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Enzymes of the arginine and polyamine biosynthesis pathways inhibited by anti-metabolite toxins mangotoxin and phaseolotoxin. Enzymes are: OAT, ornithine N-acetyltransferase; OCT, ornithine carbamoyltransferase; ODC, ornithine decarboxylase.
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Certain strains of Pseudomonas syringae pathovars phaseolicola and actinidiae and P. syringae pv. syringae strain CFBP3388 produce the chlorosis-inducing phytotoxin phaseolotoxin, which inhibits biosynthesis of arginine and polyamines. The 25 kb Pht cluster, responsible for phaseolotoxin biosynthesis, is included in a putative pathogenicity island...
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... form of the toxin in vivo is N d -(N 0 -sulfodiamino- phosphinyl) ornithine (Mitchell and Bieleski, 1977). This mole- cule is a potent inhibitor of ornithine carbamoyltransferase, which converts ornithine to citrulline in the arginine biosynthesis pathway, and also of ornithine decarboxylase, involved in the biosynthesis of polyamines ( Fig. 1) ( Bachmann et al., ...
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... ( Arrebola et al., 2003;Staskawicz and Panopoulos, 1979), strains were considered to produce phaseolotoxin when inhibition of E. coli growth was inverted on plates containing L-citrulline or L-arginine, but not on plates with L-ornithine, and to produce mangotoxin when inhi- bition was inverted with L-ornithine but not with N-acetyl orni- thine (Fig. 1); in all cases, plates were supplemented with 100 ml of a sterile 1% (wt/vol) solution of the pertinent amino ...
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... (Table 1) ( Rico et al., 2003). Also as expected, we did not detect production of phaseolotoxin by any of the remaining strains examined. However, strains ISPaVe011 and ISPaVe2056 of P. syringae pv. avellanae produced large inhibition haloes that were relieved by L-ornithine, but not by N-acetyl ornithine, indicating production of mangotoxin (Fig. 1); nevertheless, these strains might be producing another toxin, different from phaseolotoxin, since the inhibition haloes were not completely relieved with either citrulline or ...
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... strains, except for the bands that contain the borders of the clusters, that were different in P. syringae pv. actinidiae ( Supplementary Fig. 1). ...
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... strains was compatible with the absence of the complete 38 kb DNA fragment containing the Pht island in these strains. Other non-toxigenic strains of P. syringae also showed a few faint hybridization bands, probably due to cross- hybridization with transposable elements and/or to areas of homology outside of the Pht-PAI that are present in the cos- mids (Supplementary Fig. 1). ...
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... growth inhibition assays, both UPN1703 and CFBP3388 inhibited growth of E. coli. As expected, haloes produced by CFBP3388 were suppressed by the addition of L-citrulline to the medium, but not by the addition of L-ornithine, indicating production of phaseolotoxin ( Figs. 1 and 5). Haloes produced by UPN1703, however, were inverted in plates with L-ornithine, but not in media supplemented with N-acetyl-ornithine (data not shown). ...
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... synteny is conserved; this is in sharp contrast with an identity higher than 99.8% among clusters harbored by strains of pvs. phaseolicola and actinidiae (Genka et al., 2006; Navarro de la Fuente et al., 2008), especially taking into account that strain NPS3121 is phylogenetically closer to strain CFBP3388 than to strains of pv. actinidiae (Fig. 1) ( Gardan et al., 1999;Sarkar and Guttman, 2004). Remarkably, phaseolotoxin production in strain CFBP3388 occurs at 28 C, whereas this is a non-permissive temperature for other phaseo- lotoxin producers (Fig. 5). The current thermoregulation model for P. syringae pv. phaseolicola proposes that an as yet uniden- tified repressor molecule ...
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... at the permissive temperature, Fig. 5. Evaluation of anti-metabolite toxins production by the E. coli growth inhibition assay. Spent supernatants of cultures grown in Ayers minimal medium at temperatures of 18 or 28 C were absorbed in filter paper disks and overlaid on an E. coli lawn. Production of a given anti-metabolite toxin (see Fig. 1) is evidenced by the occurrence of a growth inhibition halo and its reversion in media supplemented with individual amino ...
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Background
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Results
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Citations
... actinidiae and pv. phaseolicola demonstrated that this region is highly conserved among these pathovars [26], even though these two pathogens are phylogenetically separated and could even belong to different species [15,[26][27][28][29][30]. The Pht cluster (argK-tox cluster or tox-island) of pv. ...
... actinidiae and pv. phaseolicola demonstrated that this region is highly conserved among these pathovars [26], even though these two pathogens are phylogenetically separated and could even belong to different species [15,[26][27][28][29][30]. The Pht cluster (argK-tox cluster or tox-island) of pv. ...
... actinidiae and pv. phaseolicola were confirmed to integrate site-specifically into their respective chromosomes at the homologues sites in the same direction [26,31]. Conversely, the analysis of the sequence of the Pht cluster in the P. syringae pv. ...
Phaseolotoxin is an antimetabolite toxin produced by diverse pathovars of Pseudomonas syringae which affects various plants, causing diseases of economic importance. Phaseolotoxin contributes to the systemic dissemination of the pathogen in the plant, therefore it is recognized as a major virulence factor. Genetic traits such as the Pht cluster, appear defining to the toxigenic strains phaseolotoxin producers. Extensive research has contributed to our knowledge concerning the regulation of phaseolotoxin revealing a complex regulatory network that involves processes at the transcriptional and posttranscriptional levels, in which specific and global regulators participate. Even more, significant advances in understanding how specific signals, including host metabolites, nutrient sources, and physical parameters such as the temperature, can affect phaseolotoxin production have been made. A general overview of the phaseolotoxin regulation, focusing on the chemical and physical cues, and regulatory pathways involved in the expression of this major virulence factor will be given in the present work.
... actinidiae biovars 1 (Psa1) and 6 (Psa6), as well as by P. syringae pv. syringae strain CFBP3388 (Mitchell, 1978;Tourte and Manceau, 1995;Tamura et al., 2002;Murillo et al., 2011). ...
... Evidence suggests that the Pht cluster originated from distantly related bacteria and was horizontally transferred into P. syringae after pathovar differentiation (Sawada et al., 2002); nevertheless, our study did not focus on the early events of Pht cluster assembly and distribution. Based on previous phylogenetic and structural studies (Sawada et al., 2002;Genka et al., 2006;Murillo et al., 2011;Bardaji et al., 2017;Fujikawa and Sawada, 2019), these events are hypothesized in Fig. 7 (steps 1-6). The phylogeny of the Pht clusters (Fig. 3) suggested that clades TOX2, TOX3, and TOX4 ...
... Psa (clade TOX1), despite their phylogenetic distance and assignment to different species (Genka et al., 2006;Murillo et al., 2011;Gomila et al., 2017;Sawada and Fujikawa, 2019). To date, it remains unclear whether these clusters were acquired from the same source or transferred between pathovars. ...
Genomic islands are widely distributed among bacteria, facilitating the dissemination of genes relevant to human, animal, and plant health. Herein, we explore the evolutionary history of a genomic island (Tox island) composed of a novel mobile genetic element (GInt) carrying an ∼25 kb gene cluster (Pht cluster) responsible for the biosynthesis of the phytotoxin phaseolotoxin, a virulence factor of the plant pathogen Pseudomonas syringae . The Pht cluster has been acquired, either naked or associated with a GInt, on seven independent occasions by four phylogroups of P. syringae and the distant rhizobacterium Pseudomonas sp. JAI115. The Pht cluster was independently captured by three distinct GInt elements, suggesting specific mechanisms for gene capture. Once acquired, the Tox island tends to be stably maintained, evolving with the genome. An array of molecular analyses delineated the likely evolutionary trajectory of the Pht cluster within P. syringae pv. actinidiae (Psa) and P. amygdali pv. phaseolicola (Pph), involving: 1) acquisition by Pph; 2) transfer of haplotype G to Psa biovar 1; 3) acquisition or replacement by a haplotype of haplogroup D in Psa biovar 1; 4) acquisition of haplotype C by Psa biovar 6; and 5) replacement of the Tox island in Pph by a distantly related GInt. These findings underscore the potential role of phaseolotoxin in bacterial fitness and contribute to our understanding of virulence evolution in plant pathogens. Furthermore, GInts provide a model for studying the evolutionary dynamics of mobile genetic elements and the dissemination of adaptive genes among bacterial pathogens.
... Here, we determined that gene PSPPH_4550 is included within a genomic island that is present in only a few other P. syringae strains, suggesting a possible horizontal transfer origin similar to that proposed for the Pht cluster [10,[25][26][27][28]. The chromosomal fragment containing the gene PSPPH_4550 from P. syringae pv. ...
The bean (Phaseolus vulgaris) pathogen Pseudomonas syringae pv. phaseolicola NPS3121 synthesizes phaseolotoxin in a thermoregulated way, with optimum production at 18 °C. Gene PSPPH_4550 was previously shown to be thermoregulated and required for phaseolotoxin biosynthesis. Here, we established that PSPPH_4550 is part of a cluster of 16 genes, the Pbo cluster, included in a genomic island with a limited distribution in P. syringae and unrelated to the possession of the phaseolotoxin biosynthesis cluster. We identified typical non-ribosomal peptide synthetase, and polyketide synthetase domains in several of the pbo deduced products. RT-PCR and the analysis of polar mutants showed that the Pbo cluster is organized in four transcriptional units, including one monocistronic and three polycistronic. Operons pboA and pboO are both essential for phaseolotoxin biosynthesis, while pboK and pboJ only influence the amount of toxin produced. The three polycistronic units were transcribed at high levels at 18 °C but not at 28 °C, whereas gene pboJ was constitutively expressed. Together, our data suggest that the Pbo cluster synthesizes secondary metabolite(s), which could participate in the regulation of phaseolotoxin biosynthesis.
... Some of the partial sequences from the Pss strains belonging to the P1 population and control strains were available at the NCBI database, and the rest of the partial sequences from the Pss were obtained in this study. The partial sequences of the housekeeping genes rpoD and gyrB were obtained by PCR using specific primers listed in Table 3 (54). PCR products were purified and sent for sequencing to STABvida (Portugal). ...
Copper resistance mechanisms provide an important adaptive advantage to plant-pathogenic bacteria under exposure to copper treatments. Copper resistance determinants have been described in Pseudomonas syringae pv. syringae (Pss) strains isolated from mango intimately associated with 62-kb plasmids belonging to the pPT23A family (PFP). It has been previously described that the indiscriminate use of copper-based compounds promotes the selection of copper-resistant bacterial strains and constitutes a selective pressure in the evolution of copper resistance determinants. Hence, we have explored in this study the copper resistance evolution and the distribution of specific genetic determinants in two different Pss mango populations isolated from the same geographical regions, mainly from southern Spain, with an average of 20 years of difference. The total content of plasmids, in particular the 62-kb plasmids, and the number of copper-resistant Pss strains were maintained at similar levels over time. Interestingly, the phylogenetic analysis indicated the presence of a phylogenetic subgroup (PSG) in the Pss mango phylotype mostly composed of the recent Pss population analyzed in this study that was strongly associated with a hyperresistant phenotype to copper. Genome sequencing of two selected Pss strains from this PSG revealed the presence of a large Tn7-like transposon of chromosomal location, which harbored putative copper and arsenic resistance genes (COARS Tn7-like). Transformation of the copper-sensitive Pss UMAF0158 strain with some putative copper resistance genes and reverse transcription-quantitative PCR experiments brought to light the role of COARS Tn7-like transposon in the hyperresistant phenotype to copper in Pss.
... Phaseolotoxin is a main virulence factor for the characteristic chlorotic symptoms of halo blight disease caused by Psh 1448A, and the phaseolotoxin biosynthetic gene cluster (Pht cluster) contains 23 genes organized into five transcriptional units, two monocistronic units (argK and phtL) and three operons [83,84]. Previous studies indicated that Pst DC3000 and Pss B728a have incomplete phaseolotoxin biosynthetic gene clusters and cannot produce the toxin [82,85]. Moreover, we could not detect the presence of the complete Pht cluster in NM002, and therefore NM002 was speculated to be incapable of producing phaseolotoxin (Table S12). ...
Pseudomonas amygdali pv. lachrymans is currently of important plant pathogenic bacteria that causes cucumber angular leaf spot worldwide. The pathogen has been studied for its roles in pathogenicity and plant inheritance resistance. To further delineate traits critical to virulence, invasion and survival in the phyllosphere, we reported the first complete genome of P. amygdali pv. lachrymans NM002. Analysis of the whole genome in comparison with three closely-related representative pathovars of P. syringae identified the conservation of virulence genes, including flagella and chemotaxis, quorum-sensing systems, two-component systems, and lipopolysaccharide and antiphagocytosis. It also revealed differences of invasion determinants, such as type III effectors, phytotoxin (coronatine, syringomycin and phaseolotoxin) and cell wall-degrading enzyme, which may contribute to infectivity. The aim of this study was to derive genomic information that would reveal the probable molecular mechanisms underlying the virulence, infectivity and provide a better understanding of the pathogenesis of the P. syringae pathovars.
... syringae with 74-79% certainty. To further confirm the identity of the bacterial strains, the 16S rDNA, gyrB and rpoD genes were amplified using primers 27F/1492R, gyrBFor2/gyrBRev2, and rpoDFor2/rpoDRev2 (Murillo et al. 2011 Control treatments were inoculated only with sterile water. They were then incubated at 25 °C under 80 to 90% relative humidity. ...
... In fact, the accessory genome of the P. syringae complex is characterised by genomic islands and various mobile elements, such as insertion sequences (IS elements), transposons, plasmids and integrative conjugative elements (ICEs) (Butler et al., 2013). These mobile elements include genes related to the ecological fitness and virulence, such as toxin production (Murillo et al., 2011), copper and antibiotic resistance (Butler et al., 2013;Colombi et al., 2017), siderophore production and phenolics degradation (Scortichini et al., 2012). In this sense, the possibility to characterise the "phyllobiome" through the application of high throughput, next generation sequencing technologies allows to strategically select BCAs highly specialised for the host's most sensible pathways of infection. ...
Pseudomonas syringae pv. actinidiae (Psa) is the causal agent of the bacterial canker, the most devastating disease of kiwifruit vines. Before entering the host tissues, this pathogen has an epiphytic growth phase on kiwifruit flowers and leaves, thus the ecological interactions within epiphytic bacterial community may greatly influence the onset of the infection process. The bacterial community associated to the two most important cultivated kiwifruit species, Actinidia chinensis and Actinidia deliciosa, was described both on flowers and leaves using Illumina massive parallel sequencing of the V3 and V4 variable regions of the 16S rRNA gene. In addition, the effect of plant infection by Psa on the epiphytic bacterial community structure and biodiversity was investigated. Psa infection affected the phyllosphere microbiome structures in both species, however, its impact was more pronounced on A. deliciosa leaves, where a drastic drop in microbial biodiversity was observed. Furthermore, we also showed that Psa was always present in syndemic association with Pseudomonas syringae pv. syringae and Pseudomonas viridiflava, two other kiwifruit pathogens, suggesting the establishment of a pathogenic consortium leading to a higher pathogenesis capacity. Finally, the analyses of the dynamics of bacterial populations provided useful information for the screening and selection of potential biocontrol agents against Psa.
... Another virulence factor, phaseolotoxin, was annotated, with just one gene of adenylylsulfate kinase detected in B. thermoamylovorans. Phaseolotoxin is an anti-metabolite phytotoxin that targets widely conserved biosynthetic pathways and is active against a multiplicity of organisms, including plants and bacteria (Murillo et al., 2011). Fortunately, phaseolotoxin is produced at relatively low temperatures (Aguilera et al., 2017); optimal production occurs at approximately 18°C, while production is inhibited above 28°C (Chen et al., 2015); therefore, since B. thermoamylovorans does not grow below 40°C, the risk is low that phaseolotoxin will be biosynthesized by B. thermoamylovorans. ...
... phaseolicola wild type strains NPS3121 (Tox+) and CYL233 (Tox-) containing diverse constructions with these genes. Strain CYL233 is naturally unable to synthesize phaseolotoxin because it lacks the entire Pht cluster for phaseolotoxin biosynthesis [29,32,35,36], and was used to discard the participation in this regulation of other genes from the Pht cluster. Plasmids pSAK-A, pSAK-B, pSAK-C, pSA-K-AB, pSAK-BC, pSAK-AC, and pSAK-ABC containing genes argK-phtA, argK-phtB, argK-phtC, argK-phtAB, argK-phtBC, argK-phtAC and argK-phtABC cloned into pUCP20, respectively, were used in this study ( Table 1). ...
Pseudomonas syringae pv. phaseolicola produces phaseolotoxin in a temperature dependent manner, being optimally synthesized between 18°C and 20°C, while no detectable amounts are present above 28°C. The Pht cluster, involved in the biosynthesis of phaseolotoxin, contains 23 genes that are organized in five transcriptional units. The function of most of the genes from the Pht cluster is still unknown and little information about the regulatory circuitry leading to expression of these genes has been reported. The purpose of the present study was to investigate the participation of pht genes in the regulation of the operons coded into the Pht cluster. We conducted Northern blot, uidA fusions and reverse transcription-PCR assays of pht genes in several mutants unable to produce phaseolotoxin. This allowed us to determine that, in P. syringae pv. phaseolicola NPS3121, genes phtABC are essential to prevent their own expression at 28°C, a temperature at which no detectable amounts of the toxin are present. We obtained evidence that the phtABC genes also participate in the regulation of the phtD, phtM and phtL operons. According to our results, we propose that PhtABC and other Pht product activities could be involved in the synthesis of the sulfodiaminophosphinyl moiety of phaseolotoxin, which indirectly could be involved in the transcriptional regulation of the phtA operon.
... The Pht-PAI appears to be mobile, because a nearly identical genomic island has been found syntenically inserted into the genome of certain strains of the kiwiplant pathogen Ps pv. actinidiae 13,17 . Additionally, homologues of the three putative TRase genes were found in Ps pv. ...
... phaseolicola and Ps pv. actinidiae contain nearly identical Pht-PAI islands, although they are assigned to different species 13,17,24 , suggesting that GInts are mobilized among bacterial populations. However, despite numerous attempts, we were unable to demonstrate the transfer of GInts Table S2), the cartoon shows the integration and excision of the artificial GInt pGInt0 (orange) into the genome of P. putida KT2440 (grey), with boxed sequences observed by sequencing of reaction products. ...
Integrases are a family of tyrosine recombinases that are highly abundant in bacterial genomes, actively disseminating adaptive characters such as pathogenicity determinants and antibiotics resistance. Using comparative genomics and functional assays, we identified a novel type of mobile genetic element, the GInt, in many diverse bacterial groups but not in archaea. Integrated as genomic islands, GInts show a tripartite structure consisting of the ginABCD operon, a cargo DNA region from 2.5 to at least 70 kb, and a short AT-rich 3′ end. The gin operon is characteristic of GInts and codes for three putative integrases and a small putative helix-loop-helix protein, all of which are essential for integration and excision of the element. Genes in the cargo DNA are acquired mostly from phylogenetically related bacteria and often code for traits that might increase fitness, such as resistance to antimicrobials or virulence. GInts also tend to capture clusters of genes involved in complex processes, such as the biosynthesis of phaseolotoxin by Pseudomonas syringae. GInts integrate site-specifically, generating two flanking direct imperfect repeats, and excise forming circular molecules. The excision process generates sequence variants at the element attachment site, which can increase frequency of integration and drive target specificity.
