title: DNA substrate-induced activation of the Agrobacterium VirB/VirD4 type IV secretion system.
abstract: The bitopic membrane protein VirB10 of the Agrobacterium VirB/VirD4 type IV secretion system (T4SS) undergoes a structural transition in response to sensing of ATP binding or hydrolysis by the channel ATPases VirD4 and VirB11. This transition, detectable as a change in protease susceptibility, is required for DNA substrate passage through the translocation channel. Here, we present evidence that DNA substrate engagement with VirD4 and VirB11 also is required for activation of VirB10. Several DNA substrates (oncogenic T-DNA and plasmids RSF1010 and pCloDF13) induced the VirB10 conformational change, each by mechanisms requiring relaxase processing at cognate oriT sequences. VirD2 relaxase deleted of its translocation signal or any of the characterized relaxases produced in the absence of cognate DNA substrates did not induce the structural transition. Translocated effector proteins, e.g., VirE2, VirE3, and VirF, also did not induce the transition. By mutational analyses, we supplied evidence that the N-terminal periplasmic loop of VirD4, in addition to its catalytic site, is essential for early-stage DNA substrate transfer and the VirB10 conformational change. Further studies of VirB11 mutants established that three T4SS-mediated processes, DNA transfer, protein transfer, and pilus production, can be uncoupled and that the latter two processes proceed independently of the VirB10 conformational change. Our findings support a general model whereby DNA ligand binding with VirD4 and VirB11 stimulates ATP binding/hydrolysis, which in turn activates VirB10 through a structural transition. This transition confers an open-channel configuration enabling passage of the DNA substrate to the cell surface.
authors.affiliation: Department of Microbiology and Molecular Genetics, University of Texas Medical School at Houston, Houston, Texas, USA.
authors.forename: Eric
authors.initials: E
authors.lastname: Cascales
authors.forename: Krishnamohan
authors.initials: K
authors.lastname: Atmakuri
authors.forename: Mayukh K
authors.initials: MK
authors.lastname: Sarkar
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Christie
mesh_terms: D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism; D016960:Agrobacterium tumefaciens / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D003227:Conjugation, Genetic; D004252:DNA Mutational Analysis; D004269:DNA, Bacterial / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation; D010447:Peptide Hydrolases / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D015398:Signal Transduction; D037521:Virulence Factors / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D008565:Membrane Proteins; C036483:T-DNA; D037521:Virulence Factors; D010447:Peptide Hydrolases; D000251:Adenosine Triphosphatases
references.citation: J Bacteriol. 2005 May;187(10):3486-95
references.pmid: 15866936
references.citation: J Bacteriol. 2011 May;193(10):2566-74
references.pmid: 21421757
references.citation: Nat Struct Biol. 2000 Mar;7(3):205-9
references.pmid: 10700278
references.citation: Proc Natl Acad Sci U S A. 1992 Dec 15;89(24):11837-41
references.pmid: 1465407
references.citation: Mol Microbiol. 2003 Jul;49(1):105-16
references.pmid: 12823814
references.citation: J Biol Chem. 2004 Mar 19;279(12):10955-61
references.pmid: 14699106
references.citation: J Bacteriol. 1997 Sep;179(18):5835-42
references.pmid: 9294442
references.citation: Mol Microbiol. 2002 Jul;45(1):1-8
references.pmid: 12100543
references.citation: J Bacteriol. 1994 Jun;176(12):3646-60
references.pmid: 8206843
references.citation: Mol Microbiol. 2011 Dec;82(5):1071-85
references.pmid: 22066957
references.citation: Plasmid. 2005 Jul;54(1):1-25
references.pmid: 15907535
references.citation: Mol Microbiol. 2007 Nov;66(4):948-60
references.pmid: 17919288
references.citation: EMBO J. 1996 Nov 1;15(21):5804-11
references.pmid: 8918458
references.citation: Nature. 2009 Dec 24;462(7276):1011-5
references.pmid: 19946264
references.citation: Mol Microbiol. 2009 Feb;71(3):779-94
references.pmid: 19054325
references.citation: Methods Mol Biol. 2013;966:187-204
references.pmid: 23299736
references.citation: Mol Microbiol. 1998 Jan;27(2):405-14
references.pmid: 9484895
references.citation: Cell. 1986 Nov 7;47(3):471-7
references.pmid: 3021341
references.citation: J Bacteriol. 2000 Jul;182(13):3705-16
references.pmid: 10850985
references.citation: Mol Plant Microbe Interact. 2009 Nov;22(11):1356-65
references.pmid: 19810805
references.citation: Science. 2009 Jan 9;323(5911):266-8
references.pmid: 19131631
references.citation: Proc Natl Acad Sci U S A. 2003 Sep 2;100(18):10465-70
references.pmid: 12925737
references.citation: Microbiol Mol Biol Rev. 2009 Dec;73(4):775-808
references.pmid: 19946141
references.citation: FEMS Microbiol Rev. 2012 Nov;36(6):1046-82
references.pmid: 22545799
references.citation: Cell Microbiol. 2010 Sep 1;12(9):1203-12
references.pmid: 20642798
references.citation: Mol Microbiol. 2004 Dec;54(5):1199-211
references.pmid: 15554962
references.citation: Proc Natl Acad Sci U S A. 2007 Dec 11;104(50):20019-24
references.pmid: 18056647
references.citation: Mol Microbiol. 2012 Aug;85(4):602-17
references.pmid: 22788760
references.citation: J Bacteriol. 2001 Oct;183(20):5813-25
references.pmid: 11566978
references.citation: Proc Natl Acad Sci U S A. 2005 Jan 18;102(3):832-7
references.pmid: 15644442
references.citation: Philos Trans R Soc Lond B Biol Sci. 2012 Apr 19;367(1592):1073-87
references.pmid: 22411979
references.citation: Mol Microbiol. 2003 Feb;47(4):891-901
references.pmid: 12581347
references.citation: Science. 2000 Nov 3;290(5493):979-82
references.pmid: 11062129
references.citation: Science. 2004 May 21;304(5674):1170-3
references.pmid: 15155952
references.citation: EMBO J. 2003 May 1;22(9):1969-80
references.pmid: 12727865
references.citation: Microbiology (Reading). 2005 Nov;151(Pt 11):3505-3516
references.pmid: 16272374
references.citation: J Bacteriol. 1988 Apr;170(4):1430-7
references.pmid: 2832362
references.citation: Res Microbiol. 2002 May;153(4):199-204
references.pmid: 12066890
references.citation: J Bacteriol. 2008 May;190(10):3632-45
references.pmid: 18326569
references.citation: Biochim Biophys Acta. 2010 Nov;1798(11):2160-9
references.pmid: 20647001
references.citation: Nat Rev Microbiol. 2009 Oct;7(10):703-14
references.pmid: 19756009
references.citation: J Bacteriol. 1998 Jan;180(2):430-4
references.pmid: 9440537
references.citation: Proc Natl Acad Sci U S A. 2005 Jun 7;102(23):8156-61
references.pmid: 15919815
references.citation: Mol Microbiol. 2000 Sep;37(6):1283-92
references.pmid: 10998162
references.citation: Curr Pharm Des. 2004;10(13):1551-65
references.pmid: 15134575
references.citation: Biochim Biophys Acta. 2012 Dec;1818(12):3158-66
references.pmid: 22940106
references.citation: J Mol Biol. 2006 Jun 30;360(1):56-66
references.pmid: 16730027
references.citation: J Bacteriol. 1996 Mar;178(6):1498-504
references.pmid: 8626274
references.citation: Biochemistry. 2003 Feb 18;42(6):1391-400
references.pmid: 12578351
references.citation: Nature. 2001 Feb 1;409(6820):637-41
references.pmid: 11214325
references.citation: J Biol Chem. 2002 Nov 29;277(48):46456-62
references.pmid: 12244053
references.citation: J Bacteriol. 1997 Feb;179(3):583-91
references.pmid: 9006008
references.citation: J Bacteriol. 2003 May;185(9):2867-78
references.pmid: 12700266
references.citation: EMBO J. 1998 Nov 16;17(22):6487-96
references.pmid: 9822594
references.citation: EMBO J. 1985 Apr;4(4):891-8
references.pmid: 2990912
references.citation: Mol Microbiol. 2003 Sep;49(6):1699-713
references.pmid: 12950931
references.citation: J Biol Chem. 2007 Aug 31;282(35):25569-76
references.pmid: 17599913
references.citation: Plasmid. 1982 Jan;7(1):15-29
references.pmid: 6283573
references.citation: J Bacteriol. 1997 Jan;179(1):78-89
references.pmid: 8981983
references.citation: Mol Microbiol. 2005 Oct;58(2):565-79
references.pmid: 16194240
references.citation: Nucleic Acids Res. 2003 Feb 1;31(3):860-8
references.pmid: 12560481
references.citation: Biochim Biophys Acta. 2011 Apr;1808(4):1032-9
references.pmid: 21211515
references.citation: Int J Biochem Cell Biol. 2001 Sep;33(9):839-43
references.pmid: 11461827
references.citation: J Bacteriol. 1998 Aug;180(15):3933-9
references.pmid: 9683491
references.citation: Proc Natl Acad Sci U S A. 2004 Dec 7;101(49):17228-33
references.pmid: 15569944
references.citation: Mol Microbiol. 2007 Oct;66(1):252-61
references.pmid: 17880426
references.citation: J Mol Biol. 1978 Jul 5;122(3):287-300
references.pmid: 151148
references.citation: J Bacteriol. 2000 Jul;182(14):3885-95
references.pmid: 10869063
references.citation: Plant Physiol. 2008 Feb;146(2):325-32
references.pmid: 18250230
references.citation: Nature. 1975 Jun 26;255(5511):742-3
references.pmid: 1134573
references.citation: Nature. 2009 Dec 24;462(7276):992-4
references.pmid: 20033031
references.citation: Structure. 2007 Mar;15(3):255-7
references.pmid: 17355860
references.citation: FEMS Microbiol Rev. 2010 Jan;34(1):18-40
references.pmid: 19919603
references.citation: Science. 1996 Aug 23;273(5278):1107-9
references.pmid: 8688097
references.citation: Annu Rev Microbiol. 2005;59:451-85
references.pmid: 16153176
references.citation: J Mol Biol. 2004 Aug 20;341(4):961-77
references.pmid: 15328612
references.citation: EMBO J. 2007 May 16;26(10):2540-51
references.pmid: 17505518
references.citation: J Bacteriol. 1999 Jul;181(14):4342-52
references.pmid: 10400593