HemN2 Regulates the Virulence of Pseudomonas donghuensis HYS through 7-Hydroxytropolone Synthesis and Oxidative Stress.

Compared to pathogens Pseudomonas aeruginosa and P. putida, P. donghuensis HYS has stronger virulence towards Caenorhabditis elegans. However, the underlying mechanisms haven't been fully understood. The heme synthesis system is essential for Pseudomonas virulence, and former studies of HemN have focused on the synthesis of heme, while the relationship between HemN and Pseudomonas virulence were barely pursued. In this study, we hypothesized that hemN2 deficiency affected 7-hydroxytropolone (7-HT) biosynthesis and redox levels, thereby reducing bacterial virulence. There are four hemN genes in P. donghuensis HYS, and we reported for the first time that deletion of hemN2 significantly reduced the virulence of HYS towards C. elegans, whereas the reduction in virulence by the other three genes was not significant. Interestingly, hemN2 deletion significantly reduced colonization of P. donghuensis HYS in the gut of C. elegans. Further studies showed that HemN2 was regulated by GacS and participated in the virulence of P. donghuensis HYS towards C. elegans by mediating the synthesis of the virulence factor 7-HT. In addition, HemN2 and GacS regulated the virulence of P. donghuensis HYS by affecting antioxidant capacity and nitrative stress. In short, the findings that HemN2 was regulated by the Gac system and that it was involved in bacterial virulence via regulating 7-HT synthesis and redox levels were reported for the first time. These insights may enlighten further understanding of HemN-based virulence in the genus Pseudomonas.

Pseudomonas PA14H7: Identification and Quantification of the 7-Hydroxytropolone Iron Complex as an Active Metabolite against Dickeya, the Causal Agent of Blackleg on the Potato Plant.

Soft rot Pectobacteriaceae (SRP), such as Pectobacterium and Dickeya, are phytopathogenic agents responsible for blackleg disease on several crops, such as potatoes, affecting the yield and depressing the seed production quality. However, neither conventional nor biocontrol products are available on the market to control this disease. In this study Pseudomonas PA14H7, a bacteria isolated from potato rhizosphere, was selected as a potential antagonist agent against Dickeya solani. In order to understand the mechanism involved in this antagonism, we managed to identify the main active molecule(s) produced by PA14H7. Cell-free supernatant (CFS) of PA14H7 cultures were extracted and analyzed using LC-MS, GC-MS, and NMR. We further correlated the biological activity against Dickeya solani of extracted CFS-PA14H7 to the presence of 7-hydroxytropolone (7-HT) complexed with iron. In a second time, we have synthesized this molecule and determined accurately using LC-UV, LC-MS, and GC-MS that, after 48 h incubation, PA14H7 released, in its CFS, around 9 mg/L of 7-HT. The biological activities of CFS-PA14H7 vs. synthetic 7-HT, at this concentration, were evaluated to have a similar bacteriostatic effect on the growth of Dickeya solani. Even if 7-HT is produced by other Pseudomonas species and is mostly known for its antibacterial and antifungal activities, this is the first description of its involvement as an effective molecule against pectinolytic bacteria. Our work opens the way for the comprehension of the mode of action of PA14H7 as a biocontrol agent against potato blackleg.

The Gene paaZ of the Phenylacetic Acid (PAA) Catabolic Pathway Branching Point and ech outside the PAA Catabolon Gene Cluster Are Synergistically Involved in the Biosynthesis of the Iron Scavenger 7-Hydroxytropolone in Pseudomonas donghuensis HYS.

The newly discovered iron scavenger 7-hydroxytropolone (7-HT) is secreted by Pseudomonas donghuensis HYS. In addition to possessing an iron-chelating ability, 7-HT has various other biological activities. However, 7-HT's biosynthetic pathway remains unclear. This study was the first to report that the phenylacetic acid (PAA) catabolon genes in cluster 2 are involved in the biosynthesis of 7-HT and that two genes, paaZ (orf13) and ech, are synergistically involved in the biosynthesis of 7-HT in P. donghuensis HYS. Firstly, gene knockout and a sole carbon experiment indicated that the genes orf17-21 (paaEDCBA) and orf26 (paaG) were involved in the biosynthesis of 7-HT and participated in the PAA catabolon pathway in P. donghuensis HYS; these genes were arranged in gene cluster 2 in P. donghuensis HYS. Interestingly, ORF13 was a homologous protein of PaaZ, but orf13 (paaZ) was not essential for the biosynthesis of 7-HT in P. donghuensis HYS. A genome-wide BLASTP search, including gene knockout, complemented assays, and site mutation, showed that the gene ech homologous to the ECH domain of orf13 (paaZ) is essential for the biosynthesis of 7-HT. Three key conserved residues of ech (Asp39, His44, and Gly62) were identified in P. donghuensis HYS. Furthermore, orf13 (paaZ) could not complement the role of ech in the production of 7-HT, and the single carbon experiment indicated that paaZ mainly participates in PAA catabolism. Overall, this study reveals a natural association between PAA catabolon and the biosynthesis of 7-HT in P. donghuensis HYS. These two genes have a synergistic effect and different functions: paaZ is mainly involved in the degradation of PAA, while ech is mainly related to the biosynthesis of 7-HT in P. donghuensis HYS. These findings complement our understanding of the mechanism of the biosynthesis of 7-HT in the genus Pseudomonas.

Insight into the Global Negative Regulation of Iron Scavenger 7-HT Biosynthesis by the SigW/RsiW System in Pseudomonas donghuensis HYS

7-Hydroxytropolone (7-HT) is a unique iron scavenger synthesized by Pseudomonas donghuensis HYS that has various biological activities in addition to functioning as a siderophore. P. donghuensis HYS is more pathogenic than P. aeruginosa toward Caenorhabditis elegans, an observation that is closely linked to the biosynthesis of 7-HT. The nonfluorescent siderophore (nfs) gene cluster is responsible for the orderly biosynthesis of 7-HT and represents a competitive advantage that contributes to the increased survival of P. donghuensis HYS; however, the regulatory mechanisms of 7-HT biosynthesis remain unclear. This study is the first to propose that the ECF σ factor has a regulatory effect on 7-HT biosynthesis. In total, 20 ECF σ factors were identified through genome-wide scanning, and their responses to extracellular ferrous ions were characterized. We found that SigW was both significantly upregulated under high-iron conditions and repressed by an adjacent anti-σ factor. RNA-Seq results suggest that the SigW/RsiW system is involved in iron metabolism and 7-HT biosynthesis. Combined with the siderophore phenotype, we also found that SigW could inhibit siderophore synthesis, and this inhibition can be relieved by RsiW. EMSA assays proved that SigW, when highly expressed, can directly bind to the promoter region of five operons of the nfs cluster to inhibit the transcription of the corresponding genes and consequently suppress 7-HT biosynthesis. In addition, SigW not only directly negatively regulates structural genes related to 7-HT synthesis but also inhibits the transcription of regulatory proteins, including of the Gac/Rsm cascade system. Taken together, our results highlight that the biosynthesis of 7-HT is negatively regulated by SigW and that the SigW/RsiW system is involved in mechanisms for the regulation of iron homeostasis in P. donghuensis HYS. As a result of this work, we identified a novel mechanism for the global negative regulation of 7-HT biosynthesis, complementing our understanding of the function of ECF σ factors in Pseudomonas.

High-Quality Complete Genome Resource of Tomato Rhizosphere Strain Pseudomonas donghuensis P482, a Representative of a Species with Biocontrol Activity Against Plant Pathogens.

Strain P482 was isolated from a tomato rhizosphere and classified as Pseudomonas donghuensis. The P. donghuensis species was first established in 2015 and currently consists of only four strains: P482, HYST, SVBP6, and 22G5. P. donghuensis strains antagonize plant pathogens, including bacteria, fungi, and oomycetes, and, therefore, are of high interest regarding their biological control potential to combat plant diseases. The antimicrobial activity of P. donghuensis P482 is based on the production of iron-scavenging compound 7-hydroxytropolone, antifungal volatile organic compounds, and as-yet-unidentified secondary metabolites. Here, we report a complete genome resource for P. donghuensis strain P482. The genome consists of a single chromosome (5,656,185 bp) with 5,258 open reading frames (5,158 protein-coding genes, 74 transfer RNAs, 22 ribosomal RNAs, 3 noncoding RNAs, and 1 transfer-messenger RNA) and no plasmid. We believe that information on the first high-quality, complete genome of P. donghuensis will provide resources for analyses targeting the biological control potential of this species and understanding the traits essential for plant-microbe interaction.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Pseudomonas species isolated via high-throughput screening significantly protect cotton plants against verticillium wilt.

Verticillium wilt (VW) caused by Verticillium dahliae is a devastating soil-borne disease that causes severe yield losses in cotton and other major crops worldwide. Here we conducted a high-throughput screening of isolates recovered from 886 plant rhizosphere samples taken from the three main cotton-producing areas of China. Fifteen isolates distributed in different genera of bacteria that showed inhibitory activity against V. dahliae were screened out. Of these, two Pseudomonas strains, P. protegens XY2F4 and P. donghuensis 22G5, showed significant inhibitory action against V. dahliae. Additional comparative genomic analyses and phenotypical assays confirmed that P. protegens XY2F4 and P. donghuensis 22G5 were the strains most efficient at protecting cotton plants against VW due to specific biological control products they produced. Importantly, we identified a significant efficacy of the natural tropolone compound 7-hydroxytropolone (7-HT) against VW. By phenotypical assay using the wild-type 22G5 and its mutant strain in 7-HT production, we revealed that the 7-HT produced by P. donghuensis is the major substance protecting cotton against VW. This study reveals that Pseudomonas specifically has gene clusters that allow the production of effective antipathogenic metabolites that can now be used as new agents in the biocontrol of VW.

Pseudomonas donghuensis HYS 7-hydroxytropolone contributes to pathogenicity toward Caenorhabditis elegans and is influenced by pantothenic acid.

Pseudomonas donghuensis HYS, a bacterial strain identified from Donghu Lake, has tremendous toxicity toward Caenorhabditis elegans and is characterized by high 7-hydroxytropolone siderophore production. Here, the relationship between pathogenic siderophore production and pantothenic acid was evaluated. The pathogenicity of P. donghuensis HYS was illustrated using C. elegans as a host. Based on slow-killing assay findings, a 7-hydroxytropolone deficiency-causing mutation attenuated P. donghuensis HYS pathogenicity, which was restored by the addition of extracted 7-hydroxytropolone. Moreover, data from real-time qPCR analysis and characteristic absorption assays indicated that pantothenic acid deficiency repressed transcriptional levels of orf9, which further reduced 7-hydroxytropolone production. Furthermore, slow-killing assays indicated that panB and pantothenic acid affected the virulence of P. donghuensis. These results indicate that a 7-hydroxytropolone siderophore-producing strain is virulent toward C. elegans. Our findings demonstrate that pantothenic acid is associated with P. donghuensis siderophore production-related pathogenicity.

A Complex Mechanism Involving LysR and TetR/AcrR That Regulates Iron Scavenger Biosynthesis in Pseudomonas donghuensis HYS.

7-Hydroxytropolone (7-HT) is a symmetrical seven-membered heteroatomic ring with a carboxyl group and two hydroxyl groups and was recently reported to be an iron scavenger of Pseudomonas donghuensis HYS. Cluster 1 includes 12 genes related to the synthesis of 7-HT; among these genes, those for two regulators, Orf1 and Orf12, were predicted to regulate 7-HT biosynthesis and to be LysR-type transcriptional regulators (LTTRs) and TetR/AcrR family transcriptional regulators, respectively. Data from real-time quantitative PCR and ß-galactosidase and classical siderophore assays indicated that the transcription levels of orf1 and orf12, as well as those of crucial genes orf6 to orf9, were repressed under high-iron conditions. The deletion of orf1 and orf12 led to an absence of 7-HT and a decrease in orf6-orf9 expression. Orf1 and Orf12 were essential for the production of 7-HT through orf6-orf9 These two regulators are regulated by the Gac/Rsm system; Orf1 facilitates the expression of Orf12, and Orf12 concomitantly stimulates the expression of orf6-orf9 to synthesize 7-HT. The overexpression of Orf12 decreased 7-HT yields, possibly through decreased orf6-orf9 expression. This work thus outlines a complex mechanism regulating the biosynthesis of the iron scavenger 7-HT in P. donghuensis HYS. The synergy between Orf1 and Orf12 ensures that 7-HT acts as an iron chelator despite being toxic to bacteria and provides new ideas for the novel regulation of dual-functional secondary metabolism and research on 7-HT and its derivates in other bacteria.IMPORTANCE A complex regulation mechanism including two regulators, LysR and TetR/AcrR, in the biosynthesis of the novel iron scavenger 7-hydroxytropolone (7-HT) was verified in Pseudomonas donghuensis HYS. The coaction of LysR Orf1 and TetR/AcrR Orf12 may balance the toxicity and iron chelation of 7-HT in P. donghuensis HYS to overcome iron deficiency, as well as improve the bacterial competitiveness under iron-scarce conditions because of the toxicity of 7-HT toward other bacteria, making the accurate regulation of 7-HT biosynthesis indispensable. This regulation mechanism may be ubiquitous in the Pseudomonas putida group but may better explain the group's strong adaptability.

7-Hydroxytropolone produced and utilized as an iron-scavenger by Pseudomonas donghuensis.

Pseudomonas donghuensis can excrete large quantities of iron chelating substances in iron-restricted environments. At least two kinds of iron-chelator can be found in the culture supernatant: fluorescent siderophores pyoverdins, and an ethyl acetate-extractable non-fluorescent substance. The non-fluorescent substance was the dominant contributor to the iron chelating activity of the culture supernatant of P. donghuensis. Electron ionization mass spectrometry, NMR spectroscopy, and IR spectroscopy identified the non-fluorescent iron-chelator as 7-hydroxytropolone. The stoichiometry of 7-hydroxytropolone ferric complex was determined to be 2:1 by the continuous variation method. The production of 7-hydroxytropolone was repressible by iron in the medium. Moreover, the inhibited growth of doubly siderophore-deficient strain of P. donghuensis under iron-limiting conditions could be partly restored by 7-hydroxytropolone. Thus, 7-hydroxytropolone was considered to play a previously undiscovered role as an iron-scavenger for P. donghuensis.