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PYOVERDINE PRODUCTION by <I>PSEUDOMONAS AERUGINOSA</I>

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PYOVERDINE PRODUCTION by <I>PSEUDOMONAS AERUGINOSA</I> University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln Faculty Publications in the Biological Sciences Papers in the Biological Sciences 5-1990 PYOVERDINE PRODUCTION BY PSEUDOMONAS AERUGINOSA EXPOSED TO METALS OR AN OXIDATIVE STRESS AGENT Kim-Hien Thi Dao University of Nebraska-Lincoln Katherine E. Hamer University of Nebraska-Lincoln Christine L. Clark University of Nebraska-Lincoln Lawrence G. Harshman University of Nebraska - Lincoln, [email protected] Follow this and additional works at: https://digitalcommons.unl.edu/bioscifacpub Thi Dao, Kim-Hien; Hamer, Katherine E.; Clark, Christine L.; and Harshman, Lawrence G., "PYOVERDINE PRODUCTION BY PSEUDOMONAS AERUGINOSA EXPOSED TO METALS OR AN OXIDATIVE STRESS AGENT" (1990). Faculty Publications in the Biological Sciences. 274. https://digitalcommons.unl.edu/bioscifacpub/274 This Article is brought to you for free and open access by the Papers in the Biological Sciences at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Faculty Publications in the Biological Sciences by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. Dao, Hamer, Clark & Harshman in Ecological Applications (May 1999) 9(2). Copyright 1999, Ecological Society of America. Used bypermission. May 1999 STRESS IN ECOLOGICAL SYSTEMS 441 Ecological Applications, 9(2), 1999, pp. 441-448 C) 1999 by the Ecological Society of America PYOVERDINE PRODUCTION BY PSEUDOMONAS AERUGINOSA EXPOSED TO METALS OR AN OXIDATIVE STRESS AGENT KIM-HIEN THI DAO, KATHERINE E. HAMER, CHRISTINE L. CLARK, AND LAWRENCE G. HARSHMAN' School of Biological Sciences, Universityof Nebraska-Lincoln,Lincoln, Nebraska 68588 USA Abstract. Siderophoresare low molecular mass compounds used by many microor- ganisms to scavenge dissolved iron,which is typicallyrare in environments.Pseudomonas aeruginosa (PAO1) was exposed to metals and methylviologen in low-iron medium in orderto monitorthe abundance of a specificsiderophore (pyoverdine) and monitorgrowth over time.In thisstudy it was discoveredthat cadmium can stimulatepyoverdine abundance. Cadmium may interactwith and sequester the Fur regulatoryprotein, which represses siderophoresynthesis under conditions of ironsufficiency. Mercury did not stimulatepyov- erdineproduction at concentrationsthat strongly inhibited bacterial growth, suggesting that pyoverdineproduction is not governedas partof a generalstress response. Methyl viologen is an oxygen radical generator,and it was discovered thatexposure to it decreased pyov- erdineproduction. Decreased pyoverdineproduction may be a mechanismfor reducing the iron potentiationof oxygen toxicity.It is hypothesizedthat factors stimulating production of siderophoresmay increase the susceptibilityof microorganismsto oxidativedamage. In general,our researchsuggests new opportunitiesfor predicting ecotoxicological outcomes based on understandingof molecular mechanismsand the effectof xenobiotics or stress factorson fundamentallyimportant microbial processes. Key words: cadmium,mercury; methyl viologen; oxidativestress; Pseudomonasaeruginosa; sid- erophore. INTRODUCTION are model systemsfor the studyof physiologicalpro- cesses. One example of the use of microbes for this It is generallyaccepted thatour abilityto make pre- approach is the studyof phosphoenolpyruvatecarbox- dictionsabout theecological effectof pollutantsis very ylase regulation,which is motivatedby the important limited (Moriarty1988). Studies of the effectsof xe- role of this enzyme in the metabolismof (often lim- nobiotics or other stressorson critical physiological iting) dissolved organic matterin aquatic ecosystems processes in organisms may be especially useful for by heterotrophicbacteria (Overbeck 1990). The effect thepurpose of improvingour abilityto predictthe eco- of pollutantsand stressorson critical aspects of mi- logical impact of pollutants and stressors. The as- crobial physiologyis understudiedwith respect to the sumptionis thatphysiological functionis the logical importanceof microbes in the functionof ecological point of integrationfor ecotoxicological studies. Spe- communities.Such studies are valuable for the devel- cifically,the impact of pollutants,or environmental opmentof the fieldof ecotoxicology. stress,on physiologyis relevantto populationgrowth, Iron acquisitionis an essentialphysiological process interactionbetween species, relative fitness,indicator for organisms.Iron is almost universallyrequired for gene expression,and theinteraction between organisms life, yet it is poorly soluble in the form of polyhy- and the environment.Physiological measures thatare complexes at neutralpH (Neilands 1981). fundamentallyimportant and connected with specific droxylated In some regions of the open ocean iron availabilityis environmentalstates are attractivecandidates for study. population size Microbes are obvious candidates for such studies the factor that limits phytoplankton In order to meet this demand for iron, because theyplay an essential role in ecological com- (Kerr 1994). low molecularmass munities.Moreover, they are capable of modifyingthe microbes,and otherorganisms use environmentby theirmetabolic processes. This power compounds,collectively called siderophores,to scav- is manifestin microenvironments,or on a largerscale, enge for iron (Guerinot 1994, Neilands 1995). Sider- as in geochemical change. In addition,certain bacteria ophores are importantin relationshipto the growthof aerobic and facultative anaerobic bacteria, fungal growth,plant growth,as well for theireffects on the Manuscriptreceived 20 October 1997; accepted 27 April 1998; finalversion received 8 June 1998. For reprintsof this virulenceof plant and humanmicrobial diseases (Nei- Invited Feature, see footnote1, page 429. lands 1981, Neilands and Leong 1986, Barton and 1Address corresDondenceto this author. Hemming 1993, Payne 1993). 441 Ecological Applications 442 INVITED FEATURE Vol. 9, No. 2 Pseudomonas aeruginosa is associated withaquatic low-iron medium that promotes siderophoreproduc- and otherhabitats, and is also known as an opportu- tion. Our experimentswere designed to address the nistic pathogen of humans. This species belongs to a questions of whetheradded metals or an oxidative group of microorganisms,the fluorescentpseudomo- stress agent could stimulateor suppress the existing nads, thatare widely distributedacross a range of hab- level of pyoverdineproduction in low iron medium. itats. Among the fluorescentpseudomonads, P. aeru- Growthin SSM was estimatedby measurementof tur- ginosa is the taxon best known in termsof genetics bidityat an optical densityof OD600 nm(Perkin-Elmer and physiology.P aeruginosa produces two siderop- SpectrometerLambda 3B). All solutions and media hores;pyoverdine is predominant,and has a muchhigh- were preparedusing double-distilleddeionized water. er affinityfor iron than the othersiderophore, which Chemicals were fromSigma Chemical Company, St. is called pyochelin.The structureof pyoverdineis pre- Louis, Missouri unless otherwisestated. sentedin Briskotet al. (1989). Pyoverdineis a peptide containingeight amino acids: 1 L-arginine,1 L-lysine, Metal assays 2 L-threonines,2 L-6N-hydroxyornithines,and 2 D- In orderto test the hypothesisthat pyoverdine pro- serines. The octahedral coordinationof Fe(III) is ac- ductionby P. aeruginosa is influencedby thepresence complishedby two 6N-hydroxyornithinesand an (IS)- of heavy metals,dose response assays were conducted 5-amino-2,3 -dihydro-8,9-dihydroxy-1H-pyrimido- in whichgrowth and pyoverdineproduction were mon- [1,2-a]quinoline-1-carboxylicacid providinghydrox- itored over time in samples taken fromSSM culture. ymate and catecholate donor oxygen atoms For these assays, PAO1 was inoculated froman over- (Wendenbaumet al. 1983). nightculture into 13 x 100 mmborosilicate glass tubes The presentstudy addresses the question of whether (Fisher Scientific,Pittsburgh, Pennsylvania) containing exposureto xenobioticstress factors results in a change SSM. All glassware used for cultures or assays was in pyoverdineproduction by P. aeruginosa. Two of the soaked in 0.1 mol/LHCl (MallinckrodtChemical, Par- metals employedin this study,cadmium and mercury, is, Kentucky)overnight to removecontaminating met- are significantenvironmental pollutants. In addition, als, followed by rinses withdouble-distilled water be- experimentswere conductedto determineif a nonmetal fore autoclave sterilization.Overnight cultures were oxygen radical generator,methyl viologen (paraquat), producedby inoculatinga PAO1 colony fromPAF me- could influencethe production of pyoverdine.Paraquat dium into a 50-mL long-neckculture flask with 25 mL is well establishedas a radical generator(Hassan and SSM. The growthperiod was - 16 h at 25?C withmod- Fridovich 1979), and is used routinelyto produce ox- erate shakingat 150 cycles/min. idative stress.Since iron is knownto participatein the Afterovernight growth, cells were centrifugedfor 5 generationof oxygen radicals, exposure to this com- min at 59 x 103 m/s2at room temperature.The pellet pound was motivatedby the issue of iron management was resuspendedin sterile0.85% NaCl. The centrifu- underconditions of oxidative stress. gationand resuspensionsteps were repeated. The pellet MATERIALS AND METHODS from the second centrifugationwas resuspended in 0.85% NaCl to a standardfinal OD600 nm of 0.200. and Pyoverdinemeasurement bacterial growth A 20-j,L aliquot of thisbacterial suspension was used The fluorescentchromophore group of
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