1 This article is protected by copyright. All rights reserved. The biosurfactant viscosin produced by Pseudomonas fluorescens1 SBW25 mediates in vitro spreading motility and plant growth2 promotion13 Abdullah S. Alsohim1, Tiffany B. Taylor1, Glyn A. Barrett1, Jenna Gallie2,3,4, Xue-Xian Zhang2,4 Astrid E. Altamirano-Junqueira1, Louise J. Johnson1, Paul B. Rainey2,5 and Robert W.5 Jackson1,* 6 1School of Biological Sciences, University of Reading, Whiteknights, Reading, RG6 6AJ, UK7 2New Zealand Institute for Advanced Study, Massey University, Auckland, New Zealand8 3Department of Environmental Microbiology, Eawag, 8600 Dbendorf, Switzerland9 4Department of Environmental Systems Science, ETH Zrich, 8092 Zrich, Switzerland10 5Max Planck Institute for Evolutionary Biology, Pln, Germany11 *Corresponding author12 13 14 Keywords: Plant growth-promoting rhizobacteria (PGPR); Food security; FleQ; Plant15 colonisation16 17 This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/1462-2920.12469 Accepted Article2 This article is protected by copyright. All rights reserved. Abstract18 Food security depends on enhancing production and reducing loss to pests and pathogens.19 A promising alternative to agrochemicals is the use of plant growth promoting rhizobacteria20 (PGPR), which are commonly associated with many, if not all, plant species. However,21 exploiting the benefits of PGPRs requires knowledge of bacterial function and an in-depth22 understanding of plant-bacteria associations. Motility is important for colonisation efficiency23 and microbial fitness in the plant environment, but the mechanisms employed by bacteria24 on and around plants are not well understood. We describe and investigate an atypical25 mode of motility in Pseudomonas fluorescens SBW25 that was revealed only after flagellum26 production was eliminated by deletion of the master regulator fleQ. Our results suggest this27 spidery spreading is a type of surface motility. Transposon mutagenesis of SBW25AfleQ28 (SBW25Q) produced mutants, defective in viscosin production, and surface spreading was29 also abolished.Genetic analysis indicated growth-dependency, production of viscosin and30 several potential regulatory and secretory systems involved in the spidery spreading31 phenotype. Moreover, viscosin both increases efficiency of surface spreading over the plant32 root and protects germinating seedlings in soil infected with the plant pathogen Pythium.33 Thus, viscosin could be a useful target for biotechnological development of plant growth34 promotion agents. 35 36 Accepted Article3 This article is protected by copyright. All rights reserved. Introduction37 Many strains of Pseudomonas fluorescens are proficient colonizers of soil and the38 phytosphere (plant environment). P. fluorescens can be genetically engineered to promote39 plant growth by producing hormones or inducing systemic resistance (Glick & Bashan,40 1997), but many naturally-occurring strains also significantly improve plant growth (Cook et41 al., 1995, Schippers et al., 1987, Weller, 1988) by improving the bioavailability of nutrients42 and antagonizing pathogenic fungi and oomycetes (Artursson et al., 2006, Sarathchandra et43 al., 1993). Antagonism can be conferred by the production of siderophores and of44 surfactants, such as viscosin and viscosinamide, as well as antimicrobial compounds, such as45 hydrogen cyanide, phenazines, pyrrolnitrin or 2,4-DAP (Haas & Dfago, 2005). Many strains46 harbour a type III protein secretion system that is expressed in the plant environment47 (Rainey 1999, Preston et al., 2001), and is implicated in plant growth promotion by48 antagonism of oomycete pathogens (Rezzonico et al., 2005, Weller et al., 2002, de Souza et49 al., 2003).50 Strains of P. fluorescens are attractive as plant growth promoting agents, but a major51 challenge is to ensure reliable and stable protection. Desirable qualities include production52 of a range of antagonistic molecules to target a variety of pathogens and, particularly,53 durability in the field, e.g., long-lasting colonization of plant surfaces and persistence in soil.54 There is natural variation in the ability of P. fluorescens to colonize plants, and variation in55 growth rates under different environmental conditions (Loon, 2007; Chiarini et al., 1998);56 the basis of these differences in ecological success has been the subject of intense study,57 and has been partially elucidated by genetic screens (Rainey, 1999; Gal et al., 2003; Giddens58 et al., 2007) and genome sequencing (Silby et al., 2009). Promoter-probe studies using in59 Accepted Article4 This article is protected by copyright. All rights reserved. vivo expression technology (IVET) have led to the discovery of a wide range of mechanisms60 for colonisation of plants, ranging from specific metabolism and nutrient scavenging61 systems to motility and secretion systems and the production of compounds such as62 extracellular polysaccharide (EPS; e.g. cellulose) (Rainey, 1999; Gal et al. 2003; Silby et al.,63 2009) and secreted proteins (Silby & Levy, 2004). 64 A large number of regulators have also been identified by IVET as being expressed in65 the plant environment, and the interplay between regulators and plant-induced genes was66 studied using a combination of suppressor mutagenesis and IVET, termed SPyVET (Giddens67 et al., 2007). This identified both positive and negative regulatory genes of plant-induced68 genes, and also provided insight to the complexity and hierarchy of gene regulation. For69 example, the wss gene locus is under the control of at least 7 regulators. One of these, FleQ,70 negatively regulates wss expression. Mutation of fleQ in P. fluorescens SBW25 led to71 activation of wss, but is also linked to loss of flagellum biosynthesis and swimming motility.72 An analysis of bacterial movement over the surface of semi-solid agar also revealed a73 change in the surface motility phenotype, with a switch from a radial spreading colony to a74 dendritic spreading colony. We aimed to investigate the mechanism of this motility75 phenotype (termed spidery swarming in Giddens et al., 2007; here we use spidery76 spreading because spreading encompasses any motility over the surface and therefore77 avoids assumptions about motility mechanism). 78 FleQ (sometimes called AdnA) is an RpoN-dependent enhancer binding protein and79 the master regulator of flagella biosynthesis (Dasgupta et al., 2003). As well as activating80 motility, it also regulates genes involved in attachment to surfaces and biofilm formation81 (DeFlaun et al., 1994; Robleto et al,. 2003; Mastropaolo et al., 2012). Mastropaolo et al.82 (2012) found that FleQ (AdnA) positively regulates 92 genes in P. fluorescens Pf0-1, 48 that83Accepted Article5 This article is protected by copyright. All rights reserved. are known motility or chemotaxis genes and 44 that are predicted to have a range of other84 functions, e.g. lipoproteins. Recent evidence has shown that FleQ is also a negative85 regulator of extracellular polysaccharide biosynthesis controlling pel genes in P. aeruginosa86 (Hickman & Harwood, 2008; Baraquet et al., 2012). The activity of FleQ in P. aeruginosa is87 modulated by cyclic di-GMP, a secondary messenger molecule that is synthesized by88 diguanylate cyclases these are thought to be influenced by environmental signals89 (Hickman & Harwood, 2008; Baraquet et al., 2012). Thus, FleQ plays a dual regulatory role90 and probably acts, via environmental signalling, as a switching mechanism for inverse91 expression of the flagellum and EPS common strategies for bacteria switching between92 planktonic and biofilm lifestyles (Coggan & Wolfgang, 2012). 93 Bacteria have a range of mechanisms that allow efficient motility under diverse94 environmental conditions from liquid, to semi-solid and solid surfaces. Motility mechanisms95 include flagellum-dependent swimming and swarming, or flagellum-independent96 mechanisms including twitching/gliding using type IV pili, non-social gliding (a poorly97 understood type of motility; Youderian, 1998) and sliding using reduced surface tension98 (Henrichsen, 1972; Harshey, 2003). Swimming motility is dependent on flagella and is99 observed as bacteria moving through liquids or semi-solid media, such as low percentage100 agar, and requires a high liquid presence, whereas swarming is the active spread of bacteria101 over surface like semi-solid agar, and depends on flagella, pili and the presence of a water102 film or surfactant (such as LPS or rhamnolipids) to enable motility. Sliding motility is103 independent of flagella, relies on a growing culture in combination with reduced surface104 tension between the cell and the surface (Henrichsen, 1972; Kinsinger et al., 2003), and is105 usually dependent on production of a biosurfactant.Biosurfactants are a vast and diverse106 range of metabolites of great ecological importance, as well as economic value in the food,107Accepted Article6 This article is protected by copyright. All rights reserved. drug and cosmetics industries (Mandal et al., 2013). Biosurfactants are involved in bacterial108 virulence, biofilm formation and defence against predators and pathogens in addition to109 having a role in multiple motility mechanisms. It was recently discovered that P. aeruginosa110 exhibited sliding motility over swarming plates when both pili and flagella expression were111 deleted, and biosurfactant expression was suggested as a major factor regulating this sliding112 motility (Murray & Kazmierczak, 2008). Biosurfactants have also been implicated in flagella- 113 mediated motility, perhaps by acting as a lubricant of the flagellar propeller (Burch et al.,114 2012). Although biosurfactants include many types of molecule, the lipopeptides, which115 include an oligopeptide and a lipid tail, are a particularly important and well-studied family116 (Raaijmakers et al., 2010). P. fluorescens strain SBW25 is known to produce the potent anti- 117 microbial lipopeptide viscosin (Laycock et al., 1991).118 The observation that the fleQ mutant of SBW25 retains some motility, albeit119 phenotypically different to that of the wild-type, led us to hypothesize that fleQ deletion120 exposed a FleQ-independent surface motility. To investigate the mechanism of spidery121 spreading, we used a genetic screen to identify the genes underlying it, and show that the122 phenotype requires the surfactant viscosin, consistent with its being a form of sliding123 motility. Moreover, we show that viscosin is important for plant root colonisation and plays124 a further role in protecting plant roots from an oomycete pathogen. 125 126 Accepted Article7 This article is protected by copyright. All rights reserved. Results 127 FleQ is required for radial surface spreading by SBW25128 Our first aim was to characterize the change in surface spreading motility observed by129 Giddens et al. (2007) after mutation of fleQ. In that study, the change in motility phenotype130 wasobservedwithstrainNR9.5,whichcarriedanIVETconstructintegratedintothe131 chromosomeandatransposoninsertioninfleQ.Toeliminateanypossiblegeneticside132 effectswiththepresenceoftheseconstructs,wemadeadeletionofthefleQgenein133 SBW25(SBW25Q)anddetermined thatthephenotypicconsequencesofthegenedeletion134 matchedthephenotypespreviouslyseenwithstrainNR9.5(Giddensetal.,2007).Theloss135 ofFleQactivitywasconfirmedbytheassociatedlossofswimmingmotilitythroughsemi- 136 solidagar(datanotshown)andproductionofaflagellatecells(Figure1a).Importantly,137 SBW25Qexhibitedspideryspreading(Figure1b)identicaltothatseenwithNR9.5by138 Giddens et al. (2007). 139 ToconfirmthephenotypicchangeswereduetolossofFleQactivity,thefleQ140 mutationwascomplementedbyectopicexpressionoffleQfromaplasmidresultingina141 surfacemotilityphenotypesimilartothewild-type(Figure1aandb):Inaddition,142 SBW25Q(pFleQ) was observed to produce flagella and regained the ability to swim through143 agar in the swimming assay. Twitching motility was not affected by the fleQ mutation (data144 not shown). Taken together, these experiments show that mutation offleQ alters bacterial145 surface motility. To clarify this phenotype further, we also tested the wildtype and SBW25Q146 onmediathathavebeenusedinothersurfacemotilitystudies.Theseincluded0.6%147 standardsuccinatemedium(SSM)and0.6%LB:neitherstrainwasobservedtomoveover148 the surface of the media after 3 days incubation. We also tested both strains on 0.25% SSM;149 Accepted Article8 This article is protected by copyright. All rights reserved. inbothcasesthestrainsmovedoverthesurfaceoftheagar,exhibitingasimilardendritic150 spreadwithverywhitetendril,differentfromthespideryspreadingphenotypeobserved151 withSBW25Qon0.25%LB(SupplementaryFigureS1).Takentogether,theseexperiments152 show that the SBW25Q phenotype on 0.25% LB is a novel observation that is linked to agar153 contentandmediumcomposition.Thissuggeststhatspideryspreadingisanalternative154 formofsurfacemotility,independentofFleQregulation(andflagella)thatisunmasked155 upon inactivation of fleQ. 156 157 Mutagenesis of SBW25Q reveals that spidery spreading requires the surfactant viscosin 158 Todeterminethegeneticbasisofspideryspreading,atransposonmutagenesisof159 SBW25Q with IS-O-Km/hah was performed to identify mutants that either reverted to wild- 160 typesurfacespreadingorexhibitedlossorchangeofsurfacemotility.Toensuresurface161 motilityphenotypeswerereproducibleandcomparabletothecontrolstrains(SBW25and162 SBW25Q)weperformedmotilityassayson0.25%LBagar.Duringthecourseofthese163 experiments, a clear liquid film was observed to be emanating from the point of inoculation,164 eventually forming a radial film moving in front of the bacterial colony, for both SBW25 and165 SBW25Q. Quantification of the liquid film and the spreading of the colony indicated that the166 liquid film moved across the agar surface at a similar rate for SBW25 and SBW25Q (data not167 shown),whereastheSBW25Qspideryspreadercolonyspreadmuchmoreslowlythan168 SBW25 (Supplementary Figure S2).169 A total of 2000 random mutants were individually inoculated, in duplicate, by sterile170 wireintothecentreof0.25%LBagarplatesandplateswereincubatedfor48h.Each171 mutantwasassessedevery6-12hagainstcontrolstrainsSBW25andSBW25Q.Twenty- 172 Accepted Article9 This article is protected by copyright. All rights reserved. sevenmutants(AR1-26,AR28)exhibitedalteredsurfacemotilitycomparedtothecontrols173 andall27wereretestedintriplicate:nomutantexhibitedareversiontothewild-type174 phenotype, four mutants (AR1, AR2, AR8 and AR11) were completely unable to move from175 the point of inoculation, and the remaining 23 mutants exhibited altered patterns of surface176 motility(Figure2).Someoftheselattermutantsexhibitedthesamespideryspreader177 pattern,butmovedoverthesurfacefasterthanSBW25Q,whileothersshowedaltered178 colony patterns when spreading over the surface. 179 Toidentifythelocationofthetransposoninthegenomesofthesemutants,180 Arbitrarily-PrimedPCRanalysiswasusedtopinpointtheprecisesequencesofnucleotides181 spanningtheinsertion.TheseweremappedtothechromosomeofSBW25usingthe182 available genome sequence (Table 1). A variety of different genes, including those encoding183 putativeenzymesandGGDEFsignallingregulators,accountedforsomeofthe23mutants184 withalteredsurfacemotility.Asubsetofcellmembrane/wallassociatedproteinsand185 lipoproteinswereamongtheseaswellassomegenesinvolvedinmetabolicprocesses186 includingproductionofaminoacids.Thesemayindicatethatcellreplicationisakey187 requirement for expression of the spidery spreader phenotype. However, the transposons in188 the 4 sessile mutants (AR1, AR2, AR8 and AR11) were all found to be inserted independently189 withingenesPFLU2552(viscB)andPFLU2553(viscC),whicharenon-ribosomalpeptide190 synthetase(NRPS)genesinvolvedinmakingthelipopeptidesurfactantviscosin.Ofthe27191 mutants,onlythesefourfailedtoproducealiquidfilmontheagarsurfaceorfrothin192 shaken cultures. Notably, these viscosin mutants and SBW25C, a viscosin knockout mutant,193 were affected in their ability to spread over 0.25% SSM agar, providing further evidence for194 the novel phenotype seen on 0.25% LB. While we were able to complement the deleted fleQ195 gene,thelargesizeoftheNRPSgenesprecludedindividualcloningofthegenesfor196Accepted Article10 This article is protected by copyright. All rights reserved. complementation and a genomic cosmid clone containing the genes has not been found in197 the SBW25 cosmid library. We were therefore unable to create a complemented mutant for198 viscosinproduction,butthefourindependentinsertionsintwogenesprovidecompelling199 proof of their involvement.200 201 SBW25concurrentlyemploysflagellum-dependentandflagellum-independentviscosin- 202 dependent surface motility203 ApreviousstudyreportedthatanSBW25viscosinmutant(SBW25C)wasunableto204 moveover0.6%SSMagarbyswarming(deBruijnetal.,2007)suggestingthatsurface205 motilityisdependentonbothflagellaandviscosinproduction.SinceSBW25Ccarriesa206 transposonintheviscCNRPS,themutationisnotpredictedtoaffecttheflagellum207 biosynthesis system. However, the sessile phenotype observed with SBW25C by de Bruijn et208 al.(2007)suggestedthatmutationofviscCdoesindeedimpactflagellum-dependent209 motilityi.e.viscosinisessentialforthesurfacespreadingphenotype.Wetherefore210 complementedstrainAR1(Fla-,Visc-)withplasmidpFleQtofirstexaminebacterialsurface211 motilityonLBagar(0.25%and0.6%).Thecomplementedstrain,termedAR1(pFleQ),212 exhibited surface motility producing a radial colony spreading from the inoculation point on213 0.25%LBagar,althoughthespreadwasslowerthanthatofthewild-type(Figure3).No214 liquid film was observed on the plate, the complemented strain produced flagella (data not215 shown), and we observed slow surface spreading on 0.6% LB agar (data not shown). We can216 therefore conclude that AR1(pFleQ) moves over the surfaceof 0.25% LB agarby flagellum- 217 dependentmotilitywithouttheneedforviscosin.WenexttestedSBW25Con0.25%LB218 agar; like AR1(pFleQ), this strain also spread across the agar surface (Figure 3), but without a219 Accepted Article11 This article is protected by copyright. All rights reserved. liquidfilm beingapparent.Wesuggest thatthe liquidfilm observedmovinginfrontofthe220 bacterialcolonyisviscosin,meaningthatSBW25CisFla+Visc-,andthatSBW25Ccanstill221 moveslowlyoversurfaceswithoutviscosin.Notably,therewasasubtledifferenceinthe222 structure of the colony edge of the different strains on 0.25% LB agar. The wild-type SBW25223 colonyedgewaserosewhereasAR1(pFleQ)andSBW25Ccolonyedgeswereentire(Figure224 3):SBW25Qistheonlystrainthatproducesspiderytendrils.Finally,wetestedthe225 phenotypeofSBW25ConSSM(0.25%and0.6%)comparedtotheotherstrains.On0.6%226 SSM,SBW25Cdidnotmovefromthepointofinoculation,confirmingtheprevious227 observation made by de Bruijn et al. (2007), but we also did not see movement of SBW25 on228 0.6%SSMagarincontrasttodeBruijnetal.(2007);thisisdiscussedfurtherbelow.229 However,SBW25Cdidspreadoverthesurfaceof0.25%SSMandadifferenceincolony230 phenotypewasalsoseen,withSBW25C(Fla+Visc-)exhibitingacircularcolonyspread231 comparedtothedendriticspreadofwildtype(Fla+Visc+)andSBW25Q(Fla+,Visc-)onthis232 particular medium (Supplementary Figure 1). When considered together, it is clear that agar233 concentration and medium components affect surface motility behaviour. Moreover, these234 dataindicatethatbothflagellum-andviscosin-dependentsurfacespreadingmotility235 contributetocolonyspreadingphenotype,whicharemostnotableon0.25%LB:SBW25236 wild-typehasanerosecolonyedgewhenbothflagellaandviscosinoperatetogether,237 comparedtotheentirecolonyedgephenotypewhentheflagellumaloneisoperating238 (SBW25C),orthespideryspreadercolonyphenotypewhenviscosinoperatesalone239 (SBW25Q).Wild-typecellsapparentlyusebothmotilitysystemsconcurrently;thisfavours240 anexplanationfortheobservedphenotypeoffleQdeletionmutantsinwhichdeletion241 exposesanalternativemotilitysystemindependentofFleQ.Theslowerspreadingof242 AR1(pFleQ)andSBW25CrelativetoSBW25Q(pFleQ)showsthatviscosin,whilenot243Accepted Article12 This article is protected by copyright. All rights reserved. necessary for movement, increases speed of spread over a semi-solid surface. We therefore244 investigated whether these motility differences in vitro had effects in planta. 245 246 The flagellum is essential for root surface migration while viscosin can aid surface migration247 and is a key factor for SBW25 plant growth promotion 248 Animportantquestionemergingfromthisstudyrelatestothebiologicalfunctionof249 viscosin,whichhaspreviouslybeenreportedtolysezoosporesoftheoomycetepathogen250 Phytopththorainfestansaswellasbeinginvolvedinmotility(deBruijnetal.,2007).Thus,251 we tested (i) if viscosin contributes to bacterial motility over the root surface and aimed to252 resolveitscontributionrelativetoflagellum-dependentmotilityand(ii)ifviscosinis253 antagonistic to the soil oomycete pathogen Pythium by assessing the effects of the different254 bacteria in protecting plants grown in the presence of the oomycete. We first tested aim (i)255 andgrewsugarbeetseedlingsinsterilevermiculiteandinvitroonwateragarplateskept256 vertically. After germination and seedling development (8 days), a 2 l drop of 105 bacterial257 suspension(SBW25(Fla+,Visc+),SBW25Q(Fla-,Visc+),AR1(Fla-,Visc-)andSBW25C(Fla+,258 Visc-))wasappliedtotheseedlinghypocotyl.Bacteriawererecoveredafter1and3days,259 but allstrainswerefoundto havereached the root tipattheendoftheexperiment(data260 notshown).Wethereforerepeatedtheexperiment,buttheseedlingswerelaidflatto261 preventanygravitropicormoisture-aidedbacterialdistributionovertherootsurface.We262 postulated thatifmotilityisimportantforbacterialspreadovertheroot surface, then this263 designshouldresolvetheindividualcontributionsoftheflagellumandviscosinwithout264 compounding effects of gravity or moisture. The distal 1 cm of the plant root was sampled265 at time zero, and after 1 and 3 days. Wild-type SBW25 was observed to colonize the root tip266 Accepted Article13 This article is protected by copyright. All rights reserved. after3dayswhereasneitherSBW25QnorAR1weredetected.SBW25Cwasobservedto267 colonize the root tip, but was slower to reach the tip (Figure 4; General Linear Model (GLM),268 Strain as main effect: F3, 32 = 3.353, P = 0.031). Taken together, these data clearly show that269 theflagellumisessentialforbacterialmovementovertherootsurfacewhereasviscosinis270 dispensable;however,viscosinappearstoplayaroleinaidingthespreadofmotile(Fla+)271 bacteria (GLM, Strains categorised by flagella / viscosin production as main effect: Flagella,272 F1, 32 = 9.761. P = 0.004; Viscosin, F1, 32 = 0.148, P = 0.703). 273 Oursecondhypothesispositedthatviscosinplaysaroleinplantgrowthpromotion274 throughantagonismofoomyceterootpathogens.SinceSBW25isknowntoprotectplants275 fromoosporesandhyphaeoftheoomycetePythium(Nasebyetal.,2001)andviscosinis276 known to lyse oomycete zoospores (de Bruijn et al., 2007), we focused on testing the in vivo277 effects by assessing sugar beet seedling germination and root and shoot weight and length.278 Seeds were soaked in different bacterial suspensions or buffer alone and sown into soil with279 orwithoutPythium.InsoilwithoutPythium,thepresenceofthedifferentbacterialstrains280 hadnodiscernibleeffectonseedlinggerminationordevelopmentcomparedtoseedlings281 grownintheabsenceofbacteria(datanotshown);thisdemonstratesthatviscosinalone282 doesnotpromoteplantgrowth.Whenuntreatedseedsweresownintosoilcontaining283 Pythium,therewasanegativeimpactonrootdevelopment(Figure5a).However,seeds284 soaked in bacteria before sowing showed genotype-specific effects. Specifically, the type of285 bacterialstraininoculatedsignificantlyaffectedtherootlength(GLM:F7,78 =4.520,P0.05).Inotherwords,onlytheviscosin298 producing strains (SBW25 and SBW25Q) significantly promoted root growth in the presence299 ofPythium,whereasthedifferencebetweenviscosinknockoutsandthenobacteria300 treatment was not significant (Figure 5a). This indicated viscosin production is important for301 protectionoftheplantinthepresenceofPythium.Althoughthereappearstobeaslight302 reductioninshootlengthinflagellum-viscosinmutants, this effectisnot significant(Figure303 5a; GLM: Flagellum/viscosin production as main effect, F4, 81 = 74.193; P = 0.068).304 Soaking the seeds in the different mutant types before planting also had an effect on305 thenumberofseedlingsthatsuccessfullygerminatedinsoilsupplementedwithPythium306 (SBW25 vs. No Bacteria, P = 0.035; Figure 5b). When mutants were sorted based on whether307 theyproducedviscosin,wefoundthoseseedsthathadbeensoakedinmutantsthat308 expressed viscosin (i.e. SBW25 and SBW25Q) produced on average 30% more seedlings than309 those that were unable to produce viscosin (T-test: t19 = 2.78; P = 0.012), whereas flagellum310 expression was not found to have a significant effect (P > 0.05). However, we acknowledge311 that the interaction between a PGPR and the plant host and any benefits it provides is likely312 to be more complex than one metabolite, because although the seeds treated with viscosin313 Accepted Article15 This article is protected by copyright. All rights reserved. mutants led to a reduced number of germinated seedlings, seedling germination appears to314 be better than if no bacteria were applied at all.315 316 Discussion317 Plantgrowthpromotingrhizobacteria(PGPR)areusedinagricultureandaregaining318 recognitionaskeyfuturecomponentsofintegratedpestmanagementtoreplaceor319 complement particular chemotherapeutic plant treatments (Mendes et al., 2011). However,320 there are concerns surrounding the use of some PGPR strains in terms of their durability and321 efficacy(deWegeretal.,1995).Fundamentaltothisissueisanunderstandingofthe322 dynamicsofbacterialfunctioninthenaturalenvironment.Thesynergyoffundamental323 biology with applied science helps to unravel the different mechanisms employed by PGPR,324 their relative contributions to the agricultural application, and ultimately,their contribution325 to ecological performance. This, of course, requires extensive analysis of bacterial traits and326 empirical testing in the plant environment. In this work, we extended functional analysis of327 the PGPR P. fluorescens SBW25 emanating from a previous gene regulation study(Giddens328 etal.,2007)andfoundthatviscosinplaysimportantrolesinmotilityandplantprotection.329 Our results also raise important questions concerning the use of motility nomenclature and330 the conditions used for testing different motility mechanisms.331 Although the focus for this paper is on viscosin, several other genes were identified by332 themutagenesisthataffectedsurfacespreadingmotility.Arangeofquantitativelyand333 qualitatively different spreading phenotypes were seen (Figure 2). The AR6, AR12 and AR22334 transposonsiteswereindividuallysituatedwithinputativeGGDEF/EALregulators.These335 regulators are commonly found amongst sequenced bacterial genomes andare involved in336 theproductionandbreakdownofthebacterialintracellularsecondmessengercyclic-di- 337 Accepted Article16 This article is protected by copyright. All rights reserved. GMP(Kulesekaraetal.,2006;Pauletal.,2004;Simmetal.,2004).Theyareinvolvedin338 several cellular mechanisms including the biosynthesis of exopolysaccharides, the formation339 ofbiofilmsandsurfacemotility(Ryjenkovetal.,2005).Phenotypicshiftsfromsessileto340 motilestrainsinSalmonellaentericaserovarTyphimurium,E.coliandP.aeruginosahave341 been attributed to the activity of GGDEF and EAL proteins (Simm et al., 2004). 342 WhilstAR13andAR18bothcontainedindependentmutationsinthesamegene,343 PFLU0735,themutantswerephenotypicallydistinct,withAR13displayingincreased344 motility.Thismaybeduetotheexactlocationoftransposoninsertion,butcouldbethe345 result of a secondary mutation in the chromosome. PFLU0735 is of unknown function, but is346 closelylinkedtoPFLU0734,whichencodesaputativelipoprotein.InGram-negative347 bacteria,lipoproteinsthataremostoftenlocatedatthebacterialoutermembraneare348 frequentlyassociatedwithmanyaspectsofpathogenicityincludingmotilityandinvasion349 (Kovacs-Simonetal.,2011;Zhangetal.,1998).Forexample,mutationofFlavobacterium350 johnsoniaelipoproteinGldJseverelyimpairedglidingmotilityoveragarandglass(Braun&351 McBride,2005).Interestingly,thetransposoninstrainAR9,whichmovesfasterthanthe352 SBW25Qprogenitor,sitsintergenicallybetweenaputativeLysRregulatorandPFLU0129,353 whichispartofaputativelipoproteintransportersystem.Whilethismayaffectan354 intergenictranscript,theorientationofthetransposonissuchthatitsoutwardfacing355 terminal promoters could be activating PFLU0129. Thus, AR9 may be moving faster because356 it is secreting more surfactant. This could be a good target cluster forengineering secretion357 of surfactant to maximize biocontrol activity.358 Thebiosurfactantviscosinhasbeencharacterizedbyseveralotherresearchgroups359 andhasbeenimplicatedasimportantformotility(deBruijnetal.,2007),asabiocontrol360 agent that lyses oomycete zoospores (de Bruijn et al., 2007), for protozoan grazing defence361Accepted Article17 This article is protected by copyright. All rights reserved. (Mazzola et al., 2009) and in altering soil water characteristics (Fechtner et al., 2011). In this362 study,wehavefoundthatviscosinproductionisimportantinspideryspreading,which363 operates independently of the flagellum; weconclude spidery spreading is a type of sliding364 motility (Henrichsen, 1972). We cannot rule out the possibility that pili are also employed in365 the sliding phenotype, as they have been implicated in swarming of P. aeruginosa (Kohler et366 al.,2000).However, theevidencesuggestspiliarenot involvedinspideryspreading,asno367 pilmutantswerefoundinthegeneticscreen,andwealsoobservedthenormaltwitching368 motility phenotype for SBW25Q.369 Biosurfactants have been implicated in surface spreading in other bacteriaas well as370 SBW25(Burchetal.,2012;Raaijmakersetal.,2010;Tremblayetal.,2007).Ineffect,the371 biosurfactant reduces the surface tension of the agar, and, as the bacteria divide, they slide372 overthebiosurfactantmovingoutwardfromthepointofinoculation.Slidingmotilityis373 slower than swarming motility, which we suggestaccounts for the relatively slow spread of374 SBW25Qcomparedtothewild-type(SupplementaryFigureS2),i.e.wesuggestthatits375 motility relies on cell division rather than active propulsion by the flagellum. This correlates376 withtheobservationthatsomeoftheSBW25Qtransposonmutants(e.g.inparA,hisF,377 ampD, xerD) exhibited slower surface spreading, which may be due to their function in cell378 division and metabolism. 379 SinceweobservedthatSBW25producedviscosinandSBW25C(Fla+,Visc-)wasstill380 abletomoveoverasurface,wecanconcludethatbothflagellum-dependentmotilityand381 viscosin-dependentmotilityareinoperationontheagarplates.Thiswasalsoreflectedin382 the subtle differences in the colony edges of the different strains. The microbial mechanics383 behindswarmingandslidingmotilityhavebeenapointofcontentionformanyyears,and384 surfactantshavebeenimplicatedinboth(Murray&Kazmierczak,2008).Here,weprovide385Accepted Article18 This article is protected by copyright. All rights reserved. directevidencethatbiosurfactantviscosinproductioniscrucialintheexpressionofthe386 slidingmotilityphenotypeseenwhenflagellafunctionislost.Aninterestingobservation387 thatarisesfromourworkisthatviscosinspreadsinaradialpatternforbothSBW25and388 SBW25Q.ThisraisesthequestionofwhySBW25Qspreadsdendritically.Tremblayetal.389 (2007)proposedthatthiseffectwasmediatedbyself-produceddi-rhamnolipidattractants390 and3-(3-hydroxyalkanoyloxy)alkanoicacidrepellents.Whetherasimilarmechanism391 operates in P. fluorescens in the absence of fleQ-dependent swarming motility remains to be392 seen.393 Oneoftheunexpectedoutcomesofourexperimentshasbeentheobservationof394 differentmotilityphenotypeswhenusingthesamemediumforsurfacemotility395 experimentsasusedinotherexperimentsintheliterature.Inourexperimentswehave396 observedSBW25movementthrough0.25%LBagar(whichweinterpretasswimming)and397 overthesurfaceof0.25%LBagar,butnotoverthesurfaceof0.6%LBorSSMagar.The398 literaturewouldsuggestthatlackofmovementover0.6%agarshouldresultinthe399 conclusionthatthebacteriumcannotswarm.However,weknowfromtestingAR1(pFleQ)400 and SBW25C, which do not produce viscosin and do produce flagella, that these strains can401 move over the surface of 0.25% agar. We suggest this is likely to be swarming motility rather402 thanswimmingmotilityandthusthestrainsareactuallyabletomovebyswarming,but403 clearlythisneedsfurtheranalysis.Evidently,theconditionsusedforcarryingoutmotility404 experiments must be very carefully controlled to avoid misinterpretation. For example, the405 apparentdisparitybetweenourexperimentalobservationsandthoseofdeBruijnetal.406 (2007) suggesting a viscosin mutants cannot spread over a surfacecan be explained simply407 as a difference in methodology: to investigate the spidery spreading phenotype, we used LB408 0.25% soft-agar plates rather than 0.6% standard succinate medium which is not conducive409Accepted Article19 This article is protected by copyright. All rights reserved. to either spidery spreading or swimming. However, for comparison, we performed motility410 assays on 0.25% SSM, 0.6% SSM and 0.6% LB media (Supplementary Figure S1); these assays411 confirmedthatalthoughSBW25Cdoeshavefunctioningflagella,itcannotmoveoverthe412 surface of higher agar concentration. In doing this, wildtype SBW25 (our control) swarming413 motilitywasnotobservedatagarconcentrationsof0.6%inourexperiment,butwas414 observedon0.6%SSMagarbydeBruijnetal.(2007).Themostlikelyexplanationwillbe415 duetosubtledifferencesintheculturemediumusedineachstudy.Inourinitial416 experimentsatthestartofourstudy,wefoundthatsubtlealterationsinmedium417 preparation,suchasusingdifferentagarsthathavedifferentwaterretentionproperties,418 was sufficient to change the SBW25Q phenotype. We also observed that surface motility on419 LB was reduced and eventually abolished as we increased the agar concentration (data not420 shown,fullmediadetailsgiveninSupplementaryTableS1).Thehighsensitivityofmotility421 phenotypestoculturemediumandgrowthconditionscouldexplainthedisparityin422 observations. It may be that differences in preparation of SSM medium, including source of423 agarandvolumeofmediuminplates,isenoughtodistinguishthephenotypesinthetwo424 studies.Importantly,theobservationsfromthetwostudiessuggestthatflagellum-based425 swarmingisinsufficientforsurfacemovementoverdriersurfaces,andsotheroleof426 viscosininpromotingsurfacespreadingindrierconditionsisagoodtargetforfuture427 research.428 Ourresultsalsosuggestanecologicalroleforviscosin-mediatedmotility.Mutation429 of fleQ completely abolished the ability of SBW25 to reach the root tip, confirming a result430 previouslyobservedincompetitionexperimentswithP.fluorescensF113(Capdevilaetal.,431 2004). This suggests fleQ-flagellum-dependent swarming motility plays a key role in motility432 overahorizontalrootsurface;however,asFleQplaysaregulatoryrole,itispossiblethat433Accepted Article20 This article is protected by copyright. All rights reserved. differences could be due to phenotypes other than motility that manifest in the mutant. The434 mutationofviscosindidnotpreventSBW25fromreachingtheroottip,buttheviscosin- 435 mutantwasslowertoreachtheroottip.Thissuggestsviscosinenhancesbacterialsurface436 spreadingwhenemployingflagella,butisnotessential.Theseexperimentsindicatethat437 expressionoftheflagellumandviscosinmayaidhorizontalsurfacespread,andmay438 thereforebeimportantforspreadingoverlateralroots.Indeed,slidingmotilitylinkedto439 bacterialcelldivisionhasbeenproposedasthemethodbywhichRhizobiamovewithin440 legumerootinfectionthreads(Gage&Margolin,2000;Fournieretal.,2008).However,it441 appears they are less important for spreading in a vertical root system. This difference may442 gosomewaytowardexplainingconflictingresultsofotherstudiesontheimportanceof443 flagella to P. fluorescens root colonisation; for example, while de Wegeret al. (1987) found444 flagellawereessential,andTurnbulletal.(2001)foundanadvantagetomotilityinroot445 attachment,theresultsofBowersandParke(1993)suggestedpassivemovementwith446 water flow played a more important role than motility.447 Previous studies have observed that viscosin exhibits anti-fungal and anti-oomycete448 activitybyinducingencystmentofPythiumzoosporesandadverselyaffectingmyceliaof449 Rhizoctonia solani and Pythium ultimum, both in vitro and in planta (Nielsen et al., 1999; de450 Souzaetal.,2003).SBW25isalsoknowntoprotectplantsfromtherootpathogenP.451 ultimum,althoughtheunderlyingbasisforthiswasnotdetermined(Nasebyetal.,2001);452 our results suggest that viscosin production contributes to plant protection in SBW25, but is453 not the only factor involved. 454 Ourresultspaintacomplexpictureinwhichasinglesubstance,viscosin,fulfils455 multiplefunctions,includingmotilityandplantprotection;correspondingly,eachofthese456 functions is the result of multiple, possibly partially redundant, genetic pathways. We would457Accepted Article21 This article is protected by copyright. All rights reserved. suggestthatmotilityistheprimaryfunctionofviscosin,asswiftcolonisationofgrowing458 roots provides an obvious selective advantage to the bacterium, and that the ability to lyse459 oomycetezoosporesisanincidentalby-productthathappenstobenefitplants.However,460 there may not be a clear-cut distinction between the primary and secondary functions of a461 gene:selectiononeachroleislikelytovarydependingontheenvironmentalandsocial462 conditionsexperiencedbyabacterialpopulation.Rhizospherecommunitiesarecapableof463 rapidevolutionarychange(Kiers&Denison,2008),sowemustunderstandtheseselective464 processes if we wish to optimize the agricultural benefits of PGPRs and improve the stability465 of agriculturally beneficial traits.466 Insummary,wefoundthatviscosinplaysaroleinP.fluorescensmotility,bothin467 vitro and in planta, that is additional to the flagellum-based behaviour. This showcases the468 flexibility of motility systems used by P. fluorescens and contributes to our understanding of469 themanymotilityvariantsweobserveinnaturalpopulationsofsoilmicrobes(Achouaket470 al.,2004).Wehavealsoobservedthebeneficialimpactofviscosinonplanthealthby471 suppressingthedetrimentaleffectsofarootpathogen.Thedualfunctionofviscosin472 production in motility and anti-fungal/oomycete action identifies it as a critical trait in terms473 ofP.fluorescensfunctionasabiocontrolagent.Biosurfactantsmaybeusefultargetsfor474 formulationasaseedorplanttreatmentcompound,ortheviscosingenesforasynthetic475 biology approach to creating optimal PGPR.476 477 Experimental procedures478 479 Strains, plasmids and growth conditions480 Accepted Article22 This article is protected by copyright. All rights reserved. The principal strains and plasmids used are shown in Table 2. Pseudomonas481 fluorescens strains were routinely grown in lysogeny broth (LB) or agar (1.5%) at 27oC for 16482 h or 48 h, respectively. Escherichia coli was grown in LB broth or agar at 37oC for 16 h or 24483 h, respectively. Antibiotics and supplements were included at the following final484 concentrations (g ml-1): kanamycin, 50; X-gal (5-bromo-4-chloro-3-indolyl--D- 485 galactopyranoside), 40; nitrofurantoin, 100; tetracycline, 15. CFC (Oxoid) was used486 according to manufacturers instructions. The comparison strains used in this study are:487 wild-type P. fluorescens SBW25, and its derived fleQ deletion mutant (AfleQ), hereafter488 SBW25Q (details of constructions given below; the phenotype of this strain is flagellum489 negative (Fla-) but viscosin positive (Visc+)) - these were used as control strains.In addition490 we used the mutant SBW25viscC::TnMod-OKm (hereafter SBW25C, gratefully obtained from491 Raaijmakers and de Bruijn; Fla+, Visc-), a functional viscosin knock out, for comparison492 against viscosin mutants isolated from the transposon library. Biparental matings were done493 by mixing 750 l S17-1pir carrying either plasmid pSCR001 or pFleQ and 750 l P.494 fluorescens strain, spinning cells at 13000 rpm to obtain a pellet, which was transferred to495 an LB plate. After 16 h incubation at 30oC, the pellet was streaked onto the relevant496 selective medium.497 498 Molecular analyses499 When designing deletion of fleQ, we were mindful of our lack of knowledge of500 potential cis-encoding regulatory sites located outside of fleQ, which might be important for501 flanking genes (particularly the regulators fleS and fleR); therefore, partial deletion of the502 fleQ gene PFLU4443 was done. To do this we removed the ATPase and most of the receiver503 Accepted Article23 This article is protected by copyright. All rights reserved. domain by stitching together flanking regions of fleQ by SOE-PCR (splicing by overlapping504 extension using the polymerase chain reaction). The resulting 1.64 kb PCR product was first505 cloned into pCR8/GW/TOPO using the TA cloning kit from Invitrogen. After its DNA506 sequence was verified, the DNA fragment was cloned into the BglII site of pUIC3 (ref. Rainey507 1999). The resultant construct pUIC3-140 was then conjugated into SBW25 with the help of508 pRK2013, and transconjugants were selected on LB supplemented with tetracycline and X- 509 Gal. The double crossover mutant was selected using the previously described method of510 cycloserine enrichment (Zhang and Rainey, 2007). White colonies were picked and assessed511 by PCR to confirm double-crossover deletion of fleQ. This confirmed that the entire o54512 interaction domain and most of the receiver domain were deleted in the strain, which we513 named SBW25Q (Table 2). Transposon mutagenesis of SBW25Q with IS-O-Km/hah514 transposon was carried out by conjugation of S17-7 pir (pSCR001) with SBW25Q;515 subsequent Arbitrarily-Primed PCR and genome analysis was done to determine the location516 of transposon insertions (Giddens et al., 2007).517 518 Motility assays519 Motility assays for the observation of swarming and sliding motility used full strength520 LB with 0.25% agar. Agar plates were always carefully made by pipetting 30 ml of molten521 agar into 88 mm Petri dishes and the plates allowed to set at room temperature for 4 h. 522 Plates were then placed in a laminar flow hood and lids were then removed for 30 min. 523 Assays were conducted by dipping a sterile wire into a single colony and stabbing the wire524 into the centre of an agar plate so that the wire touched the bottom of the plate. The agar525 plates were incubated on a bench without stacking in a walk-in incubator maintained at526 Accepted Article24 This article is protected by copyright. All rights reserved. 27oC and without light. Motile bacteria were observed as large colonies that moved across527 the top of the agar.Swimming was assessed by monitoring bacterial movement through528 one-tenth strength LB 0.25 % agar; bacterial movement was observed as a halo effect with529 no bacterial movement over the surface. Twitching motility was assessed by stab530 inoculation of a single colony to the bottom of a petri dish through a 1% full strength LB agar531 layer, and subsequently visualising bacterial movement across the Petri dish at the dish-agar532 interface. 533 For the root migration experiments, initial experiments were carried out by growing534 sugar beet seedlings in sterile vermiculite microcosms after Rainey (1999) and on 1.5%535 water agar plates with the plates kept vertical to encourage normal root growth on the agar.536 A 2 l drop of 105 bacterial suspension was applied directly to the hypocotyl region on the537 agar plates and to the vermiculite-root interface in the microcosms. The latter method was538 adapted so that the plates and therefore the seedlings were laid flat before bacterial539 application to the hypocotyl. In all experiments, bacterial locomotion was assessed by540 dissecting the 1cm of the root from the tip (a distance of about 5 cm from the hypocotyl) to541 enumerate the bacteria. The root tissue was crushed in 500 l phosphate buffered saline542 with an Eppendorf pestle, made up to 1ml, serially diluted and plated onto the relevant543 medium.544 545 Plant growth promotion assays546 Five 1 cm plugs of Pythium FFP1 (a kind gift from Francesco Favaron) taken from a 5- 547 day old culture on potato dextrose agar was added to sterile sand (95 g), oats (5 g) and 20548 ml 1/10-strength Czapek Dox, mixed and incubated at 27oC for 7 days. The Pythium mat549 Accepted Article25 This article is protected by copyright. All rights reserved. (oospores and hyphae) was aseptically removed from the flask, shaken to discard loose sand550 and then mixed with SHL potting compost (Lincoln, UK) to 3% (w/w) achieving circa 104 cfu551 ml-1 Pythium. A 12-pot tray module was filled with soil with or without Pythium. Sugar beet552 seeds (14 seeds in each treatment group) were washed to discard the protective storage553 coat and then soaked in 20 ml of bacterial suspension (109 cfu ml-1) for 5 min before554 planting 1-cm deep into the compost mix. The design of the seed treatment planting with555 different bacterial genotypes was randomized and 5 seeds were planted in each pot. In556 addition there were 3 biological replicates in each treatment group. The plants were557 maintained at 20oC for 14 days and the seed germination recorded daily. After 14 days the558 plants were destructively harvested and the roots and shoots separated and assessed for559 length and weight.560 561 Photography and Electron microscopy562 P. fluorescens cells were negatively stained on copper grids with 2% uranyl acetate for563 10 min before being soaked in water for 10 min. 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Table 1.Summary of surface motility mutants of SBW25Q.795 Mutant name Transposon coordinates Phenotype*Gene code Gene name Predicted gene product and function Relevant references AR12813319-2813320 SessilePFLU2553viscCNon-ribosomal peptide synthase; surfactant synthesis (De Bruijn et al., 2007) AR22798991-2798992 SessilePFLU2552viscBNon-ribosomal peptide synthase; surfactant synthesis; linked to viscC (De Bruijn et al., 2007) AR33280199-3280200 FasterPFLU3012Putative Cytochrome C oxidase subunit; linked to 3011 (Southey-Pillig et al., 2005) (Pitcher & Watmough, 2004) AR44290355-4290356 SlowerPFLU3889Acyl-CoA dehydrogenase unknown function; linked to 3890-2 (accA), lipid biosynthesis This study AR55117778-5117779 SlowerPFLU4639Hypothetical protein - Unknown function; linked to 4638 This study AR63043000-3043001 FasterPFLU2753Putative GGDEF/EAL regulator; turnover of cyclic-di-GMP (Kulesekara et al., 2006) AR76710636-6710637 AlteredPFLU6127parAATPase involved in chromosome partitioning (Lasocki et al., 2007) AR82805802-2805803 SessilePFLU2552viscBSame as AR2 (independent mutation) (De Bruijn et al., 2007) AR9139969-139970 FasterIntergenicBetween PFLU0128 and 0129; upstream 0129, lipoprotein transporter of unknown function (Zhang et al., 1998) AR103174908-3174909 FasterPFLU2912Putative membrane protein; linked to PFLU2911, ATP-dependent DNA ligase This study Accepted Article34 This article is protected by copyright. All rights reserved. AR112814686-2814687 SessilePFLU2553viscCSame as AR1 (independent mutation) (De Bruijn et al., 2007) AR125855299-5855300 FasterPFLU5329Putative GGDEF/EAL regulator; turnover of cyclic-di-GMP (Kulesekara et al., 2006) AR13836620-836621 FasterPFLU0735Hypothetical protein - Unknown function; linked to 0734 (lipoprotein) This study AR14898973-898974 FasterPFLU0798ampDN-acetylmuramyl-L-alanine amidase responsible for breakdown of MurNAc-tri-, tetra-, and pentapeptides to release the peptides for recycling; linked to 0799 (membrane prot) (Langaee et al., 2000, Jacobs et al., 1995) AR154163213-4163214 FasterPFLU3766Hypothetical protein of unknown function This study AR16363595-363596 SlowerPFLU0331hisFHistidine metabolism; linked to 0327-0330 This study AR17898973-898975 SlowerPFLU0798ampDSame as AR14 gene function (identical mutation) (Langaee et al., 2000, Jacobs et al., 1995) AR18836593-836594 AlteredPFLU0735Same as AR13 gene function (independent mutation) This study AR194791825-4791826 FasterPFLU4345Putative GTP cyclohydolase IThis study AR205503739-5503740 SlowerPFLU5008xerDSite-specific recombinase for DNA replication, recombination and repair (Martinez-Granero et al., 2005) AR215751169-5751170 AlteredPFLU5242panCPanthothenate synthetase for Coenzyme metabolism; linked to 5241 (panB) This study Accepted Article35 This article is protected by copyright. All rights reserved. AR225855327-5855328 SlowerPFLU5329Same as AR12 (independent mutation) (Kulesekara et al., 2006) AR235530422-5530423 SlowerPFLU5035Putative transglycosylase(Suvorov et al., 2008) AR241479712-1479713 FasterPFLU1343livGAmino acid transport system; linked to 1342-5 (Nazos et al., 1985, Adams et al., 1990) AR2538838-38839 FasterPFLU0036trpBTryptophan synthesis; linked to 0035 (trpA) This study AR261870065-1870066 FasterPFLU1699Putative acyl-CoA thioesterase; close to 1700 This study AR28896889-8968890 AlteredPFLU0797Hypothetical protein of unknown function; close to 0798 (AR14 and AR17) This study 796 * Motility phenotypes are categorised here relative to their progenitor strain SBW25Q; note that all mutants shown here are therefore on797 fleQ background. See Figure 2 for photographs. 798 799 Accepted Article36 This article is protected by copyright. All rights reserved. 800 Table 2 Principal bacterial strains and plasmids801 StrainRelevant characteristicsSource/reference Pseudomonas fluorescens SBW25Wild-type strain isolated from phyllosphere of sugar beet plant.Rainey & Bailey, (1996) SBW25QUnmarked deletion of fleQ; Fla-, Visc+This work SBW25CSBW25viscC::TnMod-OKm; Fla+, Visc-de Bruijn et al. (2007) AR1AfleQ viscC::IS-O-Km/hah; Fla+, Visc-This work, Table 1 E. coli DH5oF-, recA, AlacU169(u80 lacZAM15), endA, hsdR, gyrA.Gibco-BRL S17-1pirTpr, Smr, recA, thi, hsdR-M+, RP4::2-Tc::Mu::Km::Tn7, pir lysogenSimon et al. (1983) Plasmids pUIC3Tcr, Tra-, Mob+, R6K repliconRainey (1999) pRK2013Helper plasmid, Tra+, KmrDitta et al (1980) pUIC3-140pUIC3 containing 1.64 kb DNA fragment for fleQ deletion, TcrThis work pSCR001IS-O-Km/hah plasmid, Mob+, KmrGiddens et al. (2007) pBBR1MCS-5Broad host range cloning vector, Mob+, GmrKovach et al. (1995) pFleQFull length SBW25 fleQ gene in pBBR1MCS-5, GmrGiddens et al. (2007) 802 Accepted Article37 This article is protected by copyright. All rights reserved. Figure Legends803 804 Figure 1. FleQ controls production of flagella and swarming motility, but is not necessary805 for surface motility. (a) Electron microscopy shows SBW25 produces a flagellum whereas806 SBW25Q does not; ectopic expression of FleQ in SBW25Q rescues the mutation. Arrows807 show the presence of flagella. (b) Spreading motility phenotypes of the wild-type (SBW25),808 the aflagellate mutant (SBW25Q), and the complemented mutant (SBW25Q(pFleQ)).809 810 Figure 2. Surface motility phenotypes of SBW25Q and 27 transposon mutants after 30811 hours. Note mutants are derived from SBW25Q and are therefore on a fleQ background.812 AR16, AR17 and AR23 all showed slow spreading compared to AR1, AR2, AR8 and AR11,813 which never moved even after 48 h. 814 815 Figure 3. FleQ and viscosin can both promote surface spreading motility. Surface motility816 phenotype of motility mutants on LB agar plates (0.25%) after 12 hours.817 818 Figure 4. Viscosin aids bacterial spread over plant roots. Number of colony forming units819 (CFUs/ml) across the different mutant types, shown on a log scale: wild-type (SBW25),820 flagella knock-out (SBW25Q), viscosin knock-out (SBW25C) and flagella-viscosin knock-out821 (AR1), detected at the root tip 3 days after inoculation at the hypocotyl. Each data point822 represents the mean of three biological replicates and error bars are the standard error of823 the means. Note that the minimum detection limit was 100 cfu.824 825 Figure 5. Viscosin aids seedling germination and plant growth promotion. Bar shading826 represents flagella/viscosin expression profile: white bars = no bacteria; grey striped bars =827 Accepted Article38 This article is protected by copyright. All rights reserved. flagella and viscosin production; light grey bars = viscosin expression but no flagella828 expression; dark grey bars = flagella expression but no viscosin expression. (a) Root and829 shoot length of sugar beet seedlings were measured in seeds that had been grown in the830 presence of Pythium, and soaked in either the wild-type (SBW25) or one of the mutants831 (SBW25Q, SBW25C and AR1, 11, 2 and 8) or no bacteria, prior to sowing; (b) Seedling832 germination yield of sugar beet seeds soaked in the bacteria described in (a). Plants grown833 in soil without added Pythium did not show significant differences in root or shoot834 characteristics or germination yield (P>0.05). Values are the means of 3 biological replicates,835 each containing 5 seeds. Error bars are the standard error of the means. 836 837 Supplementary Figure S1. Motility phenotypes on different nutrients and agar838 composition. Motility assays were conducted on 0.25% standard succinate medium (SSM)839 to assess the surface motility phenotype on agar used by other studies. In addition, motility840 assays were performed at 0.6% agar concentrations on SSM and LB to determine whether841 spreading motility was still observable under higher agar concentrations. All strains shown842 after 48 hours. 843 844 Supplementary Figure S2. Surface spreading reliant on viscosin alone is slower than845 spreading when the flagellum is employed. 846 The area of colony spreading and growth on an LB agar plate (0.25%), for SBW25 and847 SBW25Q was measured every 2 hours over a 26 hour period. Each data point is an individual848 replicate (with 10 replicates for SBW25, and 9 replicates for SBW25Q), closed circles849 represent SBW25 and open circles SBW25Q. Interpolation lines show the mean spreading850 across the dataset at each time-point, SBW25 is represented by a solid line and SBW25Q by851Accepted Article39 This article is protected by copyright. All rights reserved. a dotted line. The data shows that the rate of spreading is faster, and the time taken to start852 rapidly spreading is quicker in SBW25 compared to SBW25Q. Therefore, this suggests that853 surface motility that is solely dependent on viscosin has a greater dependency on cell854 replication (i.e. cells have to grow to move over the surface of the surfactant). 855 856 Accepted Article40 This article is protected by copyright. All rights reserved. 857 Supplementary Table S1:858 859 Microbiological media used for motility assays860 861 MediumRecipe and Manufacturers details Luria-Bertani (LB) broth10g/l tryptone (BD, Oxford, UK) 5g/l NaCl (Oxoid Ltd., Basingstoke, UK) For agar plates, use Difco Agar (BD, Oxford, UK) at 10 g/l (twitching), 6 g/l or 2.5 g/l (spreading/swarming) depending on motility type being tested. 1/10 strength LB with 2.5 g/l agar were used for swimming plates. Standard succinate medium (SSM)6 g/l KH2PO4 (Sigma Aldrich, Dorset, UK) 3 g/l K2HPO4 (Fisher Scientific, Loughborough, UK) 1 g/l (NH4)2SO4 (Fisher Scientific, Loughborough, UK) 0.2 g/l MgSO4.7H2O (Fisher Scientific, Loughborough, UK ) 4 g/l Succinic acid (Sigma Aldrich, Dorset, UK) Correct pH to 7.0 using KOH For agar plates add 6 g/l or 2.5 g/l Difco Agar (BD, Oxford, UK) depending on motility type being tested.862 863 Accepted Article41 This article is protected by copyright. All rights reserved. 864 865 emi_12469_f1a866 867 Accepted Article42 This article is protected by copyright. All rights reserved. 868 869 emi_12469_f1b870 871 Accepted Article43 This article is protected by copyright. All rights reserved. 872 emi_12469_f2873 874 Accepted Article44 This article is protected by copyright. All rights reserved. 875 emi_12469_f3876 877 Accepted Article45 This article is protected by copyright. All rights reserved. 878 emi_12469_f4879 880 Accepted Article46 This article is protected by copyright. All rights reserved. 881 emi_12469_f5a882 883 Accepted Article47 This article is protected by copyright. All rights reserved. 884 emi_12469_f5b885 886 Accepted Article