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The secondary metabolite hydrogen cyanide (HCN) is produced by Pseudomonas fluorescens from glycine, essentially under microaerophilic conditions. The genetic basis of HCN synthesis in P. fluorescens CHA0 was investigated. The contiguous structural genes hcnABC encoding HCN synthase were expressed from the T7 promoter in Escherichia coli, resulting...
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... strains, plasmids, and growth conditions. The bacterial strains and plasmids are listed in Table 1. Strains of Escherichia coli and P. aeruginosa were routinely grown on nutrient agar (NA) plates and in nutrient yeast broth (NYB) with aeration at 37°C (18). ...
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... expression. The hcn genes of P. fluorescens were cloned under the control of the T7 promoter in pEB16, producing pME3209, pME3210, and pME3212 (Table 1 and Fig. 1). NYB cultures of P. aeruginosa ADD1976 (2) harboring any of these constructs were grown at 37°C until they reached an optical density at 600 nm of 0.8. ...
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... deduced amino acid sequence of ANR of P. fluorescens (ANR Pfl ) shows overall identities of 88% (95% similarity) with the sequence of P. aeruginosa ANR (ANR Pae ) and of 53% (76% similarity) with the sequence of E. coli FNR (Fig. 5). In FNR, three N-terminal cysteine residues Table 1. , positive for complementation of the E. coli fnr mutant JRG1728; , negative for complementation; anr Pae , anr gene of P. aeruginosa. ...
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... con- firmed that the ANR-dependent expression of the hcn genes was regulated by oxygen limitation in P. fluorescens. An hcnA- lacZ translational fusion in plasmid pME3219 (Table 1) was introduced into strains CHA0 and CHA21. On X-Gal plates, strain CHA0/pME3219 formed blue colonies, whereas strain CHA21/pME3219 remained white (corresponding to 5 Miller units of -galactosidase), indicating that ANR is needed to drive the expression of the hcn genes. ...
Citations
... In gramnegative bacteria, it is synthesized as a secondary metabolite formed from glycine that is mediated by the enzyme HCN synthase. Thielaviopsis basicola (Laville et al., 1998), which causes tobacco black root rot, was controlled by P. fuorescens strain CHAO (Voisard et al., 1989). The presence of glycine in the culture media of several rhizobacteria still seems to make them cyanogenic. ...
... Seeds may be tolerant to anoxia by reverting the central glycolytic pathways. Ethanol may either be oxidized to acetyl dehydrogenase or diffused out of the seed coat, accumulating within spaces of grains and glumes [37]. This also induces dormancy in terms of ecological consideration for submergence tolerance. ...
Submergence in rice fields creating inundation stress and realizing anoxia or hypoxia is a problem in agriculture. Seeds under this oxygen deficit are faced with fermentative respiration, where the end product would be poisoning the tissue viability. This is more aggravated in direct seeded rice cultivation with the accumulation of lactate as a poison. This review is concerned with the basic insights into anoxia tolerance in seeds and possible strategies to reduce anoxic shock through the modification of metabolism preceded by gene expression. The major concern of anoxic germination is starch metabolism and downstream physiological realization to facilitate escape or quiescence strategy, overcoming submergence stress. The coleoptiles facing hypoxic stress mated with transcripts for oxidative traits, energy metabolism, and proteins for membrane peroxidation in support of energy metabolism are the most important. Hypoxic genes are recovered from traditional indica and japonica land races of rice, and show changes in glycolytic flux and sugar sensing. Anoxic germination and seedling vigor are based on a combinational regulation of oxidative stress and fermentative catabolism. De novo antioxidant and antioxidative enzyme production can support improved seed germination in this condition. Pre-harvest spouting with seed-coat-induced dormancy, hormonal ratios, and hydrolyses would be of concern. Therefore, comprehensive analysis aimed to understand rice seed priming for better gas exchange, diffusion, temperature sensitivity, ion uptake, redox balance, and others. Still, in-depth insights are being awaited for better understanding the physiological and molecular basis using a multi-omics approach for better seed priming to overcome the anoxic/hypoxic revelation mostly acquainted with submergence stress.
... The gene hcnaA codes a hydrogen cyanide synthase that is responsible for the volatile HCN production by various bacteria [46][47][48]. HCN is an antimicrobial compound that is also released by P. protegens CHA0. The highest expression of hcnaA, in absence of salt stress, was observed in the ANE treatment (14.4-fold), followed by the treatments with fucoidan and alginate. ...
... Beneficial Pseudomonas spp. have been reported to suppress different plant diseases by producing antimicrobial metabolites, such as hydrogen cyanide [48,69]. The production of HCN by P. protegens CHA0 was not influenced by ANE, though mannitol, alginate, and fucoidan were found to increase around 3-fold this activity. ...
... The hcnaA encodes subunit A of hydrogen cyanide synthase, responsible for the volatile HCN production by bacteria. The functional protein is a heterotrimer, formed by subunits HcnA, HcnB, and HcnC [46][47][48]. It is difficult to rationalize the effects triggered by ANE, which had no effect on HCN production, but enhanced the expression of hcnaA. ...
Abiotic stresses, including salinity stress, affect numerous crops, causing yield reduction, and, as a result, important economic losses. Extracts from the brown alga Ascophyllum nodosum (ANE), and compounds secreted by the Pseudomonas protegens strain, CHA0, can mitigate these effects by inducing tolerance against salt stress. However, the influence of ANE on P. protegens CHA0 secretion, and the combined effects of these two biostimulants on plant growth, are not known. Fucoidan, alginate, and mannitol are abundant components of brown algae and of ANE. Reported here are the effects of a commercial formulation of ANE, fucoidan, alginate, and mannitol, on pea (Pisum sativum), and on the plant growth-promoting activity of P. protegens CHA0. In most situations, ANE and fucoidan increased indole-3-acetic acid (IAA) and siderophore production, phosphate solubilization, and hydrogen cyanide (HCN) production by P. protegens CHA0. Colonization of pea roots by P. protegens CHA0 was found to be increased mostly by ANE and fucoidan in normal conditions and under salt stress. Applications of P. protegens CHA0 combined with ANE, or with fucoidan, alginate, and mannitol, generally augmented root and shoot growth in normal and salinity stress conditions. Real-time quantitative PCR analyses of P. protegens revealed that, in many instances, ANE and fucoidan enhanced the expression of several genes involved in chemotaxis (cheW and WspR), pyoverdine production (pvdS), and HCN production (hcnA), but gene expression patterns overlapped only occasionally those of growth-promoting parameters. Overall, the increased colonization and the enhanced activities of P. protegens CHA0 in the presence of ANE and its components mitigated salinity stress in pea. Among treatments, ANE and fucoidan were found responsible for most of the increased activities of P. protegens CHA0 and the improved plant growth.
... The metabolite HCN is an antifungal with volatile properties and is synthesized by the action of HCN synthase from glycine. HcnA, hcnB and hcnC are genes involved in the biosynthesis of this enzyme [127]. Selected antagonistic bacteria were assessed in vitro for characteristics known to be important in promoting plant growth, siderophores production, IAA production and solubilization of phosphate [128]. ...
The soil-borne pathogen Rhizoctonia solani AG-2-2 induces both root and crown rot (RCR) and damping-off in sugar beet, which considerably reduces the productivity of this industrial crop. Most often, synthetic fungicides are commonly used to control this fungus. Biological control agents have attracted interest as an alternative to chemical fungicides for controlling several plant diseases. The current study aimed at finding antagonistic bacteria that could be used for biological control against this pathogen. A set of 198 bacterial strains were screened using an in vitro dual culture test with R. solani AG-2-2. Out of these, eleven isolates with important antifungal activity against the pathogen were chosen and characterized using 16S rRNA gene sequencing. Molecular characterization demonstrated that all selected isolates clustered under the genus Bacillus (B. velezensis B. amyloliquefaciens and B. subtilis). In addition, chosen isolates of bacteria were also characterized for their potential to synthesize antifungal metabolites and for their abilities as plant growth-stimulators. Bacterial isolates differed substantially in their capability to synthesize antifungal metabolites and for their abilities as plant growth-stimulators. Bacterial isolates differed substantially in their capability to synthesize antifungal metabolites and for their abilities as plant growth-stimulators. Bacterial isolates differed substantially in their capability to synthesize lipopeptide antibiotics. A test under greenhouse conditions revealed that sugar beet seeds soaked in an individual bacterial isolate significantly decreased Rhizoctonia damping-off. Additionally, seedlings grown from soaked seeds exhibited significant increase in assessed growth parameters. Likewise, selected bacterial isolates exhibited an antagonistic effect on this pathogen, and considerably decreased the severity of RCR caused by Rhizoctonia. For sustainable agriculture, B. velezensis SS2 seems more promising as a fungal biocontrol agent against Rhizoctonia RCR and B. velezensis BM2 can be used as a bio-fertilizer.
... Consistently, we observed an increase of the HCN signal during the transition from exponential to stationary phase, at culture densities equivalent to an optical density at 600 nm (OD 600 ) of .2 (Fig. 1C). We also confirmed that the P. aeruginosa HCN production was enhanced under microaerobic conditions (0.4 to 0.8% O 2 ) compared to aerobic conditions ( Fig. 1A and B) (33). Consistently, under these microaerobic conditions, the growth of S. aureus MW2 exposed to P. aeruginosa PAO1 was reduced by 4-fold, compared to a 1.4-fold reduction upon exposure in aerobic conditions ( Fig. 2B and D). ...
Airborne volatile compounds produced by bacteria are often only considered attractive or repulsive scents, but they also directly contribute to bacterial physiology. Here, we showed that volatile hydrogen cyanide (HCN) released by a wide range of Pseudomonas aeruginosa strains controls Staphylococcus aureus growth in low-oxygen in vitro biofilms or aggregates and in vivo lung environments.
... The genome was annotated by RASTtk version 1.073 (17). RAST annotation indicates that several potential virulence effectors against mosquitoes are present in the genome, including genes for hydrogen cyanide (hcnABC) (18,19), phospholipase haemolysin, and type III secretion systems, including a homolog of the versatile GTPase-activating protein effector yopE from Yersinia pestis that acts as both a cytotoxin and a suppressor of cellular immune responses (20). ...
Chromobacterium sp. strain IRSSSOUMB001 with potent insecticidal activity was isolated from Anopheles gambiae s.l. in Burkina Faso. The draft genome is 5,090,822 bp and encodes predicted genes for hydrogen cyanide production, haemolysin, a T3SS, and yopE , which are potential virulence factors against mosquitoes.
... These results showed that QS regulates the antifungal effect of CV 12472 and can be affected by AHL-targeting QQ. Different antifungal mechanisms could be involved such as violacein (Sasidharan et al. 2015) and cyanide production (Laville et al. 1998); both under QS regulation in CV 12472 and inhibited by SsoPox W263I. Anisomycin is also an antifungal metabolite, with known toxic activity against S. cerevisiae (Zheng et al. 2017). ...
Le phénomène de biofouling est un processus naturel qui impacte toutes les surfaces immergées en milieu marin engendrant des problèmes économiques et écologiques majeurs à l’échelle planétaire. Il est notamment induit par la formation de biofilms marins correspondant à la colonisation des surfaces immergées par des bactéries s’organisant en communautés en s’entourant d’une matrice de substances polymériques extracellulaires (EPS). L’objectif de ce travail est l’utilisation et le développement de méthodologies permettant l’étude et la compréhension de l’étape précurseur de ce phénomène. La corrélation des données récoltées à partir des méthodes appliquées (métabolomique et réseau moléculaire, protéomique, dosages colorimétriques, microscopies, spectroscopies) permet une approche multi-échelles pour la caractérisation des biofilms. Ces développements visent, en premier lieu, à caractériser la production biochimique globale de biofilms in vitro pour ensuite analyser des biofilms naturels formés in situ. L’utilisation de ce large panel de techniques a permis de répondre à certaines questions scientifiques comme l’impact des nutriments (phosphates), d’une enzyme (quorum sensing) ou de l’hydrodynamisme sur la nature de biofilms formés.
... PrnB is the second enzyme in the pyrrolnitrin biosynthesis pathway [34]. HcnB, an amino acid oxidase, is essential for cyanide production [35]. PvdL, a nonribosomal peptide synthetase (NRPS), is involved in the biosynthesis of pyoverdine [36]. ...
The bacterial pathogen Acidovorax citrulli causes the destructive fruit blotch (BFB) on cucurbit plants. Pseudomonas chlororaphis YL-1 is a bacterial strain isolated from Mississippi soil and its genome harbors some antimicrobial-related gene clusters, such as phenazine, pyrrolnitrin, and pyoverdine. Here, we evaluated the antimicrobial activity of strain YL-1 as compared with its deficient mutants of antimicrobial-related genes, which were obtained using a sacB-based site-specific mutagenesis strategy. We found that only phenazine-deficient mutants ΔphzE and ΔphzF almost lost the inhibitory effects against A. citrulli in LB plates compared with the wild-type strain YL-1, and that the main antibacterial compound produced by strain YL-1 in LB medium was phenazine-1-carboxylic acid (PCA) based on the liquid chromatography-mass spectrometry (LC-MS) analysis. Gene expression analyses revealed that PCA enhanced the accumulation of reactive oxygen species (ROS) and increased the activity of catalase (CAT) in A. citrulli. The inhibition effect of PCA against A. citrulli was lowered by adding exogenous CAT. PCA significantly upregulated the transcript level of katB from 6 to 10 h, which encodes CAT that helps to protect the bacteria against oxidative stress. Collectively, the findings of this research suggest PCA is one of the key antimicrobial metabolites of bacterial strain YL-1, a promising biocontrol agent for disease management of BFB of cucurbit plants.
... The GcvP1 and GcvP2 proteins of P. aeruginosa are also PLPdependent enzymes and GcvP2 belongs to the gcs2 cluster required for glycine catabolism (Lundgren et al., 2013). Glycine is the precursor for HCN production, first demonstrated in P. fluorescens (Laville et al., 1998), and the glycine cleavage system has been implicated in HCN production in P. aeruginosa too (Sarwar et al., 2016). Inhibition of glycine catabolism caused by increases in ammonia (a product of the reaction) has been reported to increase the abundance of the glycine substrate which in turn raises the amount utilised to make HCN via the gene cluster hcnABC (Yan et al., 2018). ...
... Transcriptional control of hcnABC, which converts glycine into HCN and CO 2 , is complex ( Figure 6). HCN formation is greater under low oxygen tension and the anaerobic regulator of arginine deiminase and nitrate reductase (ANR) protein is one of a few transcriptional regulators which influence hcnABC (Zimmermann et al., 1991;Laville et al., 1998). ANR, and the E. coli homologue FNR, sense oxygen via an iron-sulfur cluster, and DNA binding is maximal under reducing conditions (Ye et al., 1995;Lazazzera et al., 1996), and iron availability also impacts HCN expression via ANR which is abolished in P. fluorescens when iron is depleted (Blumer and Haas, 2000). ...
Pseudomonas aeruginosa is a major opportunistic human pathogen which employs a myriad of virulence factors. In people with cystic fibrosis (CF) P. aeruginosa frequently colonises the lungs and becomes a chronic infection that evolves to become less virulent over time, but often adapts to favour persistence in the host with alginate-producing mucoid, slow-growing, and antibiotic resistant phenotypes emerging. Cysteamine is an endogenous aminothiol which has been shown to prevent biofilm formation, reduce phenazine production, and potentiate antibiotic activity against P. aeruginosa, and has been investigated in clinical trials as an adjunct therapy for pulmonary exacerbations of CF. Here we demonstrate (for the first time in a prokaryote) that cysteamine prevents glycine utilisation by P. aeruginosa in common with previously reported activity blocking the glycine cleavage system in human cells. Despite the clear inhibition of glycine metabolism, cysteamine also inhibits hydrogen cyanide (HCN) production by P. aeruginosa, suggesting a direct interference in the regulation of virulence factor synthesis. Cysteamine impaired chemotaxis, lowered pyocyanin, pyoverdine and exopolysaccharide production, and reduced the toxicity of P. aeruginosa secreted factors in a Galleria mellonella infection model. Thus, cysteamine has additional potent anti-virulence properties targeting P. aeruginosa, further supporting its therapeutic potential in CF and other infections.
... A cluster of three genes, hcnABC, is responsible for HCN production from the metabolic precursor glycine (Laville et al., 1998). The hcnABC cluster appears to be ancestral in Pseudomonas strains associated with roots (Frapolli et al., 2012). ...
Most Pseudomonas biocontrol strains are associated with the rhizosphere of plants, where they control soil pathogens by antibiosis or competition, and leaf pathogens via induced systemic resistance. Genome mining and the division of the vastly heterogeneous genus Pseudomonas in phylogenomic (sub)groups has clarified the relation between biocontrol characteristics and phylogeny. Based on their activity, Pseudomonas biocontrol strains come in three types. A first type, represented by P. chlororaphis, P. protegens, P. corrugata and P. aeruginosa (sub)group strains, produces an arsenal of secondary metabolites with broad antimicrobial activity. The second type is found in the P. putida, P. fluorescens, P. koreensis, P. mandelii, and P. gessardii (sub)group. The spectrum of biocontrol properties of these strains is less diverse and involves siderophores and cyclic lipopeptides. The third type colonizes above-ground plant parts. Strains from this type mainly belong to the P. syringae group and are used to control postharvest pathogens. This chapter starts with recent advances in Pseudomonas taxonomy and a summary of its most important biocontrol traits. It then provides an overview of the most important Pseudomonas groups and subgroups harboring biocontrol strains. Examples of well-characterized and representative biocontrol strains show the links between phylogeny, ecology and biocontrol traits. The chapter concludes by reviewing commercially-available biocontrol strains.
