Overhage, J.

Strehmel, J.

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KIT – University of the State of Baden-Wuerttemberg andNational Research Center of the Helmholtz AssociationThe sensor kinase PA4398 regulates swarming motility andbiofilm formation in Pseudomonas aeruginosa PA14Janine Strehmel and Joerg OverhageInstitute of Functional Interfaces (IFG)RG Bacterial Stress Response and Process EngineeringReferences: Contact: Acknowledgements:[1] J Bacteriol. (2008), 190(8):2671-9. KIT – Campus North The Research Group Bacterial Stress Response and Process Engineering is financed[2] J Bacteriol. (2009), 191(18):5592-602. Institute of Functional Interfaces (IFG) by the concept for the future of the Karlsruhe Institute of Technology (KIT) within the. janine.strehmel@kit.edu German Excellence Initiative.INTRODUCTIONMulticellular behavior is an important process central to the pathogenesis of P. aeruginosa. In addition to biofilm formation, swarming motility represents a second surface-associated community behavior of this human pathogen. Recently, we have shown that swarming can be considered as a distinct physiological state with a tailoredmetabolic lifestyle or a complex adaptation of P. aeruginosa in response to a viscous environment leading to increased antibiotic resistance and virulence geneexpression [1]. During a screening for swarming deficient mutants [2] we identified a two-component sensor kinase transposon mutant (PA4398) in P. aeruginosa PA14with defects in the ability to swarm on semisolid surfaces. In this study, we constructed a knock-out mutant of PA4398 in P. aeruginosa PA14 and phenotypicallycharacterized this sensor kinase mutant in more detail.Growth of P. aeruginosa PA14 and PA4398 mutantP. aeruginosa PA14 cultureswere grown in a 96-wellmicrotiter plate under shakingconditions at 37 °C in BM2minimal medium.Mutation in the sensor kinase PA4398 leads to decreasedattachment and swarming motilitywtmutantP. aeruginosa (PA14 wt; PA4398 mutant) cultures were grown in LB mediumto an optical density (OD595nm) of 1. One µl of these cultures were incubatedon BM2swarm [-(NH4)2SO4 + 0.1 % CAA] agar plates [1]. Shown is arepresentative swarm plate after 18 h of incubation at 37 °C.P. aeruginosa PA14 cultures weregrown in BM2 medium containing 0.1% CAA for 1h at 37 °C. Attachmentwas determined in 96-well microtiterplates by crystal violet staining or onglass object slides by fluorescencemicroscopy [2].(*) t-test p value ≤ 0.05METHODS&RESULTSSUMMARY• The PA4398 mutant exhibited reduced swarming mobility, attachment and biofilm formation in comparison to wildtype cells whereas no differences regarding growth rate,twitching and swimming motility were observed. • Preliminary microarray analysis of mutant strain PA14 13_3 suggests a role of sensor kinase PA4398 in nitrogen and iron metabolism in P. aeruginosa PA14 (data not shown).The PA4398 mutant exhibits normal swimming and twitching motilitySwimming: One µl of overnight cultures were incubated on 0.3 % (w/v) LB agar plates for18 h at 30 °C [2].Twitching motility was performed on 1.5 % (w/v) LB agar plates using 2 µl of overnightcultures and an incubation for 48 h at 37 °C [2].CPA4398 mutant is impaired in biofilm formation(*) t-test p value ≤ 0.05Institute of Functional Interfaces , RG Bacterial Stress Response and Process EngineeringKarlsruhe Institute of Technology, Hermann-von-Helmholtz-Platz 1, 76344 Eggenstein-Leopoldshafen, GermanyBiofilm formation of PA14 Wt on glassslidesBiofilm formation of PA14 PA4398 mutanton glass slidesPA14 overnight cultures were diluted 1:100 in BM2. Biofilm formation was examinedunder static conditions on glass object slides or in 96-well microtiter plates after 16 h ofincubation at 37 °C. Visualization of biofilm formation was implemented afterimmobilization (3 % formaldehyde) and SYTO9 staining by fluorescence microscopy [A;B] or crystal violet staining [C].Attachment in 96-well microtiter platesAttachment: PA14 Wt on glass slides Attachment: PA14 PA4398 mutant on glass slidesA B00,10,20,30,40,50,60,70,01,02,03,04,05,06,07,08,09,010,011,012,013,014,015,016,017,018,019,020,021,022,023,024,0Absorbance [595nm]Time [h]BM2 medium PA14 wt PA14 PA4398 mutant0,00,51,01,52,02,53,0PA14 wt PA14 PA4398 mutantDiameter [cm]Swimming motility0,00,51,01,52,0PA14 wt PA14 PA4398 mutantDiameter [cm]  Twitching motility0,05,010,015,020,025,030,035,040,045,050,0PA14 wt PA14 PA4398 mutantArea Swarming motility*00,20,40,60,811,2PA14 wt PA14 PA4398 13-3 mutantPA14 PA4398 mutantAbsorbance [595nm]**00,10,20,30,40,50,6PA14 wt PA14 PA4398 13-3 mutantPA14 PA4398 mutantAbsorbance [595nm]Biofilm formation in 96-well microtiter plates* *

The sensor kinase PA4398 regulates swarming motility and biofilm formation in Pseudomonas aeruginosa PA14

https://publikationen.bibliothek.kit.edu/220084042/3815856

The sensor kinase PA4398 regulates swarming motility and biofilm formation in Pseudomonas aeruginosa PA14

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