ENVIRONMENTAL MICROBIOLOGY crossm Pumilacidin-Like Lipopeptides Derived from Marine Bacterium Bacillus sp. Strain 176 Suppress the Motility of Vibrio alginolyticus D PengyuanXiu,a,b,cRuiLiu,a,b DechaoZhang,dChaominSuna,b o KeyLaboratoryofExperimentalMarineBiology,InstituteofOceanology,ChineseAcademyofSciences, w n Qingdao,Chinaa;LaboratoryforMarineBiologyandBiotechnology,QingdaoNationalLaboratoryforMarine lo ScienceandTechnology,Qingdao,Chinab;UniversityofChineseAcademyofSciences,Beijing,Chinac; a DepartmentofMarineOrganismTaxonomyandPhylogeny,InstituteofOceanology,ChineseAcademyof d e Sciences,Qingdao,Chinad d f r ABSTRACT Bacterial motility is a crucial factor during the invasion and colonization o m processes of pathogens, which makes it an attractive therapeutic drug target. Here, Received22February2017 Accepted3April we isolated a marine bacterium (Vibrio alginolyticus strain 178) from a seamount in 2017 ht t Acceptedmanuscriptpostedonline7April p the tropical West Pacific that exhibits vigorous motility on agar plates and severe 2017 :/ pathogenicity to zebrafish. We found that V. alginolyticus 178 motility was signifi- /a CitationXiuP,LiuR,ZhangD,SunC.2017. e cantly suppressed by another marine bacterium, Bacillus sp. strain 176, isolated from Pumilacidin-likelipopeptidesderivedfrom m the same niche. We isolated, purified, and characterized two different cyclic lipopep- marinebacteriumBacillussp.strain176 . a suppressthemotilityofVibrioalginolyticus. tides (CLPs) from Bacillus sp. 176 using high-performance liquid chromatography, s ApplEnvironMicrobiol83:e00450-17.https:// m mass spectrometry, and nuclear magnetic resonance spectroscopy. The two related doi.org/10.1128/AEM.00450-17. . o CLPs have a pumilacidin-like structure and were both effective inhibitors of V. algi- EditorHideakiNojiri,UniversityofTokyo r g nolyticus 178 motility. The CLPs differ by only one methylene group in their fatty Copyright©2017AmericanSocietyfor / Microbiology.AllRightsReserved. o acid chains. In addition to motility suppression, the CLPs also induced cell aggrega- n AddresscorrespondencetoDechaoZhang, tion in the medium and reduced adherence of V. alginolyticus 178 to glass sub- A [email protected],orChaominSun, p strates. Notably, upon CLP treatment, the expression levels of two V. alginolyticus [email protected]. r flagellar assembly genes (flgA and flgP) dropped dramatically. Moreover, the CLPs P.X.andR.L.contributedequallytothisarticle. il 2 , inhibited biofilm formation in several other strains of pathogenic bacteria without 2 inducing cell death. This study indicates that CLPs from Bacillus sp. 176 show prom- 0 1 ise as antimicrobial lead compounds targeting bacterial motility and biofilm forma- 9 tionwithalowpotentialforelicitingantibioticresistance. b y IMPORTANCE Pathogenic bacteria often require motility to establish infections and g u subsequently spread within host organisms. Thus, motility is an attractive therapeu- e s tic target for the development of novel antibiotics. We found that cyclic lipopeptides t (CLPs) produced by marine bacterium Bacillus sp. strain 176 dramatically suppress the motility of the pathogenic bacterium Vibrio alginolyticus strain 178, reduce bio- film formation, and promote cellular aggregation without inducing cell death. These findings suggest that CLPs hold great promise as potential drug candidates target- ing bacterial motility and biofilm formation with a low overall potential for trigger- ingantibioticresistance. KEYWORDS lipopeptide,pumilacidin,Vibrioalginolyticus,motility,antibiotic, antimicrobial,antibiofilm Bacterial motility is one of several crucial factors during the initial infection and colonization processes of pathogens. In particular, it helps pathogens overcome therepulsiveforcesbetweenthebacterialcellwallandthehosttissuesandfacilitates hostattachment(1–3).Theflagellumisoneofthemainlocomotiveorganellesrespon- June2017 Volume83 Issue12 e00450-17 AppliedandEnvironmentalMicrobiology aem.asm.org 1 Xiuetal. AppliedandEnvironmentalMicrobiology sibleforbacterialmotilityandisthusconsideredanimportantvirulencefactorinmany pathogens (2, 4, 5). Flagellar motility is a complex biological process requiring the synthesis and assembly of numerous flagellar components, energetic coupling, and chemotacticcontroloverflagellarrotationtoafforddirectedmotion(3,4). VibrioalginolyticusisacommonGram-negativehalophilicbacterium.Itisanoppor- tunisticpathogenandamajorcauseofvibriosisinaquaticanimals,resultinginsevere economiclossesworldwide(6,7).RecentstudiessuggestedthatV.alginolyticusisalso apotentialhumanpathogen(8).UnlikeotherVibriospecies,V.alginolyticususually infects tissues via superficial routes (i.e., wounds, ears, and eyes) rather than the gastrointestinal tract (8). In V. alginolyticus, there are two types of flagella, polar and lateral. Polar flagella are constitutively expressed, while lateral flagellar expression is induced when polar flagellar function has been disabled (9–11). Furthermore, the flagellar assembly pathway of V. alginolyticus has been implicated in cell adhesion D processes(12,13). o Overuseofbroad-spectrumantibioticsandtheaccompanyingproliferationofdrug- w n resistant bacteria have stimulated efforts to develop environment-friendly biocontrol lo measures to reduce health hazards and environmental pollution (14, 15). In recent a d years, the antimicrobial properties of biological surfactants have been increasingly e d recognized and harnessed for antibacterial, antifungal, and antiviral applications (16– f 18). Lipopeptides are the most widely reported class of biosurfactants having antimi- ro m crobialandantiadhesiveactivityagainstpathogenicbacteria,duetotheamphipathic nature of their peptide and fatty acid components (19, 20). Many antimicrobial lipo- h t t peptidesidentifiedandcharacterizedtodatearederivedfrombacterialspeciesinthe p : / Bacillus genus. These include surfactin, fengycin, iturin, bacillomycin, mycosubtilin, / a lichenysin, and pumilacidin (21–26). To date, none of these lipopeptides have been e m reportedtopossessanyantimotilityactivity. . a Inthisstudy,wedemonstratethatthemarinebacteriumV.alginolyticusstrain178 s m exhibitsstrongmotilityonagarplatesandcauseshighlevelsofmortalityinzebrafish. . o More excitingly, we show that this motility is dramatically inhibited by two cyclic r g lipopeptides (CLPs) derived from a competing bacterium (Bacillus sp. strain 176). We / o purified and characterized the active antimotility compounds and determined their n structuralandfunctionalproperties.Inordertoexplorethemechanismofactionofthe A p CLPs, we also investigated their impact on cell aggregation, adherence, and the r expressionofflagellarassemblycomponentsinV.alginolyticus. il 2 , 2 RESULTS 0 1 The discovery and pathogenicity assays of the vigorously swarming strain V. 9 alginolyticus 178. The initial purpose of this study was to investigate the marine b y bacterial resource in the seamount area of the tropical Western Pacific. During the g courseofbacterialisolation,V.alginolyticusstrain178attractedourattention,because u e itdisplayedvigorousswarmingonthe2216Emediumwith1%agar(Fig.1A).Tofurther s t analyzetheswarmingofstrain178,weinvestigateditsmotilityonplateswithdifferent agarconcentrationsvaryingfrom0.5%to2%.Surprisingly,strain178couldcoverthe entire 9-cm plate surface within 12 h with an agar concentration up to 1% (Fig. 1A). Withtheincreaseofagarconcentrationinthemedium,themotilityofthisbacterium graduallydecreased.However,itcouldstilloccupythefullplatesurfacewithin24or36 hevenwithagarconcentrationsupto1.5%or2%,respectively(Fig.1A).Inviewofthe highhomology(99%identity)withmarinebacteriumVibrioalginolyticusby16SrRNA genesequencingandphylogeneticanalysis(seeFig.S1Ainthesupplementalmaterial), thevigorouslyswarmingstrainwasdesignatedV.alginolyticus178.Tofurthervalidate thestrongmotilityabilityofV.alginolyticus178,wecompareditsmotilityabilitywith thatofsevenotherV.alginolyticusstrains(C1,C11,C38,C48,CJ11,CJ26,andCT30).The resultsshowedthatmostV.alginolyticusstrainsweremotile;however,V.alginolyticus 178exhibitedthegreatestswarmingmotilityofalleightstrainsanalyzed(seeFig.S2). V. alginolyticus is a leading cause of vibriosis, causing opportunistic infections in humans in association with raw seafood contamination (8). To identify the possible June2017 Volume83 Issue12 e00450-17 aem.asm.org 2 LipopeptidesSuppresstheMotilityofV.alginolyticus AppliedandEnvironmentalMicrobiology D o w n lo a d e d FIG1CharacterizationofmarinebacteriumstrainV.alginolyticus178.(A)MotilityassaysofV.alginolyticus178intheplatescontaining0.5%,1%,1.5%,or2%agar f r atdifferenttimepoints.(B)ThepathogenicityassayofV.alginolyticus178tozebrafish.(C)CumulativemortalityratesofzebrafishcausedbyV.alginolyticus178. o m h t t pathogenicityofV.alginolyticus178,weperformedinjectionexperimentsinzebrafish. p : Theresultsshowedthatdeadzebrafisheswereobservedonthefirstdayafterinfection, // a andmortalitiescontinuedincreasinguntilthesixthdayoftreatmentwithhigh(3(cid:2)104 e m cells)andlow(3(cid:2)103cells)concentrationsofV.alginolyticus178cells.Comparedwith . a zebrafish in the control group, obvious hemorrhage and abdominal dropsy were s m observedinzebrafishafterinjectionwithV.alginolyticus178(Fig.1B).Thecumulative . o mortality rates in the groups challenged with high and low bacterial concentrations r reached100%and50%,respectively,andweresignificantlyhigher(P(cid:3)0.01)thanthat g/ o ofthecontrolgroup(Fig.1C).ForKoch’spostulates,87.33%(262/300)ofthereisolated n bacteriawereidentifiedassharingthesame16SrRNAgenefragmentwithstrain178. A Thus, V. alginolyticus 178 is a marine bacterium with strong motility and severe p r pathogenicity. il 2 Screening and identification of marine bacteria with antimotility activity , 2 against V. alginolyticus 178. Bacterial motility is proposed to be closely related to 0 1 virulence(4).Therefore,motilitymaybeanattractivetargetfordrugsthatcanprevent 9 the infective process. To find potential natural products inhibiting the motility of V. b y alginolyticus178,weperformedthescreeningdescribedinthe“MaterialsandMethods” g section.Notably,bacteriumstrain176wasdemonstratedtosignificantlysuppressthe u e motility of V. alginolyticus 178 based on the plate assays (Fig. 2A). Also, the colony s t FIG2 AntimotilityassayofBacillussp.176(b)againstV.alginolyticus178(a).(A)Plate-basedmotilityassayshowsazoneofinhibitionaroundthecolonyof Bacillussp.176.(B)MorphologychangesinthemarginsofV.alginolyticus178intheabsence(left)orpresence(right)ofBacillussp.176. June2017 Volume83 Issue12 e00450-17 aem.asm.org 3 Xiuetal. AppliedandEnvironmentalMicrobiology D o w n lo a d e d f r o m h t t p : / / a e m . a s FIG3 Structuralelucidationofcycliclipopeptides(CLP1,left;CLP2,right)producedbyBacillussp.176.(A)MS/MSspectraofCLP1andCLP2.(B)Fragmentions m ofCLP1andCLP2.(C)StructuresofCLP1andCLP2.ThearrowsindicatethepositionsofCLP1andCLP2thataredifferent. . o r g / o marginsofV.alginolyticus178showedcleardifferencesinmorphologywithorwithout n coculturingwithstrain176(Fig.2B).Furthermore,theexpansionofthecolonymargins A p ofV.alginolyticus178wasinhibitedevenwithouttouchingstrain176,whichledtothe r hypothesisthatbacteriumstrain176producedanextracellulardiffusibleinhibitorofV. il 2 alginolyticus178motility. , 2 It is noteworthy that strain 176 was isolated from the same niche as that of V. 0 1 alginolyticus178,andits16SrRNAgenesequencesharedhighsimilarities(greaterthan 9 98%) with Bacillus aerophilus, Bacillus aerius, and Bacillus pumilus (see Fig. S1B in the b y supplemental material). Additionally, strain 176 also clustered with these strains ac- g cordingtophylogeneticanalysis(seeFig.S1B).Strain176wasthusdefinedasBacillus u e sp.176. s t Purification,structuralelucidation,andactivityassaysoflipopeptidesderived from Bacillus sp. 176. To obtain the active compound inhibiting the motility of V. alginolyticus178,purificationwasperformedasdescribedin“MaterialsandMethods.” The filter paper disc assay was carried out to follow the trail of the bioactive com- pound(s)usingV.alginolyticus178astheindicatorbacterium.Notably,twocompounds werefoundtohaveantimotilityactivitybasedonhigh-pressureliquidchromatography (HPLC) analyses (see Fig. S3A in the supplemental material). Electrospray ionization mass spectrometry (ESI-MS) results showed that the mass-to-charge ratios (m/z ) of compounds 1 and 2 displayed the predicted molecular formulas C H N O and 57 101 7 13 C H N O , respectively (see Fig. S3B). Based on our purification scheme, we de- 58 103 7 13 duced that these two compounds were lipopeptides; therefore, the amino acid se- quencesofthesetwocompoundswerefurtheranalyzedbytandemmassspectrometry (MS/MS). In the MS/MS spectra, fragment ion sets were observed in compounds 1 and 2, and similar molecular weightsof[M(cid:4)Na](cid:4)weredetectedatm/z1114.7336 and 1128.7505 (Fig. 3A). For compound 1, the peptide chain sequence was methoxy June2017 Volume83 Issue12 e00450-17 aem.asm.org 4 LipopeptidesSuppresstheMotilityofV.alginolyticus AppliedandEnvironmentalMicrobiology glutamic acid (mGlu)-leucine (Leu)-Leu-Leu-methoxy aspartic acid (mAsp)-Leu- isoleucine(Ile)(Fig.3BandC).Thecleavagemodeofaminoacidresiduesindicatedthe existence of a cyclic structure (27). MS/MS data also showed that compound 2 con- tainedthesameaminoacidsequenceasdidcompound1(Fig.3BandC). Moreover, 1H nuclear magnetic resonance (NMR) and 13C NMR spectroscopy anal- yseswereperformedtoclarifytheexactstructureoftheseactivecompounds.Inthe1H NMRspectrum,alongalkylchain((cid:2)1.4-1.1)andapeptidebackbone(7amineprotons, (cid:2)8.22to7.76and7(cid:3)-aminoacidprotons,(cid:2)4.6to4.1)wereobservedincompound 1. A further proton next to oxygen was detected with a resonance at (cid:2)5.02, which mightbeapartoflactonering.Also,twointensesingletsat(cid:2)3.60and3.58weresimilar to those in the 1H NMR spectrum of the lipopeptide esters (28, 29), suggesting the existence of two methoxy groups in compound 1 (see Fig. S4 in the supplemental material).Inthe13CNMRspectrum,signalsof10carbonylgroups((cid:2)173.36to169.98) D weredetectedincompound1,9ofwhichwereattributedtoaminoacids.Theresonances o at (cid:2)50.11 and 51.10 in the 13C NMR spectrum also demonstrated the methoxy group w n attached on the Glu and Asp residues, respectively. In addition, a signal at (cid:2) 72.18 lo (correspondingto(cid:2)H5.02)wascharacteristicfor(cid:4)-hydroxyfattyacids(30).Thesimilar ad structureofcompound2wasidentifiedbyNMRanalysis,inwhichanexcessmethylene e d groupwaslocatedonitsalkylchain(seeFig.S5).Accordingtotheresultsofstructural f r analyses,compounds1and2weredefinedaspumilacidin-likelipopeptides(CLP1and o m CLP2)bycomparisonwithspectroscopicdatainpreviousstudies(Fig.3C)2(6). h Next, we checked the antimotility activities of these two cyclic lipopeptides (CLP1 tt p and CLP2). Notably, CLP1 and CLP2 (100 (cid:5)g/ml) showed similar antimotility activity : / / againstV.alginolyticus178,buttherewasnosignificantsynergisticantimotilityeffect a e observed (Fig. 4A). Hence, the cyclic lipopeptide used in the present study was a m mixturewithequalproportionsofCLP1andCLP2anddefinedasCLPsinthefollowing .a s study. Furthermore, the bactericidal effect of CLPs against V. alginolyticus 178 was m investigated.TheresultsshowedthatthegrowthofV.alginolyticus178inthepresence .o of CLPs with a final concentration of 100 (cid:5)g/ml was similar to that of the negative rg / control group (Fig. 4B). This indicated that CLPs from strain 176 affected only the o n motilityofV.alginolyticus178butdidnotkillit.Motilityisacrucialvirulencefactorof A Vibriospp.;thus,wecheckedthepathogenicityofV.alginolyticus178aftertreatment p r with CLPs. The results showed that CLP-treated V. alginolyticus 178 caused a signifi- il cantly lower (P (cid:3) 0.01) cumulative mortality rate than that caused by control V. 2 , alginolyticus178andkilledonly26.7%ofzebrafishesafterinjectionfor7days(Fig.4C), 2 0 which strongly suggested that CLPs could effectively reduce the pathogenicity of V. 1 9 alginolyticus178. b CellaggregationofV.alginolyticus178promotedbyCLPs.Next,wesoughtto y g investigate the effects of CLPs on V. alginolyticus 178 cells. Surprisingly, the obvious u e aggregation and sedimentation of V. alginolyticus 178 cells were observed at the s bottomofthetesttubeaftertreatmentwith300(cid:5)g/mlCLPs(Fig.5A).Theaggregation t activityofCLPswasfurtherverifiedbycrystalvioletstaining.Aftertreatmentwith300 (cid:5)g/ml CLPs for 24 h, most of the V. alginolyticus 178 cells were aggregated at the bottomof96-wellmicrotiterplatesandwerethendyedbythecrystalviolet(Fig.5B). There were fewer cells from strain 178 treated with dimethyl sulfoxide (DMSO) ob- served at the bottom of the well (Fig. 5B). Cell aggregation was quantified through measuring the crystal violet staining solution with optical density at 595 nm (OD ). 595 The results showed that the aggregation was dose dependent, and the minimum thresholdconcentrationofCLPswas25(cid:5)g/ml(Fig.5C). Moreover, the morphological features of V. alginolyticus 178 after treatment with 100(cid:5)g/mlCLPswerecheckedbyscanningelectronmicroscopy(SEM).Forthecontrol, or in the presence of DMSO, cells of strain 178 were distributed evenly with regular morphology(Fig.6AandB).However,cellsofV.alginolyticus178werefoundtogather aroundeachotherin50(cid:5)g/mlCLP-treatedgroups(Fig.6C).Morecellaggregationwas detectedinthe100(cid:5)g/mlCLP-treatedgroup(Fig.6D),andthecauseseemedtobethe June2017 Volume83 Issue12 e00450-17 aem.asm.org 5 Xiuetal. AppliedandEnvironmentalMicrobiology D o w n lo a d e d f r o m h t t p : / / a e m . a s m . o r g / o FIG4EffectsofCLP1andCLP2onthemotilityandgrowthofV.alginolyticus178.(A)Plate-basedantimotilityactivityassays n ofCLP1(a),CLP2(b),CLP1(cid:4)CLP2(c),andDMSO(d).Sterilefilterpaperdisks,eachcontaining200(cid:5)gofpurifiedCLPsora A DMSOcontrol,areindicated.(B)GrowthassayofV.alginolyticus178inthepresenceof100(cid:5)g/mlofCLPsorDMSO.(C) p PathogenicityassaysofV.alginolyticus178tozebrafishintheabsenceorpresenceofCLPs. r il 2 , 2 conglutination of flagella. The motility ability of Vibrio spp. is closely related to the 0 1 existence of flagella (31); thus, the effects of CLPs on the flagellar biosynthesis of V. 9 alginolyticus 178 were checked by transmission electron microscopy (TEM). The TEM b y resultsshowedthatonepolarflagellumandnumerouslateralflagellaexistedaround g eachcellofV.alginolyticus178(Fig.7AandB),whichmightexplainwhythisbacterium u e possesses vigorous swarming ability. Consistent with the SEM results, cells of V. s t alginolyticus178werefoundtograduallyaggregatewiththeincreaseinCLPconcen- tration(Fig.7CandD).Notably,flagellaofV.alginolyticus178couldbeclearlyobserved whentreatedbyCLPs(Fig.7CandD),andtherewerenosignificantdifferencesinthe number of flagella for cells with or without CLP treatment (Fig. 7C and D). Thus, we proposethatCLPsmightmainlyaffectthefunctionsrelatedtothemotilityofflagella ratherthantheirquantity. Cell aggregation is correlated to biofilm formation (32). Next, we investigated cell aggregation in the bacteria with or without biofilm formation capacity. Of the eight bacterial species selected, four possessed biofilm formation ability, including Vibrio anguillarum,Pseudomonasaeruginosa,Bacillussubtilis,andBacillussp.176,whileVibrio splendidus,Vibriovulnificus,Staphylococcusaureus,andPseudomonasstutzericouldnot form the biofilm (see Fig. S6 in the supplemental material). Compared with biofilm formationbynontreated,orDMSO-treated,strains,biofilmformationbyVibrioanguil- larum,P.aeruginosa,B.subtilis,andBacillussp.176wassuppresseddramaticallyafter treatmentwith300(cid:5)g/mlCLPs(seeFig.S6).Forbacteriathatcannotformbiofilm,the June2017 Volume83 Issue12 e00450-17 aem.asm.org 6 LipopeptidesSuppresstheMotilityofV.alginolyticus AppliedandEnvironmentalMicrobiology D o w n FoIbGse5rvVe.dalignincoulylttiucrues1tu7b8ecselalfatgegrrtergeaattimonenatsswayitihntehitehperreDseMnSceOoofrCL3P0s0.(A(cid:5))gC/melllaCgLgPrse.g(aBt)ioCneollfVa.gaglgreingoaltyiotincuso1f7V8. loa alginolyticus178observedinmicrotiterplatesstainedwithcrystalviolet.ThecellsweretreatedwithDMSOor300 d (cid:5)g/mlCLPs.(C)QuantitativecellaggregationassayatdifferentconcentrationsofCLPs.**,P(cid:3)0.01. e d f r o m aggregationandsedimentationofcellsweresignificantlypromotedby300(cid:5)g/mlCLPs h andhadaneffectonV.alginolyticus178(seeFig.S6). t t Effect of CLPs on cell adherence to glass slides of V. alginolyticus 178. Lipo- p : / peptideisthemostwidelyreportedclassofbiosurfactantshavingantiadhesiveaction /a e againstpathogenicbacteria(19,20).Therefore,wecheckedtheeffectofCLPsonthe m celladherenceofV.alginolyticus178.CellstreatedwithDMSOorCLP(100(cid:5)g/ml)were . a incubated with submerged glass slides for 5, 15, or 30 min in 2216E liquid medium. s m . o r g / o n A p r il 2 , 2 0 1 9 b y g u e s t FIG6SEMimagesofV.alginolyticus178cellsfollowingtreatmentwithCLPs.V.alginolyticus178cellmorphologywithoutany treatment(A),withDMSOtreatment(B),with50(cid:5)g/mlCLPs(C),andwith100(cid:5)g/mlCLPtreatment(D).Thescalebarapplies toallpanels. June2017 Volume83 Issue12 e00450-17 aem.asm.org 7 Xiuetal. AppliedandEnvironmentalMicrobiology D o w n lo a d e d f r o m h t t p : / / a e m . a s m FIG7TEMimagesofV.alginolyticus178cellsfollowingtreatmentwithCLPs.V.alginolyticus178cellmorphologywithoutany treatment(A),withDMSOtreatment(B),with50(cid:5)g/mlCLPs(C),andwith100(cid:5)g/mlCLPtreatment(D).Thescalebarapplies .o toallpanels.Thewhiteandblackarrowsindicatethepolarflagellumandperiflagella,respectively. rg / o n Thereafter, the cells attached to the glass slides were stained with crystal violet and A counted under a light microscope. Compared with the DMSO-treated group, the p r capacity of cells to adhere to slides was dramatically decreased in the CLP-treated il 2 group(seeFig.S7AandBinthesupplementalmaterial).Thecellcountsofstrain178 , 2 adherencetotheslidesintheDMSO-treatedgroupwere4.52-,3.42-,or5.37-fold(P(cid:3) 0 1 0.01)morethanthoseintheCLP-treatedgroupat5,15,or30min,respectively(seeFig. 9 S7C).TheseresultsindicatedthatCLPsfromBacillussp.176couldsignificantlyimpact b y thecapacityofV.alginolyticus178toadheretotheglassslidesurface. g TheinfluenceofCLPsontheflagellarassemblyofV.alginolyticus178.Boththe u e motility and adherence of bacteria have been mentioned in previous studies as s t correlatingwiththeflagellum(2,12).SinceCLPscouldinhibitthemotilityandadher- enceofV.alginolyticus178,theinfluenceofCLPsontheflagellarassemblywasfurther testedonthetranscriptionlevelbyquantitativereal-timePCR(qRT-PCR).Sevenimportant genes((cid:6)54,flhF,fliG,pomA,pomB,flgA,andflgP)responsiblefortheflagellarassemblyofV. alginolyticus 178 were selected for the qRT-PCR assay. Among the proteins encoded by these genes, (cid:6)54 and FlhF were located in the cytoplasm, FliG was in the cytoplasmic membrane, PomA and PomB formed a complex connecting the cytoplasmic membrane andthecellmembrane,FlgAwasseatedinthepeptidoglycanlayerofthecellmembrane, and FlgP was outside the cell membrane (Fig. 8A). The results showed that relative expression levels of the pomA, pomB, (cid:6)54, flhF, and fliG genes were all significantly upregulated after treatment with 100 (cid:5)g/ml CLPs, namely, they were 2.83-, 6.32-, 4.46-, 2.11-,and3.28-fold,respectively,greaterthanthoseofthecontrol(Fig.8B).Itisnoteworthy thatalloftheupregulatedproteinswerelocatedintheintracellularportionoftheflagellar structure (Fig. 8A). However, the mRNA expression of both flgA and flgP was decreased dramaticallyafterCLPtreatmentandwasonly0.23-and0.46-fold,respectively,lessthan June2017 Volume83 Issue12 e00450-17 aem.asm.org 8 LipopeptidesSuppresstheMotilityofV.alginolyticus AppliedandEnvironmentalMicrobiology D o w n lo a d e FIG8EffectsofCLPsontheexpressionofflagellarassembly-relatedgenesinV.alginolyticus178.(A)ProposedoverallflagellarstructureofV.alginolyticus.(B) d QuantitativeRT-PCRassaysofflagellargeneexpressioninV.alginolyticus178.CellsweretreatedwitheitherDMSOor100(cid:5)g/mlCLPs.Alldataarerelativeto f r theexpressionlevelsfoundintheDMSOcontrol(cid:5)thestandarderror(n(cid:6)4).*,P(cid:3)0.05;**,P(cid:3)0.01. o m h t t that of the control (Fig. 8B). Notably, the downregulated proteins were extracellular p : / moleculesofflagellafromV.alginolyticus178. /a e m DISCUSSION . a Bacterialswarmingmotility,afrequentlyobservedbehavior,isanimpressiveformof s m communitymotilityinwhichfoundingcoloniesbegintoconcentricallyexpandatsome . o rate (33). Swarmer cells display increased tolerance for antibiotics and increased r g virulenceininfectionmodelscomparedwithnonswarmingcells(33).Inthisstudy,the / o marine bacterium V. alginolyticus 178 showed vigorous swarming capability even on n 2% agar plates (Fig. 1A; Fig. S2A). Importantly, this bacterium could severely infect A p zebrafish and lead the fishes to die in a short time (Fig. 1B and C), which makes it a r potentialpathogeninmarineaquaculture. il 2 ToobtainpotentialcompoundsagainstV.alginolyticus178,weperformedscreening , 2 via targeting bacterial motility. Bacillus sp. 176 was found to significantly inhibit the 0 1 motilityofV.alginolyticus178(Fig.2).Correspondingly,twocycliclipopeptides(CLPs) 9 (includingCLP1andCLP2)producedbyBacillussp.176wereidentifiedwiththetypical b y aminoacidsequenceofpumilacidin(Glu-Leu-Leu-Leu-Asp-Leu-Ile)(Fig.3C),differingin g only the fatty acid chains (C H - and C H -) (Fig. 3C) with reported pumilacidins u 13 27 14 29 e (34).Interestingly,thefattyacidmoietiesofthetwolipopeptidesweredifferentinonly s t onemethylenegroup(CH -)(Fig.3C).Althoughnumerousreportshavedemonstrated 2 the mechanism of lipopeptides as biosurfactants, only a few relationships between theirchemicalstructuresandfunctionshavebeenwelldefined.Notably,bothGluand Asp in CLP1 and CLP2 are acidic amino acids and are further modified by methoxy (CH O-), an electron-donating group (35). Thus, some amino acids of CLP1 and CLP2 3 could be negatively charged at a physiological pH (7.35 to 7.45), which might be beneficial for the interaction with cations in the environment. It is well known that lipopeptidesareproducedmainlybyBacillusspeciesandhavebeenfoundtopossess various biological activities on biological controls, including the surfactin, iturin, and fengycinfamilies(26).Amongtheselipopeptides,pumilacidinwasanovellipopeptide similartosurfactinandwasfirstcharacterizedfromBacilluspumilus(26).Althoughthere havebeenonlyafewstudiesofpumilacidinafteritsdiscovery,ithasbeenreportedto possessdifferentfunctions,suchasantiviral(26),antifungal(34),andantibacterial(36) applications.Todate,noneofthelipopeptideshavebeenreportedtopossessantimo- tilityactivity. June2017 Volume83 Issue12 e00450-17 aem.asm.org 9 Xiuetal. AppliedandEnvironmentalMicrobiology CLPs from Bacillus sp. 176 were further found to significantly increase the cell aggregationofV.alginolyticus178(Fig.5).Asbiosurfactants,CLPsarealsoamphipathic moleculeswithhydrophilicandhydrophobicmoieties(18)thatinfluencethepolarityof thebacterialcellmembrane(37).Thus,theCLPssequentiallyinducedcellaggregation. ThisfunctionofCLPsseemednonspecific,becausesimilareffectsalsooccurredinother bacteriathatdidnotformabiofilm,includingV.splendidus,V.vulnificus,S.aureus,and P. stutzeri (see Fig. S6). However, biofilm-forming bacteria, such as V. anguillarum, P. aeruginosa, B. subtilis, and even Bacillus sp. 176, displayed an effective decrease in biofilm formation upon treatment with CLPs (see Fig. S6). Therefore, it would be of greatinteresttoinvestigatewhetherthequorum-sensingsystemofV.alginolyticus178 isaffectedbyCLPs. It is known that polar and lateral flagella are usually important locomotive organ- elles and virulence factors for bacterial motility and colonization (2, 4, 5). However, D basedontheTEMresults(Fig.7),theflagellarnumbersofV.alginolyticus178didnot o changeobviously,whichindicatesthatCLPsmightaffectthefunctionratherthanthe w n quantityofflagella.InVibriospecies,flagellargenesarehierarchicallyexpressedunder lo strictcontrol(5,38).TheFlhFproteindeterminesthepolarlocationandproductionof a d flagella(5).ThepomAandpomBgenesarepositivelyregulatedby(cid:6)28,expressingthe e d innermembranesodiumionchannelcomplexPomA/PomBofV.alginolyticus(39).Also, f FliG is the essential rotor component of flagella and has been reported to interact ro m withthecytoplasmicregionofPomAbyelectrostaticinteractions(40–42).FlgAisa periplasmic chaperone essential for P ring formation (43), and FlgP is an outer h t t membrane lipoprotein functioning in motility to mediate flagellar stability and p : / influenceattachmentandcolonization(31).Thesetwomoleculesareimportantfor / a motility of the flagellar complex by keeping the energy transfer and the structural e m stabilization (1). The expression levels of inner membrane genes were significantly . a upregulated after CLP treatment (Fig. 8); however, two other genes, flgA and flgP, s m were downregulated (Fig. 8). . o Altogether, this study illustrates how Bacillus sp. 176 produces pumilacidin-like r g lipopeptides to defeat piracy by V. alginolyticus 178. Going further, this study tells us / o thatsomebacteriahaveevolvedcleverstrategiesforcompetingwiththeiropponents n for better survival. Moreover, CLPs from Bacillus sp. 176 could effectively inhibit the A p biofilm formation of several other pathogenic bacteria but not kill them. Thus, this r demonstrates the potential for CLPs from Bacillus sp. 176 to become promising drug il 2 candidatesfortargetingbacterialmotilityorbiofilmformationwithalowpotentialfor , 2 antibioticresistance. 0 1 9 MATERIALSANDMETHODS b Bacterialstrainisolation,identification,andcultureconditions.Sedimentsandseawaterwere y collectedneartheYapTrenchduringtheseamountcruiseoftheR/VKexueinthetropicalWesternPacific g u inMarch2016(139°3802=E,11°44162=N).Themarinebacterialstrainsusedinthisstudywereisolated e fromtheabove-describedsamplesviathedilutionmethodasdescribedpreviously(44)andculturedin s t modified Zobell 2216E broth (5 g/liter tryptone, 1 g/liter yeast extract, 1 liter filtered seawater [pH adjustedto7.4to7.6])at28°C.Thesinglecolonieswerefurtherpurifiedin2216Eplateswith1%agar for several rounds before downstream applications. To phylogenetically classify the bacterial strains, genomicDNAwasextractedfromdifferentisolates,andPCRwasperformedtoamplifythe16SrRNA genesequenceusingtheuniversalprimers27F(5=-AGAGTTTGATCCTGGCTCAG-3=)and1541R(5=-AAG GAGGTGATCCACCC-3=).The16SrRNAgenesequencewasthencomparedwithrelatedsequencesin public databases using NCBI-BLAST (see http://www.ncbi.nlm.nih.gov/BLAST) and the phylogenetic analysisprogramMEGA6(45). Motility and pathogenicity assays of V. alginolyticus 178. Isolates of V. alginolyticus 178 were culturedovernightinZobell2216Eliquidmediumat28°Cwithshakingat170rpm.Thereafter,cellswere dilutedwithfresh2216Eliquidmedium,andtheOD wasadjustedto0.2.Onemicroliterofthecell 600 suspension was deposited in the center of 2216E plates containing 0.5%, 1%, 1.5%, or 2% agar and incubatedat28°C.Thediameteroftheresultingcolonywasmeasuredatdifferenttimepoints(6h,12h, 24 h, and 36 h after incubation) to evaluate the motility of V. alginolyticus 178 at different agar concentrations. The experiment was repeated three times with three replicates per data point in eachexperiment.ThecomparisonofmotilityactivitybetweenV.alginolyticus178andsevenother V.alginolyticusstrains(C1,C11,C38,C48,CJ11,CJ26,andCT30)wasperformedasdescribedabove. ThepathogenicityofV.alginolyticus178wasassayedusingthecumulativemortalityratesofthe zebrafish.Briefly,90zebrafisheswererandomlydividedintothreegroups(30fishpergroup,and10fish June2017 Volume83 Issue12 e00450-17 aem.asm.org 10