Biophysical characterization of OprB,a glucose-inducible porin ofPseudomonas aeruginosa

OprB, a glucose-inducible porin ofP. aeruginosa, was characterized by black lipid bilayer analysis and circular dichroism spectroscopy. Black lipid bilayer analysis of OprB revealed a single-channel conductance of 25 pS, the presence of a glucose binding site with aK _s for glucose of 380 ± 40 mM, and the formation of channels with a strong selection for anions. Analysis ofP. aeruginosa OprB circular dichroism spectra revealed a high sheet content (40%) which is within the range of that determined for other porins. Values obtained from black lipid bilayer analysis were compared to those previously obtained for OprB ofP. putida [Saravolacet al. (1991).J. Bacteriol. 173, 4970–4976] and indicated extensive similarities in the single-channel conductance and glucose-binding properties of these two porins. Immunological and amino terminal sequence analysis revealed a high degree of homology. Of the first 14 amino terminal residues, 12 were identical. A major difference between the two porins was found in their ion selectivity. WhereasP. aeruginosa OprB is anion selective,P. putida OprB and other carbohydrate selective porins are known to be cation selective.     

Characterization of OpdH, a Pseudomonas aeruginosa porin involved in the uptake of tricarboxylates

The Pseudomonas aeruginosa outer membrane is intrinsically impermeable to many classes of antibiotics, due in part to its relative lack of general uptake pathways. Instead, this organism relies on a large number of substrate-specific uptake porins. Included in this group are the 19 members of the OprD family, which are involved in the uptake of a diverse array of metabolites. One of these porins, OpdH, has been implicated in the uptake of cis-aconitate. Here we demonstrate that this porin may also enable P. aeruginosa to take up other tricarboxylates. Isocitrate and citrate strongly and specifically induced the opdH gene via a mechanism involving derepression by the putative two-component regulatory system PA0756-PA0757. Planar bilayer analysis of purified OpdH demonstrated that it was a channel-forming protein with a large single-channel conductance (230 pS in 1 M KCl; 10-fold higher than that of OprD); however, we were unable to demonstrate the presence of a tricarboxylate binding site within the channel. Thus, these data suggest that the requirement for OpdH for efficient growth on tricarboxylates was likely due to the specific expression of this large-channel porin under particular growth conditions.     

Exogenous siderophore-mediated iron uptake in Pseudomonas aeruginosa: possible involvement of porin OprF in iron translocation.

In addition to the two siderophores pyoverdine and pyochelin synthesized by Pseudomonas aeruginosa ATCC 15692 (strain PAO1), several siderophores produced by other bacteria or fungi, namely cepabactin, salicylic acid, desferriferrichrysin, desferriferricrocin, desferriferrioxamine B, desferriferrioxamine E and coprogen, were able to promote iron uptake with variable efficiencies into this bacterium. For most of these siderophores, these results were consistent with the growth stimulation produced by the same compounds in a plate bioassay. Desferriferrichrome A, enterobactin and desferriferrirubin, however, did not promote iron uptake, although enterobactin and desferriferrirubin stimulated bacterial growth. These paradoxical data are discussed in view of siderophore-inducible iron uptake systems, as demonstrated recently for enterobactin. Among the strains tested, including the wild-type PAO1, the pyoverdine-less mutant PAO6606 and the two porin-mutants P. aeruginosa H636 (oprF::omega) and P. aeruginosa H673 (oprD::Tn501), only for the porin-OprF mutant were fewer siderophores able to promote iron uptake compared to the other strains. Such results suggest that beside specific routes for iron uptake P. aeruginosa is also able to take up siderophore-liganded iron through OprF.     

Insertion mutagenesis of the Pseudomonas aeruginosa phosphate-specific porin OprP.

The gene encoding the Pseudomonas aeruginosa phosphate-specific porin OprP was subjected to both linker and epitope insertion mutageneses. Nine of the 13 linker mutant genes expressed protein at levels comparable to those obtained with the wild-type gene. These mutant proteins were shown, by indirect immunofluorescence with an OprP-specific antiserum, to be properly exposed at the cell surface. Four of the linker mutant genes expressed protein at reduced levels which were not detectable at the cell surface. A foreign epitope from the circumsporozoite form of the malarial parasite Plasmodium falciparum was cloned into the linker sites of 12 of the 13 mutant genes. Seven of the resultant epitope insertion mutant genes expressed surface-exposed protein. Two of these mutant genes presented the foreign epitope at surface-accessible regions as assessed by indirect immunofluorescence with a malarial epitope-specific monoclonal antibody. The data from these experiments were used to create a topological model of the OprP monomer.     

Pseudomonas aeruginosa porin OprF exists in two different conformations

The major nonspecific porin of Pseudomonas aeruginosa, OprF, produces a large channel yet allows only a slow diffusion of various solutes. Here we provide an explanation of this apparent paradox. We first show, by introduction of tobacco etch virus protease cleavage site in the middle of OprF protein, that most of OprF population folds as a two-domain protein with an N-terminal beta-barrel domain and a C-terminal periplasmic domain rich in alpha-helices. However, sedimentation of unilamellar proteoliposomes through an iso-osmotic gradient showed that only about 5% of the OprF population produced open channels. Gel filtration showed that the open channel conformers tended to occur in oligomeric associations. Because the open channel conformer is likely to fold as a single domain protein with a large beta-barrel, we reasoned that residues near the C terminus may be exposed on cell surface in this conformer. Introduction of a cysteine residue at position 312 produced a functional mutant protein. By using bulky biotinylation reagents on intact cells, we showed that this cysteine residue was not exposed on cell surface in most of the OprF population. However, the minority OprF population that was biotinylated in such experiments was enriched for the conformer with pore-forming activity and had a 10-fold higher pore-forming specific activity than the bulk OprF population. Finally trypsin treatment, which preferentially cleaves the C-terminal domain of the two-domain conformer, did not affect the pore-forming activity of OprF nor did it digest the minority conformer whose residue 312 is exposed on cell surface.     

Clustering of mutations affecting central pathway enzymes of carbohydrate catabolism in Pseudomonas aeruginosa

Whole metabolizing Brevibacterium linens cells were used to study the transport of aromatic amino acids. Kinetic results followed the Michaelis-Menten equation with apparent Km values for phenylalanine, tyrosine, and tryptophan of 24, 3.5, and 1.8 microM. Transport of these amino acids was optimum at pH 7.5 and 25 degrees C for phenylalanine and pH 8.0 and 35 degrees C for tyrosine and tryptophan. Crossed inhibitions were all noncompetitive. The only marked stereospecificity was for the L form of phenylalanine. Transport was almost totally inhibited by carbonyl cyanide-m-chlorophenylhydrazone. Iodoacetate and N-ethylmaleimide were much more inhibitory for tryptophan transport than for transport of the other two aromatic amino acids.     

The multicopper oxidase of Pseudomonas aeruginosa is a ferroxidase with a central role in iron acquisition

Recently it has been observed that multicopper oxidases are present in a number of microbial genomes, raising the question of their function in prokaryotes. Here we describe the analysis of an mco mutant from the opportunistic pathogen Pseudomonas aeruginosa. Unlike wild-type Pseudomonas aeruginosa, the mco mutant was unable to grow aerobically on minimal media with Fe(II) as sole iron source. In contrast, both the wild-type and mutant strain were able to grow either anaerobically via denitrification with Fe(II) or aerobically with Fe(III). Analysis of iron uptake showed that the mco mutant was impaired in Fe(II) uptake but unaffected in Fe(III) uptake. Purification and analysis of the MCO protein confirmed ferroxidase activity. Taken together, these data show that the mco gene encodes a multicopper oxidase that is involved in the oxidation of Fe(II) to Fe(III) subsequent to its acquisition by the cell. In view of the widespread distribution of the mco gene in bacteria, it is suggested that an iron acquisition mechanism involving multicopper oxidases may be an important and hitherto unrecognized feature of bacterial pathogenicity.     

Pseudomonas aeruginosa outer membrane permeability: isolation of a porin protein F-deficient mutant.

A mutant of Pseudomonas aeruginosa severely deficient in outer membrane protein F levels was isolated by screening heavily mutagenized strains for membrane protein alterations on sodium dodecyl sulphate-polyacrylamide gel electrophoresis. To provide a basis for phenotypic comparison, three independent spontaneous revertants with normal protein F levels were isolated. Neither the protein F-deficient mutant nor its revertants had gross surface alterations as judged by their sensitivities to 31 phages with diverse receptors and their low degrees of leakage of periplasmic beta-lactamase into the supernatant. Outer membrane permeability was measured in whole cells by examining the rates of hydrolysis of a chromogenic beta-lactam, nitrocefin, by periplasmic RP1-encoded beta-lactamase. It was found that the outer membrane permeabilities of wild-type and protein F revertant strains were similar, but low when compared with those of Escherichia coli and an antibiotic-supersusceptible mutant Z61 of P. aeruginosa. The loss of protein F caused a further significant decrease in outer membrane permeability. The results suggest that protein F is a pore-forming protein in vivo and that only a small proportion, as few as 1 in 400, of the protein F molecules form active functional channels in vivo.     

Pseudomonas aeruginosa porin OprF: properties of the channel

Using ion channel reconstitution in planar lipid bilayers, we examined the channel-forming activity of subfractions of Pseudomonas aeruginosa OprF, which was shown to exist in two different conformations: a minority single domain conformer and a majority two-domain conformer (Sugawara, E., Nestorovich, E. M., Bezrukov, S. M., and Nikaido, H. (2006) J. Biol. Chem. 281, 16220-16229). With the fraction depleted for the single domain conformer, we were unable to detect formation of any channels with well defined conductance levels. With the unfractionated OprF, we saw only rare channel formation. However, with the single domain-enriched fraction of OprF, we observed regular insertion of channels with highly reproducible conductances. Single OprF channels demonstrate rich kinetic behavior exhibiting spontaneous transitions between several subconformations that differ in ionic conductance and radius measured in polymer exclusion experiments. Although we showed that the effective radius of the most conductive conformation exceeds that of the general outer membrane porin of Escherichia coli, OmpF, we also found that a single OprF channel mainly exists in weakly conductive subconformations and switches to the fully open state for a short time only. Therefore, the low permeability of OprF reported earlier may be due to two factors: mainly to the paucity of the single domain conformer in the OprF population and secondly to the predominance of weakly conductive subconformations within the single domain conformer.     

Iron uptake regulation in Pseudomonas aeruginosa

The Pseudomonas genus belongs to the γ division of Proteobacteria and many species produce the characteristic yellow–green siderophore pyoverdine, and often a second siderophore, of lower affinity for iron. These bacteria are known for their ability to colonize different ecological niches and for their versatile metabolism. It is therefore not surprising that they are endowed with the capacity to take up exogenous xenosiderophores via different TonB-dependent receptors. Uptake of iron is controlled by the central regulator Fur, and via extracytoplasmic sigma factors or other types of regulators (two-component systems, AraC regulators). In this review the Fur regulon (experimentally proven and/or predicted) of P. aeruginosa will be presented. An interesting feature revealed by this analysis of Fur-regulated genes is the overlap between the iron and the sulfur regulons as well with the quorum sensing system.     

Cloning of genes specifying carbohydrate catabolism in Pseudomonas aeruginosa and Pseudomonas putida.

A 6.0-kilobase EcoRI fragment of the Pseudomonas aeruginosa PAO chromosome containing a cluster of genes specifying carbohydrate catabolism was cloned into the multicopy plasmid pRO1769. The vector contains a unique EcoRI site for cloning within a streptomycin resistance determinant and a selectable gene encoding gentamicin resistance. Mutants of P. aeruginosa PAO transformed with the chimeric plasmid pRO1816 regained the ability to grow on glucose, and the following deficiencies in enzyme or transport activities corresponding to the specific mutations were complemented: glcT1, glucose transport and periplasmic glucose-binding protein; glcK1, glucokinase; and edd-1, 6-phosphogluconate dehydratase. Two other carbohydrate catabolic markers that are cotransducible with glcT1 and edd-1 were not complemented by plasmid pRO1816: zwf-1, glucose-6-phosphate dehydrogenase; and eda-9001, 2-keto-3-deoxy-6-phosphogluconate aldolase. However, all five of these normally inducible activities were expressed at markedly elevated basal levels when transformed cells of prototrophic strain PAO1 were grown without carbohydrate inducer. Vector plasmid pRO1769 had no effect on the expression of these activities in transformed mutant or wild-type cells. Thus, the chromosomal insert in pRO1816 contains the edd and glcK structural genes, at least one gene (glcT) that is essential for expression of the glucose active transport system, and other loci that regulate the expression of the five clustered carbohydrate catabolic genes. The insert in pRO1816 also complemented the edd-1 mutation in a glucose-negative Pseudomonas putida mutant but not the eda-1 defect in another mutant. Moreover, pRO1816 caused the expression of high specific activities of glucokinase, an enzyme that is naturally lacking in these strains of Pseudomonas putida.     

The two-component sensor response regulator RoxS/RoxR plays a role in Pseudomonas aeruginosa interactions with airway epithelial cells

Pseudomonas aeruginosa is an opportunistic pathogen that infects the lungs of patients with cystic fibrosis causing aberrant and destructive neutrophil (PMN)-dominated inflammation of airways. Interaction of P. aeruginosa with the lung epithelial barrier resulting in trans-epithelial PMN migration likely represents a key event during PMN recruitment. To investigate bacterial factors involved in interactions with lung epithelial cells, a mutant library of two-component system response regulators was evaluated to identify mutants exhibiting defects in the ability to induce PMN trans-epithelial migration. Of forty-eight mutants, five reproducibly demonstrated a reduced PMN trans-epithelial migration response. All five mutants also exhibited a decreased ability to interact with lung epithelial cells. One mutant identified lacks the response regulator gene roxR, which has not previously been reported to be involved regulating factors that facilitate interactions with lung epithelial cells. This finding suggests that RoxR likely regulates genes with relevance to P. aeruginosa mediated lung disease.     

Siderophore-specific induction of iron uptake in Pseudomonas aeruginosa.

Pseudomonas aeruginosa has two siderophore-based high-affinity iron-uptake systems utilizing pyoverdin and pyochelin. Using strain IA1, a mutant deficient in production of both siderophores, we have shown that addition of purified siderophore to the growth medium induces expression of specific iron-regulated outer-membrane proteins and increases 55Fe-siderophore transport. Addition of pyoverdin from the parent strain PAO1 or from a clinical strain 0:12 induced expression of an 85 kDa IROMP and increased the rate of 55Fe-pyoverdin transport. Transport rates for 55Fe-PAO1 pyoverdin increased from 1.27 to 3.57 pmol Fe min-1 per 10(9) cells. Addition of purified pyochelin induced expression of a 75 kDa IROMP accompanied with increased 55Fe-pyochelin uptake without affecting 55Fe-pyoverdin transport. 55Fe-pyochelin transport increased from 0.3 to 10.6 pmol min-1 per 10(9) cells. Addition of pyoverdin from the parent strain or a chromatographically distinct pyoverdin caused increased reactivity with an anti-85 kDa mAb in Western blotting, indicating that the same receptor is being induced. These results suggest that P. aeruginosa can respond specifically to the presence of siderophore and moreover that not only can the pyoverdin receptor transport its cognate ferri-pyoverdin but also different ferri-pyoverdins, albeit at a reduced rate.     

Cadaverine inhibition of porin plays a role in cell survival at acidic pH

When grown at acidic pH, Escherichia coli cells secrete cadaverine, a polyamine known to inhibit porin-mediated outer membrane permeability. In order to understand the physiological significance of cadaverine excretion and the inhibition of porins, we isolated an OmpC mutant that showed resistance to spermine during growth and polyamine-resistant porin-mediated fluxes. Here, we show that the addition of exogenous cadaverine allows wild-type cells to survive a 30-min exposure to pH 3.6 better than cells expressing the cadaverine-insensitive OmpC porin. Competition experiments between strains expressing either wild-type or mutant OmpC showed that the lack of sensitivity of the porin to cadaverine confers a survival disadvantage to the mutant cells at reduced pH. On the basis of these results, we propose that the inhibition of porins by excreted cadaverine represents a novel mechanism that provides bacterial cells with the ability to survive acid stress.     

Malate plays a central role in plant nutrition

Malate occupies a central role in plant metabolism. Its importance in plant mineral nutrition is reflected by the role it plays in symbiotic nitrogen fixation, phosphorus acquisition, and aluminum tolerance. In nitrogen-fixing root nodules, malate is the primary substrate for bacteroid respiration, thus fueling nitrogenase. Malate also provides the carbon skeletons for assimilation of fixed nitrogen into amino acids. During phosphorus deficiency, malate is frequently secreted from roots to release unavailable forms of phosphorus. Malate is also involved with plant adaptation to aluminum toxicity. To define the genetic and biochemical regulation of malate formation in plant nutrition we have isolated and characterized genes involved in malate metabolism from nitrogen-fixing root nodules of alfalfa and those involved in organic acid excretion from phosphorus-deficient proteoid roots of white lupin. Moreover, we have overexpressed malate dehydrogenase in alfalfa in attempts to improve nutrient acquisition. This report is an overview of our efforts to understand and modify malate metabolism, particularly in the legumes alfalfa and white lupin.     

Mitophagy plays a central role in mitochondrial ageing

The mechanisms underlying ageing have been discussed for decades, and advances in molecular and cell biology of the last three decades have accelerated research in this area. Over this period, it has become clear that mitochondrial function, which plays a major role in many cellular pathways from ATP production to nuclear gene expression and epigenetics alterations, declines with age. The emerging concepts suggest novel mechanisms, involving mtDNA quality, mitochondrial dynamics or mitochondrial quality control. In this review, we discuss the impact of mitochondria in the ageing process, the role of mitochondria in reactive oxygen species production, in nuclear gene expression, the accumulation of mtDNA damage and the importance of mitochondrial dynamics and recycling. Declining mitophagy (mitochondrial quality control) may be an important component of human ageing.     

Membrane topology and site-specific mutagenesis of Pseudomonas aeruginosa porin OprD

Pseudomonas aeruginosa OprD is a 420-amino-acid protein that facilitates the uptake of basic amino acids, imipenem and gluconate across the outer membrane. OprD was the first specific porin that could be aligned with members of the non-specific porin super-family. Utilizing multiple alignments in conjugation with structure predictions and amphipathicity calculations, an OprD-topology model was proposed. Sixteen β-strands were predicted, connected by short loops at the periplasmic side. The eight external loops were of variable length but tended to be much longer than the periplasmic ones. Polymerase chain reaction (PCR)-based site-specific mutagenesis was performed to delete separately short stretches (4-8 amino acid residues) from each of the predicted external loops. The mutants with deletions in the predicted external loops L1, L2, L5, L6, L7 and L8 were tolerated in both Escherichia coli and P. aeruginosa. The expressed mutant proteins maintained substantial resistance to trypsin treatment in the context of isolated outer membranes. Proteins with deletions in loops L1, L5, L6, L7 and L8 reconstituted similar imipenem supersusceptibility in a P. aeruginosa OprD::Ω background. The L2-deletion mutant only partially reconstituted supersusceptibility, suggesting that loop L2 is involved in imipenem binding. These data were generally consistent with the topology model.