Two new siderophores produced by Pseudomonas sp. NCIMB 10586: The anti-oomycete non-ribosomal peptide synthetase-dependent mupirochelin and the NRPS-independent triabactin.

Introduction: Globisporangium ultimum is an oomycetal pathogen causing damping-off on over 300 different plant hosts. Currently, as for many phytopathogens, its control relies in the use of chemicals with negative impact on health and ecosystems. Therefore, many biocontrol strategies are under investigation to reduce the use of fungicides. Results: In this study, the soil bacterium Pseudomonas sp. NCIMB 10586 demonstrates a strong iron-repressed in vitro antagonism against G. ultimum MUCL 38045. This antagonism does not depend on the secretion of the broad-range antibiotic mupirocin or of the siderophore pyoverdine by the bacterial strain. The inhibitor molecule was identified as a novel non-ribosomal peptide synthetase (NRPS) siderophore named mupirochelin. Its putative structure bears similarities to other siderophores and bioactive compounds. The transcription of its gene cluster is affected by the biosynthesis of pyoverdine, the major known siderophore of the strain. Besides mupirochelin, we observed the production of a third and novel NRPS-independent siderophore (NIS), here termed triabactin. The iron-responsive transcriptional repression of the two newly identified siderophore gene clusters corroborates their role as iron scavengers. However, their respective contributions to the strain fitness are dissimilar. Bacterial growth in iron-deprived conditions is greatly supported by pyoverdine production and, to a lesser extent, by triabactin. On the contrary, mupirochelin does not contribute to the strain fitness under the studied conditions. Conclusion: Altogether, we have demonstrated here that besides pyoverdine, Pseudomonas sp. NCIMB 10586 produces two newly identified siderophores, namely mupirochelin, a weak siderophore with strong antagonism activity against G. ultimum, and the potent siderophore triabactin.

Pseudomonas arcuscaelestis sp. nov., isolated from rainbow trout and water.

Cells of strains P66T, V1 and W15Feb18 are Gram-stain-negative short rods and motile by one polar flagellum. Strain P66T was isolated from rainbow trout (Oncorhynchus mykiss) cultivated at a fish farm in Turkey. Strain V1 was isolated from sand of an intertidal shore on the Galicia coast in Spain and strain W15Feb18 was isolated from water collected at the Woluwe River in Belgium. Based on 16S rRNA sequence similarity values, the strains were grouped under the genus Pseudomonas and the Pseudomonas putida phylogenetic group of species. The DNA G+C content ranged from 58.5 to 58.9 mol%. The strains were characterized phenotypically by the API 20NE and Biolog GEN III tests, and chemotaxonomically by their whole-cell MALDI-TOF MS protein profiles and fatty acid contents. The absence of the hydrolysis of gelatin and the assimilation of arabinose, mannose and mannitol differentiated these strains from the closest species, Pseudomonas alkylphenolica. The major fatty acid components were C16:0 (29.91-31.68 %) and summed feature 3 (36.44-37.55 %). Multilocus sequence analysis with four and 83 housekeeping gene sequences and a core proteome analysis showed that these strains formed a phylogenetic cluster in the P. putida group of species. Genome comparisons by the average nucleotide identity based on blast and the Genome-to-Genome Distance Calculator demonstrated that the three strains belonged to the same genomic species and were distant from any known species, with similarity values lower than the thresholds established for species in the genus Pseudomonas. These data permitted us to conclude that strains P66T, V1 and W15Feb18 belong to a novel species in the genus Pseudomonas, for which the name Pseudomonas arcuscaelestis sp. nov. is proposed. The type strain is P66T (=CECT 30176T=CCUG 74872T). The other strains have been deposited in the CECT with the corresponding collection numbers: V1 (=CECT 30356) and W15Feb18 (=CECT 30355).

Pyoverdine and histicorrugatin-mediated iron acquisition in Pseudomonas thivervalensis.

The genome of Pseudomonas thivervalensis LMG 21626(T) has been sequenced and a genomic, genetic and structural analysis of the siderophore mediated iron acquisition was undertaken. Pseudomonas thivervalensis produces two structurally new siderophores, pyoverdine PYOthi which is typical for P. thivervalensis strains and a closely related strain, and the lipopeptidic siderophore histicorrugatin which is also detected in P. lini. Histicorrugatin consists out of an eight amino acid long peptide which is linked to octanoic acid. It is structurally related to the siderophores corrugatin and ornicorrugatin. Analysis of the proteome for TonB-dependent receptors identified 25 candidates. Comparison of the TonB-dependent receptors of P. thivervalensis with the 17 receptors of its phylogenetic neighbor, P. brassicacearum subsp. brassicacearum NFM 421, showed that NFM 421 shares the same set of receptors with LMG 21626(T), including the histicorrugatin receptor. An exception was found for their cognate pyoverdine receptor which can be explained by the observation that both strains produce structurally different pyoverdines. Mass analysis showed that NFM 421 did not produce histicorrugatin, but the analogue ornicorrugatin. Growth stimulation assays with a variety of structurally distinct pyoverdines produced by other Pseudomonas species demonstrated that LMG 21626(T) and NFM 421 are able to utilize almost the same set of pyoverdines. Strain NFM 421 is able utilize two additional pyoverdines, pyoverdine of P. fluorescens Pf0-1 and P. citronellolis LMG 18378(T), these pyoverdines are probably taken up by the FpvA receptor of NFM 421.

Draft genome sequence analysis of a Pseudomonas putida W15Oct28 strain with antagonistic activity to Gram-positive and Pseudomonas sp. pathogens.

Pseudomonas putida is a member of the fluorescent pseudomonads known to produce the yellow-green fluorescent pyoverdine siderophore. P. putida W15Oct28, isolated from a stream in Brussels, was found to produce compound(s) with antimicrobial activity against the opportunistic pathogens Staphylococcus aureus, Pseudomonas aeruginosa, and the plant pathogen Pseudomonas syringae, an unusual characteristic for P. putida. The active compound production only occurred in media with low iron content and without organic nitrogen sources. Transposon mutants which lost their antimicrobial activity had the majority of insertions in genes involved in the biosynthesis of pyoverdine, although purified pyoverdine was not responsible for the antagonism. Separation of compounds present in culture supernatants revealed the presence of two fractions containing highly hydrophobic molecules active against P. aeruginosa. Analysis of the draft genome confirmed the presence of putisolvin biosynthesis genes and the corresponding lipopeptides were found to contribute to the antimicrobial activity. One cluster of ten genes was detected, comprising a NAD-dependent epimerase, an acetylornithine aminotransferase, an acyl CoA dehydrogenase, a short chain dehydrogenase, a fatty acid desaturase and three genes for a RND efflux pump. P. putida W15Oct28 genome also contains 56 genes encoding TonB-dependent receptors, conferring a high capacity to utilize pyoverdines from other pseudomonads. One unique feature of W15Oct28 is also the presence of different secretion systems including a full set of genes for type IV secretion, and several genes for type VI secretion and their VgrG effectors.

Antimicrobial properties of Pseudomonas strains producing the antibiotic mupirocin.

Mupirocin is a polyketide antibiotic with broad antibacterial activity. It was isolated and characterized about 40 years ago from Pseudomonas fluorescens NCIMB 10586. To study the phylogenetic distribution of mupirocin producing strains in the genus Pseudomonas a large collection of Pseudomonas strains of worldwide origin, consisting of 117 Pseudomonas type strains and 461 strains isolated from different biological origins, was screened by PCR for the mmpD gene of the mupirocin gene cluster. Five mmpD(+) strains from different geographic and biological origin were identified. They all produced mupirocin and were strongly antagonistic against Staphylococcus aureus. Phylogenetic analysis showed that mupirocin production is limited to a single species. Inactivation of mupirocin production leads to complete loss of in vitro antagonism against S. aureus, except on certain iron-reduced media where the siderophore pyoverdine is responsible for the in vitro antagonism of a mupirocin-negative mutant. In addition to mupirocin some of the strains produced lipopeptides of the massetolide group. These lipopeptides do not play a role in the observed in vitro antagonism of the mupirocin producing strains against S. aureus.

The Pseudomonas community in metal-contaminated sediments as revealed by quantitative PCR: a link with metal bioavailability.

Pseudomonas bacteria are ubiquitous Gram-negative and aerobic microorganisms that are known to harbor metal resistance mechanisms such as efflux pumps and intracellular redox enzymes. Specific Pseudomonas bacteria have been quantified in some metal-contaminated environments, but the entire Pseudomonas population has been poorly investigated under these conditions, and the link with metal bioavailability was not previously examined. In the present study, quantitative PCR and cell cultivation were used to monitor and characterize the Pseudomonas population at 4 different sediment sites contaminated with various levels of metals. At the same time, total metals and metal bioavailability (as estimated using an HCl 1 m extraction) were measured. It was found that the total level of Pseudomonas, as determined by qPCR using two different genes (oprI and the 16S rRNA gene), was positively and significantly correlated with total and HCl-extractable Cu, Co, Ni, Pb and Zn, with high correlation coefficients (>0.8). Metal-contaminated sediments featured isolates of the Pseudomonas putida, Pseudomonas fluorescens, Pseudomonas lutea and Pseudomonas aeruginosa groups, with other bacterial genera such as Mycobacterium, Klebsiella and Methylobacterium. It is concluded that Pseudomonas bacteria do proliferate in metal-contaminated sediments, but are still part of a complex community.

rpoD gene pyrosequencing for the assessment of Pseudomonas diversity in a water sample from the Woluwe River.

A water sample from a noncontaminated site at the source of the Woluwe River (Belgium) was analyzed by culture-dependent and -independent methods. Pseudomonas isolates were identified by sequencing and analysis of the rpoD gene. Cultureindependent methods consisted of cloning and pyrosequencing of a Pseudomonas rpoD amplicon from total DNA extracted from the same sample and amplified with selective rpoD gene primers. Among a total of 14,540 reads, 6,228 corresponded to Pseudomonas rpoD gene sequences by a BLAST analysis in the NCBI database. The selection criteria for the reads were sequences longer than 400 bp, an average Q40 value greater than 25, and>85% identity with a Pseudomonas species. Of the 6,228 Pseudomonas rpoD sequences, 5,345 sequences met the established criteria for selection. Sequences were clustered by phylogenetic analysis and by use of the QIIME software package. Representative sequences of each cluster were assigned by BLAST analysis to a known Pseudomonas species when the identity with the type strain was greater than or equal to 96%. Twenty-six species distributed among 12 phylogenetic groups or subgroups within the genus were detected by pyrosequencing. Pseudomonas stutzeri, P. moraviensis, and P. simiae were the only cultured species not detected by pyrosequencing. The predominant phylogenetic group within the Pseudomonas genus was the P. fluorescens group, as determined by culture-dependent and -independent analyses. In all analyses, a high number of putative novel phylospecies was found: 10 were identified in the cultured strains and 246 were detected by pyrosequencing, indicating that the diversity of Pseudomonas species has not been fully described.

Analysis of the draft genome of Pseudomonas fluorescens ATCC17400 indicates a capacity to take up iron from a wide range of sources, including different exogenous pyoverdines.

All fluorescent pseudomonads (Pseudomonas aeruginosa, P. putida, P. fluorescens, P. syringae and others) are known to produce the high-affinity peptidic yellow-green fluorescent siderophore pyoverdine. These siderophores have peptide chains that are quite diverse and more than 50 pyoverdine structures have been elucidated. In the majority of the cases, a Pseudomonas species is also able to produce a second siderophore of lower affinity for iron. Pseudomonas fluorescens ATCC 17400 has been shown to produce a unique second siderophore, (thio)quinolobactin, which has an antimicrobial activity against the phytopathogenic Oomycete Pythium debaryanum. We show that this strain has the capacity to utilize 16 different pyoverdines, suggesting the presence of several ferripyoverdine receptors. Analysis of the draft genome of P. fluorescens ATCC 17400 confirmed the presence of 55 TonB-dependent receptors, the largest so far for Pseudomonas, among which 15 are predicted to be ferripyoverdine receptors (Fpv). Phylogenetic analysis revealed the presence of two different clades containing ferripyoverdine receptors, with sequences similar to the P. aeruginosa type II FpvA forming a separate cluster. Among the other receptors we confirmed the presence of the QbsI (thio)quinolobactin receptor, an ferri-achromobactin and an ornicorrugatin receptor, several catecholate and four putative heme receptors. Twenty five of the receptors genes were found to be associated with genes encoding extracytoplasmic sigma factors (ECF σ) and transmembrane anti-σ sensors.

A combinatorial approach to the structure elucidation of a pyoverdine siderophore produced by a Pseudomonas putida isolate and the use of pyoverdine as a taxonomic marker for typing P. putida subspecies.

The structure of a pyoverdine produced by Pseudomonas putida, W15Oct28, was elucidated by combining mass spectrometric methods and bioinformatics by the analysis of non-ribosomal peptide synthetase genes present in the newly sequenced genome. The only form of pyoverdine produced by P. putida W15Oct28 is characterized to contain α-ketoglutaric acid as acyl side chain, a dihydropyoverdine chromophore, and a 12 amino acid peptide chain. The peptide chain is unique among all pyoverdines produced by Pseudomonas subspecies strains. It was characterized as -L-Asp-L-Ala-D-AOHOrn-L-Thr-Gly-c[L-Thr(O-)-L-Hse-D-Hya-L-Ser-L-Orn-L-Hse-L-Ser-O-]. The chemical formula and the detected and calculated molecular weight of this pyoverdine are: C65H93N17O32, detected mass 1624.6404 Da, calculated mass 1624.6245. Additionally, pyoverdine structures from both literature reports and bioinformatics prediction of the genome sequenced P. putida strains are summarized allowing us to propose a scheme based on pyoverdines structures as tool for the phylogeny of P. putida. This study shows the strength of the combination of in silico analysis together with analytical data and literature mining in determining the structure of secondary metabolites such as peptidic siderophores.

Iron competition in fungus-plant interactions: the battle takes place in the rhizosphere.

Soilborne fungal pathogens are highly persistent and provoke important crop losses. During saprophytic and infectious stages in the soil, these organisms face situations of nutrient limitation and lack of essential elements, such as iron. We investigated the role of the bZIP transcription factor HapX as a central regulator of iron homeostasis and virulence in the vascular wilt fungus Fusarium oxysporum. This root-infecting plant pathogen attacks more than hundred different crops and is an emerging human opportunistic invader. Although iron uptake remains unaffected in a strain lacking HapX, de-repression of genes implicated in iron-consuming processes such as respiration, amino acid metabolism, TCA cycle and heme biosynthesis lead to severely impaired growth under iron-limiting conditions. HapX is required for full virulence of F. oxysporum in tomato plants and essential for infection in immunodepressed mice. Virulence attenuation of the ΔhapX strain on tomato plants is more pronounced by co-inoculation of roots with the biocontrol strain Pseudomonas putida KT2440, but not with a mutant deficient in siderophores production. These results demonstrate that HapX is required for iron competition of F. oxysporum in the tomato rhizosphere and establish a conserved role for HapX-mediated iron homeostasis in fungal infection of plants and mammals.

Evaluation of oprI and oprL genes as molecular markers for the genus Pseudomonas and their use in studying the biodiversity of a small Belgian River.

A multiplex PCR based on oprI and oprL, coding for the outer membrane lipoprotein I and the peptidoglycan-associated lipoprotein OprL, respectively, was developed for the detection of Pseudomonas strains from a bacterial collection isolated from a small river. To study the diversity of these Pseudomonas isolates, an oprI-oprL gene sequence database of 94 Pseudomonas type strains was constructed. Phylogenetic analysis of the concatenated oprI and oprL gene sequences of the Pseudomonas type strains showed that they were largely congruent with the classification based on the MLSA approach based on 16S rRNA, gyrB, rpoB and rpoD gene sequences of Mulet et al. in 2010. Identification of the isolates demonstrated a high diversity of Pseudomonas isolates at the source of the river located in a forest of which most isolates belonged to the Pseudomonas fluorescens lineage. On the other hand, the Pseudomonas population isolated at an anthropized site at the mouth of the river, receiving waste water from both households and industry, was very different and contained many Pseudomonas aeruginosa isolates.

HapX-mediated iron homeostasis is essential for rhizosphere competence and virulence of the soilborne pathogen Fusarium oxysporum.

Soilborne fungal pathogens cause devastating yield losses and are highly persistent and difficult to control. During the infection process, these organisms must cope with limited availability of iron. Here we show that the bZIP protein HapX functions as a key regulator of iron homeostasis and virulence in the vascular wilt fungus Fusarium oxysporum. Deletion of hapX does not affect iron uptake but causes derepression of genes involved in iron-consuming pathways, leading to impaired growth under iron-depleted conditions. F. oxysporum strains lacking HapX are reduced in their capacity to invade and kill tomato (Solanum lycopersicum) plants and immunodepressed mice. The virulence defect of ΔhapX on tomato plants is exacerbated by coinoculation of roots with a biocontrol strain of Pseudomonas putida, but not with a siderophore-deficient mutant, indicating that HapX contributes to iron competition of F. oxysporum in the tomato rhizosphere. These results establish a conserved role for HapX-mediated iron homeostasis in fungal infection of plants and mammals.

Promysalin, a salicylate-containing Pseudomonas putida antibiotic, promotes surface colonization and selectively targets other Pseudomonas.

Under control of the Gac regulatory system, Pseudomonas putida RW10S1 produces promysalin to promote its own swarming and biofilm formation, and to selectively inhibit many other pseudomonads, including the opportunistic pathogen Pseudomonas aeruginosa. This amphipathic antibiotic is composed of salicylic acid and 2,8-dihydroxymyristamide bridged by a unique 2-pyrroline-5-carboxyl moiety. In addition to enzymes for salicylic acid synthesis and activation, the biosynthetic gene cluster encodes divergent type II fatty acid biosynthesis components, unusual fatty acid-tailoring enzymes (two Rieske-type oxygenases and an amidotransferase), an enzyme resembling a proline-loading module of nonribosomal peptide synthetases, and the first prokaryotic member of the BAHD family of plant acyltransferases. Identification of biosynthetic intermediates enabled to propose a pathway for synthesis of this bacterial colonization factor.

A proteome analysis of the response of a Pseudomonas aeruginosa oxyR mutant to iron limitation.

In Pseudomonas aeruginosa the response to oxidative stress is orchestrated by the LysR regulator OxyR by activation of the transcription of two catalase genes (katA and katB), of the alkyl-hydroxyperoxidases ahpCF and ahpB. Next to the expected high sensitivity to oxidative stress generated by reactive oxygen species (ROS: H(2)O(2), O(2)(-)), the oxyR mutant shows a defective growth under conditions of iron limitation (Vinckx et al. 2008). Although production and uptake of the siderophore pyoverdine is not affected by the absence of oxyR, the mutant is unable to satisfy its need for iron when grown under iron limiting conditions. In order to get a better insight into the effects caused by iron limitation on the physiological response of the oxyR mutant we decided to compare the proteomes of the wild type and the mutant grown in the iron-poor casamino acids medium (CAA), in CAA plus H(2)O(2), and in CAA plus the strong iron chelator ethylenediamine-N,N'-bis(2-hydroxyphenylacetic acid) (EDDHA). Especially in the presence of hydrogen peroxide the oxyR cells increase the production of stress proteins (Dps and IbpA). The superoxide dismutase SodM is produced in higher amounts in the oxyR mutant grown in CAA plus H(2)O(2). The PchB protein, a isochorismate-pyruvate lyase involved in the siderophore pyochelin biosynthesis is not detectable in the extracts from the oxyR mutant grown in the presence of hydrogen peroxide. When cells were grown in the presence of EDDHA, we observed a reduction of the ferric uptake regulator (Fur), and an increase in the two subunits of the succinyl-CoA synthetase and the fumarase FumC1.

Synthesis of 1-substituted 1,2,3,4-tetrahydrobenz[g]isoquinoline-5,10-diones.

1,2-Disubstituted 1,2,3,4-tetrahydrobenz[g]isoquinoline-5,10-diones are prepared for the first time through an activated Pictet-Spengler reaction of the corresponding imines of 2-(1,4-dimethoxynaphth-2-yl)ethylamine in the presence of an acyl chloride and AlCl(3) followed by an oxidation with silver(II) oxide in nitric acid. Depending on the reaction conditions the N-trichloroacetyl protecting group could be cleaved off, converted to an N-methoxycarbonyl group or transformed to an N-(2-oxoacetamide) moiety. The synthesized 1,2-disubstituted 1,2,3,4-tetrahydrobenz[g]isoquinoline-5,10-diones constitute a new class of quinones, which has not been reported yet.

The Pseudomonas aeruginosa oxidative stress regulator OxyR influences production of pyocyanin and rhamnolipids: protective role of pyocyanin.

The LysR-type transcriptional regulator (LTTR) OxyR orchestrates the defence of the opportunistic pathogen Pseudomonas aeruginosa against reactive oxygen species. In previous work we also demonstrated that OxyR is needed for the utilization of the ferrisiderophore pyoverdine, stressing the importance of this regulator. Here, we show that an oxyR mutant is unable to swarm on agar plates, probably as a consequence of absence of production of rhamnolipid surfactant molecules. Another obvious phenotypic change was the increased production of the phenazine redox-active molecule pyocyanin in the oxyR mutant. As already described, the oxyR mutant could not grow in LB medium, unless high numbers of cells (>10( 8) ml(-1)) were inoculated. However, its growth in Pseudomonas P agar (King's A), a medium inducing pyocyanin production, was like that of the wild-type, suggesting a protective action of this redox-active phenazine compound. This was confirmed by the restoration of the capacity to grow in LB medium upon addition of pure pyocyanin. Although both rhamnolipid and pyocyanin production are controlled by quorum sensing, no obvious changes were observed in the production of N-acylhomoserine lactones or the Pseudomonas quinolone signal (PQS). Complementation of rhamnolipid production and motility, and restoration of normal pyocyanin levels, was only possible when the oxyR gene was in single copy, while pyocyanin levels were increased when oxyR was present in a multicopy vector. Conversely, plating efficiency was increased only when the oxyR gene was present in multicopy, but not when in single copy in the chromosome, due to lower expression of oxyR compared with the wild-type, suggesting that some phenotypes are differently affected in function to the levels of OxyR molecules in the cell. Analysis of transcripts of oxidative stress-response enzymes showed a strong decrease of katB, ahpC and ahpB expression in the oxyR mutant grown in LB, but this was not the case when the mutant was grown on P agar, suggesting that the OxyR dependency for the transcription of these genes is not total.

Use of non-porous pillar array columns for the separation of Pseudomonas pyoverdine siderophores as an example of a real-world biological sample.

We report on the first separation of a complex biomixture in pressure-driven mode using perfectly ordered pillar array columns. The separations were conducted in the reversed-phase mode using a highly aqueous mobile phase, while the outer surface of the non-porous pillars was chemically functionalized with a hydrophobic C8-layer. The samples originated from two different bacterial strains (Pseudomonas aeruginosa PAO1 and Pseudomonas sp. W15Feb38) of fluorescent pseudomonads. These produce fluorescent yellow-green pyoverdines that serve as siderophores to shuttle iron inside the cell. The pyoverdines of both strains were prepared from the supernatant through a crude solid phase extraction without any further purification step. In case of the PAO1 mixture, a separation of 15 components within a column length of 2.5 cm could be observed through the transparent cover glass of the chip. For the W15Feb38 mixture, a separation of eight components could be observed within the same distance. These fast chromatographic separations were compared with those obtained via iso-electrofocusing (IEF), which is the traditionally employed fingerprinting method to characterize pseudomonad strains based on their pyoverdine profiles (siderotyping). With this technique, and despite the injection of a 10,000 times larger sample mass, only nine bands were maximally observed for the PAO1 mixture, whereas maximally six bands were observed in case of the W15Feb38 mixture. The chromatographic pillar array method, yielding a separation in less than 1 min, was also significantly faster than the IEF method, which typically needs 1.5h. The present system can therefore be considered as a potential alternative fingerprinting tool for the fast identification of different strains of fluorescent pseudomonads, including as diagnostic tool for typing strains of the important opportunistic pathogen P. aeruginosa.

Siderophore-mediated iron acquisition in the entomopathogenic bacterium Pseudomonas entomophila L48 and its close relative Pseudomonas putida KT2440.

Pseudomonas entomophila L48 is a recently identified entomopathogenic bacterium which, upon ingestion, kills Drosophila melanogaster, and is closely related to P. putida. The complete genome of this species has been sequenced and therefore a genomic, genetic and structural analysis of the siderophore-mediated iron acquisition was undertaken. P. entomophila produces two siderophores, a structurally new and unique pyoverdine and the secondary siderophore pseudomonine, already described in P. fluorescens species. Structural analysis of the pyoverdine produced by the closely related P. putida KT2440 showed that this strain produces an already characterised pyoverdine, but different from P. entomophila, and no evidence was found for the production of a second siderophore. Growth stimulation assays with heterologous pyoverdines demonstrated that P. entomophila is able to utilize a large variety of structurally distinct pyoverdines produced by other Pseudomonas species. In contrast, P. putida KT2440 is able to utilize only its own pyoverdine and the pyoverdine produced by P. syringae LMG 1247. Our data suggest that although closely related, P. entomophila is a more efficient competitor for iron than P. putida.

The adsorption of Pseudomonas aeruginosa bacteriophage phiKMV is dependent on expression regulation of type IV pili genes.

Pseudomonas aeruginosa bacteriophage phiKMV requires type IV pili for infection, as observed from the phenotypic characterization and phage adsorption assays on a phage infection-resistant host strain mutant. A cosmid clone library of the host (P. aeruginosa PAO1) genomic DNA was generated and used to select for a clone that was able to restore phiKMV infection in the resistant mutant. This complementing cosmid also re-established type IV pili-dependent twitching motility. The correlation between bacteriophage phiKMV infectivity and type IV pili, along with its associated twitching motility, was confirmed by the resistance of a P. aeruginosa PAO1DeltapilA mutant to the phage. Subcloning of the complementing cosmid and further phage infection analysis and motility assays suggests that a common regulatory mechanism and/or interaction between the ponA and pilMNOPQ gene products are essential for bacteriophage phiKMV infectivity.

Distribution and evolution of ferripyoverdine receptors in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a ubiquitous gram-negative bacterium, which is also able to cause severe opportunistic infections in humans. The colonization of the host is importantly affected by the production of the high-affinity iron (III) scavenging peptidic siderophore pyoverdine. The species P. aeruginosa can be divided into three subgroups ('siderovars'), each characterized by the production of a specific pyoverdine and receptor (FpvA). We used a multiplex PCR to determine the FpvA siderovar on 345 P. aeruginosa strains from environmental or clinical origin. We found about the same proportion of each type in clinical strains, while FpvA type I was slightly over-represented (49%) in environmental strains. Our multiplex PCR also detected the presence or absence of an additional receptor for type I pyoverdine (FpvB). The fpvB gene was in fact present in the vast majority of P. aeruginosa strains (93%), regardless of their siderovar or their origin. Finally, molecular analyses of fpvA and fpvB genes highlighted a complex evolutionary history, probably linked to the central role of iron acquisition in the ecology and virulence of P. aeruginosa.