Separation of the cytoplasmic and outer membrane of Pseudomonas aeruginosa PAQ.

A method is described for the isolation of the cytoplasmic and outer membranes of $\text{Pseudomonas aeruginosa}$ PAO.1. The cytoplasmic membrane exhibits nicotinamide adenine dinucleotide oxidoreductase, lactate dehydrogenase DD-carboxypeptidase and succinate dehydrogenase activities. The outer membrane is rich in 2-keto-3-deoxyoctonate and exhibits phospholipase A and DD-carboxypeptidase activity. At least 25 protein species have been detected in the cytoplasmic membrane by polyacrylamide gel electrophoresis. Using the same technique, the outer membrane contains only five protein species of molecular weights, 56,000, 53,000, 38,000, 21,000 and 16,000.     

Pseudomonas aeruginosa outer membrane: peptidoglycan-associated proteins.

The Pseudomonas aeruginosa outer membrane was isolated with attached peptidoglycan and fractionated with Triton X-100, ethylenediaminetetraacetate, and lysozyme. The data suggest that major outer membrane proteins F, H2, and I are noncovalently associated with the peptidoglycan.     

Separation of gate- and channel-forming domains in the pore-forming protein of the outer membrane of Pseudomonas aeruginosa.

The domains of the pore-forming protein responsible for the gate and channel formations were separated and identified in the outer membrane of Pseudomonas aeruginosa. The proteolytic cleavage of the 46K channel protein, protein D2, yielded two major domains with apparent M(r) of 27K and 19K. We identified the 27K polypeptide to be the channel-forming domain by an in vitro permeability assay. The channel size of purified 27K domain was indistinguishable from that of native protein D2. Degradation of the 19K domain into small subfragments increases the channel activity about ten times suggesting that the 19K polypeptide forms the gate or cap.     

Linker-insertion mutagenesis of Pseudomonas aeruginosa outer membrane protein OprF

The oprF gene, expressing Pseudomonas aeruginosa major outer membrane protein OprF, was subjected to semi-random linker mutagenesis by insertion of a 1.3 kb Hincll kanamycin-resistance fragment from plasmid pUC4KAPA into multiple blunt-ended restriction sites in the oprF gene. The kanamycin-resistance gene was then removed by Pstl digestion, which left a 12 nucleotide pair linker residue. Nine unique clones were identified that contained such linkers at different locations within the oprF gene and were permissive for the production of full-length OprF variants. In addition, one permissive site-directed insertion, one non-permissive insertion and one carboxy-terminal insertion leading to proteolytic truncation were also identified. These mutants were characterized by DNA sequencing and reactivity of the OprF variants with a bank of 10 OprF-specific monoclonal antibodies. Permissive clones produced OprF variants that were shown to be reactive with the majority of these monoclonal antibodies, except where the insertion was suspected of interrupting the epitope for the specific monoclonal antibody. In addition, these variants were shown to be 2-mercaptoethanol modifiable, to be resistant to trypsin cleavage in intact cells and partly cleaved to a high-molecular-weight core fragment in outer membranes and, where studied, to be accessible to indirect immunofluorescenee labelling in intact cells by monoclonal antibodies specific for surface epitopes. Based on these data, a revised structural model for OprF is proposed.     

Insertion mutagenesis and membrane topology model of the Pseudomonas aeruginosa outer membrane protein OprM

Pseudomonas aeruginosa OprM is a protein involved in multiple-antibiotic resistance as the outer membrane component for the MexA-MexB-OprM efflux system. Planar lipid bilayer experiments showed that OprM had channel-forming activity with an average single-channel conductance of only about 80 pS in 1 M KCl. The gene encoding OprM was subjected to insertion mutagenesis by cloning of a foreign epitope from the circumsporozoite form of the malarial parasite Plasmodium falciparum into 11 sites. In Escherichia coli, 8 of the 11 insertion mutant genes expressed proteins at levels comparable to those obtained with the wild-type gene and the inserted malarial epitopes were surface accessible as assessed by indirect immunofluorescence. When moved to a P. aeruginosa OprM-deficient strain, seven of the insertion mutant genes expressed proteins at variable levels comparable to that of wild-type OprM and three of these reconstituted MIC profiles resembling those of the wild-type protein, while the other mutant forms showed variable MIC results. Utilizing the data from these experiments, in conjunction with multiple sequence alignments and structure predictions, an OprM topology model with 16 beta strands was proposed.     

A small diffusion pore in the outer membrane of Pseudomonas aeruginosa

The permeability properties of the outer membrane of Pseudomonas aeruginosa were re-examined, since the reported conclusions are conflicting [Decad, M. G. and Nikaido, H. (1976) J. Bacteriol. 128, 325-336; Caulcott, C. A., Brown, M. R. W. and Gonda, I. (1984) FEMS Microbiol. Lett. 21, 119-123]. On the basis of the experimental evidence to be described below we conclude that the exclusion limit of the outer membrane of P. aeruginosa is smaller than the size of uncharged disaccharides but larger than the size of hexose. This conclusion is based on the following evidence. Penetration of monosaccharides into the expanded periplasm was large and that of disaccharides was small, after the cells were plasmolyzed with 600 mosM NaCl. A significant amount of protein was released after osmotic down-shock of cells treated with the hypertonic monosaccharides but not of cells treated with the hypertonic saccharides larger than disaccharides. Centrifuged pellets of cells treated with hypertonic di, tri and tetrasaccharides weighed about 15-20% less than that of cells treated with the isotonic monosaccharide, suggesting that the osmotic pressure was exerted on the outer membrane causing dehydration and shrinking of the cells. By contrast, cells treated with the hypertonic pentose and hexoses weighed about 0.1% and 6% less, respectively, than cells treated with the isotonic saccharide, suggesting that pentose diffused through the outer membrane freely.     

Evidence for small pores in the outer membrane of Pseudomonas aeruginosa

Abstract Pseudomonas aeruginosa NCTC6750 and Escherichia coli K12 were used to study permeability of whole, intact cells to a series of labelled oligosaccharides. Stationary phase, oxygen depleted simple salts batch cultures were used. An efflux method was used to compare diffusion from cells of various ³H-labelled sugars (an homologous series based on isomaltitol) with diffusion of [¹⁴C]sucrose. Both plasmolysed and unplasmolysed cell suspensions were used. The data are consistent with an E. coli pore exclusion limit of approx. 833 Da for unplasmolysed cells and of about 670 Da for plasmolysed cells. For P. aeruginosa the data indicated a relatively small pore exclusion limit about the same size as sucrose with plasmolysis having little effect. These findings were confirmed with P. aeruginosa PAO1 grown in nutrient broth.     

Crystal structure of the outer membrane protein OpdK from Pseudomonas aeruginosa

In Gram-negative bacteria that do not have porins, most water-soluble and small molecules are taken up by substrate-specific channels belonging to the OprD family. We report here the X-ray crystal structure of OpdK, an OprD family member implicated in the uptake of vanillate and related small aromatic acids. The OpdK structure reveals a monomeric, 18-stranded beta barrel with a kidney-shaped central pore. The OpdK pore constriction is relatively wide for a substrate-specific channel (approximately 8 A diameter), and it is lined by a positively charged patch of arginine residues on one side and an electronegative pocket on the opposite side-features likely to be important for substrate selection. Single-channel electrical recordings of OpdK show binding of vanillate to the channel, and they suggest that OpdK forms labile trimers in the outer membrane. Comparison of the OpdK structure with that of Pseudomonas aeruginosa OprD provides the first qualitative insights into the different substrate specificities of these closely related channels.     

Secretins of Pseudomonas aeruginosa: large holes in the outer membrane

Pseudomonas aeruginosa produces a large number of exoproteins, ranging from the ADP-ribosyltransferases exotoxin A and ExoS to degradative enzymes, such as elastase and chitinase. As it is a gram-negative bacterium, P. aeruginosa must be able to transport these exoproteins across both membranes of the cell envelope. In addition, also proteins that are part of cellular appendages, such as type IV pili and flagella, have to cross the cell envelope. Whereas the majority of the proteins transported across the inner membrane are dependent on the Sec channel, the systems for translocation across the outer membrane seem to be more diverse. Gram-negative bacteria have invented a number of different strategies during the course of evolution to achieve this goal. Although these transport machineries seem to be radically different, many of them actually depend on a member of the secretin protein family for their function. Recent results show that secretins form a large complex in the outer membrane, which constitutes the actual translocation channel. Understanding the working mechanism of this protein translocation channel could open up new strategies to target molecular machineries at the heart of many important virulence factors.     

Use of steroids to monitor alterations in the outer membrane of Pseudomonas aeruginosa.

Testosterone (a strongly hydrophobic steroid) and testosterone hemisuccinate (a negatively charged derivative) were used as probes to investigate alterations in the outer membrane of Pseudomonas aeruginosa. Diffusion rates of the steroids across the lipid bilayer were measured by coupling the influx of these compounds to their subsequent oxidation by an intracellular delta1-dehydrogenase enzyme. Wild-type cells of P. aeruginosa (strain PAO1) were found to be 25 times more permeable to testosterone than to testosterone hemisuccinate. The uptake of the latter compound appeared to be partially dependent on the external pH, thus suggesting a preferential diffusion of the uncharged protonated form across the cell envelope. Using various PAO mutants, we showed that the permeation of steroids was not affected by overexpression of active efflux systems but was increased up to 5.5-fold when the outer membrane contained defective lipopolysaccharides or lacked the major porin OprF. Such alterations in the hydrophobic uptake pathway were not, however, associated with an enhanced permeability of the mutants to the small hydrophilic molecule N,N,N',N'-tetramethyl-p-phenylene diamine. Thirty-six agents were also assayed for their ability to damage the cell surface of strain PAO1, using testosterone as a probe. Polymyxins, rBPI23, chlorhexidine, and dibromopropamidine demonstrated the strongest permeabilizing activities on a molar basis in the presence of 1 mM MgCl2. These amphiphilic polycations increased the transmembrane diffusion of testosterone up to 50-fold and sensitized the PAO1 cells to hydrophobic antibiotics. All together, these data indicated that the steroid uptake assay provides a direct and accurate measurement of the hydrophobic uptake pathway in P. aeruginosa.     

Evidence for the location of OprM in the Pseudomonas aeruginosa outer membrane

Abstract OprM with a M _r of 49 K is associated with the multidrug resistance of Pseudomonas aeruginosa . Detergent fractionation of bacterial cells has demonstrated that OprM is located in the outer membrane from which it sediments with the other major outer membrane proteins. In this study we have determined the location of OprM as the P. aeruginosa outer membrane. Western immunoblots of cell fractions, obtained by sucrose density gradient centrifugation of whole cell lysates, were probed with an OprM-specific murine polyclonal antiserum.     

Interaction of TonB with the outer membrane receptor FpvA of Pseudomonas aeruginosa

Pyoverdine-mediated iron uptake by the FpvA receptor in the outer membrane of Pseudomonas aeruginosa is dependent on the inner membrane protein TonB1. This energy transducer couples the proton-electrochemical potential of the inner membrane to the transport event. To shed more light upon this process, a recombinant TonB1 protein lacking the N-terminal inner membrane anchor (TonB(pp)) was constructed. This protein was, after expression in Escherichia coli, purified from the soluble fraction of lysed cells by means of an N-terminal hexahistidine or glutathione S-transferase (GST) tag. Purified GST-TonB(pp) was able to capture detergent-solubilized FpvA, regardless of the presence of pyoverdine or pyoverdine-Fe. Targeting of the TonB1 fragment to the periplasm of P. aeruginosa inhibited the transport of ferric pyoverdine by FpvA in vivo, indicating an interference with endogenous TonB1, presumably caused by competition for binding sites at the transporter or by formation of nonfunctional TonB heterodimers. Surface plasmon resonance experiments demonstrated that the FpvA-TonB(pp) interactions have apparent affinities in the micromolar range. The binding of pyoverdine or ferric pyoverdine to FpvA did not modulate this affinity. Apparently, the presence of either iron or pyoverdine is not essential for the formation of the FpvA-TonB complex in vitro.     

A novel lipolytic enzyme located in the outer membrane of Pseudomonas aeruginosa

A lipase-negative deletion mutant of Pseudomonas aeruginosa PAO1 still showed extracellular lipolytic activity toward short-chain p-nitrophenylesters. By screening a genomic DNA library of P. aeruginosa PAO1, an esterase gene, estA, was identified, cloned, and sequenced, revealing an open reading frame of 1,941 bp. The product of estA is a 69.5-kDa protein, which is probably processed by removal of an N-terminal signal peptide to yield a 67-kDa mature protein. A molecular mass of 66 kDa was determined for (35)S-labeled EstA by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. The amino acid sequence of EstA indicated that the esterase is a member of a novel GDSL family of lipolytic enzymes. The estA gene showed high similarity to an open reading frame of unknown function located in the trpE-trpG region of P. putida and to a gene encoding an outer membrane esterase of Salmonella typhimurium. Amino acid sequence alignments led us to predict that this esterase is an autotransporter protein which possesses a carboxy-terminal beta-barrel domain, allowing the secretion of the amino-terminal passenger domain harboring the catalytic activity. Expression of estA in P. aeruginosa and Escherichia coli and subsequent cell fractionation revealed that the enzyme was associated with the cellular membranes. Trypsin treatment of whole cells released a significant amount of esterase, indicating that the enzyme was located in the outer membrane with the catalytic domain exposed to the surface. To our knowledge, this esterase is unique in that it exemplifies in P. aeruginosa (i) the first enzyme identified in the outer membrane and (ii) the first example of a type IV secretion mechanism.     

Production and characterization of monoclonal antibodies to outer membrane proteins of Pseudomonas aeruginosa grown in iron-depleted media

The iron uptake systems of pathogenic bacteria provide potential targets for immunological intervention. We have partially purified the high molecular mass, iron-regulated outer membrane proteins (IROMPs) from Pseudomonas aeruginosa and used them to prepare a panel of monoclonal antibodies (mAbs). Five mAbs reacted with an 85 kDa IROMP separated by SDS-PAGE, but gave only low-level binding to whole cells by immunogold electron microscopy. However, iodination of whole cells indicated that the 85 kDa IROMP is surface-exposed. The mAbs were only cross-reactive with clinical isolates representing eight of the 17 International Antigenic Typing Scheme serotypes of P. aeruginosa, suggesting significant heterogeneity with respect to this IROMP.     

Isolation and characterization of major outer membrane proteins of Pseudomonas aeruginosa strain PAO with special reference to peptidoglycan-associated protein.

The outer membrane of Pseudomonas aeruginosa PAO contains six major proteins (proteins D, E, F, G,H, and I). Two of them (protein F and protein H) were found to be retained by the peptidoglycan layer when cell envelopes were extracted with 2% sodium dodecyl sulfate (SDS) solution at 35 degrees C. At higher temperature (greater than 55 degrees C), no proteins were retained by peptidoglycan. By making use of this property, purification of protein F and protein H was achieved. Three other major outer membrane proteins, D, E, and I were also isolated and characterized. Their amino acids compositions were determined. Circular dichroism spectra of these isolated proteins were measured in SDS solution. Protein F was rich in beta-structure, while protein I was rich in alpha-helix. When isolated protein F was heated (100 degrees C-15 min) in SDS solution, the circular dichroism spectrum changed significantly. In parallel with the conformational change, the electrophoretic mobility of protein F on urea-SDS polyacrylamide gel also changed. These results indicate that protein F is a so-called heat-modifiable protein.     

Enolase-like protein present on the outer membrane of Pseudomonas aeruginosa binds plasminogen

Pseudomonas aeruginosa is one of the pathogenic bacteria which utilize binding of the host plasminogen (Plg) to promote their invasion throughout the host tissues. In the present study, we confirmed that P. aeruginosa exhibits binding affinity for human plasminogen. Furthermore, we showed that the protein detected on the cell wall of P. aeruginosa and binding human plasminogen is an enolase-like protein. The hypothesis that alpha-enolase, a cytoplasmatic glycolytic enzyme, resides also on the cell surface of the bacterium was supported by electron microscopy analysis. The plasminogen-binding activity of bacterial cell wall outer membrane enolase-like protein was examined by immunoblotting assay.     

Role of protein F in maintaining structural integrity of the Pseudomonas aeruginosa outer membrane.

To investigate the functional role of protein F of the outer membrane of Pseudomonas aeruginosa, we isolated mutants devoid of protein F, and the defective gene was transferred to a wild-type strain by plasmid FP5-mediated conjugation. Chemical analyses of the protein F-deficient outer membrane revealed that the amount of outer membrane protein was reduced to 72 to 74% of that of the protein F-sufficient strain and that lipopolysaccharides and phospholipids increased to 117 to 123% and 135 to 136%, respectively. The mutants and the transconjugant showed the following characteristics: (i) growth rates of protein F-deficient strains in low-osmolarity medium (e.g., L broth containing 0.1% NaCl) were less than 1/10 the rate of the protein F-sufficient strain; (ii) protein F-deficient cells were rounded, and the outer membrane formed large protruded blebs; and (iii) the outer membrane became physically fragile, since a significant amount of periplasmic proteins leaked out and the cells became highly sensitive to osmotic shock. The results suggested that protein F plays an important role in morphogenesis and in maintaining the integrity of the outer membrane. Determination of the diffusion rates of saccharides and beta-lactam antibiotics showed that the protein F-deficient outer membrane had no detectable transport defect compared with the protein F-sufficient outer membrane. The MICs of antibiotics for the protein F-deficient strains were nearly identical to those for the protein F-sufficient strain.     

Factors that influence the permeability assay of the outer membrane of Pseudomonas aeruginosa

Brief exposure of Pseudomonas aeruginosa to a temperature of 10 degrees C or lower caused a significant leakage of the periplasmic beta-lactamase into the medium. The extent of leakage increased as the incubation temperature was lowered to 4 degrees C and reached a maximum at 0 degrees C. Cells grown in the presence of beta-lactamase inducers were unsuitable for the permeability assay. It was found that the diffusion rates of beta-lactams through the outer membrane of P. aeruginosa were much lower than those previously reported, as assayed under refined conditions. The diffusion rates of beta-lactams in one of the mutants tested were an order of magnitude lower than those of the other strains, despite the fact that the outer membrane protein profile of the strain appeared to be indistinguishable from those of the others. These results suggest that beta-lactam antibiotics diffuse through the outer membrane of P. aeruginosa, at least partly, through a non-porin pathway.