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Pyoverdines are high affinity siderophores produced by most Pseudomonas with a wide role in microbial interspecies interactions. They are primarily composed of a conserved chromophore moiety, an acyl side chain and a peptide backbone which may be highly variable among strains. Upon ferric iron sequestration, pyoverdines are internalized through specialized receptors. The peptide precursor of pyoverdine, termed ferribactin, is synthesized by a set of non-ribosomal peptide synthetase (NRPS) enzymes and further modified by tailoring enzymes. While PvdL, the NRPS responsible for the synthesis of the peptide moiety that derives into the chromophore is conserved, the NRPSs for the peptide backbone are different across fluorescent Pseudomonas. Although the variation of pyoverdine is a widely recognized characteristic within the genus, the evolutionary events associated with the diversity and distribution of this trait remain mostly unknown. This study analyzed the NRPSs clusters for the biosynthesis of the peptide backbone of ferribactin in the genomes of a representative subset of strains of the Pseudomonas fluorescens complex. Bioinformatic analysis of the specificity of adenylation domains of the NRPSs allowed the prediction of 30 different pyoverdine variants. Phylogenetic reconstruction and mapping of the NRPS clusters pinpointed two different general levels of modifications. In the first level, a complete replacement of the set of NRPRs by horizontal transfer occurs. In the second level, the original set of NRPSs is modified through different mechanisms, including partial substitution of the NRPS genes by horizontal transfer, adenylation domain specificity change or NRPS accessory domain gain/loss. [ABSTRACT FROM AUTHOR]

Pyoverdines are high affinity siderophores produced by most Pseudomonas with a wide role in microbial interspecies interactions. They are primarily composed of a conserved chromophore moiety, an acyl side chain and a peptide backbone which may be highly variable among strains. Upon ferric iron sequestration, pyoverdines are internalized through specialized receptors. The peptide precursor of pyoverdine, termed ferribactin, is synthesized by a set of non-ribosomal peptide synthetase (NRPS) enzymes and further modified by tailoring enzymes. While PvdL, the NRPS responsible for the synthesis of the peptide moiety that derives into the chromophore is conserved, the NRPSs for the peptide backbone are different across fluorescent Pseudomonas. Although the variation of pyoverdine is a widely recognized characteristic within the genus, the evolutionary events associated with the diversity and distribution of this trait remain mostly unknown. This study analyzed the NRPSs clusters for the biosynthesis of the peptide backbone of ferribactin in the genomes of a representative subset of strains of the Pseudomonas fluorescens complex. Bioinformatic analysis of the specificity of adenylation domains of the NRPSs allowed the prediction of 30 different pyoverdine variants. Phylogenetic reconstruction and mapping of the NRPS clusters pinpointed two different general levels of modifications. In the first level, a complete replacement of the set of NRPRs by horizontal transfer occurs. In the second level, the original set of NRPSs is modified through different mechanisms, including partial substitution of the NRPS genes by horizontal transfer, adenylation domain specificity change or NRPS accessory domain gain/loss.

In commercial production settings, few options exist to prevent or treat angular leaf spot (ALS) of strawberry, a disease of economic importance and caused by the bacterial pathogen Xanthomonas fragariae. In the process of isolating and identifying X. fragariae bacteria from symptomatic plants, we observed growth inhibition of X. fragariae by bacterial isolates from the same leaf macerates. Identified as species of Pseudomonas and Rhizobium, these isolates were confirmed to suppress growth of X. fragariae in agar overlay plates and in microtiter plate cultures, as did our reference strain Pseudomonas putida KT2440. Screening of a transposon mutant library of KT2440 revealed that disruption of the biosynthetic pathway for the siderophore pyoverdine resulted in complete loss of X. fragariae antagonism, suggesting iron competition as a mode of action. Antagonism could be replicated on plate and in culture by addition of purified pyoverdine or by addition of the chelating agents tannic acid and dipyridyl, while supplementing the medium with iron negated the inhibitory effects of pyoverdine, tannic acid and dipyridyl. When co-inoculated with tannic acid onto strawberry plants, X. fragariae's ability to cause foliar symptoms was greatly reduced, suggesting a possible opportunity for iron-based management of ALS. We discuss our findings in the context of 'nutritional immunity,' the idea that plant hosts restrict pathogen access to iron, either directly, or indirectly through their associated microbiota.

The development of therapies that modulate or prevent pathogen virulence may be a key strategy for circumventing antimicrobial resistance. Toward that end, we examined the production of pyoverdine, a key virulence determinant, in ∼70 Pseudomonas aeruginosa isolates from pediatric cystic fibrosis patients. Pyoverdine production was heterogeneous and showed a clear correlation with pathogenicity in Caenorhabditis elegans and an acute murine pneumonia model. Examination showed pyoverdine accumulation in host tissues, including extrapharyngeal tissues of C. elegans and lung tissues of mice, where accumulation correlated with host death. Many of the isolates tested were resistant to multiple antimicrobials, so we assayed the ability of pyoverdine inhibitors to mitigate virulence and rescue pyoverdine-mediated host pathology. Representatives from three different classes of pyoverdine inhibitors (gallium, fluoropyrimidines, and LK11) significantly improved survival. Our findings highlight the utility of targeting virulence factors in general, and pyoverdine in particular, as a promising method to control bacterial pathogenesis as the utility of antimicrobials continues to diminish.

Pseudomonas aeruginosa is a clinically important pathogen that causes a variety of infections, including urinary, respiratory, and other soft-tissue infections, particularly in hospitalized patients with immune defects, cystic fibrosis, or significant burns. Antimicrobial resistance is a substantial problem in P. aeruginosa treatment due to the inherent insensitivity of the pathogen to a wide variety of antimicrobial drugs and its rapid acquisition of additional resistance mechanisms. One strategy to circumvent this problem is the use of anti-virulent compounds to disrupt pathogenesis without directly compromising bacterial growth. One of the principle regulatory mechanisms for P. aeruginosa’s virulence is the iron-scavenging siderophore pyoverdine, as it governs in-host acquisition of iron, promotes expression of multiple virulence factors, and is directly toxic. Some combination of these activities renders pyoverdine indispensable for pathogenesis in mammalian models. Here we report identification of a panel of novel small molecules that disrupt pyoverdine function. These molecules directly act on pyoverdine, rather than affecting its biosynthesis. The compounds reduce the pathogenic effect of pyoverdine and improve the survival of Caenorhabditis elegans when challenged with P. aeruginosa by disrupting only this single virulence factor. Finally, these compounds can synergize with conventional antimicrobials, forming a more effective treatment. These compounds may help to identify, or be modified to become, viable drug leads in their own right. Finally, they also serve as useful tool compounds to probe pyoverdine activity.

Pseudomonas chlororaphis HT66 is a plant-beneficial bacterium that exhibits wider antagonistic spectrum against a variety of plant pathogenic fungi due to its main secondary metabolite, i.e., phenazine-1-carboxamide (PCN). In the present study, a spontaneous phenotypic variant designated as HT66-FLUO was isolated from the fermentation process of wild-type HT66 strain. The newly isolated phenotypic variant was morphologically distinct from the wild-type strain such as larger cell size, semi-transparent, non-production of PCN (Green or yellow crystals) and enhanced fluorescence under UV light. The whole-genome, RNA-sequencing, and phenotypic assays were performed to identify the reason of phenotypic variation in HT66-FLUO as compared to the HT66. Transcriptomic analysis revealed that 1,418 genes, representing approximately 22% of the 6393 open reading frames (ORFs) had undergone substantial reprogramming of gene expression in the HT66-FLUO. The whole-genome sequence indicated no gene alteration in HT66-FLUO as compared to HT66 according to the known reference sequence. The levels of global regulatory factor gacA and gacS expression were not significantly different between HT66 and HT66-FLUO. It was observed that overexpressing gacS rather than gacA in HT66-FLUO can recover switching of the variant to HT66. The β-galactosidase (LacZ) activity and qRT-PCR results indicate the downregulated expression of rsmX, rsmY, and rsmZ in HT66-FLUO as compared to HT66. Overexpressing three small RNAs in HT66-FLUO can revert switching of colony phenotype toward wild-type HT66 up to a certain degree, restore partial PCN production and reduces the fluorescent siderophores yield. However, the origin of the spontaneous phenotypic variant was difficult to be determined. In conclusion, this study helps to understand the gene regulatory effect in the spontaneous phenotypic variant.

Pseudomonas chlororaphis HT66 is a plant-beneficial bacterium that exhibits wider antagonistic spectrum against a variety of plant pathogenic fungi due to its main secondary metabolite, i.e., phenazine-1-carboxamide (PCN). In the present study, a spontaneous phenotypic variant designated as HT66-FLUO was isolated from the fermentation process of wild-type HT66 strain. The newly isolated phenotypic variant was morphologically distinct from the wild-type strain such as larger cell size, semi-transparent, non-production of PCN (Green or yellow crystals) and enhanced fluorescence under UV light. The whole-genome, RNA-sequencing, and phenotypic assays were performed to identify the reason of phenotypic variation in HT66-FLUO as compared to the HT66. Transcriptomic analysis revealed that 1,418 genes, representing approximately 22% of the 6393 open reading frames (ORFs) had undergone substantial reprogramming of gene expression in the HT66-FLUO. The whole-genome sequence indicated no gene alteration in HT66-FLUO as compared to HT66 according to the known reference sequence. The levels of global regulatory factor gacA and gacS expression were not significantly different between HT66 and HT66-FLUO. It was observed that overexpressing gacS rather than gacA in HT66-FLUO can recover switching of the variant to HT66. The b-galactosidase (LacZ) activity and qRT-PCR results indicate the downregulated expression of rsmX, rsmY, and rsmZ in HT66-FLUO as compared to HT66. Overexpressing three small RNAs in HT66-FLUO can revert switching of colony phenotype toward wild-type HT66 up to a certain degree, restore partial PCN production and reduces the fluorescent siderophores yield. However, the origin of the spontaneous phenotypic variant was difficult to be determined. In conclusion, this study helps to understand the gene regulatory effect in the spontaneous phenotypic variant. [ABSTRACT FROM AUTHOR]

Extracytoplasmic function (ECF) sigma factors control expression of large numbers of genes in bacteria. Most ECF sigma factors are inhibited by antisigma proteins, with inhibition being relieved by environmental signals that lead to inactivation of the antisigma protein and consequent sigma factor activity. In cell surface signaling (CSS) systems in Gram negative bacteria antisigma activity is controlled by an outer membrane protein receptor and its ligand. In Pseudomonas aeruginosa one such system controls expression of genes for secretion and uptake of a siderophore, pyoverdine. In this system the activities of two sigma factors σFpvI and σPvdS are inhibited by antisigma protein FpvR20 that binds to the sigma factors, preventing their interaction with core RNA polymerase. Transport of ferripyoverdine by its outer membrane receptor FpvA causes proteolytic degradation of FpvR20, inducing expression of σFpvI- and σPvdS-dependent target genes. Here we show that degradation of FpvR20 and induction of target gene expression was initiated within 1 min of addition of pyoverdine. FpvR20 was only partially degraded in a mutant lacking the intracellular ClpP protease, resulting in an FpvR20 subfragment (FpvR12) that inhibited σFpvI and σPvdS. The translation inhibitor chloramphenicol did not prevent induction of an σFpvI-dependent gene, showing that degradation of FpvR20 released pre-existing σFpvI in an active form. However, chloramphenicol inhibited induction of σPvdS-dependent genes showing that active σPvdS is not released when FpvR20 is degraded and instead, σPvdS must be synthesized in the absence of FpvR20 to be active. These findings show that sigma factor activation occurs rapidly following addition of the inducing signal in a CSS pathway and requires ClpP protease. Induction of gene expression that can arise from release of active sigma from an antisigma protein but can also require new sigma factor synthesis.

Pseudomonas aeruginosa is a re-emerging, multidrug-resistant, opportunistic pathogen that threatens the lives of immunocompromised patients, patients with cystic fibrosis, and those in critical care units. One of the most important virulence factors in this pathogen is the siderophore pyoverdine. Pyoverdine serves several critical roles during infection. Due to its extremely high affinity for ferric iron, pyoverdine gives the pathogen a significant advantage over the host in their competition for iron. In addition, pyoverdine can regulate the production of multiple bacterial virulence factors and perturb host mitochondrial homeostasis. Inhibition of pyoverdine biosynthesis decreases P. aeruginosa pathogenicity in multiple host models. To better understand the regulation of pyoverdine production, we developed a high-throughput genetic screen that uses the innate fluorescence of pyoverdine to identify genes necessary for its biosynthesis. A substantial number of hits showing severe impairment of pyoverdine production were in genes responsible for early attachment and biofilm formation. In addition to genetic disruption of biofilm, both physical and chemical perturbations also attenuated pyoverdine production. This regulatory relationship between pyoverdine and biofilm is particularly significant in the context of P. aeruginosa multidrug resistance, where the formation of biofilm is a key mechanism preventing access to antimicrobials and the immune system. Furthermore, we demonstrate that the biofilm inhibitor 2-amino-5,6-dimethylbenzimidazole effectively attenuates pyoverdine production and rescues Caenorhabditis elegans from P. aeruginosa-mediated pathogenesis. Our findings suggest that targeting biofilm formation in P. aeruginosa infections may have multiple therapeutic benefits and that employing an unbiased, systems biology-based approach may be useful for understanding the regulation of specific virulence factors and identifying novel anti-virulence therapeutics or new applications for existing therapies for P. aeruginosa infections.

Extracytoplasmic function (ECF) sigma factors control expression of large numbers of genes in bacteria. Most ECF sigma factors are inhibited by antisigma proteins, with inhibition being relieved by environmental signals that lead to inactivation of the antisigma protein and consequent sigma factor activity. In cell surface signaling (CSS) systems in Gram negative bacteria antisigma activity is controlled by an outer membrane protein receptor and its ligand. In Pseudomonas aeruginosa one such system controls expression of genes for secretion and uptake of a siderophore, pyoverdine. In this system the activities of two sigma factors σFpvI and σPvdS are inhibited by antisigma protein FpvR20 that binds to the sigma factors, preventing their interaction with core RNA polymerase. Transport of ferripyoverdine by its outer membrane receptor FpvA causes proteolytic degradation of FpvR20, inducing expression of σFpvI - and σPvdS-dependent target genes. Here we show that degradation of FpvR20 and induction of target gene expression was initiated within 1 min of addition of pyoverdine. FpvR20 was only partially degraded in a mutant lacking the intracellular ClpP protease, resulting in an FpvR20 subfragment (FpvR12) that inhibited σFpvI and σPvdS. The translation inhibitor chloramphenicol did not prevent induction of an σFpvI-dependent gene, showing that degradation of FpvR20 released pre-existing σFpvI in an active form. However, chloramphenicol inhibited induction of σPvdS-dependent genes showing that active σPvdS is not released when FpvR20 is degraded and instead, σPvdS must be synthesized in the absence of FpvR20to be active. These findings show that sigma factor activation occurs rapidly following addition of the inducing signal in a CSS pathway and requires ClpP protease. Induction of gene expression that can arise from release of active sigma from an antisigma protein but can also require new sigma factor synthesis. [ABSTRACT FROM AUTHOR]

Bacterial traits that contribute to disease are termed ‘virulence factors’ and there is much interest in therapeutic approaches that disrupt such traits. What remains less clear is whether a virulence factor identified as such in a particular context is also important in infections involving different host and pathogen types. Here, we address this question using a meta-analytic approach. We statistically analyzed the infection outcomes of 76 experiments associated with one well-studied virulence factor – pyoverdine, an iron-scavenging compound secreted by the opportunistic pathogen Pseudomonas aeruginosa. We found that this factor is consistently involved with virulence across different infection contexts. However, the magnitude of the effect of pyoverdine on virulence varied considerably. Moreover, its effect on virulence was relatively minor in many cases, suggesting that pyoverdine is not indispensable in infections. Our works supports theoretical models from ecology predicting that disease severity is multifactorial and context dependent, a fact that might complicate our efforts to identify the most important virulence factors. More generally, our study highlights how comparative approaches can be used to quantify the magnitude and general importance of virulence factors, key knowledge informing future anti-virulence treatment strategies.

In commercial production settings, few options exist to prevent or treat angular leaf spot of strawberry, a disease of economic importance and caused by the bacterial pathogen Xanthomonas fragariae (Xfr). In the process of isolating and identifying Xfr bacteria from symptomatic plants, we observed growth inhibition of Xfr by bacterial isolates from the same leaf macerates. Identified as species of Pseudomonas and Rhizobium, these isolates were confirmed to suppress growth of Xfr in agar overlay plates and in microtiter plate cultures, as did our reference strain Pseudomonas putida KT2440. Screening of a transposon mutant library of KT2440 revealed that disruption of the biosynthetic pathway for the siderophore pyoverdine resulted in complete loss of Xfr antagonism, suggesting iron competition as a mode of action. Antagonism could be replicated on plate and in culture by addition of purified pyoverdine or by addition of the chelating agents tannic acid and dipyridyl, while supplementing the medium with iron negated the inhibitory effects of pyoverdine, tannic acid and dipyridyl. When co-inoculated with tannic acid onto strawberry plants, Xfr’s ability to cause foliar symptoms was greatly reduced, suggesting a possible opportunity for iron-based management of angular leaf spot. We discuss our findings in the context of ‘nutritional immunity’, the idea that plant hosts restrict pathogens access to iron, either directly, or indirectly through their associated microbiota.

Bacteria living in oxic environments experience iron deficiency due to limited solubility and slow dissolution kinetics of iron-bearing minerals. To cope with iron deprivation, aerobic bacteria have evolved various strategies, including release of siderophores or other organic acids that scavenge external Fe(III) and deliver it to the cells. This research investigated the role of siderophores produced by Pseudomonas aeruginosa in the acquisition of Fe(III) from two iron-bearing colloidal nontronites (NAu-1 and NAu-2), comparing differences in bioavailability related with site occupancy and distribution of Fe(III) in the two lattices. To avoid both the direct contact of the mineral colloids with the bacterial cells and the uncontrolled particle aggregation, nontronite suspensions were homogenously dispersed in a porous silica gel before the dissolution experiments. A multiparametric approach coupling UV-vis spectroscopy and spectral decomposition algorithm was implemented to monitor simultaneously the solubilisation of Fe and the production of pyoverdine in microplate-based batch experiments. Both nontronites released Fe in a particle concentration-dependent manner when incubated with the wild-type P. aeruginosa strain, however iron released from NAu-2 was substantially greater than from NAu-1. The profile of organic acids produced in both cases was similar and may not account for the difference in the iron dissolution efficiency. In contrast, a pyoverdine-deficient mutant was unable to mobilise Fe(III) from either nontronite, whereas iron dissolution occurred in abiotic experiments conducted with purified pyoverdine. Overall, our data provide evidence that P. aeruginosa indirectly mobilise Fe from nontronites primarily through the production of pyoverdine. The structural Fe present on the edges of Nau-2 rather than Nau-1 particles appears to be more bio-accessible, indicating that the distribution of Fe, in the tetrahedron and/or in the octahedron sites, governs the solubilisation process. Furthermore, we also revealed that P. aeruginosa could acquire iron when in direct contact with mineral particles in a siderophore-independent manner.

Pseudomonas putida strains are frequent inhabitants of soil and aquatic niches and they are occasionally isolated from hospital environments. As the available iron sources in human tissues, edaphic, and aquatic niches are different, we have analyzed iron-uptake related genes in different P. putida strains that were isolated from all these environments. We found that these isolates can be grouped into different clades according to the genetics of siderophore biosynthesis and recycling. The pyoverdine locus of the six P. putida clinical isolates that have so far been completely sequenced, are not closely related; three strains (P. putida HB13667, HB3267, and NBRC14164T) are grouped in Clade I and the other three in Clade II, suggesting possible different origins and evolution. In one clinical strain, P. putida HB4184, the production of siderophores is induced under high osmolarity conditions. The pyoverdine locus in this strain is closely related to that of strain P. putida HB001 which was isolated from sandy shore soil of the Yellow Sea in Korean marine sand, suggesting their possible origin, and evolution. The acquisition of two unique TonB-dependent transporters for xenosiderophore acquisition, similar to those existing in the opportunistic pathogen P. aeruginosa PAO, is an interesting adaptation trait of the clinical strain P. putida H8234 that may confer adaptive advantages under low iron availability conditions. [ABSTRACT FROM AUTHOR]

Bacterial traits that contribute to disease are termed virulence factors and there is much interest in therapeutic approaches that disrupt such traits. What remains less clear is whether a virulence factor identified as such in a particular context is also important in infections involving different host and pathogen types. Here, we address this question using a meta-analytic approach. We statistically analyzed the infection outcomes of 81 experiments associated with one well studied virulence factor pyoverdine, an iron scavenging compound secreted by the opportunistic pathogen Pseudomonas aeruginosa. We found that this factor is consistently involved with virulence across different infection contexts. However, the magnitude of the effect of pyoverdine on virulence varied considerably. Moreover, its effect on virulence was relatively minor in many cases, suggesting that pyoverdine is not indispensable in infections. Our works supports theoretical models from ecology predicting that disease severity is multifactorial and context dependent, a fact that might complicate our efforts to identify the most important virulence factors. More generally, our study highlights how comparative approaches can be used to quantify the magnitude and general importance of virulence factors, key knowledge informing future anti-virulence treatment strategies. [ABSTRACT FROM AUTHOR]

Pseudomonas putida GB-1 is a Mn(II)-oxidizing bacterium that produces pyoverdine-type siderophores (PVDs), which facilitate the uptake of Fe(III) but also influence MnO2 formation. Recently, a non-ribosomal peptide synthetase mutant that does not synthesize PVD was described. Here we identified a gene encoding the PVDGB-1 (PVD produced by strain GB-1) uptake receptor (PputGB1_4082) of strain GB-1 and confirmed its function by in-frame mutagenesis. Growth and other physiological responses of these two mutants and of wild type were compared during cultivation in the presence of three chemically distinct sets of PVDs (siderotypes no1, no2 and no4) derived from various pseudomonads. Under iron-limiting conditions, Fe(III) complexes of various siderotype no1 PVDs (including PVDGB-1) allowed growth of wild type and the synthetase mutant, but not the receptor mutant, confirming that iron uptake with any tested siderotype no1 PVD depended on PputGB1_4082. Fe(III) complexes of a siderotype no2 PVD were not utilized by any strain and strongly induced PVD synthesis. In contrast, Fe(III) complexes of siderotype no4 PVDs promoted the growth of all three strains and did not induce PVD synthesis by the wild type, implying these complexes were utilized for iron uptake independent of PputGB1_4082. These differing properties of the three PVD types provided a way to differentiate between effects on MnO2 formation that resulted from iron limitation and others that required participation of the PVDGB-1 receptor. Specifically, MnO2 production was inhibited by siderotype no1 but not no4 PVDs indicating PVD synthesis or PputGB1_4082 involvement rather than iron-limitation caused the inhibition. In contrast, iron limitation was sufficient to explain the inhibition of Mn(II) oxidation by siderotype no2 PVDs. Collectively, our results provide insight into how competition for iron via siderophores influences growth, iron nutrition and MnO2 formation in more complex environmental systems.

When iron-starved, the Mn(II)-oxidizing bacteria Pseudomonas putida strains GB-1 and MnB1 produce pyoverdines (PVDGB-1 and PVDMnB1), siderophores that both influence iron uptake and inhibit manganese(II) oxidation by these strains. To explore the properties and genetics of a PVD that can affect manganese oxidation, LC-MS/MS and various siderotyping techniques were used to identify the peptides of PVDGB-1 and PVDMnB1 as being (for both PVDs): chromophore-Asp-Lys-OHAsp-Ser-Gly-aThr-Lys-cOHOrn, resembling a structure previously reported for P. putida CFML 90-51, which does not oxidize Mn. All three strains also produced an azotobactin and a sulfonated PVD, each with the peptide sequence above, but with unknown regulatory or metabolic effects. Bioinformatic analysis of the sequenced genome of P. putida GB-1 suggested that a particular non-ribosomal peptide synthetase, coded by the operon PputGB1_4083-4086, could produce the peptide backbone of PVDGB-1. To verify this prediction, plasmid integration disruption of PputGB1_4083 was performed and the resulting mutant failed to produce detectable PVD. In silico analysis of the modules in PputGB1_4083-4086 predicted a peptide sequence of Asp-Lys-Asp-Ser-Ala-Thr-Lsy-Orn, which closely matches the peptide determined by MS/MS. To extend these studies to other organisms, various Mn(II)-oxidizing and non-oxidizing isolates of P. putida, P. fluorescens, P. marincola, P. fluorescens-syringae group, P. mendocina-resinovorans group and P. stutzerii group were screened for PVD synthesis. The PVD producers (12 out of 16 tested strains) were siderotyped and placed into four sets of differing PVD structures, some corresponding to previously characterized PVDs and some to novel PVDs. These results combined with previous studies suggested that the presence of OHAsp or the flexibility of the pyoverdine polypeptide may enable efficient binding of Mn(III).

When iron-starved, the Mn(II)-oxidizing bacteria Pseudomonas putida strains GB-1 and MnB1 produce pyoverdines (PVDGB?1 and PVDMnB1), siderophores that both influence iron uptake and inhibit manganese(II) oxidation by these strains. To explore the properties and genetics of a PVD that can affect manganese oxidation, LC-MS/MS, and various siderotyping techniques were used to identify the peptides of PVDGB?1 and PVDMnB1 as being (for both PVDs): chromophore-Asp-Lys-OHAsp-Ser-Gly-aThr-Lys-cOHOrn, resembling a structure previously reported for P. putida CFML 90-51, which does not oxidize Mn. All three strains also produced an azotobactin and a sulfonated PVD, each with the peptide sequence above, but with unknown regulatory or metabolic effects. Bioinformatic analysis of the sequenced genome of P. putida GB-1 suggested that a particular non-ribosomal peptide synthetase (NRPS), coded by the operon PputGB1_4083-4086, could produce the peptide backbone of PVDGB?1. To verify this prediction, plasmid integration disruption of PputGB1_4083 was performed and the resulting mutant failed to produce detectable PVD. In silico analysis of the modules in PputGB1_4083-4086 predicted a peptide sequence of Asp-Lys-Asp-Ser-Ala-Thr-Lsy-Orn, which closely matches the peptide determined by MS/MS. To extend these studies to other organisms, various Mn(II)-oxidizing and non-oxidizing isolates of P. putida, P. fluorescens, P. marincola, P. fluorescens-syringae group, P. mendocina-resinovorans group, and P. stutzerii group were screened for PVD synthesis. The PVD producers (12 out of 16 tested strains) were siderotyped and placed into four sets of differing PVD structures, some corresponding to previously characterized PVDs and some to novel PVDs. These results combined with previous studies suggested that the presence of OHAsp or the flexibility of the pyoverdine polypeptide may enable efficient binding of Mn(III). [ABSTRACT FROM AUTHOR]