Pyoverdin production by the plant growth beneficial Pseudomonas strain 7NSK2: Ecological significance in soil

The P. aeruginosa-related plant growth beneficial strain 7NSK₂ was able to increase the yield of barley, wheat, 4 varieties of maize, cucumber, spinach and corn salad from 10 to 24%. Siderophore deficient mutants of 7NSK₂, obtained by Tn5-mutagenesis were never able to stimulate plant growth, although their root colonizing capacities were not impaired. The effect of 7NSK₂ inoculation on cucumber dry weight was more pronounced in EDDHA-treated soil (+18%) than in the control soil (+10%). Soil inoculation with 7NSK₂ in a FeEDDHA-treated soil had no effect on plant growth.The 7NSK₂ strain and a sid^- mutant MPFM1 could make use of a variety of Fe(III) siderophore complexes, while the pyoverdin produced by 7NSK₂ was only used by a limited number of other fluorescent Pseudomonas strains. Many more root than soil bacteria were able to use Fe-desferal or Fe-pyoverdin to overcome iron-shortage. It is postulated that pyoverdin production plays an important role in plant growth stimulation, but is not the only factor involved. Pyoverdin production might be very strategic, especially in the rhizosphere, not only to supply the cell with iron, but also to keep other microorganisms, which might be harmful to 7NSK₂ and to the plant, at bay.     

Survival and Plant Growth Promotion of Detergent-Adapted Pseudomonas fluorescens ANP15 and Pseudomonas aeruginosa 7NSK2

Four detergents were tested as selective C sources for the plant growth-promoting rhizobacteria Pseudomonas aeruginosa 7NSK2 and Pseudomonas fluorescens ANP15. CO-720 (Igepal CO-720) or DOS (dioctyl sulfosuccinate), applied at 0.2% to the soil, increased the number of detergent-adapted, inoculated strains by almost 1.5 log units after 25 days, accounting for virtually the entire increase in total bacteria. The same dose of Tween 80 or N-laurylsarcosine, on the other hand, increased the indigenous populations by almost 2.5 log units, with only minor increases in the number of detergent-adapted inoculated strains. When CO-720 or DOS was initially supplied, the number of detergent-adapted 7NSK2 organisms was about 2 log units higher after 3 months of incubation than for the detergent-unadapted strain. This better survival resulted in a significantly higher root colonization of maize in a pot experiment with soil inoculation, with a significantly (P <= 0.05) higher shoot dry weight (18 to 33%). In a first field experiment with rhizosphere inoculation of 1-month-old maize plants, no effects on the height of two maize cultivars could be observed 1 month after inoculation. In a second field experiment, leaf and stem dry weights of yellow mustard and grass dry weight were increased in the treatments with seed and soil inoculation of the detergent-adapted 7NSK2 in combination with CO-720 application by, respectively, 7 to 8%, 19 to 23%, and 20 to 31%, although only the increases in grass dry weight were statistically significant at P <= 0.1. To some extent, 7NSK2 and DOS application also positively affected the mineral content of yellow mustard.     

Zinc affects siderophore-mediated high affinity iron uptake systems in the rhizosphere Pseudomonas aeruginosa 7NSK2

Zinc concentrations ranging between 0.1 and 1 mm only slightly reduced maximal growth of wild-type Pseudomonas aeruginosa 7NSK2 in iron-limiting casamino acid medium, but had a clear negative effect on the growth of mutant MPFM1 (pyoverdin negative) and especially mutant KMPCH (pyoverdin and pyochelin negative). Production of pyoverdin by wild-type strain 7NSK2 was significantly increased in the presence of 0.5 mm zinc and could not be repressed by iron even at a concentration of 100 m. Siderophore detection via isoelectrofocusing revealed that mutant KMPCH did not produce any siderophores, while mutant MPFM1 overproduced a siderophore with an acidic isoelectric point, most likely pyochelin. Pyochelin production by MPFM1 was stimulated by the presence of zinc in a similar way as pyoverdin for the wild-type. Analysis of outer membrane proteins revealed that three iron regulated outer membrane proteins (IROMPs) (90, 85 and 75 kDa) were induced by iron deficiency in the wild-type, while mutants were found to have altered IROMP profiles. Zinc specifically enhanced the production of a 85 kDa IROMP in 7NSK2, a 75 kDa IROMP in MPFM1 and a 90 kDa IROMP in KMPCH.     

Candida albicans Inhibits Pseudomonas aeruginosa Virulence through Suppression of Pyochelin and Pyoverdine Biosynthesis

Bacterial-fungal interactions have important physiologic and medical ramifications, but the mechanisms of these interactions are poorly understood. The gut is host to trillions of microorganisms, and bacterial-fungal interactions are likely to be important. Using a neutropenic mouse model of microbial gastrointestinal colonization and dissemination, we show that the fungus Candida albicans inhibits the virulence of the bacterium Pseudomonas aeruginosa by inhibiting P. aeruginosa pyochelin and pyoverdine gene expression, which plays a critical role in iron acquisition and virulence. Accordingly, deletion of both P. aeruginosa pyochelin and pyoverdine genes attenuates P. aeruginosa virulence. Heat-killed C. albicans has no effect on P. aeruginosa, whereas C. albicans secreted proteins directly suppress P. aeruginosa pyoverdine and pyochelin expression and inhibit P. aeruginosa virulence in mice. Interestingly, suppression or deletion of pyochelin and pyoverdine genes has no effect on P. aeruginosa’s ability to colonize the GI tract but does decrease P. aeruginosa’s cytotoxic effect on cultured colonocytes. Finally, oral iron supplementation restores P. aeruginosa virulence in P. aeruginosa and C. albicans colonized mice. Together, our findings provide insight into how a bacterial-fungal interaction can modulate bacterial virulence in the intestine. Previously described bacterial-fungal antagonistic interactions have focused on growth inhibition or colonization inhibition/modulation, yet here we describe a novel observation of fungal-inhibition of bacterial effectors critical for virulence but not important for colonization. These findings validate the use of a mammalian model system to explore the complexities of polymicrobial, polykingdom infections in order to identify new therapeutic targets for preventing microbial disease.     

Organotin Decomposition by Pyochelin, Secreted by Pseudomonas aeruginosa Even in an Iron-Sufficient Environment

A triphenyltin (TPT)-decomposing strain, Pseudomonas aeruginosa CGMCC 1.860, was screened out. It secreted an unknown TPT-decomposing factor into the medium, later shown to be pyochelin, even in the presence of 100 μM iron. To our knowledge, this is the first report of organotin decomposition by pyochelin.     

Nanogram amounts of salicylic acid produced by the rhizobacterium Pseudomonas aeruginosa 7NSK2 activate the systemic acquired resistance pathway in bean

Root colonization by specific nonpathogenic bacteria can induce a systemic resistance in plants to pathogen infections. In bean, this kind of systemic resistance can be induced by the rhizobacterium Pseudomonas aeruginosa 7NSK2 and depends on the production of salicylic acid by this strain. In a model with plants grown in perlite we demonstrated that Pseudomonas aeruginosa 7NSK2-induced resistance is equivalent to the inclusion of 1 nM salicylic acid in the nutrient solution and used the latter treatment to analyze the molecular basis of this phenomenon. Hydroponic feeding of 1 nM salicylic acid solutions induced phenylalanine ammonia-lyase activity in roots and increased free salicylic acid levels in leaves. Because pathogen-induced systemic acquired resistance involves similar changes it was concluded that 7NSK2-induced resistance is mediated by the systemic acquired resistance pathway. This conclusion was validated by analysis of phenylalanine ammonia-lyase activity in roots and of salicylic acid levels in leaves of soil-grown plants treated with Pseudomonas aeruginosa. The induction of systemic acquired resistance by nanogram amounts of salicylic acid is discussed with respect to long-distance signaling in systemic acquired resistance.     

Redox-active pyocyanin secreted by Pseudomonas aeruginosa 7NSK2 triggers systemic resistance to Magnaporthe grisea but enhances Rhizoctonia solani susceptibility in rice

Pseudomonas aeruginosa 7NSK2 induces resistance in dicots through a synergistic interaction of the phenazine pyocyanin and the salicylic acid-derivative pyochelin. Root inoculation of the monocot model rice with 7NSK2 partially protected leaves against blast disease (Magnaporthe grisea) but failed to consistently reduce sheath blight (Rhizoctonia solani). Only mutations interfering with pyocyanin production led to a significant decrease in induced systemic resistance (ISR) to M. grisea, and in trans complementation for pyocyanin production restored the ability to elicit ISR. Intriguingly, pyocyanin-deficient mutants, unlike the wild type, triggered ISR against R. solani. Hence, bacterial pyocyanin plays a differential role in 7NSK2-mediated ISR in rice. Application of purified pyocyanin to hydroponically grown rice seedlings increased H202 levels locally on the root surface as well as a biphasic H202 generation pattern in distal leaves. Co-application of pyocyanin and the antioxidant sodium ascorbate alleviated the opposite effects of pyocyanin on rice blast and sheath blight development, suggesting that the differential effectiveness of pyocyanin with respect to 7NSK2-triggered ISR is mediated by transiently elevated H202 levels in planta. The cumulative results suggest that reactive oxygen species act as a double-edged sword in the interaction of rice with the hemibiotroph M. grisea and the necrotroph R. solani.     

Pyochelin biotransformation by Staphylococcus aureus shapes bacterial competition with Pseudomonas aeruginosa in polymicrobial infections

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The sss gene product, which affects pyoverdin production in Pseudomonas aeruginosa 7NSK2, is a site-specific recombinease

Pyoverdin production by Pseudomonas aeruginosa strain 7NSK2 was induced by Zn(II) in the presence of iron. A mutant was isolated in which Zn(II) no longer induced pyoverdin production. The sss gene which was inactivated in this mutant was cloned and sequenced. Its protein sequence showed 50% identity to the XerC protein of Escherichia coli, which is a member of the lambda integrase family of site-specific recombinases. An open reading frame was found upstream of sss whose protein sequence showed strong identity to DapF, the diaminopimelate epimerase. In E. coli, xerC is part of a multicistronic unit that also contains dapF. The sss gene of P. aeruginosa could restore site-specific recombination at cer in an E. coii xerC mutant and the E. coii xerC gene could complement a genomic sss mutation in P. aeruginosa.     

Induction of systemic resistance to Botrytis cinerea in tomato by Pseudomonas aeruginosa 7NSK2: role of salicylic acid,pyochelin, and pyocyanin

The rhizobacterium Pseudomonas aeruginosa 7NSK2 produces secondary metabolites such as pyochelin (Pch), its precursor salicylic acid (SA), and the phenazine compound pyocyanin. Both 7NSK2 and mutant KMPCH (Pch-negative, SA-positive) induced resistance to Botrytis cinerea in wild-type but not in transgenic NahG tomato. SA-negative mutants of both strains lost the capacity to induce resistance. On tomato roots, KMPCH produced SA and induced phenylalanine ammonia lyase activity, while this was not the case for 7NSK2. In 7NSK2, SA is probably very efficiently converted to Pch. However, Pch alone appeared not to be sufficient to induce resistance. In mammalian cells, Fe-Pch and pyocyanin can act synergistically to generate highly reactive hydroxyl radicals that cause cell damage. Reactive oxygen species are known to play an important role in plant defense. To study the role of pyocyanin in induced resistance, a pyocyanin-negative mutant of 7NSK2, PHZ1, was generated. PHZ1 is mutated in the phzM gene encoding an O-methyltransferase. PHZ1 was unable to induce resistance to B. cinerea, whereas complementation for pyocyanin production or co-inoculation with mutant 7NSK2-562 (Pch-negative, SA-negative, pyocyanin-positive) restored induced resistance. These results suggest that pyocyanin and Pch, rather than SA, are the determinants for induced resistance in wild-type P. aeruginosa 7NSK2.     

Suppression of fluorophenylalanine resistance by mutation to streptomycin resistance in Pseudomonas aeruginosa

Waltho, Judith A. (University of Melbourne, Victoria, Australia), and B. W. Holloway. Suppression of fluorophenylalanine resistance by mutation to streptomycin resistance in Pseudomonas aeruginosa. J. Bacteriol. 92:35-42. 1966.-Fluorophenylalanine-resistant mutants (fpa-r) of Pseudomonas aeruginosa have been isolated. By cotransduction analysis, the mutations were shown to have at least two chromosomal locations. One locus (fpaA) showed linkage to three other markers, str, try-3bi, and arg-3, and the order of these four linked markers was found to be try-3bi, arg-3, fpaA, str. The linkage relationships of the other fpa loci are not yet known. The phenotypic expression of resistance at the fpaA locus can be suppressed by mutation of the str locus from str-s to str-r, whereas that at an unlinked fpa locus cannot.     

Involvement of DNA helicases in chromate resistance by Pseudomonas aeruginosa PAO1

Chromate-hypersensitive mutants of the Pseudomonas aeruginosa PAO1 strain were isolated using transposon insertion mutagenesis. Comparison of the nucleotide sequences of the regions interrupted within the PAO1 genome showed that mutant strains GGP-64 and AJ-22 were affected in open reading frames PA0967 and PA5345, which correspond to the ruvB and recG genes, respectively. These genes encode helicases RuvB and RecG involved in DNA replication, recombination and repair. The chromate resistance phenotype in mutants GGP-64 and AJ-22 was restored by cosmids bearing wild type ruvB or recG genes, respectively. Also, both mutant strains showed an increased susceptibility to the toxic oxyanions tellurite and selenite as well as to mitomycin C, but not to arsenite, paraquat and hydrogen peroxide. It was concluded that P. aeruginosa RuvB and RecG helicases are involved in repairing DNA damage caused by chromate or its derivatives.     

Involvement of chromosomally-encoded genes in malathion utilization by Pseudomonas aeruginosa AA112

Malathion is an organophosphate insecticide that has been widely used for both domestic and commercial agricultural purposes. However, malathion has the potential to produce toxic effects in mammalian systems. In this study, Pseudomonas aeruginosa AA 112 which was isolated from soil using enrichment technique could utilize the malathion as a sole carbon source and a source of energy. Pseudomonas aeruginosa AA112 was able to grow in MSMPY medium containing 42.75 mg/ml malathion. However, the optimum concentration of malathion which supported the maximum bacterial growth was found to be 22. 8 mg/ml. Malathion was used as an initial source of energy and carbon when it was found without additional carbon sources (in MSM medium) while it was utilized as second source of energy and carbon in a nutrient-supplemented medium (in MSMPY medium). Moreover, lead acetate test indicated that malathion was first attacked at a sulphur site 1-2 hours after the start of incubation. TLC and IR analysis indicated that malathion was completely degraded into diethyl succinate, hydrogen sulphide and phosphates. Therefore a malathion degradation pathway was proporsed. The degradation of malathion is attributed to the genes located on the chromosome and at least three proteins of high molecular size might be involved in malathion utilization. Bacteria able to use malathion as a food source or metabolize its residues in the environment to inactive, less toxic, and harmless compounds, could be used in bioremediation of an environmental pollution caused by the pesticide.     

Involvement of nitric oxide in biofilm dispersal of Pseudomonas aeruginosa

Bacterial biofilms at times undergo regulated and coordinated dispersal events where sessile biofilm cells convert to free-swimming, planktonic bacteria. In the opportunistic pathogen Pseudomonas aeruginosa, we previously observed that dispersal occurs concurrently with three interrelated processes within mature biofilms: (i) production of oxidative or nitrosative stress-inducing molecules inside biofilm structures, (ii) bacteriophage induction, and (iii) cell lysis. Here we examine whether specific reactive oxygen or nitrogen intermediates play a role in cell dispersal from P. aeruginosa biofilms. We demonstrate the involvement of anaerobic respiration processes in P. aeruginosa biofilm dispersal and show that nitric oxide (NO), used widely as a signaling molecule in biological systems, causes dispersal of P. aeruginosa biofilm bacteria. Dispersal was induced with low, sublethal concentrations (25 to 500 nM) of the NO donor sodium nitroprusside (SNP). Moreover, a P. aeruginosa mutant lacking the only enzyme capable of generating metabolic NO through anaerobic respiration (nitrite reductase, DeltanirS) did not disperse, whereas a NO reductase mutant (DeltanorCB) exhibited greatly enhanced dispersal. Strategies to induce biofilm dispersal are of interest due to their potential to prevent biofilms and biofilm-related infections. We observed that exposure to SNP (500 nM) greatly enhanced the efficacy of antimicrobial compounds (tobramycin, hydrogen peroxide, and sodium dodecyl sulfate) in the removal of established P. aeruginosa biofilms from a glass surface. Combined exposure to both NO and antimicrobial agents may therefore offer a novel strategy to control preestablished, persistent P. aeruginosa biofilms and biofilm-related infections.