Christine Baysse

The pyocins of Pseudomonas aeruginosa.

Pyocins are produced by more than 90% of Pseudomonas aeruginosa strains and each strain may synthesise several pyocins. The pyocin genes are located on the P. aeruginosa chromosome and their activities are inducible by mutagenic agents such as mitomycin C. Three types of pyocins are described. (i). R-type pyocins resemble non-flexible and contractile tails of bacteriophages. They provoke a depolarisation of the cytoplasmic membrane in relation with pore formation. (ii). F-type pyocins also resemble phage tails, but with a flexible and non-contractile rod-like structure. (iii). S-type pyocins are colicin-like, protease-sensitive proteins. They are constituted of two components. The large component carries the killing activity (DNase activity for pyocins S1, S2, S3, AP41; tRNase for pyocin S4; channel-forming activity for pyocin S5). It interacts with the small component (immunity protein). The synthesis of pyocins starts when a mutagen increases the expression of the recA gene and activates the RecA protein, which cleaves the repressor PrtR, liberating the expression of the protein activator gene prtN. R and F-pyocins are derived from an ancestral gene, with similarities to the P2 phage family and the lambda phage family, respectively. The killing domains of S1, S2, AP41 pyocins show a close evolutionary relationship with E2 group colicins, S4 pyocin with colicin E5, and S5 pyocin with colicins Ia, and Ib.

Characterization of a new efflux pump, MexGHI-OpmD, from Pseudomonas aeruginosa that confers resistance to vanadium.

Vanadium has an antibacterial activity against Pseudomonas aeruginosa, especially under conditions of iron limitation. Some degree of resistance to V is inducible by prior exposure to the metal. One mutant (VS1) with a higher sensitivity to V was obtained by transposon mutagenesis of P. aeruginosa PA 59.20, a clinical isolate. This mutant had an insertion in a non-coding region, upstream of a cluster of four genes. Three of them show similarities to genes corresponding to known P. aeruginosa antibiotic efflux systems, including an efflux protein, a membrane fusion protein and an outer-membrane porin. This cluster was named mexGHI-opmD. By allelic exchange, three mutants, ncr (for non-coding region), mexI and opmD were constructed in P. aeruginosa PAO1. Next to V sensitivity, the ncr, mexI and opmD mutants also showed reduced production of elastase, rhamnolipids, pyocyanine, pyoverdine and had reduced swarming motility, phenotypes that are known to be regulated by quorum sensing. All wild-type phenotypes, including growth in the presence of V, were restored by complementation with the complete cluster. The production of N-acyl-homoserine lactones (AHLs) was detected using the Chromobacter violaceum bioassay. Total extracts from the three mutants failed to induce the production of violacein by C. violaceum, although AHLs were detected by TLC and C. violaceum overlay. Violacein production was restored by complementation with mexGHI-opmD. The opmD mutant grew very slowly in LB or CAA medium, indicating that OpmD has an important physiological function for the cell. In conclusion, it is believed that the MexGHI-OpmD pump is probably involved in AHL homeostasis in P. aeruginosa.

Identification of new, conserved, non‐ribosomal peptide synthetases from fluorescent pseudomonads involved in the biosynthesis of the siderophore pyoverdine

Pyoverdines, the main siderophores of fluorescent pseudomonads, contain a peptide moiety, different for each pyoverdine, and an identical chromophore. While it has been shown that non‐ribosomal peptide synthetases (NRPSs) are involved in the biosynthesis of the peptide chain of pyoverdines, this was not demonstrated for the biosynthesis of the chromo‐phore part. We found that PvsA, from Pseudomonas fluorescens ATCC 17400, and PvdL (PA2424), from Pseudomonas aeruginosa are similar NRPSs and functional homologues, necessary for the production of pyoverdine. Transcriptional lacZ fusions showed that pvdL is co‐transcribed with the upstream PA2425 gene, encoding a putative thioesterase, and is iron‐regulated via PvdS. Similarly, RT‐PCR analysis revealed that expression of pvsA is repressed by iron. Analysis of the adenylation domains of PvsA, PvdL and their homologues, revealed that their N‐terminus starts with an acyl‐CoA ligase module, followed by three amino acid activation domains. Computer modelling of these domains suggests that PvsA in P. fluorescens and PvdL in P. aeruginosa are orthologues involved in the biosynthesis of the pyoverdine chromophore.

Vanadium interferes with siderophore-mediated iron uptake in Pseudomonas aeruginosa

Vanadium is a metal that under physiological conditions can exist in two oxidation states, V(IV) (vanadyl ion) and V(V) (vanadate ion). Here, it was demonstrated that both ions can form complexes with siderophores. Pseudomonas aeruginosa produces two siderophores under iron-limiting conditions, pyoverdine (PVD) and pyochelin (PCH). Vanadyl sulfate, at a concentration of 1-2 mM, strongly inhibited growth of P. aeruginosa PAO1, especially under conditions of severe iron limitation imposed by the presence of non-utilizable Fe(III) chelators. PVD-deficient mutants were more sensitive to vanadium than the wild-type, but addition of PVD did not stimulate their growth. Conversely, PCH-negative mutants were more resistant to vanadium than the wild-type strain. Both siderophores could bind and form complexes with vanadium after incubation with vanadyl sulfate (1:1, in the case of PVD; 2:1, in the case of PCH). Although only one complex with PVD, V(IV)-PVD, was found, both V(IV)- and V(V)-PCH were detected. V-PCH, but not V-PVD, caused strong growth reduction, resulting in a prolonged lag phase. Exposure of PAO1 cells to vanadium induced resistance to the superoxide-generating compound paraquat, and conversely, exposure to paraquat increased resistance to V(IV). Superoxide dismutase (SOD) activity of cells grown in the presence of V(IV) was augmented by a factor of two. Mutants deficient in the production of Fe-SOD (SodB) were particularly sensitive to vanadium, whilst sodA mutants deficient for Mn-SOD were only marginally affected. In conclusion, it is suggested that V-PCH catalyses a Fenton-type reaction whereby the toxic superoxide anion O(2)- is generated, and that vanadium compromises PVD utilization.

The Pseudomonas siderophore quinolobactin is synthesized from xanthurenic acid, an intermediate of the kynurenine pathway

To cope with iron deficiency fluorescent pseu‐domonads produce pyoverdines which are complex peptidic siderophores that very efficiently scavenge iron. In addition to pyoverdine some species also produce other siderophores. Recently, it was shown that Pseudomonas fluorescens ATCC 17400 pro‐duces the siderophore quinolobactin, an 8‐hydroxy‐4‐methoxy‐2‐quinoline carboxylic acid (Mossialos, D., Meyer, J.M., Budzikiewicz, H., Wolff, U., Koedam, N., Baysse, C., Anjaiah, V., and Cornelis, P. (2000) Appl Environ Microbiol 66: 487–492). The entire quinolobactin biosynthetic, transport and uptake gene cluster, consisting out of two operons comprising 12 open reading frames, was cloned and sequenced. Based on the genes present and physiological complementation assays a biosynthetic pathway for quinolobactin is proposed. Surprisingly, this pathway turned out to combine genes derived from the eukaryotic tryptophan‐xanthurenic acid branch of the kynurenine pathway and from the pathway for the biosynthesis of pyridine‐2,6‐bis(thiocarboxylic acid) from P. stutzeri, PDTC. These results clearly show the involvement of the tryptophan‐kynurenine‐xanthurenic acid pathway in the synthesis of an authentic quinoline siderophore.

Quinolobactin, a New Siderophore of Pseudomonas fluorescens ATCC 17400, the Production of Which Is Repressed by the Cognate Pyoverdine

ABSTRACT Transposon mutant strain 3G6 of Pseudomonas fluorescensATCC 17400 which was deficient in pyoverdine production, was found to produce another iron-chelating molecule; this molecule was identified as 8-hydroxy-4-methoxy-quinaldic acid (designated quinolobactin). The pyoverdine-deficient mutant produced a supplementary 75-kDa iron-repressed outer membrane protein (IROMP) in addition to the 85-kDa IROMP present in the wild type. The mutant was also characterized by substantially increased uptake of 59Fe-quinolobactin. The 75-kDa IROMP was produced by the wild type after induction by quinolobactin-containing culture supernatants obtained from the pyoverdine-negative mutant or by purified quinolobactin. Conversely, adding purified wild-type pyoverdine to the growth medium resulted in suppression of the 75-kDa IROMP in the pyoverdine-deficient mutant; however, suppression was not observed when Pseudomonas aeruginosa PAO1 pyoverdine, a siderophore utilized by strain 3G6, was added to the culture. Therefore, we assume that the quinolobactin receptor is the 75-kDa IROMP and that the quinolobactin-mediated iron uptake system is repressed by the cognate pyoverdine.

Impaired maturation of the siderophore pyoverdine chromophore in Pseudomonas fluorescens ATCC 17400 deficient for the cytochrome c biogenesis protein CcmC.

Pyoverdines are the main siderophores of fluorescent pseudomonads. They comprise a quinoline chromophore, a peptide chain, and a dicarboxylic acid or a dicarboxylic acid amide side chain. Each Pseudomonas species produces a pyoverdine with a different peptide chain. A cytochrome c biogenesis ΔccmC mutant of Pseudomonas fluorescens ATCC 17400 produces multiple pyoverdine forms, showing differences at the level of the chromophore or the side chain. When grown in the presence of L‐cysteine, ΔccmC produces only ferribactin, a non‐fluorescent precursor of pyoverdine, while addition of oxidized glutathione improves pyoverdine production. We suggest that the conversion of ferribactin to pyoverdine does not take place in the ΔccmC mutant because of lack of oxidizing power in the periplasm.