changed significantly its structural organization. In due course, the hypothetical complex pyocyanin--cellular wall decomposes to yield an "oxidized" pigment similar to that liberated into the cultural broth. the cell wall of the mutant possesses the properties of "oxidized" pyocyanin immediately after isolation of the fraction. The pigment cannot be identified in the fractions of cytoplasmic membranes; pyocyanin is present in the "oxidized" state in the fractions of cytoplasm for the cells of both types. The paper discusses the role of the permeability of cytoplasmic membranes in the transport of pyocyanin from the cytoplasm into the cellular wall of the bacterium and then into the surrounding medium.     

Use of synthetic peptides to confirm that the Pseudomonas aeruginosa PAK pilus adhesin and the Candida albicans fimbrial adhesin possess a homologous receptor-binding domain

Pseudomonas aeruginosa PAK pili and Candida albicans fimbriae are adhesins present on the microbial cell surfaces which mediate binding to epithelial cell-surface receptors. The receptor-binding domain (adhesintope) of the PAK pilus adhesin has been shown previously to reside in the carboxy-terminal disulphide-bonded region of P. aeruginosa pilin (PAK128-144). The delineation of the C. albicans fimbrial adhesintope was investigated in these studies using synthetic peptides which correspond to the whole (PAK128-144) or part of (PAK134-140) adhesintope of the PAK pilus and their respective anti-peptide antisera and biotinylated PAK pili (Bt-PAK pili), fimbriae (Bt-fimbriae), P. aeruginosa whole cells (Bt- P. aeruginosa ) and C. albicans whole cells (Bt- C. albicans ). The results from these studies confirmed that a structurally conserved motif akin to the PAK(128-144) peptide sequence is present in C. albicans fimbrial adhesin and that the seven-amino-acid residue PAK(134-140) sequence plays an important role in forming the adhesintope for both P. aeruginosa PAK pilus and C. albicans fimbrial adhesins.     

Cloning and expression of the pilin gene of Pseudomonas aeruginosa PAK in Escherichia coli.

Many strains of Pseudomonas aeruginosa possess pili which have been implicated in the pathogenesis of the organism. This report presents the cloning and expression in Escherichia coli of the gene encoding the structural subunit of the pili of P. aeruginosa PAK. Total DNA from this strain was partially digested with Sau3A and inserted into the cloning vector pUC18. Recombinant E. coli clones were screened with oligonucleotide probes prepared from the constant region of the previously published amino acid sequence of the mature pilin subunit. Several positive clones were identified, and restriction maps were generated. Each clone contained an identical 1.1-kilobase HindIII fragment which hybridized to the oligonucleotide probes. Western blot analysis showed that all of the clones expressed small amounts of the P. aeruginosa pilin subunit, which has a molecular mass of ca. 18,000. This expression occurred independently of the orientation of the inserted DNA fragments in the cloning vector, indicating that synthesis was directed from an internal promoter. However, subclones containing the 1.1-kilobase HindIII fragment in a specific orientation produced an order of magnitude more of the pilin subunit. While the expressed pilin antigen was located in both the cytoplasmic and outer membrane fractions of E. coli, none appeared to be polymerized into a pilus structure.     

In vitro demonstration by the rate assay of the presence of small pore in the outer membrane of Pseudomonas aeruginosa

Determination of the rates of saccharide diffusions by the proteoliposomes showed that the outer membrane of Pseudomonas aeruginosa only possesses small diffusion pores and that protein F might have not been involved in the pore formation. Proteoliposomes containing stachyose or Dextan T-10 showed the same relative diffusion rates as measured by the liposome swelling method. Slopes of the lines, diffusion rate vs saccharide Mr, in the liposomes made of the P. aeruginosa and E. coli B outer membranes appeared to be -7.4 and -3.5, respectively. Intercepts of the lines with x-axis in the liposomes containing the P. aeruginosa and E. coli B outer membrane appeared to be about Mr, 220 and 320, respectively. Relative diffusion rates of saccharides through the liposome membranes reconstituted from the protein F-deficient outer membrane were superimposable with that of the protein F-sufficient outer membrane.     

大鼠心肌线粒体内、外膜磷脂动态结构的研究

我们以DPH为荧光探针.用毫微秒荧光分光光度计测定了大鼠心肌线粒体及线粒体内、外膜的动态微细结构;用HPLC分析了磷脂组成.实验结果提示.完整线粒体膜流动性主要反映了线粒体外膜的运动状态.线粒体内膜微粘度及磷脂分子摇动角大于外膜,扩散速率小于外膜.除去了蛋白质的线粒体内、外膜磷脂脂质体膜流动性无明显差异.提示线粒体内膜的高微粘度与膜中所含有的多量蛋白有关.