Polyvalent Proteins, a Pervasive Theme in the Intergenomic Biological Conflicts of Bacteriophages

31 Pages·2017·1.26 MB·English

RESEARCH ARTICLE
crossm
Polyvalent Proteins, a Pervasive Theme
in the Intergenomic Biological Conflicts
of Bacteriophages and Conjugative
Elements
LakshminarayanM.Iyer,aA.MaxwellBurroughs,aSwadhaAnand,a D
RobsonF.deSouza,bL.Aravinda o
w
NationalCenterforBiotechnologyInformation,NationalLibraryofMedicine,NationalInstitutesofHealth, n
Bethesda,Maryland,USAa;MicrobiologyDepartment,BiomedicalSciencesInstitute,UniversityofSaoPaulo, lo
CidadeUniversitária,SãoPaulo,Brazilb a
d
e
ABSTRACT Intensebiologicalconflictsbetweenprokaryoticgenomesand their genomic d

f
parasites have resulted in an arms race in terms of the molecular “weaponry” de- Received4April2017 Accepted17May2017 ro
ployed on both sides. Using a recursive computational approach, we uncovered a Acceptedmanuscriptpostedonline30May m
remarkable class of multidomain proteins with 2 to 15 domains in the same poly- 2017 h
peptide deployed by viruses and plasmids in such conflicts. Domain architectures CitationIyerLM,BurroughsAM,AnandS,de ttp
SouzaRF,AravindL.2017.Polyvalentproteins,a :
and genomic contexts indicate that they are part of a widespread conflict strategy pervasivethemeintheintergenomicbiological //
involving proteins injected into the host cell along with parasite DNA during the conflictsofbacteriophagesandconjugative jb.
elements.JBacteriol199:e00245-17.https://doi a
earliest phase of infection. Their unique feature is the combination of domains with s
.org/10.1128/JB.00245-17. m
highly disparate biochemical activities in the same polypeptide; accordingly, we
EditorIgorB.Zhulin,UniversityofTennessee .
o
term them polyvalent proteins. Of the 131 domains in polyvalent proteins, a large atKnoxville r
g
fraction are enzymatic domains predicted to modify proteins, target nucleic acids, al- Copyright©2017Iyeretal.Thisisanopen- /

accessarticledistributedunderthetermsof o
ter nucleotide signaling/metabolism, and attack peptidoglycan or cytoskeletal com- theCreativeCommonsAttribution4.0 n
ponents. They further contain nucleic acid-binding domains, virion structural do- Internationallicense. A
p
mains, and 40 novel uncharacterized domains. Analysis of their architectural network AddresscorrespondencetoL.Aravind, r
reveals both pervasive common themes and specialized strategies for conjugative el- [email protected]. il
1
ements and plasmids or (pro)phages. The themes include likely processing of multi- ,
2
domain polypeptides by zincin-like metallopeptidases and mechanisms to counter 0
1
restriction or CRISPR/Cas systems and jump-start transcription or replication. DNA- 9

binding domains acquired by eukaryotes from such systems have been reused in b
y
XPC/RAD4-dependent DNA repair and mitochondrial genome replication in kineto-
g
plastids. Characterization of the novel domains discovered here, such as RNases and u
e
peptidases, are likely to aid in the development of new reagents and elucidation of s
t
thespreadofantibioticresistance.
IMPORTANCE This is the first report of the widespread presence of large proteins,
termed polyvalent proteins, predicted to be transmitted by genomic parasites such
as conjugative elements, plasmids, and phages during the initial phase of infection
along with their DNA. They are typified by the presence of multiple domains with
disparate activities combined in the same protein. While some of these domains are
predicted to assist the invasive element in replication, transcription, or protection of
their DNA, several are likely to target various host defense systems or modify the
host to favor the parasite’s life cycle. Notably, DNA-binding domains from these sys-
tems have been transferred to eukaryotes, where they have been incorporated into
DNArepairandmitochondrialgenomereplicationsystems.
KEYWORDS DNAreplication,DNA-bindingproteins,RNases,antirestriction,
bacteriophages,biologicalconflicts,effectors,metallopeptidase,plasmids,
transcription
August2017 Volume199 Issue15 e00245-17 JournalofBacteriology jb.asm.org 1
Iyeretal. JournalofBacteriology
Cellulargenomesarepittedinmultilevelconflictsinvolvingadiversearrayofnonself
genomes,includingbacteriophages,plasmids,andconjugativetransposons(1,2).
At one level of conflict, these elements utilize cellular resources to further their own
replication, thereby reducing the fitness of the host genome. At another level, they
mightencodedeterminantsthatenhancehostfitnessagainstrivalinvasiveelementsor
competingorganisms.Suchconflictsareparticularlywidespreadinprokaryotes,where
the DNA, unlike that in eukaryotes, is not sequestered in a separate organelle. As a
result, extensive molecular “weaponry” and repair systems, which counter damage
caused by such weaponry, have evolved in both prokaryotic cellular genomes and
invasive elements that exploit them. In the past 2 decades, comparative genomic
analyseshavebeenparticularlysuccessfulinbringingtolightsuchadaptationsinboth
cellulargenomes(1,3–5)andinvasiveelements(2,6–8)andpointtoaveritable“arms
race” between the two. A key commonality in the conflict-related adaptations across
thesesystemsisthepresenceofastrikingarrayofeffectors(sometimestermedtoxins) D
that,withvariousdegreesofspecificity,targetthemacromoleculesofthecompeting o
w
entity,suchasDNA,variousRNAs,proteinsfromcoresystems,cellmembranes,andcell n
walls.Mostwell-characterizedeffectorsachievetheirendresultthroughtheactionof lo
a
oneormorecatalyticdomainsthatcovalentlymodifyorcleavethetargetmacromol- d
e
ecule.However,othersmightachievethesameendresultthroughacatalyticactivity d
that generates a low-molecular-weight product or via noncovalent interactions with f
r
o
thetargetmolecule(9–11). m
Whileconflictanddamagerepairsystemsencodedbycellulargenomeshavebeen
h
subject to intense scrutiny in several recent studies (2, 11), much less is known of tt
p
mechanisms deployed by invasive nonself genomes against these host responses. :
/
/
Althoughtargetingofinvasiveelementsoccursduringseveraldistinctphasesofthelife jb
.
(infection) cycle of the parasitic element (9), we were particularly interested in the a
s
conflictmechanismstheyhaveevolvedtosurvivetheinitialphaseinvolvingtheentry m
oftheelementassingle-strandedDNA(ssDNA)ordouble-strandedDNA(dsDNA)and .o
r
the period shortly thereafter (2, 7, 8). Invasive elements are particularly vulnerable at g
/
thisstagebecausetheyaretypicallyinasinglecopyandmightnothavehadthetime o
n
for the synthesis of defensive components by using host resources. Moreover, an
A
invadingelementisnotjustattackedbyhostdefensesbutmightalsofacecompetition p
r
from other invasive elements that are already resident in the host (e.g., immunity il
1
conferred by lysogenic phages). These competing elements too are likely to deploy
,

their weaponry right at the time of invasion to prevent establishment of the new 2
0
element in the host (12). Consistent with the proposal that this is indeed a phase of 1
9
intense biological conflict, several studies have shown that invasive elements often
b
transfer,alongwiththeirgenomicDNA,adiversearrayofproteinsthatfacilitatetheir y

g
survival. In conjugative transposons and mobile plasmids using the type IV secretion u
system (T4SS) and related secretion systems for DNA transfer, protein effectors either e
s
“escort” the DNA or are independently transferred through the T4SS (13–17). Phages t
packageeffectorproteinsintotheircapsidheadandinjectthemalongwithDNAinto
thehostcell(18–20).
Proteins identified in prior studies that are transferred during invasion can be
groupedintofivebroadcategories.(i)DNAprocessingandtransferproteinsareseen
primarily in conjugative transposons and plasmids and are intimately involved in the
mechanicsofconjugation.Thekeyproteinhereisarelaxasewithacatalyticdomainof
therolling-circlereplication(RCR)superfamilythatnickstheelement’sDNA,forminga
covalentlinkagewithasinglestrandoftheelement’sDNA.TheDNAisthenunwound,
transferred along with the linked relaxase through the T4SS, and religated by the
relaxase to form a single-stranded circle in the new host (14, 15). (ii) Host-modifying
enzymeshavebeenstudiedprimarilyinphages,andtheprototypeisthephageT4Alt
protein, an ADP-ribosyltransferase (ART) that is packaged into the phage head and
injected into the host cell along with viral DNA. Alt ADP-ribosylates the host RNA
polymerase(RNAP)subunitsandprobablyotherproteins,includingtranslationfactors,
metabolicenzymes,andchaperones(21),withthehostRNAPconsequentlyswitching
August2017 Volume199 Issue15 e00245-17 jb.asm.org 2
PolyvalentProteinsinConflict JournalofBacteriology
specificity to transcribe phage DNA (22, 23). Further evidence comes from the MuF
domain proteins (e.g., phage Mu F [gp30] and Bacillus phage SPP1 gp7), which are
structuralcomponentsoftheheadinphagesutilizingtheportalterminasepackaging
system(24,25).TheMuFdomainisfusedtoanumberofenzymaticdomainsthatare
likely to function as effectors delivered into the host cell by phages (10, 26). (iii)
Antirestrictionproteinsaredeployedbybothphagesandmobileelementsandtarget
thebarrierimposedoninvasiveDNAbyrestriction-modification(R-M)systemsfromthe
host or other resident elements. One mode of action, typified by the phage T7 OCR
(overcome classical restriction), phage T4 IPI (internal protein I), phage lambda Ral
(restrictionalleviation),andArdAandArdB(alleviationofrestrictionofDNA)proteinsof
conjugative transposons and self-transmissible plasmids, is physical interaction with
restrictionenzymestoinhibittheiractivity(27–30).Incontrast,thephageP1DarAand
DarB (defense against restriction) proteins and the plasmid ArdC proteins bind the
transferred DNA, protecting it against R-M systems (31, 32). (iv) Anti-CRISPR mecha- D
nisms have been identified primarily in phages and inhibit the CRISPR/Cas immunity o
w
system by binding various proteins in the CRISPR/Cas complex (33, 34). (v) Early life n
cycle components directly initiate early events of the posttransfer life cycle of both lo
a
phagesandmobileelements.Oneexampleisthevirion-packagedRNAPofcoliphage d
e
N4andrelatedPseudomonasphages,whichtranscribesearlygenes(35,36).Examples d
frommobileelementsincludetheToprimdomainprimases,TraC1ofIncPplasmidRP4 f
r
o
and SogL of Inc1 plasmid R64 (37, 38), which are transferred during conjugation and m
primeposttransferDNAreplicationinthenewhost.
h
Given our interest in biological conflicts during invasion by nonself elements, we tt
p
usedcomparativegenomicanalysistobetterunderstandthecomponentsanddiversity :
/
/
ofsystemssuchasthosedescribedabove.Inthecourseofthisanalysis,weuncovered jb
.
a remarkable class of proteins that are widely distributed in phages, prophages, a
s
plasmids, and conjugative transposons and characterized by the fusion in the same m
polypeptide of multiple protein domains with a striking diversity of biochemical .o
r
activities.Accordingly,wetermthempolyvalentproteins.Theyencompassatleast131 g
/
distinct domain types, many of which we predict to mediate an array of functions o
n
neededtoestablishtheinvasiveelementsinhostcells,promotetheirreplication,and
A
overcome host defenses directed against them. We propose that these polyvalent p
r
proteinsrepresentahithertoundescribedgeneralstrategyusedbyinvasivegenomes il
1
in the face of the ongoing arms race with their prokaryotic hosts and rival invasive
,

elements. 2
0
1
9
RESULTSANDDISCUSSION
b
Searchstrategyforrecoveryofearly-phaseeffectorsininvasiveDNAelements. y

g
Todeterminethedistributionanddiversityofproteinsthataretransferredduringthe u
invasion of hosts by various DNA elements (phages, plasmids, and conjugative trans- e
s
posons),wefirstinitiatedsequenceprofilesearchesbyusingacomprehensivelibrary t
ofpreviouslyidentifiedexemplarsofsuchproteinsasqueries(Fig.1A).Theseincluded
relaxase,phageT4Alt,phageT7OCR,phageT4IPI,Ardproteins,phageP1DarAand
DarB,N4virionRNAP,MuF,andtherecentlyreportedanti-CRISPRproteins.Preliminary
searches were carried out iteratively in the nonredundant (nr) database with the
PSI-BLASTandJACKHMMERprograms.Severalqueries,suchasT7OCR,T4IPI,andthe
anti-CRISPR proteins, recovered a phyletically limited set of proteins, suggesting that
they probably represent lineage-specific adaptations. In contrast, searches with relax-
ase, phage T4 Alt, Ard proteins, phage N4 RNAP, MuF, and phage P1 DarA and DarB
recoveredalarge,diversearrayofhits.Wethenevaluatedthegenecontextsofthehits
recovered to check for the presence of either (pro)phage packaging systems (late
genes) or associations with the conjugative element transfer components. Thus, we
distinguished proteins that are transferred during invasion from those that are either
not linked to invasive elements or unlikely to be transferred with DNA. Through this
analysis,werecoveredadistinctandnotableclassofproteinswithoneormoreofthe
above-mentioneddomains,wheretheywerecombinedinmultidomainpolypeptides,
August2017 Volume199 Issue15 e00245-17 jb.asm.org 3
Iyeretal. JournalofBacteriology
A C
Domains/proteins Nterm T7-RNApolymerase MPTase C-terminal
transferredduringinvasion
byDNAelements gp50__Escherichia_phage_N4__YP_950528.1
sneoadvrPocemrhloelafiinsilnekwseidth (PSSeI-qBsuLeeAanSrccTeh/epHsrMofiMleer) ViDrEiNonnAzsybtmirnuadctitinucgrdadolomdmoamainiansisns Ngtepr2m2_MDPinToarsoeseobTa7c-RteNr_Apphoalgyem_eDraFsLe12pMhiP1_TaYsPe_009C0-t4e3r7m0in2a.1l
Domainarchitecture
analysis
Lysozyme T7-RNApolymerase MPTasePBECR1 C-terminal
Polyvalentproteins
Sequence/Structural DR90_1081__Moraxella_catarrhalis__WP_038519592.1
/Profile-profileanalysis
ofnovellinked
Geneneighborhoodanalysis domains Lysozyme T7-RNApolymerase LPD3MPTasePBECR1 C-terminal
MOVS_05240__Moraxella_ovis__WP_063514060.1
D
Polyvalentproteins Domainsnotunifiedto o
ofphagesandT4SS knowndomains:LPDs NtermART T7-RNApolymerase MPTase C-terminal w
orf62_Roseovarius_Plymouth_podovirus_1_CBX87992.1 n
B lo
a
LGPNDA3T9 Top 50 domains in polyvalent proteins GGDEFLPD9 SWI2/SNF2 meNth6yAlaseLPD12MPTase LPD3ARTLPD4 de
LPD26 d
PBMLLEPPuPCDDtaSR31r_B532I ALO_12301__Acetonema_longum__WP_004096050.1 fro
PolB_LNPTDa2se7 m
RadC D
zf−CHC2 ConjugativetransposonswithT4SScomponents
LPD40 h
LPD21
PhagPeB−LLLLEpPPPLPACoPDDDDrrDRd1112taA987742l VirB4/TrsEORNFLP2C5__OERFn2te2roOcRoFc23cus_Tfoapeoc IaIIlis_AD_rUdAFA4+A3A1Fr6d+C7L-NP2D+3M85+PLT2PaD.s1e6++YD(oTUdFLr4+a3n16s(cid:21111)poOsRoFn2 6 TnO1R5F24 79)Relaxase ttp://jb
LLLPPPDDD13361 Relaxase ArdC+MPTase+MPTase ART DNAG .a
PBEACRRT2 Enterococcus_faecium_ERV1__EJY11698.1(plasmid-deriv ed) s
InPase m
DarA_N
RelADL−aPLLSrYAPPDpo−DDod2CTL552 BaAcRteTr+oPidBeEsC_Rfr5a+gMilPisT_a_seWP_0V0i5rD840/T0r3a0G5.1 .org
PBLELLPCPPDDRD38362 TraJ_RHH TraI-RelaxaseMeVtirhDy4lo/TmraoGnas_spT_oMpoK II1I__WP_NA0r62dAC0-M+4Me8tPh5Tyal8ass2ee+8+GS.N1NAFT+ o/
Toprim−Primase n
LPD1
PMBEuFC−RC1 Phageorprophagemorphogenesis/packagingsystems A
ddrB−ParB
MuF p
N6A−MeMAtLrhPdPyTSClDaaN−3ssNFee6 Major Coat Protein gp5g5p50__viEriosnc ghpe5r4ichia_gpp5h3age_Nvi4rio_n_ gYp5P2_9505g2p581.1 N+RNAP+MPTase+C ril 1
0 500 1000 1500 2000 2500 3000 3500 ,
Counts Terminase_SS Terminase_LS GNAT+ART 2
uncultured_Mediterranean_phage_uvMED__BAR32501.1 0
Top 50:100 domains in polyvalent proteins 1
DDUUFgFp4R4121e216lE962 HaTeermminoaspe_hLiSlus_inPflouretanlzaedA_dRrFTB+-3PPa0BrEB4C+7MR_5u+F_L-CCP+DBM40PY+TLa8Ps7eD+03828.1 9 b
ZnR
Terminase_LS y
PhageF_TiSntKNe−gUHrDaTsIXHe E 4.0 Number proteins vs Number of domains per protein g
ZeDtUa−FT3o6x3in1 u
PentapeptideT−orperpimea_tNs 3.5 y=−0.29x+4.297; r2=0.952 e
NADAR s
DUFD1C9M2 p = 2.891656e−09 t
Assemblin
SpoIID 3.0
LPD13
SnoC
UvrD−hSeKLLLLlPPpPPiMincHSoDDDDSaaCVAT2n2313ssMGHHPoee8740 mber) of proteins22..05
CbtALL_PPTDoDx31i5n1 (Nu1.5
priSmMpoSl og
DnaJ l
LLPPDD1358 1.0
LPD9
PriCT2
PRTase
RNApol
LPD4 0.5
LPD23
LPD29
LPD20
Lysozyme
LPD34 0.0
0 10 20 30 40 50 60 70 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Counts Number of domains per protein
FIG1(A)Schematicshowingtherecursiveprocessusedtofinddomainsinpolyvalentproteins.(B)Frequencydistributionofthetop100domainsobserved
inpolyvalentproteins.(C)Examplesofrecoveredpolyvalentproteinsandtheirdomainarchitectures.(D)Examplesofneighborhoodsofpolyvalent-protein-
encodinggenesillustratingthebroadtypesofgenomecontexts.(E)Plotillustratingthenumberofpolyvalentproteinswithagivennumberofdomains.
Notethattheyaxisisonalogscale.
August2017 Volume199 Issue15 e00245-17 jb.asm.org 4
PolyvalentProteinsinConflict JournalofBacteriology
MAeMtNhuy6FMlAa−uMP-stCSPNeBu_NAIBFEUDP1100001100E11DA00C10201B0RI01CX1030RP1E00MR1B0C10P51120E00R0TCaP1341e1s0R00n01Pte0a0B4p1Ee30P1p0C0Rt0iRPdTPeh5ah1Pa−s0agar20ePgee1reaAp0_10−r0eTPB0pa4oot_lsBrGt_a11pRN1l020T00Re0a1NslLA0yeAs0p−oRozaS1ly1Ld0o0pmC0n0eo_LRCTPe1SlDaLF09xI0a−P1shS0eeDAl0icP8asSeM1SL0P0D61S0N10FL0P0D401LP0D301L7P1D305L0PD3130Sn0oCTSeprmoIinIDase_LSToprim−PL11rPimD10a3s2e000ToxN_toxinUvrD−helicase10010LPD310YodL100LPD3Zeta−1Toxin0100LPD2801LPD2600ddrB−ParB11LPD02501001L0PD2p30rim11polL00PzDf2−002CHC21BILDP0D2D01C1MDL0DP−DP0D2e0UptiFd11a1LLs5PP0e6DD2019P9hag1HeLT_P0HmDiL0n1P_8cDa3Pp4h2La1gRPee0l_iDZE0nnt1eRggpr7a1sD2L1e9U10PTF0o10pD09ri21mL_P6NDLP11L5D11P100D4L001P3DLL1P12L1DPP021D011D01003L61P0100D0SK111n00ino00I1aAn0sr0H1ePL0dHs0PadiGsADLM1stG2N1o00F_07enA0PGLNTDeT1LPD0S_0D1PDEH0KDa03D23FN−L10D0r3H0SDaC9AUPTLN2rb1FDH0tP_0AADP4ACnD110r_T10N01i0H_a−cL54l1nC1iL0T0JgdR1N0P06DpoCPTHAaANDHHVxLU2iDAsENDAKP2HnAFeYrDADAFM2Ss3APoD4U_fordvsH83ITrIUpUviCARRRrBCaebF2e−oDmCFF_smMHS97STlrVM−43eTey65_1bG7−M26lp0Ctl6if3eneaN21atshtesylase AIRASIRS_C CDLPRcLUTdDPenFAlD1p1aB5V9x3Ta24SosxpeNoV_tGLoPxPiDnrLi3CP0TPD1h2La7PgDDAePn1_read0innJAtLteaPgpDDrLeaCUP2psb4tFDeBitd4A2IeD16_L-1HrTP6eosDPLpxdPre2iMinmaDLL_t2psPPN9oDDl42Y01oLADdLPPTrLPNMDdoNaDALC1uprAP12L_AtLr-SiND0PlDPLmi_UgPDAD1L_IavD8R11PNsr7D62eD5_-2LhLAP8PeP_olDDDiMlzcB3fUL8a-_2PCsFLNeDH4PPLTD23CBDaPL63UsL2ED1P2SLeNPF9C6DPDnD1SARoD3aN1MD3C41r536R6AaS65ADsH-CP-eLCHMrPTiMPeCRoDDatpThMa1rUr2ydBG2iPSmFlCaLNTN3-syaFLPA2FessSPTT7roeinSD5mzoyKM2aHmK2-LsuHEfeePLFrHTABPD-THMCTD1H-Cr31eDRLAdMpHPaNRdeirDrATsaABt9pto--sPNLPoSnLPBPlaeFIPBELDnr_IB-DPEPHC3hDLZ3DCa7eNRPUn9slL2RS2iRDFecP31a31Ds643e3L1KPPiDnBDLUaREPL4sFPeCPDDe9lRRDA5D6PgLT-370-pPSaBPR1sDpEe2eeoM3pC9lTE8tuRidF5SaspZeoPeIhItDaa-gPTeho_a2GxHNTgHinG4NUe_D-HDpGEIoXpFr2ta0lTFeormrSmiAnAyPalstsfsaees_meLPYbShvlaibngHeN_moviLneo_l-ncM_aeCpt2hylase Down
lo
a
LPD5 ParB LPD13 de
RelA-SpoT d
C LPD9 InPase PBECR2 D fr
Histone_HNSLPD7 o
LMPuDtS32-ILPD33 LPD31 ArdC-N LPD36 SNF N6A-MeMthPylTaasseeLPD1 ddrB-ParBARMTuF-CPBECRL1PD3 PPBBEECCRR53 LPDM23uF LPD22 PLPhNaDgA3e4D_AinRtegBrIaDDCseUbtFA4_1T1To6oxMzpinfru-iCmtSHU_-CNIvpr2DrPi-mhBpeEoliCRclAaRLHrsdP4HeCDL-3PNLZ3DPn3RDL2TP1o1DpSL3rPni5moD-3P1rNimS6nAaos-RMCeaeLdthPCyDSla3NsG6FeNAFTTLSMPDKLDLPa-PHPr1TDATDa_9sHM3Ne7uFLP-PCLaDdPLrAdB2yDRrs3B3To9-zIPnyaPmPrLaRBePsPTeDLaBPPs3EBLeDCEP2CDRRP2R42NBPR1AEBepCEloARCl-5RS3poLTPDKM3iZn8ueaFtsae-Toxginp129AssePmhbalginNTeeU-rpmDoPIriXnthaaalsgee__LTS4_Gp20 http://jb.m
LPD25 LPD4 LPD15 ArdA LPD18 LPD25LPD8 LPD6 DarA-CLPD12 LPD13 LPD5 2H SAP as
YodL LPD16 LPD18 LPD6 Toprim-Primase LPD37 LPD39 LPD27 LPD40LPDL2P6D20YoLdPLDL2PLPDD2L12P9DLP1L7DP1D6L2P8DP19entapePpotliBde_-NreTpaeseatSsMSDCMHTH LPD14 SFI-helicase m.org
Conjugnaettivweo-reklement Phagnee/tpwrooprkhage ParA o/
LPD17 n

FIG2Polyvalentproteindomainnetworks.(A)Chorddiagramofdomaincooccurrencesinpolyvalentproteins.Theplotincludesallcooccurrencesofdomains. A
p
Thus,anedgeisdrawnbetweentwodomainsinaproteinwhethertheyareadjacentornot.(B)Domainarchitecturenetworkofpolyvalentproteins.Domains
r
linkedinthesameproteinareconnectedbyarrowswitheacharrowheadpointingtotheC-terminaldomain.Hereandinthesubsequentnetworkimages,an il
edgeisdrawnonlybetweentwoadjacentdomains.(C)Cliquesubnetworkofthedomainarchitecturenetworkmerginglargecliqueswithsevenoreight 1
,
nodes.Thenetworkrevealstwodistinctsubgraphsasdescribedinthetext.(D)Largestbiconnectedsubnetworkofthedomainarchitecturenetwork. 2
In all of the networks, the node size and color are scaled on the basis of the number of connections per node (degree). Nodes with two or fewer 0
connectionsaregray.Edgethicknessisbasedonthenumberofedgeoccurrences.Edgesoccurring(cid:3)15timesaregray,thoseoccurring16to90times 1
9
arecadetblue,andthoseoccurring(cid:2)90timesaremaroon.
b
y

g
u
often of large size ((cid:2)2,000 amino acids [aa]), with several other known or novel e
s
domains (hence, polyvalent proteins). Their linkage in a single polypeptide gave us t
contextualinformation,suggestingthattheyarelikelytoacttogetheratthesametime.
We then comprehensively investigated the other domains found in such proteins by
seeding iterative profile searches similar to the initial ones with each of the newly
isolated domains. This was followed by a further detailed analysis of their gene
neighborhood and domain architectural contexts. Profile-profile searches with the
HHPRED program seeded with hidden Markov models generated from sequence
alignments of these domains were used to recover distant homologs and determine
their protein fold (Fig. 1A). Globular domains that could not be unified in any of our
searches to known domains were assigned a code consisting of an LPD (large
polyvalent-protein-associated domain) prefix followed by a number. By this iterative
method,weassembledacomprehensiveinventoryof(cid:2)9,928polyvalentproteinswith
131 domains (see Files S1 and S5 at ftp://ftp.ncbi.nih.gov/pub/aravind/polyvalent/
polyvalent.html) whose domain architectural and gene neighborhood linkages are
presentedasacontextualnetwork(Fig.2A).The100mostfrequentlyoccurringofthese
domainsarefoundinanywherebetween3,900and3distinctproteins(Fig.1B).
August2017 Volume199 Issue15 e00245-17 jb.asm.org 5
Iyeretal. JournalofBacteriology
We illustrate the above-described search procedure with a typical example. The
largevirionpolymeraseofcoliphageN4(gp50;accessionno.YP_950528.1)isamulti-
domain protein with an N-terminal alpha-helical region (998 aa) involved in protein
injection into the host, a middle polymerase domain that provides the core DNA-
dependent RNAP activity (1,100 aa; Protein Data Bank [PDB] code 2PO4), and a
C-terminal 1,400-aa region required for encapsidation (Fig. 1C )3(6, 39, 40). Sequence
and profile-profile searches using the N-terminal region and middle polymerase do-
main recovered only a T7 RNAP module that is also found in related phages and
prophages.However,wefoundthattheC-terminalregioniscomposedoftwopotential
domains, of which one is associated in other proteins with a greater diversity of
domains.TheupstreamdomainofthispaircontainsahighlyconservedHEXXHmotif
(region, aa 2130 to 2448). Profile-profile searches with an alignment of this region
unifiedittothezincin-likemetallopeptidase(MPTase)superfamily(e.g.,thelethalfactor
endopeptidase;PDBcode4dv8;P(cid:4)4.1(cid:5)10(cid:6)4inHHPRED).Domaincontextanalysis D
showedthattheN4-likeRNAPsarealwaysfusedtooneormoreMPTasedomainsand o
w
canadditionallybefusedincomparablelargeproteinstoalysozyme/transglycosylase n
domain (e.g., Moraxella prophage protein DR90_1081; GenBank accession no. lo
a
WP_038519592.1), a novel predicted RNase of the barnase/EndoU/colicin E5/D-RelE d
e
(BECR)-like fold (10) (see below for details; PBECR1, Moraxella prophage DR90_1081; d
accession no. WP_038519592.1), and a NAD(cid:7)-dependent ART domain (Roseovarius f
r
o
Plymouth podovirus 1 vRNAP; accession no. CBX87992.1 )4(1) (Fig. 1C). The MPTase m
domainisalsoindependentlyfusedinlargeproteinstootherglobulardomainssuchas
h
lysozyme, ART, PBECR1 (see below), SWI2/SNF2 ATPase, DNA adenine methylase, a tt
p
distinct ParB domain, and a variety of LPDs (Fig. 2; see Fig. 3A). Thus, a network of :
/
/
domainconnectionscenteredontheRNAP,theMPTase,andthelinkeddomainswas jb
.
obtainedandthiswasfurtheriterativelyextendedasmentionedabove(Fig.1Aand2). a
s
Polyvalent proteins encoded by phages and mobile elements combine func- m
tionallydiversedomains.Weobservedthatthepolyvalentproteinsretrievedbythe .o
r
above-describedsearchprocedureoccurinthreebroadgenomiccontexts.(i)Thefirst g
/
isthepresenceinaplasmidorconjugativetransposonencodedalongsideT4SS-related o
n
components(Fig.1C).Examplesincludeintegratingconjugativeelements,prototyped
A
by the vancomycin resistance transposon Tn1549-like elements of Firmicutes (42) and p
r
the Tn5253-like elements of Streptococcus pneumoniae (43), and several conjugative il
1
plasmids from diverse prokaryotic lineages (Fig. 1D). This is the largest subset of
,

polyvalentproteinsinourcollectionandincludesupto40%oftheproteinsrecovered. 2
0
(ii)Inthesecondcontext,upto35%ofthepolyvalentproteinsareencodedbyphages 1
9
orprophagesandarelikelytobepackagedinthecapsid(seebelow).Theseencompass
b
agreatdiversityofphages,butmostofthemareunifiedbythepresenceofaportal y

g
terminasepackagingsystem(26)(Fig.1D).(iii)Inthethirdcontext,theproteins,while u
not showing any distinguishing genome-contextual associations, show homologous e
s
domains and domain architectures syntactically identical to one of the above two t
types. We interpret these as being polyvalent proteins acquired by the host genome
from remnants of one of the above-described invasive elements that might be de-
ployeddefensivelyagainstotherinvasiveelements.
Polyvalent proteins contain 2 to 15 domains in a polypeptide (Fig. 1D) with the
numberofproteinswitheachadditionalnumberofdomainsabove2fallingexponen-
tially(r2(cid:4)0.95,P(cid:4)2.89(cid:5)10(cid:6)9)(Fig.1E).Although(cid:2)75%oftheproteinshaveonly
two or three domains, the same domains are often found together or separately in
larger polyvalent proteins with other domains, suggesting that, irrespective of their
domain architectural complexity, they belong to the same functional system. Large
multidomain proteins with variable domain architectures have been previously re-
portedinmultipleconflict-relatedcontextsinprokaryotes.(i)Proteinsinvolvedinthe
biosynthesis of secondary metabolites (e.g., antibiotics and siderophores), especially
basedonpeptideorpolyketideskeletons,combinenonribosomalpeptidesynthetase
or polyketide synthetase domains with several other enzymatic and nonenzymatic
domains (44, 45). These domains usually act sequentially in the biosynthesis of the
August2017 Volume199 Issue15 e00245-17 jb.asm.org 6
PolyvalentProteinsinConflict JournalofBacteriology
secondary metabolite, and the variations in the combined domains contribute to the
diversityofthesecondarymetabolitesproducedbythem.(ii)Secretedtoxinproteins,
which are some of the largest proteins in bacteria, display multidomain architectures
typically with N-terminal domains related to particular secretion systems; middle
domainsusuallycomposedofsequencerepeatsinvolvedinpackaging,presentation,or
autoproteolytic processing; and C-terminal toxin domains (10, 46, 47). Most of the
variabilityhereisseenintheC-terminaltoxindomains.However,thepolyvalentproteins
recovered in this study, while having certain domains in common with the above-
describedsystems,didnotconformtotheabove-describedorganizationalthemes.The
domains included in these proteins broadly belong to four types, (i) known and
predicted enzymatic domains with diverse activities, (ii) DNA-binding domains, (iii)
domains with structural roles in phage virions, and (iv) domains for which we were
unable to predict a definitive function. Strikingly, a common theme often seen in
polyvalent proteins is the linking of enzymatic domains with entirely unrelated bio- D
chemical activities in the same polypeptide. A dramatic example of this is seen in a o
w
gigantic protein of prophage origin encoded by the genome of the firmicute Ac- n
etonemalongum(accessionno.WP_004096050.1)containingatleastninedomains,of lo
a
whichtheknownenzymaticdomainsincludethecyclic-diguanylate-generatingGGDEF, d
e
SWI2/SNF2 ATPase, DNA adenine methyltransferase (MTase), zincin-like MPTase, and d
ART domains (Fig. 1B). This observation indicates that the domains of the polyvalent f
r
o
proteinsarelikelytotargetmultiplemacromoleculesorsystems,probablysimultane- m
ously,upondeliveryduringinvasion.
h
Wenextpresentasystematicanalysisandfunctionalprediction(wherepossible)of tt
p
thedomainsthatarecommonlyfoundinpolyvalentproteins,followedbyananalysis :
/
/
oftheircontextualassociations. jb
.
Catalytic domains in polyvalent proteins predicted to act on other proteins. a
s
Oneofthemostfrequentlyfoundclassesofcatalyticdomainsobservedinpolyvalent m
proteinsarepredictedtooperateonproteinsandpeptides.Theseincludepeptidases .o
r
and four families of protein-modifying domains, namely, the ARTs, serine/threonine/ g
/
tyrosine-type protein kinases, GCN5-like acetyltransferases (GNATs), and the polymer- o
ase (cid:2) (Pol-(cid:2)) superfamily nucleotidyltransferases (NTases). These domains are also n
A
encountered in diverse conflict systems such as polymorphic toxin, toxin-antitoxin, p
r
antibioticresistance,andbacterialeffectorsystemstargetingeukaryotichostproteins il
(10, 41, 48, 49). This favors a similar conflict-related role for their counterparts in 1
,

polyvalentproteins. 2
0
Peptidases.Polyvalentproteinspossessfourdistinctsuperfamiliesofpeptidases.Of 1
9
these, the zincin-like MPTase is by far the most widespread and is overall the most
b
common type of domain in polyvalent proteins. It is also the most prominent hub y

g
domainintheoveralldomainnetworkofpolyvalentproteins(Fig.2A).About36%of u
the polyvalent proteins in our data set have one to five MPTase domains, of which e
s
about 8% have more than one copy of the domain in the same polypeptide. A t
significantpercentageoftheseproteins(52%)areassociatedwiththeArdC-Ndomain
(see below) and make up about 32% of the 514 distinct nonredundant domain
architectural types with MPTases. The MPTase domains are found in the N- or
C-terminal or middle region of the proteins, and in those with two or more MPTase
domains,theyareofteninterspersedbetweenotherdomains.
Thecorefoldofthezincin-likeMPTasedomainischaracterizedbythepresenceof
athree-stranded(cid:2)sheetflankedbyanN-terminalhelixandtwoC-terminalhelices(10,
50). The first of the two conserved C-terminal helices contains the catalytic HEXXH
motif,whilethesecondcontainsaglutamateresidue(Fig.3A).Thetwohistidines,the
glutamatefromtheC-terminalhelix,andawatermoleculecoordinateaZn2(cid:7)ioninthe
active-sitepocket,whereastheglutamateintheHEXXHmotifispredictedtofunction
asthegeneralbasethatactivatesawatermoleculeforproteolysis(50).Beyondthese
conservedresidues,theMPTasesfrompolyvalentproteinsshowmultiplevariationsin
theconfigurationsoftheiractivesite,additionalconservedresidueslikelytocontribute
to the active-site pocket, and variable inserts between the two C-terminal conserved
August2017 Volume199 Issue15 e00245-17 jb.asm.org 7
Iyeretal. JournalofBacteriology
A
MPTase Secondary Structure
MSCBCUXBHPSgSoFCHHAQBBWTDTLHANCTatcRaa8N8N7NMBEpUr48MMC0NNP4ecTMLMGArroAqm050S27PPL5Ff10PPI246_3sa_POESSdanZua400W88RRS0G600RRN4760_rrGR_NHICCsW___2_2_9EBP|_2__EEW_262CuDEE19i_|-eK410_0_1_F0UE0|g0FFC0__0H_RWF23R0I1n31550014005Op0Rp0017600001AL13034p|sB8901709137Lh0P07604210361F_90006lpu_4604353227_a2l73623755440TW81422aRs1||5123036|0|0y9262_983743_P5|_1||P/6MS|0|||0_P0Y||||__A|320||1__A0_TA486haL|BCM|0p0PSCEB000P||6BD200l91aA|03ymbAvceC2r0_pBrv107aTB.tr920o41sDC%0daamuosl2o39hXpu114eaa1hu310n80i5A|||culnps7||5a8hl555rss|um|15||0|2ARYY|c|||p2YS0|7||23||ppV||T76WN2Y14oPPW|WWW||Ps5Y9YW||AW||bYYc0|Pm|P62s__PWPPPWD_p2P9PPPDcPWWaPPa1M_eH_04p99_P___Pe1u8_2__res_PPc__r8i0ni0.5|520_000_s3|.0.00ssp0__|00|2s0|n015W67101220p2W10101pp|200W00W.p4Wf4.P94626132|2P776||W322P54P1|0P|81_57222081W5_602WWP112_55_|W9_W7003817929P00707PP_2930740SP60P40..390380_.0550__01382040o_00_27116834950168830002030667v052068002130210120092780262a202001271731098519951603748|1.65.5.0...590...038.374..9W7116161.111.951111051..7115P77761_1011101100_0921133345690997.3.350..20.613151.111810...11..1117110182.1 211141131321211222225399419696295883240286722344714111408341752461783249122748986665545567821400566780833275382715629446629DDEDDDVAHNDQQETETAFNNDSRGEKDDGIIGT.LGAAYYLGSEQAAKKKSTAEAYSQTATLLPQNRTW.RFLFLVLKAYDIASQALPDRHVVEIVIKTTQPDAS.KMAHDKERATITTITLHQDFYIIETKSTATHEAAA.DDDDYDHLQLKILVVFTAVGGFFFQRRNENAIVDV.MLLFSEKRKRQRASDTPKRKANNDKSKEEEEQDPE.RAAAENEAASQEEVIRIAGESEEAKDQMRQSRRER.AKLRLLLELLLMILLLELVTVKSLLIIANRIAALA.ELVVYFFLALSLQKKAKQVIVDDLMVMLLIKQTDE.LLLLDKKDQENAKEPPIADNSLIKDKDSSIKDGAH.RDGGATARSDQGSYLATKGETKRESLKEADRIKFI.KVLIYYYLLVIVLLLLSMILLIITFLILILLLHFLhIPPQPPPGGNAKAPGSELAEETTGRKNGGAARKAA.G@@@@87744443423221765332277644444322GERMIIIILGDDDNAPPKRIVRRRVTARKNGGNDT.TRRSRKSGRGGGYLRIVLLSRTTTPPKLALAAVTK.LLLVVILLFNVKKQRRHENFFGGGVVAAAIDTRRK.GNSYEKDRERTTIVRVVIILFRRRRVEEESIIVVL.LLIVIKIILFLYIYIVVILKRMYLFWIQHRRRVITbAAAFYIRSVFNYYFESQQKDSGPGFMIVIATHVFK.FFFKNSDEDQQQGGSDNDSNSVTVIDDDEDSGGGK.@@@@@@@@@@@@@@@@@@@@@@@@@12211111111@@@@@@755333816131112222307094227769--DSE.RQGRSGPHSGGELDAQKEDGGQDGRATESDRAGEH.AGKQNEPATQNNRVNRRGLIEKAQGSSRDTQEVPDhAVAETKLAAIIVARIAAVIISVVVKLLVVVRRLRR.LMAAGLDDSQYPRFKKEESYYYSLRHREENGGGAAuAAAGAGGGGGGGGNGGAGGGGGGGGGGGGAAEWYF.HHHWTYYSSPWMTAWWLFLAFAIVFFFWWYIIFYYhFYYYYYFYYFTYYDYWYYSTTVYFHHYFFFSKDNR.ESEDYDLFESNDDASSDYVVMYKKVSMDND@@@@44222466744476663333333333366655PPPSLKVLVSFFTTIAYVWAVIILEEETTLTTNII.ASMIIKIIVVQVIIIILIMVTYYYVVVYFYLLVHHhNERAREGSRTISYFYFYLVLLLILIIIILLVIVML.NREIVIVILSTFLLILMLAVINDNRRRNNNAAYPP.ATVNNGSSNIFFVTTRPPDASGPPTSTRRREDNPD.ILILEIALIFFESSTVNNNSNSRSRRKNRNNTKIK.NANSFNSDNKKAPTKATNINMSILDTGGSGLISAG.LLLSVNAASDNASAS-TAELLLAEYAYDENTNNTQ.@@@@@@@@@@@@@@@@@@@@@@@@@@@@@1@@@@@@@@@044465232221444441112223566D-K-NTN.NGGGQFDPPIDDAVATVDDDTLLALAYYEMVNPAF.NAAARRKMDLLLQAVVALLFEPEVQAYYSEEEEEN.EGGGSSSASQSSANQPGEPRWQWWFFAATTTTTTTsTSASTITIVTTTTTVTVTTAAVTVVATTFLVIPPPhLMVLLLLMLAPLIAWFFLIIFFLAAALALILLIIIhFAAGLAMRDSILFVALLAAMWWGWWWALHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEETLMMYYFhAYFLIVVVALGSVVQLVTVISSFLMLALAVVVTTGIGWFQQQIITAGVVIGGGGGLFTGAGTG..---------------AGVGE---------------HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH.AAAAAAAAAFVMKNHAAHVYWYWRRYWWIAAALLI.GFFLIVILLIFYGGGGGLLLIMLGGGTTFSGAWWW.IADDQQQRRQIFLLLFLDDDKRAFIFGGYTTTDAMpQNHDESENDDNERRRQAKKKQAEANRHHDFYYTDN.NYHIYIIKVYDIEKGNRKRGQNNAVLKPDEKGAMV.GLLLEEEEGALLLLMMMFLLDNRDGPSSLESKCVA@@@@@@@@@@@@@@@@@@@@@@@1122212111112.@@@55243623195409183484322211222233.IAMTWFDEALSENRRNDRVQDIQSLLRLLVRRGYR.TSTGSAAWDYITLLLMFFLYYNDANQNYYYFDFKS.NEEDMRGVEASAEDEQDDESGPSPDNDQGKSLAGQ.VLYIIGIDLRNEGADVTDSEREREPPADEARAKTE.MWSIESMRVEDDLLLLFFLLVLAIKARLMMFHYYY.DLKAEGRYRILLMMAMLLWMVWWYFHFYVRSPRDK.RNKKFEIENTKAETRRDGDRARQKDDDNHSDFARV.QDPTAKVIYVTKQEQQNESQQRDKDGNAHARGELV.LVARDGEAEVVVFFFFVVMFFGIGEEEVVVKSLML.QPSRKERDVNRDRMVMYYGDDVVMLLLNESSEEAD.SL-RRIDTRKDASNENNEGKQESDMMINRKYGRKT.A--SEYYYYDWWLTILNHMLLLLLKKSTAVAYAVA@@@@@@@@@545477322212222223111322333@@5599412730362566995211204225121220R.WFWANEWDVTEAAAAIQLVHDDDEEEDAEEEEEELASSRERVQRTGAASSRQLRNEDYQQRDDAEEEEE.STHSQLKHITEAAAAATKAQIILVIVLIVVAAALL.HIPPASTVWLQALLILANVQPAAQRRRIIIVIIIR.GESCIGEEDQHKNNNNTAELSGDEQAGEEEVAVAA.EEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEELLMLVAGIVQYFFFFEATYRVVAGGGMMMLVIIIA.hMFVFEMLLLVILAFLEVVNMMVYDLHLMFIYMFGEAASADAAAAAAASALAAAAAAAMRPSAAGGAAANAsRKRIHAWYWAMYRTEDDAAACRRRDRTYAYREEDM.AAGGPALLFFFRIAENMAGGATDELLAFEEQHSMN.FGTFWLAVSMIMLFYVFYFLLLLSLTIMMVREQLV.EAGEASEEGRHQGENQQAEASASHTGGRAGTTTCQ.ACMQMNKRQLDDDTKSQDANNN-KLKDRDNGGGAN.AYMYEQSVHYY-EYLRWWYRNQEGS-KY--DKDSR.ILLLFGKAGLTVLVVMNRMAQNRVSGGLIGWWFLL.ENNRAVANATTKGNALVLFELFNDSANRGTHASGN.SDDEARKDADNEIVQMGGEARQPSMSSNKGGEHLL.EREGRRSARTKRGADTARGASAARQLFPRSILLIG.248812753594212241122225232522111211439761257355508060313900707676244357774091888032789908426212777216913 1 153462226 7335895 3528 //// /// \\\\\\\\\\ \ / /\/ //ffff fff f f f f f aaaaaaaaa a a a mmmmmmmmmm m m iiiiiiiiii i i llllllllll l l yyyyyyyyyy y y 1234678111 9 5 012
D
B o
Helix-1 Str-1 Str-2 Helix-2 Str-3 Helix-3 Str-4 w
P112BBRHCRCRCBCCHECCCBMOMULR8OC8MKK88NWRNNHoPSPBOA11U035102OOF15OlRIRHNS7__90__845TF67M-ENEAE3_20_900__|||52_βFTFLX80880_8300HTH__11L9__45730R8824its105 014003nha6261S3135003N88500_fep700810026434T42797R7||30||14025a___82S|RC|3RR||05|000350DioL5ouLC6|Rs391||0onsb5bsbl7Che113ET3stpa|epas|lo 572hn0p||cC||cpCsmS490ae0|YY|cYY||dp||||lx|WPPElPPWWi|YeCRD||BP__Oo__PPfWPcbifWWr_00S|00__|P_ounoPPs0005W0000C_0nttr__p1773P7710C00d|||00|6772_8764K24WWW00W279300820918aPPP54P295804297494r___36_209.23350140y000410242157785931 000513467890818402653657..4..83.07S583589.11811..15.t8532081411.1r298..071u124114.64431c9590t....u1111re11111112112445419861336210332404199858952322079238107183720697614 KMEEEEEEELEEEEEDEEEEISDELLYRREKESEEKQQEEERELTNTEGGTLTMTTTTTTSTQEEAIVVIVVVVVIVVVVVVVILTKYYLLLLKWRKKYWWLLLQALCAAFYYCEASCCAAASCCSIRHYYHHHYIIHHHYYYQYYVVAVAVVVVVLLLLAAAAAAAVKRQQQRQQQEQEQQQQQQQQNTRASSAAAAEADAASSSIAAKIEQKKVVVQVEAKKQKKIEQALAAILILLIILIILLLVLIIGQVDDDEEEDSIHEDDDDDEDRQLEEEEDDESETEEEEEEEE-LSSYYMMMMFNFYYYYYLAM-@26255555555557777677DALIVIIIILLLLLLLLLLL-YVEQVVIVVVVVVILLILLL-TREADDDDDDDDDGAAGAGGPVVVVVVAAVVVMVAAAAAAALWRYRVAVAVVVAARRRRRRREAVIVLVVVVVVVVVVVVVVVVFFFYVTVASSSSSHYYYYYFIGGGGGGGGGGGGGGGGGGGGGSSSSSSSSSSSSSSSSSSSSSRRRRRRRRRRRRRRRRRRRVAVATCCCCCCCCCSSSSTSSAAKRRRRRRRRRRRRRRRRRRRR@@@@@@@@@@@@@11111111GGGGGGGGGGGGGDDDGTGGGKEDLLLLILLLLLLLLLLLMQAYEEEEEEEEEAYYYYYYYYRKLHHKQKKKGQRKRRRRRKHKKEDEEDEEAAAEQAEEPEEEPYDTDGSGNGGGNDDDDDDGGTSSSSSSSSSSSSSSSSSSSSSDDDDDDDDDDDDDDDDDDDDDLIVLLLLLLLLLVLLLVLIIIDDDDDDDDDDDDDDDDDDDDDLIVVVVVVVVVLVVVVVVVVYALVVVVVVVVVLVVVVAVAVLIFIVVVVVVVVVILLLLLVVVIVEEEEEEEEEAASSSSSSSPSFYLLLYYYYYFYYYYYYYYH@32233333333333333333LGIVEEEETEEEEIIIIIFL-DRKRKRRRRHHNTRRRSRRK-FTEEEEEEEEEEEEEEEEEE-LLMDDDDDDDDDDDDDDDDD-ALNDADDADDDASSSSSVAA-RDVLLLLLLLLLLFFFFFFFTDHFFFFFFFFFFFFFFFFFFSRADSNNNDNNNNNNNNNNKNLLRRAIAAMMAVATEEAYLATPKLMLLFFFLFFFFLLLLLLLYEKYNNNNNHNNNNNNNKQHHYALMEEEEEEEEEESAEEEEEKFA-AEDDSDDDDDYHSDNDG-SLVGGGGGSGGGGGGGMGGGGEESYLLLLILLFLMIILMLLLSGRKSHHHYMMKSAAAYRTKEDLFVIIIIIIIIIIMMVIIIRLLIGGGGGAGGGGAAAKAAAEPGRNGEEGGGGGGGGGEGGGLWVGMRVVVIVVVVIIIILIIPRRNRNKKKQKKKKKKKPKPLGVVVVIVVVVVVVVVVVIIVVLDDDDDDDDDDDDDDDDDDDDDLIYMMIIIIIIIIIIIIIIIPLVINNNNNNNNNNNNNNNNNEDSVPPPPPPPPPPPPPPPPPHWELIIIIIIIIIIIIIIIIIG@52722222222221112222IDLVDDQQQGGGGGEEETEEEVFAKKKRHRRKKKKEEEKKKKDTTTTTTTTRRRSTTTPRPTSKGGGGGGGGIIIGGGGGERGGVTTSTTTTTTTTTTTTVILTTLLLLLLLLLLLLLLLLGIRLLFEEEEAAGGAAAPQEENREEEWSTTTTTEQEEEEEEEPKQDTKYYYYYYYYYYYYYYYEVVFYRLLLLLLLLIMILMLLIYLLLEPPPPPPPPKKKEKEKGVREPKQQQEREGGEEERRSSPVEKEGMAVVVVVVSASAAAAFIASVFEEEEEEEEEDEEEEEIQIEEVEKDTSSPAAAKYQKEREPRRLYYYYYYIYYYYYYYYFKLYY119172411111471311952108384222354326260888041064032149633354 62 71369477 //\ \\ NsMMpttNNoorTTlxusy4c v5tfa-uollrueiennksdte itnoxins hnloaded from
HHSHMMMAPPPMRRRNEEE0FFF50914700467646381___41006334_201519915127|||2SCB|sanFpsojipro|||hWWW|PPPW___P000_00002971944291804472244568968474...5111.1 11022221912767 LLLLLLLTRRRRIIIIEEQENNNNAAAAAAAATTNTRRRRIIIIGGGGKKRK--------@@@@--------PPPPIIIINTTNVVVVVVVVGGGGSSSSRRRRAAAASSAS@@@@GGGGTTKTAAAAGKGKAAPPMYYTSSSSDDDDWWWWDDDDYYYYVVVVIIIIEEEEGGKG@@@@-GG-LLLLNNNTSSSNRKKKSNEQWWWWSSSKKKKQIIIIKKKKNNNN----SSSSLLILPPPPGGGGAAAAKKKPSSSSTTFRIIIIDDDDNNNNTMTLLAPPRRRR@@@@----NNRMIIIIDDDDLIIIIFFFRTDKTGGGPANPLVIVDRNDKTRPTESTKLKKPPPPYHYF12310972954994 Pol-β NTases ttp
WS7_18736|Xaxo|EKQ58962.1 230 LKRIQNAANRSGT--@--TITVVGSRAA@GTARSSSDWDYVVNA@--NSKTRNNLSR-SLPGSGNMKEAERA@-NMDIFRGEVDKNRPH306 :
cFYTTIMXXOBo6EP9KADDsDn1RE31L66c0s1MB11B09i1e_PL_2___l_nR_ZR821062sS0_S|65330u0000T43807s4221k48041/9281o|||_|86509dIAC3T00|27|abb6n%|T|8Wlaa8aSbC5Pbcc5utaM|_|||||sreA0WKKOAp|tfl1PUUaH|A|o1_KKcLWDK|2024u2PTPW5161|3_8QP0435W6034_0506P652207621_7205291..0.6297.411118.2418521.081813.42719918.271.31.1 21181511134351764949 RFELLIILLLbAEEPEKEERDbRALTAEEEDEpIILLFVFFFFlQGLRLEKKKIbNRPTELARRR.ARFYELKEGR.ALRFLSVLVVhNREQKRQREEpREQTGENGEA.IAILKILIIR.GFLYFFYYYF.KGKGGRGGGG.PDIKDDKPHD.IRAKRNRRRS.@@@@@@@@@14VAVLILLFLVhVYEEEEKKKE.GNEQEKNQRE.SMVLIVILIIhRRIIIIIIVI.TVILILLVLLlLFFYFFYYYF.GGGGGGGGGGuSSSSSSSPSSSRRVRQYYWYHRVASAAVAAAAsRSRRRRRRRR+@@@@@@@@@@3GKGGuGGGGGGEGDD.DDDEDNAWHA.HYFQAATHRKcDDDKERPGEP.EESEEEDEDD.EEHGDESSSSSSSSSSSDDDDDDDDDDDIWLIIIIILVlDDDDDDDDDDDhFYVILVVLLIlLVLLLMAALLlVILVIIVIVL.ETVIVVLLVI.LGLLGGLLLL@@@@2376666-QINGLAWDN.-SKNKADNVV.-SAYEEEEDSp-LEFIRRIFF.-FDDDEADDD.IDWERRKRGEbNYDIMLLTILhSIESISSSMIpRGIQNEEQEN.KLGVIVLFVIhKMKSIIIIVS.HQLTTFLATTp-DSDDKEDNK.SLALLLLLIV.VAESQACCLLhSALLMLLLLL.SLTKKKEEEK.SLLYYYHHYY.LGEGNNDTGG.PRLIAIVVEE.KKGMLVVVLL.AVIIILLIIIlDDDSSSSSSSS.VIVIVVVTPP.TAMLYLLAII.LEILPPPFVI.G-MVVIIVLI@@247976777-.RKRDVRGER-KNpRVKPKKRP.QLSKQSDDDNsDPPPPPSSSN.IFLLFFFLFLhFYLLLFIYIH.TKILRLRNKEpGNNNNNTTTLhPFIVVVVAVIhLIHKRHLKKR.DKKRRRSTRDpTEEEEEEEEKsSGGGGGGGGVbRKIIVVKIIL.PVTAVPVAKShHYAVVLLVV92111111921215110142 6142160 6 8 // \ PHaoEnlPd-N β-r NaeTslasasotecesidated //jb.asm
.
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/

Str-1 Helix-(OSA) Helix-1 Str-2 Str-3 Helix-2 Str-4 o
ParB Secondary Structure n
WWjGHHTWHPHOpdPpBSkcSdh1APnCoPPMMIHMSaaapoRdp7S_dEr__PPCAPArrronXr17I0B_B00RRO0R|BBB0sNB0__1|0|20EEN0ES|||Je1|4g00S3P05FF_6FflPPT|n|E4p41l9R4210150ehftBsHv|169i2K0006871|PuhsusiH127v|224762|7P1reusaPp||1|B|79064R92|||b/p|yHT2AcC94__0l_9Y1Y|7|Ylpa1AeA9800|e07PVPN52P|hs|ZrA..21Hg57_K_P%R_NKiP2|61191i|32010_I0P||s9A8||05nW0.0_03I0_YYs0A5GA92fP616A59_62PPp4S0tc|||_|551Y42__|133rsBPY0B2799300W.9.oporP2w7371700P131||vs_0a..6.674_2WWap00d11.112808PP||05|1.371.__WW41W1740100PP19P92120__31_869050085090742007.1..1004901611224303317921.6.945111198730..63211259...111 1111166335241958629561142229734144038634388473438932554YYYYVHLFYYYIELYVIVPhVFDEAATDEAEEEVERVVEEI.HDIIIIVVTLVVLYVLFRKEE.NIIKVVINIVVVLVIIILIILlVVEDKKDNEEDDENPPEPKKDpPDALAAAAPAAAAGPKLLTILhLARNKKSYIKTADLNIDAKAD.SSDDDDLKANTDDAQFKSQDL.VALLLLLPPLLLVFVLMILLIlLLIIKKQTIISVRYEFKRIRDbILPTPPPEETPTARSDPPPPEsRVSSSSSGPSTSSADEHNANDsPSHNFFHFKHQHHWTDEPEPP.LNNDTTIPEDQNLLFE-RYYH.PTKATTQIPAKDPAVL-----.-LDLGGGDISDDSLDA-----.-DCYGGVEEGDLRGQP-----.-GSATTRNGVNGGVHE-----.-KQV--NGNA-VFENV-----.-INN--S-KN-NQDNR-----.-NSP--L-IP-PKSGA-----.-PKR--H-NK-ANQR------.-DHY--F-EF-FPAD------.-YLP--I-SP-PAC-------.-PIA--D-IQ-QYP-------.-QSE--E-KE-EGL-------.-EKL--A-DL-LLK-------.-LA-----N-------------.-QQQQQQQQQQQQQQQQQQSENQVPPKKPSRPFPDARLPPPNP.P152232RRRRKRRRVRRRER+NRKTKPRDDTTEDDDQKKTRKpDDDPGADRRDDRRRRRRHARD+RRRKRKRSTMMNSDS-FFDYL.DTK---TGRNNDDRR---NHK.YT----RAEEEEETA---PDP.EK----ADAEEAKAM---GE-.RQ----SLS--SSSS-ADF-G.DS----SCR--VVQQ-EDS-D.EQ----LNQ--LESD-EEP-S.DA----LQQQQSSQQESASPE.-QYS-VQSIIIAIIILLLIGYhAVPLLQVKEKKRKNTEKAASEpRISKPSNLSSSKANGDEEERKpKNSSALQMMIIINIIFLLI-LhIIAILVIAAAAASAAIAKGAKsSSARTDSQDEEEKRSEDEAKRcRKHSRTKKDNNNNNNSSSTVSsSNFTYIDLLFFILLLVIVIMLlILAILRLNRDDRVDQTRLKEEpADVKVERPPPPPPPPGEQENE.DPGHDDPDDKKEERESKHRAFpNDIQNPALWKKEKKRGGGGAG.YRDQPDQILIIILLLILIVKYhDVS-TSL12@@@@@@@@47888778588881LLKV.GGGDDGSGGGGGRWPFVQQSE.AAALLAVSAAAAAKVYPPPPPuPPPPPPTPPPPPGPPPPLLIVlIVIIITVIILIVQMLAILISIlITVIIVVVILVIILAIDVVVW.GLGLLNDEGAGDVLDGVRRRN.ESLHHENEDLPHIAVVLPKEKpDDDDDRNNDDDDSKSRW@@@@@@@@@@@51853864247.KKAIRYYYGYYNGNGGGGLGNETVEFFLERDI.AQVQQEIILNAICILYILVIIVVVVVVTVVVlAIFLISVVIVNEIEIIILEELbLATDNFDAGAHSQSAAQSSSGs111GGGGGGGGGGGGGGGGGGGGGuNNMSQCHEHEANNNNNNNNNNp-GTRHHHRQRRGGGHHNGGGG.RRRRRRRRRRRRRRRRRRRRRRDSSRRYWYLFYTSTIVSNAVA.IMMAAAARKRR.MILQADIILMAAWSAAGAVAGuGAAGGAAVASVLLAIYLALASILLMMLGILIhDRLIEQQLLKKIKNLLREKRAbGQALQQKMSLWKIKNNLLSKR.NAHEALLAMAAhAAAFFMAAAAGYENLQGGGGGYYYTTWAYYY.GQVVKRALILKQDAPPEKDQE.KEA-EEKQDDKDEDKKSQNQSp@@@@@@@@@3565373355522--------.DNREGVDEDEEYFIA--------.RKRAAENGNQEYY--------.YYYYYAYYYY---------.RRVRINRRRR-NE--------.ENNQDDAQNE-KR--------.YYDYYLWFFA-AA--------.LLFLLLLLLL-II--------.ILAKRPVKRK-KK--------.DKNDESNDAA-EE--------.HNTHFHNNNN-YY1111111@@@@@@@@@@446644468667TSNPPQPQQEAHVGTREETS.TARNDDSRFQNSSFLIAVIVI.IPEPEEPVRPPPPPRPPPEPP.AVYVLLVHVVVVMVVASACAAhVLVLLLLFLLLLLLLKVVVIFlVVDVVVVVAVVIVSVLIVVVIlFRRRRRNPGRRARRTVLKVRD.FRFTVLMDNIRRRDDQDDEENpHRSRPPLEGRLRLEQSYLMLD.D116411177741119997911606924568142623299931735780476326196931479 \/ OPftaahrmeBsr April 1, 2019 b
y

g
D u
e
Str (-1) Helix-1 Str-1 Str-2 Str-3 Str-4 s
PpBNPchNMsoBi4EanEH7NNsCP29ZeR3_84n3005s1_10_u g582sSp8_7/e132282|195c|T15%oHa1|nps|PdhpNsp|mya|WerrYPn|yP_|W _0WP02P_S01_0t4901r9307u50267728c6634t2382u0.47r.164e12..11 12164765512802GGGGuDE.DWIlIIID.YSDLhLLFV.RVVWhAWWuGGGG.NKNG.KHET@@@@1@@@51DAGK.YMFA.GGGGuLLVLlEANQ.HHHKHIVIIlLIWIhKEQK.RSGK.RRHHR@@111@@822MGQ.PSMD.EVLVhVVIS.LKGA.GTRF.NIVVlIP[e-mail][e-mail]a111280754539620 t
PBECR3 Secondary Structure
pDtBHchtrNMoioa6PnHxN9RsP_|3Ee33H_Fn37i09s_0n15ug9f45sp||74/1DY5_82aP|05|c_P2%He2r8p|4s7hY9p3pP1|||_9WTY00PpP0._h_310a012|092W431P900_542070406.1221570.0.111002.1 1189298992742719TKWDF.RSQEK.AIHNH.VISGE.ILIKI.DDDGNpYSEYS.QDS-N.ASSGD.IVILL.SRRVR.HHHHHHhIVIIALbMLEMKpNKAKEKpQG-HEHHH+KAGGG.NpHNSDLNEQS.SLKIL.FEARK.@@@@11@@1141EKKKE.DDDSD.IhLIVLAKLEK.NALDY.YI[e-mail][e-mail].@@@23FEEGN.IIIFT.hYFTFLYSFVFhVVVV-.EQEVI.VEEEE.VVFQM.NRRVS.SKASR.KRKND.KHRAN.---K-.GKGNS.IKQEY.LILLWhSAEMN.GVLhLVKKVKN.TTTTSTMMCMAhWWYYGbKIRKI.131120869013068233
PBECR5 Secondary Structure
HICON_18240|Hinf|YP_004138703.1 1615YQRDLIVTHNIRADGIMHADK MGGLPLASVAVAKQSNPLTNFGEVTLIGSRNYIDPKGVNKAQVFGSDIYSPRYP1689
HHMMPPRREEFF10095955__0021957016||BLfbraac||WWPP__000059729577286299..11 1421645GEGTKDSKLLVLGALLHHNNIKSDEEENKSLILLKAAALIKK LLGGGGLLAPNMPPSSAIAAVIIVDK1AARRQDSGHHETGKYYGGEPIISSLLIVLFPSSKSDMTIIDDKPRQTLGFRRNAANGKTVFYSGTGDDAAWWTTPPTVYAPP4183399
BN580_00518|Fbac|WP_022294475.1 1065QTKDLIAVHNMTTAELEKSLD LGGLPMPSIAIIKAADGHSEYGDVSLVFGKDTIDPKKSARNKVYGGDAWTPTFP1139
consensus/85% ..+plhshHNls.pplbpu.chuuls.uShul.cspps.p.au-loll.spp.lD.p....s.sausDhaTu.hu
MuF-C Secondary Structure
MEHvHMcuCMbIOop0P_CMn38R2OAs0BE4N_e|KFB_0nET0_12sv76486ui76823sM26|40/|1_S0|8N90c|M0P_1oHm%_21nia005|nc562Af|03|D|W6|PNYP3Er6P_4cs8_0.op4001l|709|W247WP.16P_137_08000782905103.41.19119393156..11 411370733491554130561S.LVIVTLhMLLLVApKKKHRTcKKDAKT.AIADA21432KLKKKApDHHHHHH15.GVNNAAlFVVVLLsAPTTKT.PMPAPPpKDDEEE.EVTDDDlLILLLFpEEKKQGpNRQQRRlLLLLLLuPPPPPPpAEKKSA.ALQQLMhVMILML.AHNEAAsDDDSKKuPPPPPP.IDVIQKhAAAAAAlVIVIVVhFYMMLLpDRKNWWSSSpNDD3336.RKTRQP.ERDNNNuNHNAGA.HNRVYY.QNSVVVhLLIMVVlLMLLLL.YYTLTTpVIEDEShLALLVV.KVMVAEpTDEETS33KKRK.GNDNDDpIKGGGGuNENDTSpKKFPAVsPPLVRQlVIVVIIlVISSVAsSILAVVsAATVRNlLLIHTApHDGMSPhLVKKQYpKI44133QQIKQpT.NPDRGNDDVII.KRKDEE.GALFIL.ADDNNIsIpVVIKNELIIVIlVVVAAAsTSNSSSSSVlIAAVVYYSFYYYGRGGRGu441144318018493836295
PBECR4 Secondary Structure
NBLcALio3IPn0GAs__5e000n400s38_u74gs01p/||28SB65il|%noLinp||hWWaPP|__Y00P10_770700951414596648194..1112.1 [e-mail]@@@[e-mail].SHS.23PEH.RKG.TIF.RSI..GDDKRKKVAVhEQKpVVS.LLYhSPY.NLF.ALLhLQH.DEDpLIV.ETF.GMI.NSH.VKK.DSS.VFI.VVQhVFI.DDCp191SSY.LMD.KKKpALAhSDEpHLV.VVVhIFYhQYY.SRL.224KEKpNAC.LWIhICV.VLLlAGG.LLAhKDQ.TSKpVDT.234VNT.TRY.FMRhPRL.ASC.STGTLLLhCLH.NKV.GSK.251675079
PBECR2 Secondary Structure
BSGAcNA1Xo4M7Fn7N71s20_3e_2g_n07p0s1416u5517s87|0/32H58|9p|5T_hD%a1Kfs0|ap1Yi|2P|W8_WP|0P_M0_0g702e261o329|723S950D827Z9169.43310.8.1917.1 211286152207307GLGF.ITTN.VVVG.KGKV.MILE.GTGN.NAQK.NANE.QSQN.ILVFhEGEI.KEKK.LHLH.FLLLhEKQK.KTRS.GDGK.5RRRRRSHLA.QKAS.QEYLbFALVhGAHP.MMLLhIVVIlKAEP.PPPE.TTTI.LLLIlEEKEbQREEpPPPPuLLDF.ALIE.VVTIhIVLWhEAIL.VEFM.PHKP.@@[email protected]+TSAA.FFFLhLE-A.G[e-mail].211139874090372
FIG3Multiple-sequencealignmentsofMPTase(A),Pol-(cid:2)NTase(B),DdrB-ParB(C),andPBECR(D)domains.Secondarystructureandelementlabelingis
providedatthetopline,andresidueconsensusatvariouspercentagesisprovidedatthebottomline.Sequencesareidentifiedontheleftbygenename,
(Continuedonnextpage)
August2017 Volume199 Issue15 e00245-17 jb.asm.org 8
PolyvalentProteinsinConflict JournalofBacteriology
helices(Fig.3A).Forexample,oneofthewidelydistributedMPTasedomainsfoundin
thephage-typepolyvalentproteinscontainsahighlyconservedhistidineattheendof
strand 2 of the conserved core and an aspartate residue three residues downstream
from the HEXXH motif, both of which are predicted to project into the active-site
pocket(Fig.3A).InyetanothersetofpolyvalentproteinMPTases,theactive-sitemotif
is of the form HEXXXH, with four residues instead of the usual three between the
conserved histidines and a potential bend in the helix bearing the HEXXXH motif to
accommodatetheadditionalresidue.AphylogenetictreeoftheMPTasesofpolyvalent
proteins suggests that they belong to 12 distinct clades (Fig. 3A; see File S6 at
ftp://ftp.ncbi.nih.gov/pub/aravind/polyvalent/polyvalent.html). None of these clades
are specifically related to other previously defined clades of zincin-like MPTases,
suggesting that they are likely to constitute a distinct group that diversified in the
contextofpolyvalentproteins.
AcluetothepotentialfunctionoftheseMPTasesisofferedbypriorstudiesonthe D
phage P1 DdrB protein (Fig. 4A) encoded by the darA operon. This protein has been o
w
shown to be proteolytically processed into a 76-kDa ((cid:8)700-aa) polypeptide before n
being packaged into the phage head (51, 52). On the basis of the C-terminal MPTase lo
a
domain that we identified in this protein, it is likely that the domain catalyzes the d
e
autoproteolysis of the protein before packaging. Although it has not been shown d
whethertheN-orC-terminalfragmentisincorporatedintothephagehead,wepredict f
r
o
that it is likely to be the N-terminal fragment with the DdrB-like ParB domain (see m
below). In contrast to phage P1 DdrB, which is cleaved before encapsidation, the
h
bacteriophage N4 virion RNAP, which also contains an MPTase domain (Fig. 1B), is tt
p
packaged as an unprocessed protein even though the region encompassing the :
/
/
MPTase domain is required for encapsidation (53, 54). Similarly, the ArdC protein of jb
.
IncWplasmidpSaandtheTraC1primaseofIncPplasmidRP4(Fig.4A),bothofwhich a
s
contain a C-terminal MPTase, are transferred as full-length polypeptides during the m
conjugation process (16, 31). These examples suggest that the MPTase domains in .o
r
polyvalentproteinsmightalsofunctioninprocessingeventsotherthanprepackaging g
/
processing. In phage N4 virion RNAP-like polyvalent proteins, the MPTase domain o
n
mightautoproteolyticallyprocesslargeproteinsoncetheyareinsidethephagehead
A
to enable them to be suitably accommodated and/or readied for release during p
r
invasion.Alternatively,assuggestedbytheplasmidpolyvalentproteins,suchMPTase il
1
domainsmightalsoactafterbeinginjectedintothehostcell.Insuchcases,theycould
,

again autoproteolytically release specific domains of the polyvalent protein (e.g., the 2
0
C-terminal Toprim primase domain seen in TraC1) or convert the inactive polyvalent 1
9
protein into active products once inside the host cell. The diversity of enzymatic
b
mechanisms of the domains combined into the polyvalent proteins indeed supports y

g
thisscenario.Finally,itisalsopossiblethatsomeoftheMPTasedomainsactaseffectors u
that proteolytically target host proteins. Both of these functions are compatible with e
s
previously reported MPTase domains in conflict systems; they function either as t
autoproteolyticagentstoreleaseothereffectordomains,asseeninthePhotorhabdus
virulencecassettesystems(20),orthemselvesactaseffectors,asseeninpolymorphic
toxinsystems(10,46,47).
Theotherpeptidasesuperfamilies,i.e.,theassemblin-like,DD,andLonCpeptidase
superfamilies,areratherlimitedintheirdistribution.IncontrasttotheMPTasedomains,
theassemblin-likepeptidaseisalwaysfoundattheCterminiofthepolyvalentproteins
(Fig. 4A). Given the previously described role of the assemblin-like peptidases in the
processingofproteinsduringvirionassembly(55,56),itislikelythattheyplaythesame
FIG3Legend(Continued)
organismabbreviation,andNCBIaccessionnumberseparatedbyverticallinesandtotherightbyfamilyname.Sequencesofstructuresarelabeledwith
PDBcodes,shadedinorange.Poorlyconservedsecondarystructureelementsarecoloredwhite.Family-specificconservedresiduesdescribedinthetext
aredenotedbyblueboxes.Alignmentsarecoloredasfollows:h(hydrophobic),l(aliphatic),anda(aromatic)areshadedyellow;p(polar),(cid:7)(positively
charged),(cid:6)(negativelycharged),andc(charged)areshadedblue;s(small)andt(tiny)areshadedgreen;b(big)isshadedgray;absolutelyconserved
residuesareinwhiteletteringandshadedinblack.Fororganismabbreviations,seeFileS4atftp://ftp.ncbi.nih.gov/pub/aravind/polyvalent/polyvalent.html.
August2017 Volume199 Issue15 e00245-17 jb.asm.org 9
Iyeretal. JournalofBacteriology
A. Peptidases in polyvalent proteins E. NTases in polyvalent proteins
ddrB-ParB//MPTase hdf darA ddrA IddB ddrB ArdC-NMPTase DNAG ArdC-NMPTaseLPD17YodLLPD16NTase LPD19LPD2 TraJ-RHH Relaxase+LPD7 A+LrdPCD-1N6++MNPTTaassee++LLPPDD1197++LYPoDd2L
ddrB__P(γh-apgroet ePo1b_a_cYtePr_ia0)06491.1 traC-1__plasmid RP4__CAA42455.1 C824_04619__Clostridium_sp__WP_004070629.1 (Firmicutes)
LPD5MPTaseLPD1GNATAARRTTMPTaseMPTaseMPTaseMPTaseAARRTT LPD32RuLmPinDo3c6occmuesNth_6ysAlaps_eSLRP1D/51_8_LCPBDL11687L0P7D.11 6(FiNrmTiacsuteesL)PD19 HerbinLiPxD_2hemiceTrllauJl-oRsHilHyticRae_la_xCaRseZ+3L5P0D971.1N (tFasirem siocluotes)
Thioalkalivibrio_sp_ALE12__WP_026329813.1 (γ-proteobacteria)
F. Helicases in Polyvalent proteins
LPD5RMAZPWTaKs3eB_m1e6Nth66y3Ala0s_e_RoseSoWbaIc2t/eSr_NsFp_2_WP_0M0P78T1a5s6e61L.1P(Dα-1proteobDacatreBri-aC) LPD36meNth6yAlase SWI2/SNF2 LPD36meNth6yAlase SWI2/SNF2 LPD8 UvrD-helicase TopNPTroimpraimse
DarB__Phage P1__YP_006479.1 Streptomyces_viridifaciens_plasmid_OJH72069.1 (Actinobacteria)
InPaseLPD5MPTaseLPD1MPTaseMPTaseMPTaseAARRTT DarB-C
ACINWC743_A0745__Acinetobacter_sp__WP_009585050.1 (γ-proteobacteria) LPD36meNth6yAlase SWI2/SNF2 LPD6 UvrD-helicase VirB4-like VirD4/TraG LSPWDI23/6S+NNF62A+-LMPeDth6y+laUsver+D
Streptomyces_aureus__WP_037620140.1 (Actinobacteria)
PBECR1NUDIXAssemblin LPD3Assemblin
Saccharibacter_floricola Neisseria_bacilliformis G. DdrB-like ParB in Polyvalent proteins
__W(Pα_-p0r1o8te9o8b1a0c4t4e.r1ia) __(βW-pPro_t0e0o7b3a4c2te1r4ia1).1 ddrB-ParBMPTase Hdf DarA DdrAIddB DdrB LysozymeddrB-ParB
Terminase_SS Terminase_LS Portal+LPD3 MuF LPD3+Assemblin Capsid DdrB__Phage P1__YP_006491.1 Acinetobacter__WP_005204948.1
Neisseria_bacilliformis__WP_007342141.1 ((cid:1)-proteobacteria) (γ-proteobacteria)
B. ARTs in polyvalent proteins SMS/RadAArdC-NMPTaseLPD6LPD31ddrB-ParB D
MPTaseAARRTTAARRTTAARRTTAARRTTAARRTTAARRTTAARRTTLPD23MPTaseAARRTTAARRTTAARRTTAARRTTAARRTT ParB PBECR1ARTMPTase Gracilimonas_tropica__WP_020402799.1 (Balneolaeota) o
w
Pseudomonas_aeruginosa__WP_023121221.1 (γ-proteobacteria) Akkermansia_muciniphila H. RadC in Polyvalent proteins
WP_022198070.1 n
ddrB-ParBMuF-CMPTaseAARRTTPBECR5 LPD4 LPD38(Verrucomicrobia) MuF RadC Terminase_LS Portal MuF+RadC ParB RadC ARTMPTaseMuF-C LPD4 LPD38 lo
HHaaeemmoopTpehhrimilluuisnsa__siinenf_flluLuSeennzzaaeeP__oFFr3t3a00l4477__dA__dRCCrTBB+B-PPYYaBr8E8B7C+7MR005u22+F88L-C.P.11+D M(4(γ+Pγ-L-TpPparsDroeo3+t8teeoobbaacctteerriiaa)) SStotampahtyolRobcaaocdcuCcluums__AalroduCnreg-NuusmM___P_WTWaPPs__e0000L19P85D336138959439.1.1 ( F(Firimrmicicuutetess)) MRaPdTCaSsM+eAt+oarLdmmPCD-aeN1tl+9ioebllaac_uallubmaL__P(_FlDoKi2rnmHgQiucm5u1t_eT0_rsa9W)J5-P.R1_H (0Hα0-9pR5roe3lt3aex5oa4bs9ae.+c1LtePrDia7) aded

TenacibaculuAmrd_Cd-iNceMntPraTrcahsie__AAWRRPTT_L0P58D8685S3M89S.1/R (aBdaActeroidetes) SpoIIDInPaseLPDA9gPrBoEbCaRct1erLiuPmD_3tu6mmefeaNthc6yieAlansse__WPS_W04I622/0S5N53F22.1 (α-proRteaodbCacteriaL)PD39 LPD4 from
LPD14 KinaseInPaseLPD9LPD36meNth6yAlase SWI2/SNF2 MPTase I. SMS/RadA in Polyvalent proteins h
Bacteroides_cellulosilyticus__WP_060408190.1 (Bacteroidetes) ArdC-NMPTaseSMS/RadA LPD8LPD6MuF-CSMS/RadA ArdC-NMPTaseARTLPD6SMS/RadA tt
C. Kinases in polyvalent proteins p
FWlaPv_o0b4a7c7te7r9iu0m8_4s.1p Capn_o_cWytPop_h0a4g1a9_9c1y4n9o5d.1e gmi TenacWibPa_c0u5lu8m88_5d3ic8e9n.t1r archi :/
MPTaCshelamMyPdTiaa_streachKoimnaatsise__CRHS6F55I-6h1e.1li c(CashelamydiaMe)PTaseVerruLcPomDi3cr7obiu(MmVe_PrsTrpuaicnsooemsuicmKro_inb_aiWas)Pe_02K9in19a0s8e12.1 J. Primas(Beasct eirno idPeotelsy)valent proteins (Bacteroidetes) (Bacteroidetes) /jb.
KinaseV+MerPruTacsoemicrKoibnaiusme+_MsPpTinaosesum_LK_PinWDa3sP7e++_MK0iPn2aT9ases1e9+081M2P.1T a(VseerrucomPBicEroCbRi+aM)PTase RelaxaseEnteTroocproimcc-Pursim_faaseecaTolipso_is_oWmPer_a0s1e6627S3S5B1.1 (FNir6AmArd-MiCce-uNtht+eyLlasPs)De3+6S+NF BacterVoiirdEes_fraTogpilriism_-_PWrimPa_se045T8ra9J9-3R6H6H.1 (BaRcetlearxoaisdeetes) asm
D. GNATs in polyvalent proteins (solo Toprim-Primase related to polyvalent form) (solo Toprim-Primase related to polyvalent form) .
o
GNATART Terminase_SS Terminase_LS GNAT+ART PrimpolArdC-NLPD32LPD32LPD25 LPD36meNth6yAlase SWI2/SNF2 rg
uncultured_Mediterranean_phage_uvMED__BAR32501.1 (Type 1 GNAT) Streptococcus_equi__WP_050320114.1 (Firmicutes) /

meNth6yAlase Sno-like SWI/SNF SnoCArdC-NMPTasePTroimpraimseGNAT TraJ-SRtHreHptocoRceclauxsa_seequi__WP_05032PL0Pr1imD12p45o+l.+1LAP r Dd(C3F6-iN+rmN+L6iPAcD-uM3t2ee+thsLy)PlaDs3e2++SNF on
Methylomonas_sp_MK1__WP_020485828.1 (γ-proteobacteria) (Type 2 GNAT) A
A18R/UvsW helicase SSB Primpol+D5ATPase VRR-NUC
TraJ-RHH Relaxase+LPD7 VirD4/TraG Topoisomerase N+M6AP-TMaeseth+yTlaosperi+mS-nPori+mSansoeC++GANrAdCT-N LP101_065__Listeria_phage_LP-101__YP_009044866.1 p
FIG4DomainMeathryclohmiotneasc_tsup_rMeKs1_a_nWdP_g02e04n8e582n8e.1i (gγ-hprboteoorbhacoteoriad) (sTyopef 2d GoNmATa)insfoundinpolyvalentproteinsg(Prriomupopl reeldatebd tyo otnhees fopunrde isne pnolycvealeontf pvroaterinisousprincipaldomainsorgroups ril 1
ofdomains,includingpeptidases(A),ARTs(B),kinases(C),GNATs(D),Pol-(cid:2)NTases(E),helicases(F),DdrB-likeParB(G),RadC(H),SMS/RadA(I),andToprimfold ,
andPrimpolprimases(J).Theseincludeonlyasmallsampleoftheentirediasporaofassociations.Forthecompletesetofdomainarchitecturesandoperons, 2
0
see ftp://ftp.ncbi.nih.gov/pub/aravind/polyvalent/polyvalent.html. Proteins and gene neighborhoods are shown with their species names and GenBank 1
accessionnumbers.Forgeneneighborhoods,theaccessionnumberofthegeneindarkpinkisused.Genenamesareshownonlyforwell-studiedproteins. 9

Genesinneighborhoodsareshownasboxedarrows,withthearrowheadpointingtothe3=gene.Domainsarenotdrawntoscale. b
y

g
u
role when found in polyvalent proteins. The DD peptidase is likely to be involved in e
s
breachingofthecellwall(seebelow). t
ARTs. ARTs use NAD(cid:7) as a substrate to catalyze the transfer of one or more
ADP-ribosemoietiestodiversetargets,resultinginN-,O-,orS-glycosidiclinkageswith
the 1=’ position of ribose (57). The substrate range of ARTs includes a variety of side
chains and terminal positions in proteins, nucleic acids, and nucleotides. Previous
sequence and structural analyses showed that the primary diversification of ARTs
occurredinbacterialandviralbiologicalconflictsystems(41).ARTsarebyfarthemost
commonpredictedprotein-modifyingenzymaticdomainsfoundinpolyvalentproteins.
They are prototyped by the one found in the polyvalent protein from Roseovarius
Plymouth podovirus 1 (accession no. CBX87992.1) along with a phage N4-like RNAP
module(Fig.1B).WefoundthatthisfamilyofARTdomainshasanactive-siteconfig-
urationwithahistidine(H)instrand1,atyrosine(Y)instrand2,andanaspartate(D)
in strand 5. Identification of distinct shared sequence features allowed us to group
these with a clade of ART domains, the Tox-ART-HYD2 clade we had previously
described in polymorphic toxin systems (10), and a novel class of toxin-antitoxin
systems (41, 48). ART domains found in polyvalent proteins are both (i) of phage
August2017 Volume199 Issue15 e00245-17 jb.asm.org 10