Roles of the Carboxy-Terminal Half of Pseudomonas aeruginosa Major Outer Membrane Protein OprF in Cell Shape, Growth in Low-Osmolarity Medium, and Peptidoglycan Association

OprF, the major outer membrane protein of Pseudomonas aeruginosa, is multifunctional in that it can act as a nonspecific porin, plays a role in the maintenance of cell shape, and is required for growth in a low-osmolarity environment. The latter two structural roles of OprF, and OprF’s association with the peptidoglycan, have been proposed to be localized in the carboxy terminus of the protein, based on this region’s similarity to members of the OmpA family of proteins. To determine if this is correct, we constructed a series of C-terminally truncated OprF derivatives and examined their effects on P. aeruginosa cell length and growth in low-osmolarity medium. While the C terminus of OprF was required for wild-type cell length and growth in low-osmolarity medium, expression of the N terminus (first 163 amino acids [aa]) also influenced these phenotypes (compared with OprF deficiency). The first 154 to 164 aa of OprF seemed required for stable protein expression, consistent with the existence of a β-barrel domain in the N terminus of OprF. Greater than 215 aa of the protein were required for strong peptidoglycan association, confirming that residues in the C-terminal end of OprF are required for peptidoglycan binding. OprF deficiency did not affect the in vivo growth of an OprF-deficient strain in a mouse chamber model. Collectively, these data suggest that the C terminus of OprF plays a role in cell length, growth of P. aeruginosa in low-osmolarity media (but not in vivo), and peptidoglycan association, while the N terminus has an influence on the first two characteristics and is additionally important for stable protein expression.     

Specific Association of Lectin LecB with the Surface of Pseudomonas aeruginosa: Role of Outer Membrane Protein OprF

The fucose binding lectin LecB affects biofilm formation and is involved in pathogenicity of Pseudomonas aeruginosa. LecB resides in the outer membrane and can be released specifically by treatment of an outer membrane fraction with fucose suggesting that it binds to specific ligands. Here, we report that LecB binds to the outer membrane protein OprF. In an OprF-deficient P. aeruginosa mutant, LecB is no longer detectable in the membrane but instead in the culture supernatant indicating a specific interaction between LecB and OprF.     

Pore Size Dependence on Growth Temperature Is a Common Characteristic of the Major Outer Membrane Protein OprF in Psychrotrophic and Mesophilic Pseudomonas Species

Pseudomonas species adapt well to hostile environments, which are often subjected to rapid variations. In these bacteria, the outer membrane plays an important role in the sensing of environmental conditions such as temperature. In previous studies, it has been shown that in the psychrotrophic strain P. fluorescens MF0, the major porin OprF changes its channel size according to the growth conditions and could affect outer membrane permeability. Studies of the channel-forming properties of OprFs from P. putida 01G3 and P. aeruginosa PAO1 in planar lipid bilayers generated similar results. The presence of a cysteine- or proline-rich cluster in the central linker region is not essential for channel size modulations. These findings suggest that OprF could adopt two alternative conformations in the outer membrane and that folding is thermoregulated. In contrast, no difference according to growth temperature was observed for structurally different outer membrane proteins, such as OprE3 from the Pseudomonas OprD family of specific porins. Our results are consistent with the fact that the decrease in channel size observed at low growth temperature is a particular feature of the OprF porin in various psychrotrophic and mesophilic Pseudomonas species isolated from diverse ecological niches. The ability to reduce outer membrane permeability at low growth temperature could provide these bacteria with adaptive advantages.     

重组绿脓杆菌外膜蛋白OprF的表达、纯化及多克隆抗体制备与鉴定

绿脓杆菌外膜蛋白Opr F可有效激活机体的免疫机制对抗细菌的感染,在疫苗上有很好的应用前景。采用分子克隆方法获得绿脓杆菌外膜蛋白Opr F表达菌株;利用SDS-PAGE电泳切胶纯化、尿素梯度复性获得Opr F蛋白,免疫小鼠制备Opr F蛋白多克隆抗体。ELISA法表明,Opr F抗血清滴度达1∶12 800倍,Western blotting证实抗血清具有很好的特异性。通过DNAMAN软件对Opr F序列同源性分析发现其C端在不同细菌间存在较高同源性;采用MEGA软件对Opr F的系统发生分析发现假单胞菌属细菌的亲缘关系高于其它属细菌。     

Outer Membrane Protein Heterogeneity within Pseudomonas fluorescens and P. putida and Use of an OprF Antibody as a Probe for rRNA Homology Group I Pseudomonads

The electrophoretic patterns of outer membrane proteins of strains representing the biovars of Pseudomonas fluorescens and Pseudomonas putida were analyzed by gel electrophoresis. The outer membrane protein profiles were variable, and they were not useful for assigning strains to a specific biovar. However, three or four predominant outer membrane proteins migrating at 42 to 46 kDa, 33 to 38 kDa, and 20 to 22 kDa were conserved among the strains. They could be tentatively identified as OprE (44 kDa), OprF (38 kDa), OprH (21 kDa), and OprL (20.5 kDa), which are known proteins from Pseudomonas aeruginosa. A 37-kDa OprF-like protein was purified from P. fluorescens DF57 and used to raise a polyclonal antibody. In Western blot (immunoblot) analysis, this antibody reacted with OprF proteins from members of Pseudomonas rRNA homology group I but not with proteins from nonpseudomonads. The heterogeneity in M(infr) of OprF was greater among P. fluorescens strains than among P. putida strains. Immunofluorescence microscopy of intact cells demonstrated that the antibody recognized epitopes that were accessible only after unmasking by EDTA treatment. The antibody was used in a colony blotting assay to determine the percentage of rRNA homology group I pseudomonads among bacteria from the rhizosphere of barley. The bacteria were isolated on 10% tryptic soy agar, King's B agar, and the pseudomonad-specific medium Gould S1 agar. The estimate of OprF-containing CFU in rhizosphere soil obtained by colony blotting on 10% tryptic soy agar was about 2 and 14 times higher than the values obtained from King's agar and Gould S1 agar, respectively, indicating that not all fluorescent pseudomonads are scored on more specific media. The colonies reacting with the OprF antibody were verified as being rRNA homology group I pseudomonads by using the API 20NE system.     

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     

Membrane Topology of Outer Membrane Protein AlgE,Which Is Required for Alginate Production in Pseudomonas aeruginosa

The ubiquitous opportunistic human pathogen Pseudomonas aeruginosa secretes a viscous extracellular polysaccharide, called alginate, as a virulence factor during chronic infection of patients with cystic fibrosis. In the present study, it was demonstrated that the outer membrane protein AlgE is required for the production of alginate in P. aeruginosa. An isogenic marker-free algE deletion mutant was constructed. This strain was incapable of producing alginate but did secrete alginate degradation products, indicating that polymerization occurs but that the alginate chain is subsequently degraded during transit through the periplasm. Alginate production was restored by introducing the algE gene. The membrane topology of the outer membrane protein AlgE was assessed by site-specific insertions of FLAG epitopes into predicted extracellular loop regions.Pseudomonas aeruginosa is an ubiquitous opportunistic human pathogen responsible for chronic infections of the lungs of patients with cystic fibrosis (CF), in whom it is the leading cause of mortality and morbidity (9). The establishment of a chronic infection in the lungs of patients with CF coincides with the switch of P. aeruginosa to a stable mucoid variant, producing copious amounts of the exopolysaccharide alginate; this is typically a poor prognostic indicator for these patients (24, 31). Alginate is a linear unbranched exopolysaccharide consisting of 1,4-linked monomers of β-d-mannuronic acid and its C-5 epimer, α-l-guluronic acid, which is known to be produced by only two bacterial genera, Pseudomonas and Azotobacter (34). The switch to a mucoid phenotype coincides with the appearance of a 54-kDa protein in the outer membrane; this protein has been identified and has been designated AlgE (13, 31).The genes encoding the alginate biosynthesis machinery are located within a 12-gene operon (algD-alg8-alg44-algK-algE-algG-algX-algL-algI-algJ-algF-algA). AlgA and AlgD, along with AlgC (not encoded in the operon), are involved in precursor synthesis (34). Alg8 is the catalytic subunit of the alginate polymerase located at the inner membrane (35). AlgG is a C-5 mannuronan epimerase (19). AlgK contains four putative Sel1-like repeats, similar to the tetratricopeptide repeat motif often found in adaptor proteins involved in the assembly of multiprotein complexes (3, 10). AlgX shows little homology to any known protein, and its role is unclear (14). Knockout mutants of AlgK, AlgG, and AlgX have nonmucoid phenotypes, although they produce short alginate fragments, due to the activity of the alginate lyase (AlgL), which degrades the nascent alginate (1, 14, 19-21, 36). AlgF, AlgI, and AlgJ are involved in acetylation of alginate, but they are not ultimately required for its production (12). The membrane-anchored protein, Alg44, is required for polymerization and has a PilZ domain for the binding of c-di-GMP, a secondary messenger essential for alginate production (16, 25, 33). The periplasmic C terminus of Alg44 shares homology with the membrane fusion proteins involved in the bridging of the periplasm in multidrug efflux pumps (11, 43). The periplasmic alginate lyase, AlgL, appears to be required for the translocation of intact alginate across the periplasm (1, 26). AlgE is an outer membrane, anion-selective channel protein through which alginate is presumably secreted (30). A protein complex or scaffold through which the alginate chain can pass and be modified and which spans the periplasm bridging the polymerase located (Alg8) at the outer membrane pore (AlgE) has been proposed (21). Indeed, it has been demonstrated that both the inner and the outer membranes are required for the in vitro polymerization of alginate (35).The requirement of AlgE for the biosynthesis of alginate in P. aeruginosa was first observed by complementation of an alginate-negative mutant derived by chemical mutagenesis with a DNA fragment containing algE (8) Secondary structure predictions suggested that AlgE forms an 18-stranded β barrel with extended extracellular loops. Several of these loops show high densities of charged amino acids, suggesting a functional role in the translocation of the anionic alginate polymer (29, 30). Preliminary analysis of AlgE crystals has been reported (48).In this study, the role of AlgE in alginate biosynthesis was investigated and the membrane topology of AlgE was assessed by site-directed insertion mutagenesis.     

Outer Membrane Protein I of Pseudomonas aeruginosa Is a Target of Cationic Antimicrobial Peptide/Protein

Cationic antimicrobial peptides/proteins (AMPs) are important components of the host innate defense mechanisms against invading microorganisms. Here we demonstrate that OprI (outer membrane protein I) of Pseudomonas aeruginosa is responsible for its susceptibility to human ribonuclease 7 (hRNase 7) and α-helical cationic AMPs, instead of surface lipopolysaccharide, which is the initial binding site of cationic AMPs. The antimicrobial activities of hRNase 7 and α-helical cationic AMPs against P. aeruginosa were inhibited by the addition of exogenous OprI or anti-OprI antibody. On modification and internalization of OprI by hRNase 7 into cytosol, the bacterial membrane became permeable to metabolites. The lipoprotein was predicted to consist of an extended loop at the N terminus for hRNase 7/lipopolysaccharide binding, a trimeric α-helix, and a lysine residue at the C terminus for cell wall anchoring. Our findings highlight a novel mechanism of antimicrobial activity and document a previously unexplored target of α-helical cationic AMPs, which may be used for screening drugs to treat antibiotic-resistant bacterial infection.     

Protein secretion systems of Pseudomonas aeruginosa and P fluorescens

Gram-negative bacteria have evolved numerous systems for the export of proteins across their dual-membrane envelopes. Three of these systems (types I, III and IV) secrete proteins across both membranes in a single energy-coupled step. Four systems (Sec, Tat, MscL and Holins) secrete only across the inner membrane, and four systems [the main terminal branch (MTB), fimbrial usher porin (FUP), autotransporter (AT) and two-partner secretion families (TPS)] secrete only across the outer membrane. We have examined the genome sequences of Pseudomonas aeruginosa PAO1 and Pseudomonas fluorescens Pf0-1 for these systems. All systems except type IV were found in P. aeruginosa, and all except types III and IV were found in P. fluorescens. The numbers of each such system were variable depending on the system and species examined. Biochemical and physiological functions were assigned to these systems when possible, and the structural constituents were analyzed. Available information regarding the mechanisms of transport and energy coupling as well as physiological functions is summarized. This report serves to identify and characterize protein secretion systems in two divergent pseudomonads, one an opportunistic human pathogen, the other a plant symbiont.     

A Novel Role for an ECF Sigma Factor in Fatty Acid Biosynthesis and Membrane Fluidity in Pseudomonas aeruginosa

Extracytoplasmic function (ECF) sigma factors are members of cell-surface signaling systems, abundant in the opportunistic pathogen Pseudomonas aeruginosa. Twenty genes coding for ECF sigma factors are present in P. aeruginosa sequenced genomes, most of them being part of TonB systems related to iron uptake. In this work, poorly characterized sigma factors were overexpressed in strain PA14, in an attempt to understand their role in the bacterium´s physiology. Cultures overexpressing SigX displayed a biphasic growth curve, reaching stationary phase earlier than the control strain, followed by subsequent growth resumption. During the first stationary phase, most cells swell and die, but the remaining cells return to the wild type morphology and proceed to a second exponential growth. This is not due to compensatory mutations, since cells recovered from late time points and diluted into fresh medium repeated this behavior. Swollen cells have a more fluid membrane and contain higher amounts of shorter chain fatty acids. A proteomic analysis was performed to identify differentially expressed proteins due to overexpression of sigX, revealing the induction of several fatty acid synthesis (FAS) enzymes. Using qRT-PCR, we showed that at least one isoform from each of the FAS pathway enzymes were upregulated at the mRNA level in the SigX overexpressing strain thus pointing to a role for this ECF sigma factor in the FAS regulation in P. aeruginosa.     

Influence of O Polysaccharides on Biofilm Development and Outer Membrane Vesicle Biogenesis in Pseudomonas aeruginosa PAO1

Pseudomonas aeruginosa is a common opportunistic human pathogen known for its ability to adapt to changes in its environment during the course of infection. These adaptations include changes in the expression of cell surface lipopolysaccharide (LPS), biofilm development, and the production of a protective extracellular exopolysaccharide matrix. Outer membrane vesicles (OMVs) have been identified as an important component of the extracellular matrix of P. aeruginosa biofilms and are thought to contribute to the development and fitness of these bacterial communities. The goal of this study was to examine the relationships between changes in the cell surface expression of LPS O polysaccharides, biofilm development, and OMV biogenesis in P. aeruginosa. We compared wild-type P. aeruginosa PAO1 with three chromosomal knockouts. These knockouts have deletions in the rmd, wbpM, and wbpL genes that produce changes in the expression of common polysaccharide antigen (CPA), O-specific antigen (OSA), or both. Our results demonstrate that changes in O polysaccharide expression do not significantly influence OMV production but do affect the size and protein content of OMVs derived from both CPA⁻ and OSA⁻ cells; these mutant cells also exhibited different physical properties from wild-type cells. We further examined biofilm growth of the mutants and determined that CPA⁻ cells could not develop into robust biofilms and exhibit changes in cell morphology and biofilm matrix production. Together these results demonstrate the importance of O polysaccharide expression on P. aeruginosa OMV composition and highlight the significance of CPA expression in biofilm development.     

Culturability and Expression of Outer Membrane Proteins during Carbon, Nitrogen, or Phosphorus Starvation of Pseudomonas fluorescens DF57 and Pseudomonas putida DF14

Changes in culturability and outer membrane protein profiles were investigated in Pseudomonas fluorescens DF57 and Pseudomonas putida DF14 during starvation for carbon, nitrogen, and phosphorus. P. fluorescens DF57 remained fully culturable for 4 days in all starvation regimes. The cell mass increased during starvation for nitrogen and phosphorus, indicating the accumulation of storage compounds, whereas it decreased slightly in carbon-starved cells. P. putida DF14 lost culturability during phosphorus starvation, and the mass of phosphate-starved cells did not increase. Analysis of additional P. fluorescens and P. putida strains, however, showed that the ability to preserve culturability during phosphorus starvation was not species but strain dependent. In DF57, an outer membrane protein of 55 kDa appeared during starvation for phosphorus, while another protein of 63 kDa was seen during all starvation conditions. DF14 induced two outer membrane proteins of 28 and 29 kDa during starvation for carbon and nitrogen, but no phosphorus-specific starvation protein could be detected. Therefore, starvation-induced outer membrane proteins do not seem to be conserved among the fluorescent pseudomonads and a unique starvation response might be found in individual strains.     

Sialylation of Outer Membrane Porin Protein D: A Mechanistic Basis of Antibiotic Uptake in Pseudomonas aeruginosa

Pseudomonas aeruginosa (PA) is an environmentally ubiquitous, extracellular, opportunistic pathogen, associated with severe infections of immune-compromised host. We demonstrated earlier the presence of both α2,3- and α2,6-linked sialic acids (Sias) on PA (PA^+Sias) and normal human serum is their source of Sias. PA^+Sias showed decreased complement deposition and exhibited enhanced association with immune-cells through sialic acid binding immunoglobulin like lectins (Siglecs). Such Sias-siglec-9 interaction between PA^+Sias and neutrophils helped to subvert host immunity. Additionally, PA^+Sias showed more resistant to β-lactam antibiotics as reflected in their minimum inhibitory concentration required to inhibit the growth of 50% than PA^−Sias. Accordingly, we have affinity purified sialoglycoproteins of PA^+Sias. They were electrophoresed and identified by matrix-assisted laser desorption-ionization time-of-flight/time-of-flight mass spectrometry analysis. Sequence study indicated the presence of a few α2,6-linked, α2,3-linked, and both α2,3- and α2,6-linked sialylated proteins in PA. The outer membrane porin protein D (OprD), a specialized channel-forming protein, responsible for uptake of β-lactam antibiotics, is one such identified sialoglycoprotein. Accordingly, sialylated (OprD^+Sias) and non-sialylated (OprD^−Sias) porin proteins were separately purified by using anion exchange chromatography. Sialylation of purified OprD^+Sias was confirmed by several analytical and biochemical procedures. Profiling of glycan structures revealed three sialylated N-glycans and two sialylated O-glycans in OprD^+Sias. In contrast, OprD^−Sias exhibit only one sialylated N-glycans. OprD^−Sias interacts with β-lactam antibiotics more than OprD^+Sias as demonstrated by surface plasmon resonance study. Lyposome-swelling assay further exhibited that antibiotics have more capability to penetrate through OprD^−Sias purified from four clinical isolates of PA. Taken together, it may be envisaged that sialic acids on OprD protein play important role toward the uptake of commonly used antibiotics in PA^+Sias. This might be one of the new mechanisms of PA for β-lactam antibiotic uptake.Sialic acids (Sias)¹ are nine carbon atom containing acidic residues characteristically found in the terminal position of glycoproteins and glycolipids (1–4). Structural diversity of sialic acids is because of the modification of one or more hydroxyl groups in various positions of the core structure by different groups like acetyl-, methyl-, sulfate-, lactyl-, or phosphate (1, 5–7). More than fifty derivatives of Sias has been reported both in vertebrate and invertebrate systems. It functions as ligand for various cellular communications and also act as masking element for glycoconjugates (8–12).Sialic acid binding immunoglobulins (Ig)-like lectins (siglecs) selectively expressed on the hematopoetic cells and interact with an array of linkage-specific Sias on a glycan structure express on the same cells or other cells (13). Siglecs can also recognize terminal sialylated glycoconjugates on several pathogens (14–16). After recognizing, they carry out various functions like internalization, attenuation of inflammation, restraining cellular activation along with inhibition of natural killer cell activation (17).Pseudomonas aeruginosa (PA) is a Gram-negative, rod-shaped bacterium. This human pathogen has remarkable capacity to cause diseases in immune compromised hosts. This colonizing microbial pathogen is responsible for infection in chronic cystic fibrosis, nosocomial infections; severe burn, transplantation, cancer, and AIDS and other immuno-supressed patients (18).We have reported earlier the presence of linkage-specific Sias on PA. Normal human serum (NHS) is possibly one of the sources of these Sias (19). PA utilizes these Sias to interact through siglecs present on the surface of different immune cells. PA^+Sias showed enhanced association with neutrophils through α2,3-linked Sias-siglec-9 interaction which facilitated their survival by subverting innate immune function of host (20).The treatment of PA-infected patient depends upon the extent of the disease and the concerned organs. Conventional β-lactam, cephalosporins, and aminoglycosides group of antibiotics are most common for such treatment (21). β-lactam antibiotics inhibit cell wall synthesis by disrupting the synthesis of the peptidoglycan layer of bacterial cell walls (22). When PA showed resistant to β-lactam antibiotics, new generation of β-lactam with increased doses or other broad spectrum antibiotics like tetracyclines or fluoroquinolones are prescribed (23). PA isolates from intensive care unit (ICU) patients in general showed higher rates of β-lactam resistance among other hospitalized patients (24). The increasing frequency of resistance to ceftazidime, piperacillin, imipenem, fluoroquinolone, and aminoglycoside were 36.6%, 22.3%, 22.8%, 23.8%, and 17.8% respectively in PA (25).The outer membrane of Gram-negative bacteria is, in general, semipermeable through which hydrophilic molecules including antibiotics of below exclusion limit size (0.6 kDa) can pass through the channel-forming proteins generally called porins e.g. OprD, OprF, OprG etc. (26, 27). PA shows lower outer membrane permeability with respect to many other Gram-negative bacteria like Acinetobacter baumannii, Stenotrophomonas maltophilia, Burkholderia cepacia, hence the diffusion rate of β-lactam antibiotics is decreased (27).Additionally, PA uses MexA-MexB-OprM, MexC-MexD-OprJ, MexE-MexF-OprN, and MexX-MexY-OprM as efflux pumps along with important regulatory factors MexR/NalB, NfxB, NfxC/MexT, and MexZ respectively on their membrane to pump out undesirable chemicals, detergent and antibiotics (28–32). Other Gram-negative bacteria also uses similar types of efflux pumps for such purposes. Moreover, PA produces antibiotic-resistance genes by some mutation (33). Furthermore, β-lactamase and aminoglycoside-modifying enzymes produced by PA are capable of breaking down the antibiotics (34). Alternatively, these enzymes can directly modify the drug. Hence these antibiotics become functionally ineffective (27).The presence of lipopolysaccharides (LPS) containing O-specific polysaccharides with tri-saccharide repeats of 2-acetamido-2,6-dideoxy-d-glucose, 2-acetamido-2,6-dideoxy-d-galactose, and 5-acetamido-3,5,7,9-tetyradeoxy-7-[(R)-3-hydroxybutyramidol]-3-l-glycerol-l-manno-nonulosonic acid are known for PA serogroup O11 (35). The genes for key enzymes required for complex protein glycosylation are found in the genome of PA14 (36). Moreover, glycosylation in PA1244 has been reported in the form of an O-linked glycan in pilin (37). A cluster of seven genes known as the pel genes, encode proteins with similarity to components involved in polysaccharide biogenesis. Among these genes, PelF is a putative glycosyltransferase (GT) of the type IV glycosyltransferase (GT4) family (36). PA secreted sialidase in culture medium (38). Genome search reveals that PA14 has the sialidase gene, which may be responsible for cleaving sialic acids (39). PA1 also has sialic acid transporter gene, which possibly transport sialic acids inside the cells (Gene ID: 17688338, Source: http://www.ncbi.nlm.nih.gov/gene/17688338). Additionally, CMP-sialic acid transferase, which is responsible for converting sialic acids to CMP-sialic acid, was purified from PAO12 (40). This enzyme shows close similarity with the enzyme found in E. coli.However, PA being such a notorious organism, it might have many other different mechanisms to fight against antibiotics for their survival. Therefore, it is worthwhile to explore newer mechanism to understand how antibiotics penetrate inside this bacterium. Here we addressed the following questions. Does sialylation of glycoproteins demonstrated on PA play any role in the entry of antibiotics that might facilitate their survival within host?Accordingly, we have affinity purified a few sialoglycoproteins from PA. Sequence analysis identified twenty six α2,3- and α2,6-linked sialoglycoproteins. One such identified sialoglycoprotein is OprD porin protein. The presence of Sias on OprD was conclusively confirmed. We have demonstrated that Sias on OprD protein isolated four different clinical isolates hampered its interaction with β-lactam antibiotics. This might be one of the new mechanisms for β-lactam antibiotic resistance of PA and thereby facilitates their survival in host.     

Electron Microscopy of the Major Outer Membrane Protein of Campylobacter jejuni

The surfaces of the disrupted-cell surfaces of the Campylobacter jejuni strains FUM158432 and M1 were examined using the negative-staining technique and electron microscopy. The surfaces of the whole cells and the outer membranes were covered with small dark dots which, in some areas, were arranged in hexagonal patterns. The hexagonal arrangement was more clearly seen in extracted outer membrane. The size of each structure was measured based on a center-to-center distance with the adjacent structure, and was determined to be 9.9±0.9 nm. A profile of the proteins in the outer membrane by SDS-PAGE, performed in 0.1% SDS and at 100 C, showed 42 kDa proteins to comprise the major outer membrane protein of this bacterium. Digestion of the outer membrane materials with proteinase reduced this protein band in the SDS-PAGE, and the amount of dark dots on the electron micrograph indicated the structure to be the major outer membrane protein (porin) of this bacterium. The power spectrogram of a computer-assisted Fourier transformation of the hexagonally arranged porin proteins suggests that the porin has a trimeric structure rather than a monomeric one.     

Membrane and Chaperone Recognition by the Major Translocator Protein PopB of the Type III Secretion System of Pseudomonas aeruginosa

The type III secretion system is a widespread apparatus used by pathogenic bacteria to inject effectors directly into the cytoplasm of eukaryotic cells. A key component of this highly conserved system is the translocon, a pore formed in the host membrane that is essential for toxins to bypass this last physical barrier. In Pseudomonas aeruginosa the translocon is composed of PopB and PopD, both of which before secretion are stabilized within the bacterial cytoplasm by a common chaperone, PcrH. In this work we characterize PopB, the major translocator, in both membrane-associated and PcrH-bound forms. By combining sucrose gradient centrifugation experiments, limited proteolysis, one-dimensional NMR, and β-lactamase reporter assays on eukaryotic cells, we show that PopB is stably inserted into bilayers with its flexible N-terminal domain and C-terminal tail exposed to the outside. In addition, we also report the crystal structure of the complex between PcrH and an N-terminal region of PopB (residues 51–59), which reveals that PopB lies within the concave face of PcrH, employing mostly backbone residues for contact. PcrH is thus the first chaperone whose structure has been solved in complex with both type III secretion systems translocators, revealing that both molecules employ the same surface for binding and excluding the possibility of formation of a ternary complex. The characterization of the major type III secretion system translocon component in both membrane-bound and chaperone-bound forms is a key step for the eventual development of antibacterials that block translocon assembly.     

Differential Contributions of the Outer Membrane Receptors PhuR and HasR to Heme Acquisition in Pseudomonas aeruginosa

Pseudomonas aeruginosa PAO1 encodes two outer membrane receptors, PhuR (Pseudomonas heme uptake) and HasR (heme assimilation system). The HasR and PhuR receptors have distinct heme coordinating ligands and substrate specificities. HasR is encoded in an operon with a secreted hemophore, HasAp. In contrast the non-hemophore-dependent PhuR is encoded within an operon along with proteins required for heme translocation into the cytoplasm. Herein we report on the contributions of the HasR and PhuR receptors to heme uptake and utilization. Employing bacterial genetics and isotopic [¹³C]heme labeling studies we have shown both PhuR and HasR are required for optimal heme utilization. However, the unique His-Tyr-ligated PhuR plays a major role in the acquisition of heme. In contrast the HasR receptor plays a primary role in the sensing of extracellular heme and a supplementary role in heme uptake. We propose PhuR and HasR represent non-redundant heme receptors, capable of accessing heme across a wide range of physiological conditions on colonization of the host.