Different Ancestries of R Tailocins in Rhizospheric Pseudomonas Isolates

19 Pages·2015·1.42 MB·English

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Different Ancestries of R Tailocins in Rhizospheric
Pseudomonas Isolates
Maarten G.K. Ghequire1,*, Yo¨rg Dillen2, Ivo Lambrichts2, Paul Proost3, Ruddy Wattiez4, and Rene´ De Mot1
1CentreofMicrobialandPlantGenetics(CMPG),UniversityofLeuven,Heverlee,Belgium
2GroupofMorphology,BiomedicalResearchInstitute(BIOMED),HasseltUniversity,Diepenbeek,Leuven,Belgium
3LaboratoryofMolecularImmunology,DepartmentofMicrobiologyandImmunology,RegaInstitute,UniversityofLeuven,Belgium
4ProteomicsandMicrobiologyLaboratory,ResearchInstituteforBiosciences,UniversityofMons,Mons,Belgium
*Correspondingauthor:E-mail:[email protected].
Accepted:September 10, 2015 D
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Datadeposition:ThisprojecthasbeendepositedatGenBankundertheaccessionnumbersKP698091,KP698092,andKP698093. n
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Abstract from
h
Bacterialgenomesaccommodateavarietyofmobilegeneticelements,includingbacteriophage-relatedclustersthatencodephage ttp://g
tail-likeproteincomplexesplayingaroleininteractionswitheukaryoticorprokaryoticcells.Suchtailocinsareunabletoreplicateinside b
e
targetcellsduetothelackofaphageheadwithassociatedDNA.Asubsetoftailocinsmediateantagonisticactivitieswithbacteriocin- .ox
fo
likespecificity.FunctionalcharacterizationofbactericidaltailocinsoftwoPseudomonasputidarhizosphereisolatesrevealednotonly rd
extensivesimilaritywiththetailassemblymoduleofthePseudomonasaeruginosaR-typepyocinsbutalsodifferencesingenomic jou
rn
integrationsite,regulatorygenes,andlyticreleasemodules.Conversely,thesethreefeaturesarequitesimilarbetweenstrainsofthe a
ls
P.putidaandPseudomonasfluorescensclades,althoughphylogeneticanalysisoftailgenessuggeststhemtohaveevolvedseparately. .o
rg
UnlikeP.aeruginosaRpyocinelements,thetailocingeneclustersofotherpseudomonadsfrequentlycarrycargogenes,including a/
bacteriocins.ComparedwithP.aeruginosa,thetailocintailfibersequencesthatactasspecificitydeterminantshavedivergedmuch t K
U
moreextensivelyamongtheotherpseudomonadspecies,mostlyisolatesfromsoilandplantenvironments.ActivityoftheP.putida L
e
u
antibacterialparticlesrequiresafunctionallipopolysaccharidelayerontargetcells,butcontrarytoRpyocinsfromP.aeruginosa,strain v
e
n
susceptibilitiessurpassspeciesboundaries. U
n
Key words: bacteriocin, phage tail, antibacterial, pyocin. ive
rs
ity
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ib
Introduction ra
ry
o
Bacteria are able to secrete very diverse secondary TheEscherichiacolicolicinsandthePseudomonasaerugi- n
O
metabolites and ribosomally synthesized antimicrobials to nosa pyocins are among the best-characterized “classical” cto
antagonize competitors with similar niche preferences. bacteriocins of Gram-negative bacteria. In the latter species, be
Molecularsizesoftheseantibacterialsvarylargely,ranging fivetypeshavebeendescribed(GhequireandDeMot2014). r 21
from small peptides to multiprotein complexes (Hibbing S-type pyocins are bacteriocins with a modular organization , 20
1
et al. 2010). Historically, the (poly)peptides which exhibit similartocolicins.Theyconsistofareceptor-bindingdomain 5
high specificity and selectively kill close phylogenetic typicallytargetingoutermembranereceptorsinvolvediniron
relatives of the producer are collectively referred to as uptake(Baysseetal.1999;Denayeretal.2007;Elfarashetal.
bacteriocins.Inrecentyears,ithasbecomeclearthatbac- 2012,2014),atranslocationdomain,andacarboxy-terminal
teriocin-likeactivityofGram-negativebacteriacanalsobe toxin domain with nuclease or pore-forming activity. Self-
mediatedbytypeVIsecretionsubstrates,proteinscausing intoxication of a producer is prevented by tightly regulated
contact-dependent inhibition, or Rhs (rearrangement hot coexpression of an immunity gene (Ghequire and De Mot
spot) proteins (Braun and Patzer 2013; Ruhe et al. 2013; 2014). Such accessory immunity partner appears not to be
Durandetal.2014;Hayesetal.2010,2014;Russelletal. requiredforlipidII-degradingM-typepyocinsthatcauseinhi-
2014). bition of peptidoglycan biosynthesis similarly to colicin
(cid:2)TheAuthor(s)2015.PublishedbyOxfordUniversityPressonbehalfoftheSocietyforMolecularBiologyandEvolution.
ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommonsAttributionNon-CommercialLicense(http://creativecommons.org/licenses/by-nc/4.0/),whichpermits
non-commercialre-use,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.Forcommercialre-use,[email protected]
2810 GenomeBiol.Evol.7(10):2810–2828. doi:10.1093/gbe/evv184 AdvanceAccesspublicationSeptember26,2015
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TailocinsfromRoot-AssociatedPseudomonads
M(Barreteauetal.2012).Lpyocinscarryingatandemoflectin relativelystablePrtRexpressionlevels(Sunetal.2014).Inad-
domainshavenocounterpartamongcolicins(Ghequireetal. dition,PrtRalsocontrolsexpressionofptrB,encodingaTypeIII
2014; McCaughey et al. 2014). The carboxy-terminal lectin secretionsystemrepressor,whichislocatedinoppositedirec-
domain,recognizingD-rhamnose,allowsanchoringontothe tionofprtRjustaheadofthepyocincluster(WuandJin2005).
cellularsurface(McCaugheyetal.2014),buttargetspecificity Also in soil-dwelling and plant-associated pseudomonads
ismainlyconferredbytheamino-terminaldomain(Ghequire differenttypesofbacteriocins,oftensubjecttostress-induced
etal.2013). production, have beencharacterized: Pyocin Mhomologs in
Two morphologically distinct types of bactericidal PseudomonasfluorescensandpathovarsofPseudomonassyr-
“tailocins”,high-molecular-weight(HMW)bacteriocinsshow- ingae(Barreteauetal.2009;Grinteretal.2012);L-typebac-
ingstructuralsimilaritieswithbacteriophage tails, havebeen teriocins in Pseudomonas protegens, Pseudomonas putida
described in P. aeruginosa (Michel-Briand and Baysse 2002). andP.syringae(Parretetal.2003,2005;delosSantosetal.
The R-type pyocins are rigid and contractile, whereas the 2005;Ghequireetal.2012);agyrase-inhibitingB-typemicro-
F-type pyocins are flexible but noncontractile. Based on cininP.syringae(Metelevetal.2013);andaphagetail-like Do
w
gene cluster similarities, a common ancestry with tailed bacteriocininP.fluorescensSF4c(Fischeretal.2012).Using n
lo
enterobacteriophage, P2 (P2likevirus genus of the hybridizationof genomic DNAfrom 30P. fluorescensstrains ad
e
Myoviridae family) for the R-type and with phage (cid:2) d
(Lambdalikevirus genus of the Siphoviridae) for the F-type, wesictehnDsNSBAWp2ro5baensdd(cid:2)e-rliivkeedtafirlogmenPe2s-ilnikeP.tflauilogreesnceesnisnQP8.rfl1u-9o6r-, from
was proposed by Nakayama et al. (2000). To date, five R- h
thewidespreadoccurrence,eitherindividuallyorcombined,of ttp
type and three F-type pyocins have been described such phage-like genes was demonstrated (Mavrodi et al. ://g
(GhequireandDeMot2014).AlthoughtheFpyocinreceptors 2009). Comparative genomics of the P. fluorescens clade be.o
remainunrevealed,itwasdemonstratedthatanL-rhamnose revealed that in most strains the mutS–cinA intergenic xfo
and two D-glucose residues of the lipopolysaccharide (LPS) regionisoccupiedbymosaicP2-and/or(cid:2)-typephage-related rdjo
outer core account for the O-serotype-specific character of u
clustersdifferinginsizeandgenecomposition,buttheirexact rn
secuotsracele.op2lti0gib1oi0lsit)a.ycIcnfohtrahrReid1ce,aRisse2in,ovafonpldvyeoRdc5ininpRyb3oa,cciatnesgr,ilourceciosnpseseucrsteicvseeidplyutieb(Kiilnio¨tyhthlaeesr nreamtuariens(einlutasicvte(pLrooppehraegteal.o2r0r1e2m).nant, HMW bacteriocin) aals.org/
In this study we characterized HMW bacteriocins in two t K
well.AdeletioninthewapBpromoterpreventstheaddition U
ofthisterminalcarbohydrateresidueandaccountsforbacte- P. putida strains, isolated from the rhizosphere of banana Le
(strain BW11M1) and rice (strain RW10S2), and identified u
riocin resistance (Kocı´ncova(cid:2) and Lam 2013). Atomic-resolu- ve
thecorrespondinggeneclusters.Phylogeneticanalysisofreg- n
tion structures of pyocin R2 in extended configuration U
(sheath and tube) and in the contracted state (sheath) ob- ulatory,tailassemblyandlysisgenesofHMWbacteriocinsand niv
e
taaninisemdbfoyrcrreyloe-aesleectorfonstmoriecdrosecnoeprygysutgogedsrtivaeppoesrsfiobrlaetimonecohf- raenladtaendc(epsrtor)ypohfagtheessweamsoubsieledetolemaceqnutisreuibnisqiguhitotuinsltyhperdeisveenrstitiny rsity L
pseudomonad genomes. Based on this genomic perspective ib
bacterial membranes by the inner tube (Ge et al. 2015). ra
weproposeaphylogeny-basedclassificationofRtailocinsin ry
With its inner surface being lined by negative charges, the o
tubethenfunctionsasacation-conductingchannelthatdis- pseudomonads. n O
c
sipatestheprotonpotentialofthetargetcell. to
b
e
TheexpressionofP.aeruginosaS-type,R-type,andF-type r 2
pyocingenesiscontrolledbythePrtNactivatorthatbindsto Materials and Methods 1, 2
0
regulatoryPboxesintheirpromoterregions.Innoninducing 1
Strains,Media,andGrowthConditions 5
conditions, expression of prtN is repressed by PrtR. Upon
Bacterial strains used in this study are listed in table 1.
exposuretostressconditions,suchasDNAdamagebychem-
PseudomonasstrainswereculturedinTrypticSoyBroth(TSB
icalsorUVirradiation,activatedRecAtriggersauto-proteolytic
cleavageofPrtR, whichabrogates prtNrepressionandleads 3%,BDBiosciences)at30(cid:2)C,onarotaryshakerat200rpm.
to pyocin production (Ghequire and De Mot 2014). EscherichiacoliandP.aeruginosastrainsweregrownat37(cid:2)C
RecA-mediated pyocin production can also be triggered by in LB (2.5%; MP Biomedicals). Media were solidified with
the CRISPR/Cas phage immunity system in sessile P. aerugi- 1.5% agar (Invitrogen) and supplemented with kanamycin
nosacells(Heussleretal.2015).Underdenitrifyingconditions, (50mg/ml; Sigma-Aldrich) or spectinomycin (100mg/ml;
pyocinsareenclosedwithinP.aeruginosamembranevesicles, Sigma-Aldrich)whenrequired.
equallydependentontheSOSresponseroute(Toyofukuetal. Genomic DNA from Pseudomonas strains was extracted
2014). The P. aeruginosa prtR and prtN genes are located using the Puregene Yeast/Bact. Kit B (Qiagen). Plasmid DNA
upstream of the pyocin clusters, in opposite orientation was isolated using the QIAprep Spin Miniprep Kit (Qiagen).
(Ghequire and De Mot 2014). PrtR also binds its own pro- Bacterial stocks were stored in the appropriate medium at
moter,pointingtoautorepressiveregulationandresultingin (cid:3)80(cid:2)Cin25%(v/v)glycerol(VWRInternational).
GenomeBiol.Evol.7(10):2810–2828. doi:10.1093/gbe/evv184 AdvanceAccesspublicationSeptember26,2015 2811
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Ghequireetal.
Large-ScaleProductionandPurificationofRTailocin tested by heating the tailocins in a heat block at 75(cid:2)C for
10 min. After cooling to room temperature, samples were
One-milliliter volumes of overnight test tube cultures of
spottedontocelllawnsasdescribedabove.Plateswereincu-
P. putida BW11M1 were transferred to 500ml erlenmeyers.
bated overnight at 30(cid:2)C (37(cid:2)C for P. aeruginosa). The fol-
After the OD reached 0.5, filter-sterilized mitomycin C
600
lowingday,plateswereevaluatedforthepresenceofhalos.
(Sigma-Aldrich) was added to a final concentration of
TN50bufferwasusedasanegativecontrol.
0.1mg/ml. The culture was incubated further until cell lysis
occurred. Subsequently, benzonase nuclease (2 U/ml;
ProteinElectrophoresisandAmino-TerminalAminoAcid
Sigma-Aldrich)wasaddedtothelysedculturetoreducethe
Sequencing
viscosity,followedbya30-minincubationat37(cid:2)C.Debrisand
membrane fragments were removed by centrifugation at Samples with bacteriocin activity collected after gel filtration
8,000(cid:4)gfor30minat4(cid:2)C.Supernatantswerefiltersteril- chromatography were analyzed through SDS-PAGE
ized(0.22mm;Millipore)andsupplementedwithpolyethylene (Invitrogen) at 200V for 0.7h using the Precision Plus
D
glycol (PEG) 6000 (Sigma-Aldrich) and NaCl (VWR Protein Kaleidoscope ladder (Bio-Rad) as a size standard. o
w
International) at final concentrations of 10% (w/v) and Afterseparation,thegelswereelectroblottedontopolyvinyli- nlo
a
0.5M, respectively. This mixture was stirred overnight dene difluoride (PVDF) membranes (Invitrogen) and stained d
e
d
(200rpm, 4(cid:2)C) and centrifuged the following day at with Coomassie blue R-250 (Bio-Rad) to visualize proteins. fro
8,000(cid:4)g for 1h at 4(cid:2)C. The precipitate was resuspended Protein bands were cut from the membrane, destained in m
h
in10mlTN50buffer(50mMNaCl,Tris–HCl10mM,pH7.5) methanol(VWRInternational)andamino-terminalsequencing ttp
and centrifuged at 4,000(cid:4)g for 15 min at 4(cid:2)C to remove was performed by automated Edman degradation, using a ://g
b
small undissolved debris. Finally, bacteriocin particles were Procise 491 cLC protein sequencer (Applied Biosystems, e.o
sedimented at 48,400(cid:4)g for 3h at 4(cid:2)C, resuspended in FosterCity,CA). xfo
2mlTN50bufferandfiltered(0.45mm;Millipore)andfurther rdjo
u
purified by gel filtration using a Sephacryl S-500 HR column RecombinantDNAMethods rn
a
(16/60, GE Healthcare Life Sciences; fractionation range for ls
Standardmethodswereusedforthepreparationofcompe- .o
dextrans: 4(cid:4)104–2(cid:4)107Da) atanelutionrateof0.7ml/ rg
tent E. coli andP. putida cells, heat shock transformation of a/
min. Collected fractions were tested for antibacterial activity E.coliandDNAelectrophoresis(GreenandSambrook2012). t K
andanalyzedbytransmissionelectronmicroscopy,SDS-PAGE U
SMARTladder(Eurogentec)wasusedasastandardforDNA L
(sodium dodecyl sulfate polyacrylamide gel electrophoresis), size estimation. Restriction enzymes were used according to euv
a4n(cid:2)dCmunatsilsusspee.ctrometry. Bacteriocin samples were kept at twhaesspueprpfoliermr’sesdpuecsiinfigcaTti4onDsN(ARolcighaeseDi(aIngvnitorsotgicesn).).DSNeAquliegnactiinogn en Univ
e
ofplasmidDNAwasperformedbyGATCBiotech(Constance, rs
TransmissionElectronMicroscopy Germany). ity L
PurifiedtailocinsfromP.putidaBW11M1weretransferredto ibra
0.7%formvar-coatedcoppergrids(Aurion,Wageningen,The ConstructionofTailocinMutants ry
o
Netherlands)byplacingthegridsinthetailocinemulsionfor TerminalregionsofptbJ(encodingphagetailsheathprotein) n O
30s. Consequently, the tailocins were incubated in 0.25% andptbF(encodingbaseplateprotein)fromtheRtailocingene cto
b
phosphotungstenicacid(pH7)for30satroomtemperature clusters of P. putida BW11M1 and RW10S2, respectively, er 2
and washed in distilled water. After sample drying, analysis 1
were amplified by polymerase chain reaction (PCR) with Pfx , 2
wasperformedwithaPhillipsEM208Stransmissionelectron 0
DNApolymerase(Invitrogen),usinggenomicDNAasatem- 1
5
microscope (80kV), equipped with a Morada Soft Imaging
plate. Primers used are listed in supplementary table S1,
System camera. Digital processing of the obtained images
Supplementary Material online. Amplicons were purified
was performed with the iTEM-FEI software (Olympus SIS,
with the QIAquick PCR Purification kit (Qiagen), digested
Mu¨nster,Germany).
with HindIII and BamHI for the upstream, and BamHI and
EcoRI for the downstream fragments, and subsequently li-
BacteriocinAssay
gated in suicide plasmid pAKE604 (El-Sayed et al. 2001).
Spot activity assays with purified tailocins from P. putida Sequence-verified plasmids pCMPG6226 and pCMPG6227,
BW11M1 and RW10S2, and extracts of derived tailocin carrying the fused upstream and downstream fragments of
mutants, were performed as follows. First, cell lawns were BW11M1 ptbJ and RW10S2 ptbF, respectively, were trans-
prepared by overlaying TSA (Tryptic Soy Agar) plates with formedtoE.coliS17-1(cid:2)pirandtransferredtothecorrespond-
5mlofTSBsoftagar(0.5%),seededwith25mloftheindi- ingP.putidastrainsthroughbiparentalconjugation.Selection
catorstrain(~108CFU/ml).Next,10-mlvolumesofcollected withkanamycinandspectinomycinyieldedtransformantsthat
bacteriocinwerespottedontopoftheseplatesandallowed were individually grown in a test tube without antibiotic for
to air-dry. Heat sensitivity of the purified bacteriocins was 12h. Serial dilutions were plated out on TSA containing
2812 GenomeBiol.Evol.7(10):2810–2828. doi:10.1093/gbe/evv184 AdvanceAccesspublicationSeptember26,2015
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TailocinsfromRoot-AssociatedPseudomonads
Table1
Susceptibility of Pseudomonas SpeciesGroupstoRTailocinsof PseudomonasputidaStrainsBW11M1andRW10S2
Species Strain Genotype/Characteristics Susceptibilityto Susceptibilityto OriginorReference
TailocinR TailocinR
BW11M1 RW10S2
Pseudomonasaeruginosa PA14 Opportunisticpathogen, (cid:3) (cid:3) PseudomonasgenomeDB
humanisolate
Pseudomonasaeruginosa PAO1 Opportunisticpathogen, (cid:3) (cid:3) PseudomonasgenomeDB
humanisolate
Pseudomonasagarici LMG2112 Mushroomisolate,type (cid:3) (cid:3) BCCMcollection
strain
Pseudomonasaureofaciens LMG1245 Riverclay,typestrain (cid:3) (cid:3) BCCMcollection
Pseudomonaschlororaphis LMG5004 Contaminatedplate,type (cid:3) (cid:3) BCCMcollection
strain
D
Pseudomonascichorii LMG2162 Cichorium,typestrain (cid:3) (cid:3) BCCMcollection o
w
Pseudomonasentomophila L48 Entomopathogenicstrain (cid:3) + PseudomonasgenomeDB nlo
thatkillsDrosophila ad
e
Pseudomonasfluorescens 13-79 Whmeealtanrhoigzaosstpehrere (cid:3) (cid:3) WellerandCook(1983) d from
Pseudomonasfluorescens 2-79 Wheatrhizosphere (cid:3) (cid:3) WellerandCook(1983) h
PPsseeuuddoommoonnaassflfluuoorreesscceennss BAW50161P2 PBeaanranisaolrahteizosphere (cid:3)+ (cid:3)(cid:3) PVslaesusdaokmeotnaal.s(g1e9n9o2)meDB ttp://gb
e
Pseudomonasfluorescens DR54 Viscosinamideproducer, + (cid:3) Nielsenetal.(1999) .o
x
sugarbeetrhizosphere fo
rd
isolate jo
u
Pseudomonasfluorescens F113 Sugarbeetrhizosphere (cid:3) (cid:3) PseudomonasgenomeDB rn
a
Pseudomonasfluorescens LMG1794 Prefilterwater-workstank, (cid:3) (cid:3) BCCMcollection ls.o
typestrain rg
Pseudomonasfluorescens OE28.3 Wheatrhizosphere (cid:3) (cid:3) CMPGcollection at K/
Pseudomonasfluorescens Pf0-1 Soilisolate + (cid:3) PseudomonasgenomeDB U
CMPG2247 RBW11M1tailocin-resistant (cid:3) (cid:3) Thisstudy Leu
Pf0-1mutant ve
n
CMPG2248 RBW11M1tailocin-resistant (cid:3) (cid:3) Thisstudy U
n
Pf0-1mutant iv
e
CMPG2249 RBWPf101-M11mtauiltoacnint-resistant (cid:3) (cid:3) Thisstudy rsity L
CMPG2250 RBW11M1tailocin-resistant (cid:3) (cid:3) Thisstudy ibra
Pf0-1mutant ry
Pseudomonasfluorescens SBW25 Sugarbeetleaves (cid:3) (cid:3) PseudomonasgenomeDB on
O
Pseudomonasfluorescens WCS365 Potatorhizosphere (cid:3) (cid:3) GeelsandSchippers(1983) c
Pseudomonasgingeri LMG5327t1 Mushroom,typestrain (cid:3) (cid:3) BCCMcollection tob
e
Pseudomonasmarginalis LMG2210 Cichoriumisolate,pathovar (cid:3) (cid:3) BCCMcollection r 2
1
referencestrain , 2
0
Pseudomonasmendocina LMG1223 Isolatefromethanol-en- (cid:3) (cid:3) BCCMcollection 1
5
richedsoil
Pseudomonasprotegens Pf-5 Soilisolate (cid:3) (cid:3) PseudomonasgenomeDB
Pseudomonasputida GB-1 Freshwaterisolate,manga- (cid:3) (cid:3) PseudomonasgenomeDB
neseoxidizer
Pseudomonasputida OE47.1 Maizerhizosphere (cid:3) (cid:3) CMPGcollection
Pseudomonasputida OE53.2 Maizerhizosphere (cid:3) (cid:3) CMPGcollection
Pseudomonasputida OE55.1 Maizerhizosphere (cid:3) (cid:3) CMPGcollection
Pseudomonasputida OE55.7 Maizerhizosphere (cid:3) (cid:3) CMPGcollection
Pseudomonasputida WCS358 Potatorhizosphere (cid:3) (cid:3) PseudomonasgenomeDB
Pseudomonassavastanoi LMG6768 Neriumoleanderisolate (cid:3) + BCCMcollection
Pseudomonassyringae DC3000 Modelpathogen,bacterial (cid:3) + PseudomonasgenomeDB
speckontomatoand
Arabidopsis
(continued)
GenomeBiol.Evol.7(10):2810–2828. doi:10.1093/gbe/evv184 AdvanceAccesspublicationSeptember26,2015 2813
GBE
Ghequireetal.
Table 1 Continued
Species Strain Genotype/Characteristics Susceptibilityto Susceptibilityto OriginorReference
TailocinR TailocinR
BW11M1 RW10S2
Pseudomonassyringae GR12-2R3 Grassrhizosphere (cid:3) (cid:3) Lifshitzetal.(1987)
Pseudomonassyringae LMG1247 IsolatefromSyringavulga- (cid:3) + BCCMcollection
ris,pathovarreference
strain
Pseudomonassyringae LMG5192 Nicotianatabacumisolate (cid:3) + BCCMcollection
Pseudomonassyringae LMG5295 Raphanussativusisolate (cid:3) (cid:3) BCCMcollection
Pseudomonassyringae LMG5456 Cucumissativusisolate (cid:3) + BCCMcollection
CMPG2251 R tailocin-resistant (cid:3) (cid:3) Thisstudy
RW10S2
LMG5456mutant
CMPG2252 R tailocin-resistant (cid:3) (cid:3) Thisstudy
RW10S2 D
LMG5456mutant o
w
CMPG2253 RRW10S2tailocin-resistant (cid:3) (cid:3) Thisstudy nlo
LMG5456mutant ad
e
CMPG2254 RRWLM10GS25ta4i5lo6cimn-uretasinsttant (cid:3) (cid:3) Thisstudy d from
Pseudomonastolaasii CH36 Mushroom (cid:3) (cid:3) CMPGcollection h
Pseudomonastolaasii LMG2342 Mushroom,typestrain (cid:3) (cid:3) BCCMcollection ttp://g
Pseudomonasviridiflava LMG2352 Phaseolussp.isolate,type (cid:3) + BCCMcollection b
e
strain .o
x
fo
NOTE.—“+,”sensitive;“(cid:3),”insensitive.PseudomonasgenomeDB,Pseudomonasgenomedatabase(Winsoretal.2011). rd
jo
u
rn
a
ls
.o
sucrose(7%;VWRInternational)toselectforthelossofthe LPSPurificationandSDS-PAGEAnalysis rg
a/
cassette. Allelic exchanges of the recombinants were LPS was prepared by the method of Hitchcock and Brown t K
checked by PCR with Taq polymerase (BIOKE´), using U
(1983). Briefly, overnight bacterial cultures were diluted to L
pBWrim1e1rMs 1,PGanPdRBP-G10P0R2B2-101a2n0d anPdGPPRGBP-R1B0-01205123fofror ssttrraaiinn O(SDig6m00a0-A.5ldarnicdh)w.Casehllepdetlwleticseowfwithildp-htyopsephstartaei-nbsuafnfedreredssisatlainnet euven U
RW10S2. Putative mutants were sequence verified for mutantsweredissolvedin250mlHitchcockandBrownlysis niv
in-frame deletions following PCR amplification with e
buffer(SDS20mg/ml[Sigma-Aldrich],glycerol10%v/v,bro- rs
Pfx. Resulting deletion mutants were CMPG2245 (deletion mophenol blue 0.02mg/ml [Sigma-Aldrich], 1M Tris–HCl ity L
oCfMiPnGte2r2n4a6l 7(d8e0le-btiponfroafgminetenrtnaolf68p1tb-bJpinfraBgWm1en1tMo1f) patnbdF A[Inftveitrrocgoeonli]ngp,H1.65.8-m),l vaonldumheesatoefdpartot1ei0n0as(cid:2)eCKfo(r203m0gm/minl.; ibrary
inRW10S2). Sigma-Aldrich)wereaddedandthemixturesincubatedover- on O
nightat55(cid:2)C.Thefollowingday,thesampleswereheated cto
b
GenerationofSpontaneousTailocinResistantMutants for5minat100(cid:2)Cbeforeloading5-mlvolumesonthegel e
r 2
forSDS-PAGEanalysis.Gelswereelectrophoresedfor1hat 1
A bacteriocin assay was performed on 24-h-old cultures of , 2
150V and LPS was visualized with the SilverQuest Silver 0
Rtailocin-susceptiblestrainsPf0-1(fortheBW11M1Rtailocin) 1
5
StainingKit(Invitrogen)usingthebasicstainingprotocol.
and LMG 5456 (for the RW10S2 R tailocin), using the spot-
on-lawn assay. After 48-h incubation, tailocin-resistant
LiquidChromatography-TandemMassSpectrometry
mutantscanbeisolatedascoloniesgrowinginsidethetailocin
Analysis
halos. One mutant (if present) per halo per cell culture was
streaked to single colony and verified for resistance to the The identification of proteolytic peptides resulting from in-
tailocin by spot assay (Pf0-1 mutants CMPG2247– solution digestion of HMW fractions was performed on an
CMPG2250; LMG 5456 mutants CMPG2251–CMPG2254). UHPLC-HRMS (ultra-high-pressure liquid chromatography -
To exclude the isolation of bacteriocin-tolerant contaminant high resolution mass spectrometry) platform combining a
bacteria,genomicDNAofthepresumedmutantswasisolated NanoLC-Ultra system (Eksigent) with a TripleTOF 5600
and the boxA element was amplified using primer BOXA1R System (AB SCIEX). Tryptic peptides were separated in a
(PGPRB-10181; supplementary table S1, Supplementary 25-cm C18 reverse-phase column (Acclaim PepMap 100,
Material online) as described (Koeuth et al. 1995). After gel 3mm; Dionex) by a linear acetonitrile gradient (4–35% v/v,
electrophoresis,thebandingpatternsofthewild-typestrains flowrateof300nl/min,20min)inwatercontaining0.1%(v/
andresistantmutantswerecompared. v)formicacid.Thetime-of-flightanalyzerwasregularlyand
2814 GenomeBiol.Evol.7(10):2810–2828. doi:10.1093/gbe/evv184 AdvanceAccesspublicationSeptember26,2015
GBE
TailocinsfromRoot-AssociatedPseudomonads
automaticallycalibratedwithtrypticpeptidesofb-galactosi- GenomeSequencingandNucleotideAccessionNumbers
dase from E. coli, which maintains an average mass error
GenBank accessionnumbersofthe nucleotidesequences of
below10ppmacrossallinjections.Massspectra(MS)were
theRtailocinclustersfromP.putidastrainBW11M1,RW10S1
acquired across 400–1,500m/z with 0.5-s accumulation
and RW10S2 are KP698091, KP698092 and KP698093,
time. A maximum number of 50 precursors per cycle was
respectively.
selected according to an intensity threshold of 200 counts
per second. Each selected precursor was accumulated for
Results
50msandsubmittedtofragmentationwithN asthecolli-
2
siongas.Tandemmassspectrometry(MS/MS)spectrawere
Strain-SpecificAntibacterialActivitiesofPhage-Related
acquiredacross100–1,800m/zandanexclusiontimeof30s
ComplexesPurifiedfromTwoP.putidaRhizosphere
was applied. The acquired data were analyzed using
Isolates
ProteinPilotSoftwarev.4.1(ABSCIEX)andsearchedagainst
the UniProt TrEMBL database or a local database covering Pseudomonas putida strain BW11M1 produces a 30-kDa D
o
lectin(LlpA)withbacteriocinactivityagainstanumberoffluo- w
the BW11M1 (Li et al. 2013) and RW10S2 (Rokni-Zadeh n
rescentpseudomonads(Ghequireetal.2012),andthecyclic lo
etal.2012)genomes.Thesearchparametersincludedami- ad
nanodcaarbmaimnoidaocmidet(hAyAl)csyusbtestiniteu,tioanlls,baionldogoicnaelmmisosdeidfictarytiposnins lxipanotpheopmtidoenaxdasnathnodlyfsuinngtih(aLti einthailb.it2s01th3e).gThroewctuhltuorfesseuvpeerar-l ed from
colfedaivsatignec.tSpeeqputiednecsehdapvienpgtiadtelsea(9st59%5)%recpornesfiednetntchee.Mnuumltibpeler nsoamtaentPosefutdhoismsotrnaains iasolsloatsehsotwhastaanrteaginosneinstsiictivaecttivoitLylpaAgaainnsdt http://g
xantholysin,suggestingthatBW11M1iscapableofproducing b
modifiedandcleavedstatesofthesameunderlyingpeptide e
smeoqlueecnuclaeraforermcuolnassi.dSeereqduedniscteinccotvpeerapgtiede(9s5d%ue)ctoorrdeisfpfeornednst sattanlecaes.tTohniseanottahgeor,niysetitceufnfiedcetnwtiafiseadbaonlistih-Pesdeuupdoonmhoenaatstrseuabt-- .oxford
tothepercentageofmatchingAAsfromidentifiedpeptides mentoftheculturesupernatant(seefurther)and,compared journ
with LlpA, resulted in relatively small inhibition zones which a
havingconfidencegreaterthanorequalto95%,dividedby ls
aretypicallyobservedforlarge,slowlydiffusinginhibitorypro- .o
thetotalnumberofAAsinthesequence. rg
teincomplexessuchasphage-likebacteriocins(Ghequireand a/
DeMot2014). t K
U
PhylogeneticAnalysis Increased production of the presumed bacteriocin activity L
e
u
The predicted AA sequences of the P. putida BW11M1 and by strain BW11M1 was obtained by exposing the culture to v
e
n
RW10S2 tail proteins (PtbJ, PtbK, and PtbH), lysis proteins mitomycinC,whichwasaccompaniedbycelllysis.Aftercel- U
n
(PtbB and PtbR), and regulator protein (PtbA) were used as lular debris removal, HMW particles were collected by PEG iv
e
BLAST (BasicLocal Alignment SearchTool) homologysearch precipitationandsubsequentgelfiltration.SDS-PAGEanalysis rsity
queriesagainstthenonredundantproteindatabaseanddraft ofthisHMWbacteriocin-activefractionofP.putidaBW11M1 L
ib
genomesofpseudomonadstoidentifyanddelineaterelated revealedthepresenceofseveralproteins(fig.1A).Afterblot- ra
tailocingeneclustersandprophages.Thesameapproachwas tingtoaPVDFmembrane,amino-terminalsequencesofthe ry o
n
usedtoscreenbacteriophagegenomes(subsetCaudovirales) mostprominentbandsweredeterminedthroughEdmandeg- O
c
fordistanthomologs.Sequencealignmentsandphylogenetic radation.Peptidesequencesmatchedtophage-likestructural to
b
e
analyses were performed with Geneious 7.1.7 (http://www. proteins (predicted tail sheath, baseplate, and tail tube pro- r 2
1
geneious.com). Synteny between related gene clusters was tein)encodedbyaptb(phagetail-likebacteriocin)genecluster , 2
0
evaluated by pairwise and multiple alignment using MAFFT identifiedinthedraftgenomeofP.putidaBW11M1(fig.2). 1
5
v7.017(KatohandStandley2013).Completemultiplealign- Genomic regions highly similar to this cluster were also
ments of AA sequences performed with MUSCLE (imple- retrieved in the draft genomes of rice rhizosphere isolates
mented in Geneious 7.1.7) were used to construct P. putida RW10S1 (Li et al. 2011) and RW10S2 (Rokni-
maximum-likelihood trees using PhyML (JTT [Jones, Taylor, Zadehetal.2012).Ofthese,P.putidaRW10S2wasequally
and Thorton] substitution model; version 2.2.0) (Guindon examined for HMW bacteriocin production, resulting in an
and Gascuel 2003). The following settings were used for activeparticulatefractionwithaproteinbandingpatternsim-
PhyML (implemented in Geneious 7.1.7): Estimated propor- ilar to the one observed for the purified BW11M1 sample
tion of variable sites, optimization of topology/length/rate, (datanotshown).
best topology of NNI and SPR searches. For identification Activityofbothbacteriocinswasassayedagainstadiverse
and classification of predicted holins, homology searches setofpseudomonadisolatesbyapplying10-mlspotsofpuri-
were conducted against the Transporter Classification (TC) fied HMW complexes on trypticase soy agar plates, overlaid
database(http://www.tcdb.org).Proteindomainswerelocal- with soft agar seeded with cells of the respective indicator
ized using SMART (http://smart.embl-heidelberg.de/) and strains. After overnight incubation, plates were scored
InterPro(https://www.ebi.ac.uk/interpro)analyses. for the presence of growth inhibition halos (fig. 1B and C).
GenomeBiol.Evol.7(10):2810–2828. doi:10.1093/gbe/evv184 AdvanceAccesspublicationSeptember26,2015 2815
GBE
Ghequireetal.
D
o
w
n
lo
a
d
e
d
fro
m
h
ttp
://g
b
e
.o
x
fo
rd
jo
u
rn
a
ls
.o
rg
a/
FIG.1.—(A)Silver-stainedSDS-PAGEgelofpurifiedRtailocinfromP.putidaBW11M1.Lane1,ladderwithindicatedsizes;lane2,bandingpatternofR t KU
particlewithproteinannotationsresultingfromamino-terminalsequencing.Sequencesobtainedwere:SGFFHGVTVTNVDTGARSI,correspondingtoPtbJ L
e
(~43kDa,predictedsize41.8kDa);S(Q)VDLSKLPA,correspondingtoPtbF(~33kDa,predictedsize31.8kDa);AMIPETLANLNLFVD,correspondingtoPtbK uv
e
(~21kDa,predictedsize18.3kDa).(B–E)Antagonisticactivityoftailocins.Spot-on-lawnassayofpurifiedHMWextractsof(B)P.putidaBW11M1,(C) n U
P.putidaRW10S2,(D)CMPG2245(ptbJmutantofBW11M1),(E)CMPG2246(ptbFmutantofRW10S2)againstindicatorstrainsP.fluorescensPf0-1(Band n
iv
D)andP.syringaeLMG5456(CandE).(F)ElectronmicroscopystructureoftheRtailocinfromP.putidaBW11M1.Contractedparticlesarerecognizableas ers
electron-densesheath-coreregions.Averagetailocindimensionswere166.95nm(length,SD:32.73nm;65particles)and13.22nm(width,SD:2.74nm;72 ity
L
particles)wheninextendedform,and65.96nm(length,SD:11.18nm;43particles)and19.64nm(width,SD:2.94nm;44particles)whencontracted.The ib
scalebarisindicated.(G)LPSprofileoftailocin-susceptiblewild-typestrainsandtailocin-resistantmutants.ExtractedLPSwasseparatedbySDS-PAGEand ra
ry
silver-stained.Lane1,Pf0-1wild-type;lanes2–5,Pf0-1mutantsresistanttotailocinRBW11M1(lanes2–5:CMPG2247–CMPG2250);lane6,LMG5456wild- on
type;lanes7–10,LMG5456mutantsresistanttotailocinR (lane7–10:CMPG2251–CMPG2254). O
RW10S2 c
to
b
e
r 2
ThetestpanelconsistedofasetofPseudomonasculturecol- nonmarked BW11M1 and RW10S2 mutants were con- 1
, 2
lectionstrains,togetherwithfluorescentPseudomonasrhizo- structed by deletions in ptbJ and ptbF, respectively, that 01
5
sphere isolates from our own collection. Results are encode predicted structural proteins of the phage-like com-
summarized in table 1. For the phage-like complex from plexes (putative tail sheath protein and baseplate protein,
strainBW11M1,3of38testedisolatesprovedtobesuscep- respectively). The PEG-precipitated HMW fractions derived
tible,whereas7ofthemwerekilledbytheproteincomplex from culture supernatants of mitomycin-induced BW11M1
from strain RW10S2, together covering about 26% of the andRW10S2ptbmutantsdidnotdisplaythewild-typeanti-
strainstested.Susceptiblestrainsbelongtodifferentspecies, bacterial activity (fig. 1D and E), hereby confirming that the
includingP.fluorescensandP.syringae,whereasnoP.putida observedbacteriocinactivitiesweresolelyattributabletothe
indicator was found. None of the strains was susceptible to respectivepurifiedparticles.
both particles. As initially no sensitive P. aeruginosa strain
couldbeidentified,thisspeciespanelwasexpandedtostrains
BiochemicalCharacterizationRevealsLPS-Targeting
belongingto15differentOserotypes,butagainnosuscepti-
R-TypeTailocins
blestraincouldberetrieved(datanotshown).
To verify that the observed antagonistic activities are not BasedonextensivegenesyntenyandsignificantAAsequence
caused by other copurified bacteriocins or phages, defined homologies of encoded proteins with the R-type pyocin
2816 GenomeBiol.Evol.7(10):2810–2828. doi:10.1093/gbe/evv184 AdvanceAccesspublicationSeptember26,2015
GBE
TailocinsfromRoot-AssociatedPseudomonads
D
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a
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fro
m
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ttp
FIG.2.—GeneorganizationoftheRtailocinclustersfromP.putidastrainsBW11M1andRW10S2.ThegenomicregionofstrainLESB58isincludedto ://gb
illustratesyntenywithacanonicRpyocinclusterfromP.aeruginosa.Arrowsrepresentcodingregionswhicharecoloredaccordingtotheirpredictedfunction e.o
x
(colorlegendinboxes).FlankinggenepairsmutS–cinA(black)andtrpE–trpG(gray)areincluded.TheptbgenesaredenotedptbAthroughptbT.Thecargo fo
genesofstrainsBW11M1(bacteriocinLlpA)andRW10S2(unknown;markedwithasterisk)arehighlighted.ThescalebarrepresentsaDNAregionof rdjo
2,000bp. urn
a
ls
.o
backboneofP.aeruginosa(fig.2),thePtbsystemlikelyrep- To isolate spontaneous tailocin-resistant mutants, station- arg/
resentsanR-typeofphagetail-likebacteriocin.Thiswasfur- aryphaseculturesofP.fluorescensPf0-1andP.syringaeLMG t K
U
therqueriedbyanalyzingthepurifiedBW11M1andRW10S2 5456 were subjected to spot-on-lawn assays with RBW11M1 L
e
particles through MS/MS. The samples were digested with and R particles, respectively. Colonies emerging inside u
RW10S2 v
e
trypsin and the resulting peptides were analyzed using an the halos were picked, and bacteriocin resistance of these n
U
UHPLC-nanoESI-MS/MS approach. Multiple proteins were suspected mutants was confirmed by the absence of halos niv
identifiedasptbgeneproductsencodingphagetailproteins: inasubsequentspot-on-agartest(table1).Giventheinvolve- ers
PtbC, PtbE, PtbF, PtbG, PtbH, PtbJ, PtbK, PtbN, PtbO, PtbP, mentofLPSincellularkillingbyRpyocins,LPSbandingprofiles ity L
and PtbQ (fig. 2, supplementary table S2, Supplementary oftheisolatedcloneswereanalyzedandcomparedwiththe ib
ra
Material online). In the RW10S2 sample, PtbL was detected wild-type indicator strains (fig. 1G). Major differences in the ry
o
aswell.Noindicationwasfoundforthepossiblepresenceof LPSpatternswereobserved,oftenincombinationwithaltered n O
phageheadcompartmentproteins. constitution of the coreregions, pointing towardthe loss of cto
Transmission electron microscopy on the purified HMW (partof)themutant’sLPSfunctionality. ber 2
fraction of BW11M1 (fig. 1F) revealed structures consisting 1
, 2
ofadoublecylinder,quitesimilartopreviouslyobtainedstruc- TailocinGeneClustersofP.putidaandP.aeruginosa: 0
1
5
tures of R-type pyocins (Michel-Briand and Baysse 2002). LocatedatDifferentHotSpots,CarryingSimilarTail
MeasureddimensionsoftheBW11M1tail-likecomplexesor CassettesbutEquippedwithDistinctLysisandRegulatory
tailocins were 167nm (length) and 13nm (width) when Modules
sheathsareinextendedform.Partofthecomplexesdisplaying The overall organization of the P. putida BW11M1 and
higher electron density apparently represent contracted RW10S2tailocingeneclustersissimilartothoseofP.aerugi-
forms, resulting in thickened sheaths of 66nm long and nosaforR-typepyocinproduction(fig.2).However,although
20nmwide.AscomparedwiththeP.aeruginosapyocinR2 theirtailassemblymodulesexhibitextensivesynteny,thelysis
complexes studied by cryo-electron microscopy (Ge et al. cassettes differ substantially. Although both species share
2015),slightlyhigherlengthandwidthratiosofuncontracted homologous genes encoding a cytoplasmic membrane-
versuscontractedsheathsareobservedfortheBW11M1tai- permeabilizingholin(PtbB;64%AAidentity)andapeptido-
locin(2.53vs.2.4and1.48vs.1.3,respectively).Asexpected, glycanhydrolase(PtbR;50%AAidentity),positionedproximal
bacteriocinactivityoftheBW11M1andRW10S2tailocinswas anddistaltothetailregion,respectively,theP.putidasystems
lostafterheattreatment,duetodisassemblyofthemultipro- encode different accessory proteins predicted to destabilize
teincomplexes(datanotshown). the cell envelope. The gene products PtbS and PtbT belong
GenomeBiol.Evol.7(10):2810–2828. doi:10.1093/gbe/evv184 AdvanceAccesspublicationSeptember26,2015 2817
GBE
Ghequireetal.
to the same protein family as, respectively, Rz (an integral Pseudomonas nitroreducens HBP1 (Garcia et al. 2014),
membraneprotein;PfamPF03245)andRz1(anoutermem- P.nitroreducensTX1(Huangetal.2014),andPseudomonas
branelipoprotein;PfamPF06085),thespaninsthatarepartof sp. M1 (Soares-Castro and Santos 2013). A pyocin cluster
thephage(cid:2) lysiscassette(Catala~oetal.2013).Theremark- nearly identical to the latter, except for the tail fiber region,
able“Siamesetwin”spaningeneorganization,withtheRz1 ispresentintwootherstrains,Pseudomonassp.AACandPI1.
coding region being embedded entirely within the Rz gene
buttranscribedinadifferentreadingframe(+1),isconserved
Spanin-LikeProteinsArePartoftheLyticCassettesof
in both P. putida strains, as well as in P. fluorescens SF4c
MostP.putidaandP.fluorescensTailocins
(Fischeretal.2012).However,inthelatterstrainthe“hidden”
TheBW11M1andRW10S2PtbBproteins,togetherwiththe
rz1 homolog was not annotated due to the overlap by the
largemajorityofthepseudomonadtailocinholins,belongto
open reading frame specifying the Rz-like gene product
the same TC family (http://www.tcdb.org) (Saier and Reddy
(Fischeretal.2012).Anotherdeviationfromtheprototypical
R-typepyocingeneorganizationistheabsenceofequivalents 2015) as theP. aeruginosa pyocin R holins (TC(cid:3)1.E.20) and D
o
are not related to the P2 type (TC(cid:3)1.E.3) or (cid:3)CTX type w
oftheP.aeruginosaregulatorygenesprtNandptrB,located n
(TC(cid:3)1.E.56). Apart from a short, highly charged carboxy- lo
downstream and upstream of prtR, respectively (fig. 2). A ad
pcortuRn,tietrpisarttraonfsctrhibeedprftrRomgetnhee(spatmbAe)DisNApresstreanntdbaust,thuenltikaeil tceornmseinraveldstraemtcohn(g<2p0seuAdAo),mtohneaPdtsbB(suspepqlueemnecnestaaryrefisletroSn1gAly, ed from
a(3n8d%ly)siwsigthentehse.PN.oatearbulyg,inPotbsaArsehpareresssorratphreorteloinw.YAeAtaidneonthtietyr SPdu.eprflepudloerrmaepsecindetnacsreylQlMl8ysra1ist-e9(Mr6iaalPvtorbonBdliniheeot)m.aIoln.ldo2ug0c0ei9nd).Ee.xcporleiscseiollns eonfgtehne- http://gb
strikingdifferencewiththeP.aeruginosasystemconcernsthe e
tgaeilnoocminicgepnoesiticolnustoefrsthtehaPt.apruetidfoauBnWd 1b1eMtw1eeanndthReWs1t0reSs2s droTlahseespr(ePdtbicRtes)dlaBcWk1a1Mde1teacntdabRleWs1e0cSre2topreypstiidgongallycpaenpthidye- .oxford
response genes mutS and cinA–recA–recX. Partial sequence aenxpdopseredsubmyathbelyaaccttioanscoafntohneicreaslpeencdtoivleyshinoslionns(pCeapttaidla~oogleytcaanl. journa
analysisofthegenomicregionsupstreamofmutSandcinAin ls
2013). This type of endolysin appears to be common to the .o
P.fluorescensSF4crevealedthesamelocation(Fischeretal. rg
2012), whereas R-type pyocin genes are consistently posi- pessceeundsomQ8orn1a-d96taPiltobcRinhsoamndolothgewlyatsicdceampaocnitsytraotfetdheprPe.viflouuoslry- at K/
tionedbetweentheAAanabolicgenestrpEandtrpG. U
(Mavrodi et al. 2009). Belonging to the glycoside hydrolase L
e
u
family19(PfamPF00182;CAZY-GH19;http://www.cazy.org), v
e
AbundanceofR-TypeTailocinsinRhizospheric n
theseenzymesaredifferentfromthelysozymedeployedbyP2 U
Pseudomonads n
(glycosidehydrolasefamily24;PF00959)andthepeptidogly- iv
e
To explore the occurrence of R-type tailocins, we searched can-binding endolysin of (cid:3)CTX (carrying domains PF01471 rs
ity
Pseudomonas genomes for regions carrying phage tail-like and PF11860). Remarkably, among characterized L
ib
genes homologous to those identified in P. putida Caudovirales phages the most similar homologs are not ra
BW11M1/RW10S2 and exhibiting organizational similarity. found among Myoviridae members, with only less than ry o
n
This analysis revealed several tailocin-type gene clusters 40% AA identity to the endolysin of enterobacteriohage O
c
(~47),consistentlysandwichedbetweenmutSandcinA(sup- 4MG (Kim et al. 2014). Significantly better matches are to
b
e
plementary table S3, Supplementary Material online). Their detected for a strain of the Podoviridae lysing P. aeruginosa r 2
1
diversityisillustratedbythefactthatonlytwoofthesehave (Jeon et al. 2012 ) and a strain of theSiphoviridae infecting , 2
0
an identical nucleotide sequence: The strains Pseudomonas P.syringaepv.actinidiae(DiLalloetal.2014)(supplementary 1
5
simiaeWCS417andPseudomonassp.R81werebothisolated fileS2,SupplementaryMaterialonline).
fromwheatrootsbutoriginatefromremoteagriculturalareas TheRz/Rz1moduleiswidespreadamongphagesinfecting
(the Netherlands and India, respectively). In addition, near Gram-negativebacteria(Young2014)andisalsopartofmost
identity (99.5%) is displayed between the corresponding ofthepseudomonadtailocins.However,itisabsentfromthe
genomic regions in the biocontrol strains P. fluorescens gene clusters of P. aeruginosa and a few other isolates
A506,originatingfrompearphyllosphere(USA),andP.fluor- (P.fluorescensstrainsA506andFH5;Pseudomonassp.strains
escens WCS374, retrieved from potato rhizosphere (the CF150,CFT9,WCS347,andWCS417).Therz/rz1genepairis
Netherlands).Apparently,occurrenceofstrainswithapartic- equally absent from (cid:3)CTX that carries partially overlapping
ulartailocinelementisnotgeographicallyrestricted. spanin genes (Summer et al. 2007). Close to their carboxy-
ThissearchfurtherrevealedthattheP.aeruginosa-typeof terminalend,theRzproteinsofBW11M1andRW10S2carry
pyocinRgeneclusterisnotonlyconfinedtothispathogenic a cysteine residue that is conserved in the other inspected
species but also occurs in several environmental isolates pseudomonad tailocins (supplementary file S1B,
degradingxenobioticsorplant-derivedmetabolites:Forexam- Supplementary Material online). The smaller Rz1 proteins
ple, Pseudomonas knackmussii B13 (Miyazaki et al. 2014), have,inadditiontothecharacteristicamino-terminalcysteine
2818 GenomeBiol.Evol.7(10):2810–2828. doi:10.1093/gbe/evv184 AdvanceAccesspublicationSeptember26,2015
GBE
TailocinsfromRoot-AssociatedPseudomonads
required for lipoprotein formation, two other cysteines that (Hattman 1999). This gene organization is conserved in
areperfectlyconservedamongthesetailocins(supplementary Pseudomonas mosselii SJ10. Also elsewhere in the tailocin
file S1C, Supplementary Material online). This suggests that clusters of P. fluorescens DSM 8569 and F113,
disulphide bond formation could be important for the func- Pseudomonas parafulva CRS01-1, P. putida RW10S2, and
tionofthesespanins(Berryetal.2012,2013). Pseudomonas sp. GM67, a com homolog is apparently
linked to a (different) cargo gene of unknown function (fig.
DedicatedTailocinRegulatoryProteinsDifferbetween 3).Ratherunexpectedly,wedetectedLlpAintheMS-analyzed
P.aeruginosaandOtherPseudomonads purified R tailocin sample (supplementary table S2,
BW11M1
The regulatory proteins of pseudomonad tailocins, PrtR Supplementary Material online). LlpA is readily detectable in
(P.aeruginosa)andPtbA(mainlyP.putidaandP.fluorescens theculturesupernatantofBW11M1stationaryphasecellsbut
groups)appeartobespecificforthesebacteriocinsasnoclose its production can be significantly enhanced by UV-induced
homologswereidentifiedin(pro)phages.Thebestmatchesto DNAdamage(Parretetal.2003).Apparently,whenLlpAand
D
PtbAs (~55% AA identity) and PrtRs (~42% identity) are thetailocinarecoexpressed,(partof)thelectinremainsasso- o
w
shownby,respectively,lambdoidprophage06ofP.protegens ciatedwiththeparticles.AlsointheRW10S2tailocinprepa- nlo
a
Pf-5(Mavrodietal.2009)andP.aeruginosatemperatephage ration,thecom-linkedcargoproteinwasdetected(fig.2). d
e
d
PAN70 (Rangel et al. 2013). The PrtR and PtbA sequences Although the tail region of the tailocins in the various fro
show strong sequence conservation within each subfamily strainsiswellconserved,significantsequencevariationisap- m
h
(>80%AAsequenceidentity)butthereisonlylowsimilarity parent near either end of it, but in particular in the regions ttp
between members from the respective subfamilies (<40% flanking the repressor and holin genes (fig. 3). Most of the ://g
b
AA sequence identity). Although in P. aeruginosa PrtR predicted gene products are hypothetical proteins of e.o
x
repressesprtNexpression,nohomologofthelatterisfound unknownfunctions,buttosomeatentativefunctioncanbe fo
incombinationwithptbA. assigned: A peptidoglycan peptidase (Lee et al. 2013) and a rdjo
u
YafO-like protein synthesis inhibitor (Zhang et al. 2009 ) in rn
a
ABacteriocinCargoEmbeddedintheRTailocinGene P. fluorescens SS101, a putative Zeta family toxin inhibiting ls.o
ClusterofP.putidaBW11M1 peptidoglycan synthesis in P. fluorescens strains BBc6R8 and arg/
ComparisonofthetailocingeneclustersofP.putidaBW11M1 ATCC17400(MutschlerandMeinhart2011),aputativeLPS- t K
U
andP.putidaRW10S2revealedthepresenceoftheBW11M1 modifying acyltransferase in P. putida UASWS0946, and a L
e
structuralgeneencodingthe lectin-likebacteriocin LlpA(fig. potential UV radiation-protective RulA–RulB module in uv
e
2).Asecond“cargo”genelocatedupstreamofllpAencodes P. putida BW11M1 (Sundin et al. 2000). Pseudomonas n U
ahomologofbacteriophageMutranslationalregulatorCom putida MTCC 5279 carries tailocin-associated metabolic niv
e
rs
ity
L
ib
ra
ry
o
n
O
c
to
b
e
r 2
1
, 2
0
1
5
FIG.3.—GeneralorganizationofPseudomonasRtailocinclusterswithinsertionsitesofcargogenes(teal).Arrowsillustratecodingregionsandare
coloredasinfigure2,exceptforthetailassemblymodule(ingray),andtheirrespectivefunctionsareshownindottedboxes.Cargogenesinsertedatoneof
thefoursites,togetherwiththeirPseudomonasstrainoforigin,areshowninseparateboxes.The(predicted)functionofthecargogeneproductsisspecified
byacolorlegend.Homologousgenesareshowninthesamecolor.
GenomeBiol.Evol.7(10):2810–2828. doi:10.1093/gbe/evv184 AdvanceAccesspublicationSeptember26,2015 2819