The PvdRT-OpmQ efflux pump controls the metal selectivity of the iron uptake pathway mediated by the siderophore pyoverdine in Pseudomonas aeruginosa

Pyoverdine (PVD) is the major siderophore produced by Pseudomonas aeruginosa for iron acquisition. PvdRT-OpmQ is an ATP-dependent efflux pump involved in the secretion of newly synthesized pyoverdine (PVD) and of PVD that has transported and released its iron into the bacterium from the periplasm into the extracellular medium. This iron uptake pathway also involves an outer membrane transporter, FpvA, for PVD-Fe uptake from the extracellular medium into the periplasm. In binding assays, FpvA bound PVD in complex with many different metals, with affinities from 2.9?nM for PVD-Fe to 13?μM for PVD-Al. Uptake assays with various FpvA and PvdRT-OpmQ mutants, monitored by inductively coupled plasma-atomic emission spectrometry (ICP-AES) for metal detection, and by fluorescence for PVD detection, showed that both metals and PVD accumulated in P.?aeruginosa, due to the uptake of these compounds via the FpvA/PVD pathway. Higher levels of accumulation were observed in the absence of PvdRT-OpmQ expression. Thus, FpvA has a broad metal specificity for both the binding and uptake of PVD-metal complexes, and the PvdRT-OpmQ efflux pump exports unwanted metals complexed with PVD from the bacterium. This study provides the first evidence of efflux pump involvement in the export of unwanted siderophore-metal complexes and insight into the molecular mechanisms involved controlling the metal selectivity of siderophore-mediated iron uptake pathways.     

Study on pyoverdine and biofilm production with detection of LasR gene in MDR Pseudomonas aeruginosa

In cystic fibrosis individuals, chronic lung infections and hospital-acquired pneumonia are caused by Pseudomonas aeruginosa. P. aeruginosa generates siderophores such as pyoverdine (PVD) as iron uptake systems to cover its needs of iron ions for growth and infection. lasR quorum sensing (QS) gene has a crucial function in PVD production and biofilm generation in P. aeruginosa. Fifty isolates of P. aeruginosa were obtained from clinical specimens of sputum (collected from individuals suffering from pulmonary infections). Antibiotic sensitivity test was performed for 50P. aeruginosa isolates by using 10 different types of antibiotics. All isolates of P. aeruginosa showed resistance for all 10 using antibiotics in this study. Ten multidrug resistant isoloates of P. aeruginosa were selected for next tests. Virulence factors of ten multidrug resistant isolates of P. aeruginosa, such as biofilm generation, PVD production, and lasR gene were detected. From results, all 10P. aeruginosa isolates can produce biofilm, PVD, and contain lasR gene. The produced amplicon for the lasR gene was 725 bp. After mice injection by fresh and heated PVD produced by P. aeruginosa PS10 LC619328.2, the fresh PVD caused 100 % mortality within five days using 0.3 ml of its concentration (37.4 µM), while (15.3 µM) of heated PVD (toxoid) caused 50 % mortality.     

The Ferrichrome Uptake Pathway in Pseudomonas aeruginosa Involves an Iron Release Mechanism with Acylation of the Siderophore and Recycling of the Modified Desferrichrome

The uptake of iron into Pseudomonas aeruginosa is mediated by two major siderophores produced by the bacterium, pyoverdine and pyochelin. The bacterium is also able of utilize several heterologous siderophores of bacterial or fungal origin. In this work, we have investigated the iron uptake in P. aeruginosa PAO1 by the heterologous ferrichrome siderophore. ⁵⁵Fe uptake assays showed that ferrichrome is transported across the outer membrane primarily (80%) by the FiuA receptor and to a lesser extent (20%) by a secondary transporter. Moreover, we demonstrate that like in the uptake of ferripyoverdine and ferripyochelin, the energy required for both pathways of ferrichrome uptake is provided by the inner membrane protein TonB1. Desferrichrome-⁵⁵Fe uptake in P. aeruginosa was also dependent on the expression of the permease FiuB, suggesting that this protein is the inner membrane transporter of the ferrisiderophore. A biomimetic fluorescent analogue of ferrichrome, RL1194, was used in vivo to monitor the kinetics of iron release from ferrichrome in P. aeruginosa in real time. This dissociation involves acylation of ferrichrome and its biomimetic analogue RL1194 and recycling of both modified siderophores into the extracellular medium. FiuC, an N-acetyltransferase, is certainly involved in this mechanism of iron release, since its mutation abolished desferrichrome-⁵⁵Fe uptake. The acetylated derivative reacts with iron in the extracellular medium and is able to be taken up again by the cells. All these observations are discussed in light of the current knowledge concerning ferrichrome uptake in P. aeruginosa and in Escherichia coli.Iron is essential for life for practically all living organisms and plays a number of key roles in biology. DNA and RNA synthesis, glycolysis, energy generation by electron transport, nitrogen fixation, and photosynthesis are examples of processes in which iron-containing enzymes play vital roles. However, under physiological conditions iron forms highly insoluble ferric hydroxide complexes, which severely limits its bioavailability. To overcome the problem of iron inaccessibility, bacteria excrete high-affinity iron chelators termed siderophores, which are able to solubilize iron and deliver it into the cells (3, 64).Pseudomonas aeruginosa is a ubiquitous environmental bacterium that is capable of infecting a wide variety of animal, insects, and plants. As a human pathogen, P. aeruginosa is the leading source of Gram-negative nosocomial infections (59) and causes chronic lung infections in approximately 90% of individuals suffering from cystic fibrosis (40). Under iron-limited conditions, P. aeruginosa produces two major siderophores, pyoverdine (PVD) (62) and pyochelin (PCH) (15). P. aeruginosa is also capable of utilizing numerous siderophores secreted by other microorganisms: pyoverdins from other pseudomonas, enterobactin (49), cepabactin (45), mycobactin and carboxymycobactin (38), fungal siderophores (ferrichrome [39]; deferrioxamines [39, 60]; and desferrichrysin, desferricrocin, and coprogen [44]), and natural occurring chelators such as citrate (14, 23) (for a review, see reference 47).In Gram-negative bacteria, the uptake of ferrisiderophores always involves a specific transporter at the level of the outer membrane (4). The energy required for this process is provided by the proton motive force (PMF) of the inner membrane by means of an inner membrane complex comprising TonB, ExbB, and ExbD (21, 51, 63). In silico analysis of the P. aeruginosa genome (http://www.pseudomonas.com) revealed 32 genes encoding putative TonB-dependent transporters (13), of which only 12 are involved in metal (mostly iron) uptake (38). FpvA and FpvB are the outer membrane transporters involved in the uptake of PVD-Fe (19, 48), and FptA transports PCH-Fe (25). Concerning the heterologous siderophores, there are two transporters, FoxA and FiuA, involved in the transport of ferrioxamine B and ferrichrome (39). The mechanism involved in the translocation of ferrisiderophores across the outer membrane by the TonB-dependent transporters has been studied mostly in E. coli (for a review, see reference 5) and in the case of P. aeruginosa has been studied only for the FpvA/PVD and the FptA/PCH systems. The structures of FpvA (8, 11, 65) and FptA (12) have been solved and their interactions with PVD and PCH investigated at the molecular level (26, 27, 45, 53). Three tonB genes, encoding the energy coupler TonB, have been found in the P. aeruginosa genome, i.e., tonB1, tonB2, and tonB3. Disruption of tonB1 abrogates PVD- and PCH-mediated iron uptake (50, 58) and heme uptake (67). Inactivation of tonB2 has no adverse effect on iron or heme acquisition, but tonB1 tonB2 double mutants are more compromised with respect to growth in iron-restricted medium than is a single tonB1 knockout mutant (67). Mutation of tonB3 appears to result in defective twitching motility (28), and the gene product is most likely not involved in iron uptake.In P. aeruginosa, many ferrisiderophore outer membrane transporters are also involved in a signaling cascade regulating the expression of genes involved in iron uptake. This is the case for FpvA (PVD uptake), FoxA (ferrioxamine), and FiuA (ferrichrome) (38, 39, 43, 61). Such a signaling cascade involves an extracytoplasmic function (ECF) sigma factor and an inner membrane anti-sigma factor. Equivalent cell surface signaling is present in Escherichia coli for ferricitrate uptake by FecA but not for ferrichrome, ferrioxamine, and enterobactin uptake by FhuA, FhuE, and FepA, respectively.Little is known about the translocation of ferrisiderophores across the inner membrane in P. aeruginosa. In E. coli, this step involves a specific ABC transporter for almost every siderophore used by this bacterium: FhuBCD for the uptake of ferrichrome and ferrioxamine (33-36), FecBCD for the uptake of ferricitrate (6, 56) and, FepBCDG for the uptake of ferrienterobactin (9). In P. aeruginosa, the only characterized inner membrane siderophore transport protein is FptX, a proton motive force-dependent permease, which functions in PCH-Fe utilization (16). The inner membrane FoxB is involved in the utilization of ferrichrome and ferrioxamine B, but it remains to be determined whether this protein functions in the transport of ferrisiderophore or in the release of iron from ferrichrome or ferrioxamine (17). The genome clearly shows a fepBCDG homologue for the transport of ferrienterobactin. For the other iron uptake pathways present in P. aeruginosa, the transporters involved at the level of the inner membrane have not been identified. An import ABC transporter is present in the pvd locus (PA2407 to PA2410; http://www.pseudomonas.com), but its mutation does not affect PVD-Fe uptake (46).In P. aeruginosa, the mechanism of ferrisiderophore dissociation has been investigated only for the PVD pathway. This step occurs in the periplasm by a mechanism involving no chemical siderophore modification but involving a reduction of iron and a recycling of the siderophore into the extracellular medium by the PvdRT-OpmQ efflux pump (20, 54, 66). In E. coli, the mechanism of ferrisiderophore dissociation has been investigated for the ferrichrome and ferrienterobactin pathways. Iron release from ferrichrome occurs in the cytoplasm and probably involves iron reduction (41) followed by acetylation of the siderophore and its recycling into the growth medium (24). For the ferrienterobactin pathway, a cytoplasmic esterase hydrolyzes the siderophore (7).In the present work, we have investigated the ferrichrome pathway in P. aeruginosa using both ferrichrome and a fluorescently labeled biomimetic ferrichrome analogue. We evaluated the siderophore properties of the fluorescent analogue and identified the different transporters involved in the uptake across the outer and inner membranes. Furthermore, we demonstrated that following ferrichrome uptake, iron is released from the siderophore by a mechanism involving an acetylation of the chelator and the modified desferrichrome is secreted back into the growth medium.     

Mechanism of ferripyoverdine uptake by Pseudomonas aeruginosa outer membrane transporter FpvA: no diffusion channel formed at any time during ferrisiderophore uptake

To get access to iron, Pseudomonas aeruginosa produces the siderophore pyoverdine (PVD), composed of a fluorescent chromophore linked to an octapeptide, and its corresponding outer membrane transporter FpvA. This transporter is composed of three domains: a β-barrel inserted into the membrane, a plug that closes the channel formed by the barrel, and a signaling domain in the periplasm. The plug and the signaling domain are separated by a sequence of five residues called the TonB box, which is necessary for the interaction of FpvA with the inner membrane TonB protein. Genetic deletion of the plug domain resulted in the presence of a β-barrel in the outer membrane unable to bind and transport PVD-Fe. Expression of the soluble plug domain with the TonB box inhibited PVD-(55)Fe uptake most likely through interaction with TonB in the periplasm. A reconstituted FpvA in the bacterial outer membrane was obtained by the coexpression of separately encoded plug and β-barrel domains, each endowed with a signal sequence and a signaling domain. This resulted in polypeptide complementation after secretion across the cytoplasmic membrane. The reconstituted FpvA bound PVD-Fe with the same affinity as wild-type FpvA, indicating that the resulting transporter is correctly folded and reconstituted in the outer membrane. PVD-Fe uptake was TonB-dependent but 75% less efficient compared to wild-type FpvA. These data are consistent with a gated mechanism in which no open channel with a complete removal of the plug domain for PVD-Fe diffusion is formed in FpvA at any point during the uptake cycle.     

Effects of carbon and nitrogen sources on the proteome of Pseudomonas aeruginosa PA1 during rhamnolipid production

The PA1 strain of Pseudomonas aeruginosa isolated from oil waste produces rhamnolipid, a biodegradable surfactant with applications in several industrial and environmental fields. The metabolic pathway and genetic regulation of rhamnolipid production in P. aeruginosa are poorly understood. Herein, several proteins directly or indirectly related to rhamnolipid production and their genetic regulations were identified by comparative proteomic. We compared the proteome of P. aeruginosa PA1 after fermentation in two different conditions of carbon and nitrogen sources: condition A allowed rhamnolipid production and condition B prevented it. Protein extracts from cellular pellets were compared using 2D-PAGE stained with colloidal Coomassie followed by MALDI-TOF/TOF mass spectrometry. We identified 21 differentially expressed proteins, including those involved in secretion, quorum sensing, oxidative response and metabolism.     

An efflux pump is involved in secretion of newly synthesized siderophore by Pseudomonas aeruginosa

Pseudomonas aeruginosa secretes the fluorescent siderophore, pyoverdine (PVD), to enable iron acquisition. Epifluorescence microscopy and cellular fractionation were used to investigate the role of an efflux pump, PvdRT-OpmQ, in PVD secretion. Bacteria lacking this efflux pump accumulated PVD, or a fluorescent precursor, in the periplasm, due to their inability to efficiently secrete into the media newly synthesized PVD. PvdRT-OpmQ is only the second system identified for secretion of newly synthesized siderophores by Gram negative bacteria.     

The Proprotein Convertase KPC-1/Furin Controls Branching and Self-avoidance of Sensory Dendrites in Caenorhabditis elegans

Animals sample their environment through sensory neurons with often elaborately branched endings named dendritic arbors. In a genetic screen for genes involved in the development of the highly arborized somatosensory PVD neuron in C. elegans, we have identified mutations in kpc-1, which encodes the homolog of the proprotein convertase furin. We show that kpc-1/furin is necessary to promote the formation of higher order dendritic branches in PVD and to ensure self-avoidance of sister branches, but is likely not required during maintenance of dendritic arbors. A reporter for kpc-1/furin is expressed in neurons (including PVD) and kpc-1/furin can function cell-autonomously in PVD neurons to control patterning of dendritic arbors. Moreover, we show that kpc-1/furin also regulates the development of other neurons in all major neuronal classes in C. elegans, including aspects of branching and extension of neurites as well as cell positioning. Our data suggest that these developmental functions require proteolytic activity of KPC-1/furin. Recently, the skin-derived MNR-1/menorin and the neural cell adhesion molecule SAX-7/L1CAM have been shown to act as a tripartite complex with the leucine rich transmembrane receptor DMA-1 on PVD mechanosensory to orchestrate the patterning of dendritic branches. Genetic analyses show that kpc-1/furin functions in a pathway with MNR-1/menorin, SAX-7/L1CAM and DMA-1 to control dendritic branch formation and extension of PVD neurons. We propose that KPC-1/furin acts in concert with the ‘menorin’ pathway to control branching and growth of somatosensory dendrites in PVD.     

Fate of ferrisiderophores after import across bacterial outer membranes: different iron release strategies are observed in the cytoplasm or periplasm depending on the siderophore pathways

Siderophore production and utilization is one of the major strategies deployed by bacteria to get access to iron, a key nutrient for bacterial growth. The biological function of siderophores is to solubilize iron in the bacterial environment and to shuttle it back to the cytoplasm of the microorganisms. This uptake process for Gram-negative species involves TonB-dependent transporters for translocation across the outer membranes. In Escherichia coli and many other Gram-negative bacteria, ABC transporters associated with periplasmic binding proteins import ferrisiderophores across cytoplasmic membranes. Recent data reveal that in some siderophore pathways, this step can also be carried out by proton-motive force-dependent permeases, for example the ferrichrome and ferripyochelin pathways in Pseudomonas aeruginosa. Iron is then released from the siderophores in the bacterial cytoplasm by different enzymatic mechanisms depending on the nature of the siderophore. Another strategy has been reported for the pyoverdine pathway in P. aeruginosa: iron is released from the siderophore in the periplasm and only siderophore-free iron is transported into the cytoplasm by an ABC transporter having two atypical periplasmic binding proteins. This review presents recent findings concerning both ferrisiderophore and siderophore-free iron transport across bacterial cytoplasmic membranes and considers current knowledge about the mechanisms involved in iron release from siderophores.     

Direct glutaminyl-tRNA biosynthesis and indirect asparaginyl-tRNA biosynthesis in Pseudomonas aeruginosa PAO1

The genomic sequence of Pseudomonas aeruginosa PAO1 was searched for the presence of open reading frames (ORFs) encoding enzymes potentially involved in the formation of Gln-tRNA and of Asn-tRNA. We found ORFs similar to known glutamyl-tRNA synthetases (GluRS), glutaminyl-tRNA synthetases (GlnRS), aspartyl-tRNA synthetases (AspRS), and trimeric tRNA-dependent amidotransferases (AdT) but none similar to known asparaginyl-tRNA synthetases (AsnRS). The absence of AsnRS was confirmed by biochemical tests with crude and fractionated extracts of P. aeruginosa PAO1, with the homologous tRNA as the substrate. The characterization of GluRS, AspRS, and AdT overproduced from their cloned genes in P. aeruginosa and purified to homogeneity revealed that GluRS is discriminating in the sense that it does not glutamylate tRNA^Gln, that AspRS is nondiscriminating, and that its Asp-tRNA^Asn product is transamidated by AdT. On the other hand, tRNA^Gln is directly glutaminylated by GlnRS. These results show that P. aeruginosa PAO1 is the first organism known to synthesize Asn-tRNA via the indirect pathway and to synthesize Gln-tRNA via the direct pathway. The essential role of AdT in the formation of Asn-tRNA in P. aeruginosa and the absence of a similar activity in the cytoplasm of eukaryotic cells identifies AdT as a potential target for antibiotics to be designed against this human pathogen. Such novel antibiotics could be active against other multidrug-resistant gram-negative pathogens such as Burkholderia and Neisseria as well as all pathogenic gram-positive bacteria.     

Development of phenylthiourea derivatives as allosteric inhibitors of pyoverdine maturation enzyme PvdP tyrosinase

Infections caused by Pseudomonas aeruginosa become increasingly difficult to treat because these bacteria have acquired various mechanisms for antibiotic resistance, which creates the need for mechanistically novel antibiotics. Such antibiotics might be developed by targeting enzymes involved in the iron uptake mechanism because iron is essential for bacterial survival. For P. aeruginosa, pyoverdine has been described as an important virulence factor that plays a key role in iron uptake. Therefore, inhibition of enzymes involved in the pyoverdine synthesis, such as PvdP tyrosinase, can open a new window for the treatment of P. aeruginosa infections. Previously, we reported phenylthiourea as the first allosteric inhibitor of PvdP tyrosinase with high micromolar potency. In this report, we explored structure-activity relationships (SAR) for PvdP tyrosinase inhibition by phenylthiourea derivatives. This enables identification of a phenylthiourea derivative (3c) with a potency in the submicromolar range (IC₅₀ = 0.57 + 0.05 µM). Binding could be rationalized by molecular docking simulation and 3c was proved to inhibit the bacterial pyoverdine production and bacterial growth in P. aeruginosa PA01 cultures.     

Functional characterization of ExFadLO,an outer membrane protein required for exporting oxygenated long-chain fatty acids in Pseudomonas aeruginosa

Bacterial proteins of the FadL family have frequently been associated to the uptake of exogenous hydrophobic substrates. However, their outer membrane location and involvement in substrate uptake have been inferred mainly from sequence similarity to Escherichia coli FadL, the first well-characterized outer membrane transporters of Long-Chain Fatty Acids (LCFAs) in bacteria. Here we report the functional characterization of a Pseudomonas aeruginosa outer membrane protein (ORF PA1288) showing similarities to the members of the FadL family, for which we propose the name ExFadLO. We demonstrate herein that this protein is required to export LCFAs 10-HOME and 7,10-DiHOME, derived from a diol synthase oxygenation activity on oleic acid, from the periplasm to the extracellular medium. Accumulation of 10-HOME and 7,10-DiHOME in the extracellular medium of P. aeruginosa was abolished by a transposon insertion mutation in exFadLO (ExFadLO¯ mutant). However, intact periplasm diol synthase activity was found in this mutant, indicating that ExFadLO participates in the export of these oxygenated LCFAs across the outer membrane. The capacity of ExFadLO¯ mutant to export 10-HOME and 7,10-DiHOME was recovered after complementation with a wild-type, plasmid-expressed ExFadLO protein. A western blot assay with a variant of ExFadLO tagged with a V5 epitope confirmed the location of ExFadLO in the bacterial outer membrane under the experimental conditions tested. Our results provide the first evidence that FadL family proteins, known to be involved in the uptake of hydrophobic substrates from the extracellular environment, also function as secretion elements for metabolites of biological relevance.     

Importance of the lysine cluster in the translocation of anions through the pyrophosphate specific channel OprO

Pseudomonas aeruginosa is a Gram-negative bacterium with an intrinsic resistance towards antibiotics due to the lack of a large diffusion pores. Exchange of substances with the environment is done mainly through a set of narrow and substrate-specific porins in its outer membrane that filter molecules according to their size and chemical composition. Among these proteins are OprP and OprO involved in the selective uptake of mono- and pyrophosphates, respectively. Both proteins are homotrimers and each monomer features an hourglass-shaped channel structure including a periplasmic cavity with a lysine cluster. In this study, we focus on the characterization of this lysine cluster in OprO. The importance of these lysine residues was shown with alanine substitutions in single channel conductance experiments, by titration of mono- and pyrophosphate in multi-channel analysis and by molecular dynamics simulations. All obtained data demonstrated that the closer the mutated lysine residues are to arginine 133, the lower gets the single channel conductance. It was found that the ion flow through each monomer can follow two different lysine paths indicating that phosphate ions have a larger freedom on the periplasmic side of the constriction region. Our results emphasize the important role of the lysine residue 121 in the binding site together with arginine 133 and aspartic acid 94. An improved understanding of the ion mobility across these channels can potentially lead to an optimized permeation of (phosphonic acid containing) antibiotics through the outer membrane of P. aeruginosa and the development of new drug molecules.     

Rhamnolipid Stimulates Uptake of Hydrophobic Compounds by Pseudomonas aeruginosa

The biodegradation of hexadecane by five biosurfactant-producing bacterial strains (Pseudomonas aeruginosa UG2, Acinetobacter calcoaceticus RAG1, Rhodococcus erythropolis DSM 43066, R. erythropolis ATCC 19558, and strain BCG112) was determined in the presence and absence of exogenously added biosurfactants. The degradation of hexadecane by P. aeruginosa was stimulated only by the rhamnolipid biosurfactant produced by the same organism. This rhamnolipid did not stimulate the biodegradation of hexadecane by the four other strains to the same extent, nor was degradation of hexadecane by these strains stimulated by addition of their own biosurfactants. This suggests that P. aeruginosa has a mode of hexadecane uptake different from those of the other organisms. Rhamnolipid also enhanced the rate of epoxidation of the aliphatic hydrocarbon α,ω-tetradecadiene by a cell suspension of P. aeruginosa. Furthermore, the uptake of the hydrophobic probe 1-naphthylphenylamine by cells of P. aeruginosa was enhanced by rhamnolipid, as indicated by stopped-flow fluorescence experiments. Rhamnolipid did not stimulate the uptake rate of this probe in de-energized cells. These results indicate that an energy-dependent system is present in P. aeruginosa strain UG2 that mediates fast uptake of hydrophobic compounds in the presence of rhamnolipid.     

Insights into Mechanisms and Proteomic Characterisation of Pseudomonas aeruginosa Adaptation to a Novel Antimicrobial Substance

Antibiotic resistance has been reported since the introduction of synthetic antibiotics. Bacteria, such as one of the most common nosocomial pathogens P. aeruginosa, adapt quickly to changing environmental conditions, due to their short generation time. Thus microevolutional changes can be monitored in situ. In this study, the microevolutional process of Pseudomonas aeruginosa PAO1 resistance against a recently developed novel antibacterial zinc Schiff-base (ZSB) was investigated at the proteome level. After extended exposure to ZSB the passaged strain differed in tolerance against ZSB, with the adapted P. aeruginosa PAO1 exhibiting 1.6 times higher minimal inhibitory concentration. Using Two-dimensional Difference Gel Electrophoresis, the changes in the proteome of ZSB adapted P. aeruginosa PAO1 were examined by comparison with the non-adapted P. aeruginosa PAO1. The proteome of the adapted P. aeruginosa PAO1 strain differed significantly from the non-adapted in the abundance of two proteins when both strains were grown under stressing conditions. One protein could be identified as the outer membrane protein D that plays a role in uptake of basic amino acids as well as in carbapeneme resistance. The second protein has been identified as alkyl peroxide reductase subunit F. Our data indicated a slight increase in abundance of alkyl peroxide reductase F (AhpF) in the case of ZSB passaged P. aeruginosa PAO1. Higher abundance of Ahp has been discussed in the literature as a promoter of accelerated detoxification of benzene derivatives. The observed up-regulated AhpF thus appears to be connected to an increased tolerance against ZSB. Changes in the abundance of proteins connected to oxidative stress were also found after short-time exposure of P. aeruginosa PAO1 to the ZSB. Furthermore, adapted P. aeruginosa PAO1 showed increased tolerance against hydrogen peroxide and, in addition, showed accelerated degradation of ZSB, as determined by HPLC measurements.     

Identification of QuiP, the Product of Gene PA1032, as the Second Acyl-Homoserine Lactone Acylase of Pseudomonas aeruginosa PAO1

The relevance of the acyl homoserine lactone (acyl-HSL) quorum signals N-3-oxododecanoyl-homoserine lactone (3OC12HSL) and N-butanoyl-homoserine lactone to the biology and virulence of Pseudomonas aeruginosa is well investigated. Previously, P. aeruginosa was shown to degrade long-chain, but not short-chain, acyl-HSLs as sole carbon and energy sources (J. J. Huang, J.-I. Han, L.-H. Zhang, and J. R. Leadbetter, Appl. Environ. Microbiol. 69:5941-5949, 2003). A gene encoding an enzyme with acyl-HSL acylase activity, pvdQ (PA2385), was identified, but it was not required for acyl-HSL utilization. This indicated that P. aeruginosa encodes another acyl-HSL acylase, which we identify here. A comparison of total cell proteins of cultures grown with long-acyl acyl-HSLs versus other substrates implicated the involvement of a homolog of PvdQ, the product of gene PA1032, for which we propose the name QuiP. Transposon mutants of quiP were defective for growth when P. aeruginosa was cultured in medium containing decanoyl-HSL as a sole carbon and energy source. Complementation with a functional copy of quiP rescued this growth defect. When P. aeruginosa was grown in buffered lysogeny broth, constitutive expression of QuiP in P. aeruginosa led to decreased accumulations of the quorum signal 3OC12HSL, relative to the wild type. Heterologous expression of QuiP was sufficient to confer long-chain acyl-HSL acylase activity upon Escherichia coli. Examination of gene expression patterns during acyl-HSL-dependent growth of P. aeruginosa further supported the involvement of quiP in signal decay and revealed other genes also possibly involved. It is not yet known under which “natural” conditions quiP is expressed or how P. aeruginosa balances the expression of its quorum-sensing systems with the expression of its acyl-HSL acylase activities.