NIOA IA I AIALC.ALLLFILFRMIRAVDAL.K.RSKDACLADLLNYA. PTor fD. SHLSTS,.LITHCADLVRSWRVOCIAFESAEPELVSIORHEOLNCL;WRA ISC. VirB.
\./ 1993 Oxford University Press
Nucleic Acids Research, 1993, Vol. 21, No. 2 353-354
The virB operon of the Agrobacterium tumefaciens virulence regulon has sequence similarities to B, C and D open reading frames downstream of the pertussis toxinoperon and to the DNA transfer-operons of broad-hostrange conjugative plasmids Ken Shirasu and Clarence l.Kado* Davis Crown Gall Group, Department of Plant Pathology, University of California, Davis, CA 95616, USA Received December 1, 1992; Accepted December 4, 1992 The 28.6 kDa virulence regulon of te pTiC58 plasmid is comprised of six major operons (1), of which the virB operon is required for the transfer of the T-DNA from the resident Ti plasmid in Agrobacterium tumefaciens to plant cells. A growing body of indirect evidence suggests tat this operon encodes proteins that may simulate a bacterial conjugative transfer A Vi rB2
IPLKDLRASLPRLAFMAAC .........TLLS5ATLPDLAO. II.: .1. -:: .. 1. II I..: .1. MRCFERYRtVNIIRL.SLSNAVI"tVSCYAPSVVGAMGWS IFSS9G.PAAAOS .: It II .:,1: :11 1:.
Tr,bC
WMTAPFRLSWMNRXILFYIAVFFVLALAL.SAH .......PAASE
PTortB
PTor."B
VirB2 TrbC
1A11IAWAaYKLLFR
.HA
CSVIV I..;CSGN A00SIPASKTIMICTFILCIPF.C0
RA
ACCCLOR
VMAIVVVLRCAkSV
CGTCCLPYESWtLTNLRNSVTCPV
.AFALSIICIWVA
CCVLIFCC.ELNA
PT.rfS DVLDNVRVVLACLLICMASAIARYLLT 102 VirB2
S LLVA
, 121 'VCIVIUSLCTLSCCC
T rbC
rLFatLTVLIGFVLVVLVMNAILLVGASNVNRTC(AE
TrbC
AVRAVAAGltLA 140
IAAAISNGALHOVOVAAAD
B PTor fC V i,rB3
TrbD
PSor fC V irB3
...MRDPLFICOCTRPAlIAMCPATPLAVCSCTrIALLG IWFS IAFLALr NNDRLEEATLYLAATR PALFIA
PLSLACLLVKFACW IVIVONPLY
..4LRT IPIRRGRNM).. DRELVNFSCl^FALI FSAOELRtA PVALL1.. R IlURRDDOOtR IWL LRM§tLSRDRTHAFWOSMVAP EVVLVPLWrG M
v
r
CR
V
C
IbICCASVSPN P
TrbD TVYGLILWFGA.LYAFRIMAKADPKMFRVYLRHRRYKPYY.. PARSTP
PT.rfC LRYAERRRRLRKP 104 Vir,B3 TrbD
lltVPMAROtA 1011
FRNMS=CAYR 103
PSor.fD
NRRCOSWAFAAIARNERAIAAFIPYS
TrbE
NIOA IA I AIALC.ALLLFILFRMIRAVDAL.K.RSKDACLADLLNYA
PTor fD SHLSTS,.LITHCADLVRSWRVOCIAFESAEPELVSIORHEOLNCL;WRA ISC V i rB
CHSD IVLLECCSIU-''11W-lv_
Ar
TLRA lN IAD
Tr,bE AVVDDC;VIVCKNCSrFAWLYKGDNHASSSDOOREVVSAR I NALAGLGS
PSorfD
DOVALWIHCIRRKTOACLDAR YENPFCRALDASYNA
DHVS IYAH LVRHDDYVPPS PARNHFRSArSASLSEAFEE ..RVLSGK LLR.N
TrbE
GWM. IHVDVRR .PAPNYAERCLSAFPDRLTAAIEE.Er.RMNRr ESLGTMY
DHFLTLIVSP.RAALCKVM"FTKRYROKEN. DLTAQRNLEDLWHLVA
TrbE
EGYFVLT LTW rFPPLLAORKr'VELMFDMDATAPDRtBARTRGL IDO FKRDV
Comparative sequence analyses between the virB operon and those present in the data bank revealed striking similarities to the Tra2 operon of RP4 (8) and to the PilW operon of R388 (9-11) that are required for conjugative transfer between bacteria. Here, we report that these DNA transfer related proteins have striking sequence similarities to the open reading frames downstream of the PT operon (12) involved in pertussis toxin biosynthesis in Bordetella pertussis (Figure 1). The amino acid sequence similarity (and identity) between VirB2 and PT-orfB, between VirB3 and PT-orfC, and between the amino terminal portion of VirB4 and PT-orfD are 54% (31%), 54% (26%) and 53 % (25 %), respectively (calculated by the BESTFIT program, GCG, Wisconsin). Noteworthy is that the genetic arrangement are also alike (Figure 2). Although the function of each of the genes of the above operons (virB, Tra2, PilW and PTdownstream orfs') remain to be identified, these genes (and orfs') are likely encoding accessory proteins to facilitate the secretion of either DNA or polypeptide, or both as complexes. That VirBi 1 protein sequence is similar to secretory proteins of Gram-negative bacteria (13) support this concept that the virB operon encodes the secretion machinery necessary for the transfer of T-DNAprotein complexes from A. tumefaciens to plant cells.
ACKNOWLEDGEMENTS We thank the collaborative efforts of Dr Fernando de la Cruz. This work was support by NIH Grant GM-45550-22.
GALE...AYGLRRLG1RE1KoD LrrEVGEA ...LRLIPMTRrTPVP RS IES RLS SAVSLTR LKGHK
C
D
B1
B2
B3
B4
C
D
E
VirB
VNEDS1TTVTH DDFLRWLOFCVrGLMHH W
B
Trb
iSgume 1. Amino acid sequence comparisons between VirB, PT and Trb proteins. A. VirB2 compared withi PTorfB and TrbC. B. VirB3 compared withi PTorfC and TrbD. C. VirB4 compared wit PTorfD and TrbE. *
B
PTorf
PT.rfD TTLR ...SHGE-NH ..EO ITV GETDSA ...CR ...RYSRTL
T rbE
J03320
apparatus to facilitate T-DNA transfer to plants (2-7).
OLLAHVRRMDIGSLI E
VirB4
VirB4
no.
RLOARO
V irB4
PTor fD EFYLTLVWRPGHAALCKRAIHHCOAEVRR
GenBank accession
To whom correspondence should be addressed
LU~~~~~~~Am~~~
Figure 2. Identical genetic arrangement of the ORFs of P, VirB and Trb operons.
354 Nucleic Acids Research, 1993, Vol. 21, No. 2
REFERENCES 1. 2. 3. 4.
5. 6. 7. 8.
Rogowsky,P.M., et al. (1990) Plasmid 23, 85106. Ream,W. (1989) Annu. Rev. Phytopathol. 27, 583-618. Kado,C.I. (1991) Crit. Revs. Plant Sci. 10, 1-32. Zambryski,P. (1992) Annu. Rev. Plant Physiol. Plant Mol. Biol. 43, 465-490. Winans,S.C. (1992) Microbiol. Rev. 56, 12-31. Beijersbergen,A., et al. (1992) Science 256, 1324-1327. Kado,C.I. (1993) In: Clewell,D.B., Bacterial Conjugation. Plenum Publ. NY. Lessl,M., et al. (1992) J. Biol. Chemn. 267, 20471-20480.
9. Bolland,S., et al. (1990) J. Bacteriol. 172, 5795-5802. 10. Shirasu,K., et al. (1993) Proc. Natl. Acad. Sci. USA, submitted. 11. Bolland,S., Llosa,M. and de la Cruz,F. unpublished. 12. Nicoisa,A., et al. (1986) Proc. Natl. Acad. Sci. USA 83, 4631-4635. 13. Dums,F., et al. (1991) Mol. Gen. Genet. 229, 357-364.
