title: Chimeric Coupling Proteins Mediate Transfer of Heterologous Type IV Effectors through the Escherichia coli pKM101-Encoded Conjugation Machine.
abstract: Bacterial type IV secretion systems (T4SSs) are composed of two major subfamilies, conjugation machines dedicated to DNA transfer and effector translocators for protein transfer. We show here that the Escherichia coli pKM101-encoded conjugation system, coupled with chimeric substrate receptors, can be repurposed for transfer of heterologous effector proteins. The chimeric receptors were composed of the N-terminal transmembrane domain of pKM101-encoded TraJ fused to soluble domains of VirD4 homologs functioning in Agrobacterium tumefaciens, Anaplasma phagocytophilum, or Wolbachia pipientis A chimeric receptor assembled from A. tumefaciens VirD4 (VirD4At) mediated transfer of a MOBQ plasmid (pML122) and A. tumefaciens effector proteins (VirE2, VirE3, and VirF) through the pKM101 transfer channel. Equivalent chimeric receptors assembled from the rickettsial VirD4 homologs similarly supported the transfer of known or candidate effectors from rickettsial species. These findings establish a proof of principle for use of the dedicated pKM101 conjugation channel, coupled with chimeric substrate receptors, to screen for translocation competency of protein effectors from recalcitrant species. Many T4SS receptors carry sequence-variable C-terminal domains (CTDs) with unknown function. While VirD4At and the TraJ/VirD4At chimera with their CTDs deleted supported pML122 transfer at wild-type levels, ΔCTD variants supported transfer of protein substrates at strongly diminished or elevated levels. We were unable to detect binding of VirD4At's CTD to the VirE2 effector, although other VirD4At domains bound this substrate in vitro We propose that CTDs evolved to govern the dynamics of substrate presentation to the T4SS either through transient substrate contacts or by controlling substrate access to other receptor domains.
  
  
  
  Bacterial type IV secretion systems (T4SSs) display striking versatility in their capacity to translocate DNA and protein substrates to prokaryotic and eukaryotic target cells. A hexameric ATPase, the type IV coupling protein (T4CP), functions as a substrate receptor for nearly all T4SSs. Here, we report that chimeric T4CPs mediate transfer of effector proteins through the Escherichia coli pKM101-encoded conjugation system. Studies with these repurposed conjugation systems established a role for acidic C-terminal domains of T4CPs in regulating substrate translocation. Our findings advance a mechanistic understanding of T4CP receptor activity and, further, support a model in which T4SS channels function as passive conduits for any DNA or protein substrates that successfully engage with and pass through the T4CP specificity checkpoint.
authors.affiliation: Department of Microbiology and Molecular Genetics, McGovern Medical School, Houston, Texas, USA.
authors.forename: Neal
authors.initials: N
authors.lastname: Whitaker
authors.affiliation: Department of Microbiology and Molecular Genetics, McGovern Medical School, Houston, Texas, USA.
authors.forename: Trista M
authors.initials: TM
authors.lastname: Berry
authors.affiliation: Department of Microbiology and Molecular Genetics, McGovern Medical School, Houston, Texas, USA.
authors.forename: Nathan
authors.initials: N
authors.lastname: Rosenthal
authors.affiliation: Department of Microbiology and Molecular Genetics, McGovern Medical School, Houston, Texas, USA.
authors.forename: Jay E
authors.initials: JE
authors.lastname: Gordon
authors.affiliation: Department of Microbiology and Molecular Genetics, McGovern Medical School, Houston, Texas, USA.
authors.forename: Christian
authors.initials: C
authors.lastname: Gonzalez-Rivera
authors.affiliation: Department of Biology, Indiana University, Bloomington, Indiana, USA.
authors.forename: Kathy B
authors.initials: KB
authors.lastname: Sheehan
authors.affiliation: Department of Microbiology and Immunology, Virginia Commonwealth University Medical Center, School of Medicine, Richmond, Virginia, USA.
authors.forename: Hilary K
authors.initials: HK
authors.lastname: Truchan
authors.affiliation: Department of Microbiology and Immunology, Virginia Commonwealth University Medical Center, School of Medicine, Richmond, Virginia, USA.
authors.forename: Lauren
authors.initials: L
authors.lastname: VieBrock
authors.affiliation: Department of Biology, Indiana University, Bloomington, Indiana, USA.
authors.forename: Irene L G
authors.initials: IL
authors.lastname: Newton
authors.affiliation: Department of Microbiology and Immunology, Virginia Commonwealth University Medical Center, School of Medicine, Richmond, Virginia, USA.
authors.forename: Jason A
authors.initials: JA
authors.lastname: Carlyon
authors.affiliation: Department of Microbiology and Molecular Genetics, McGovern Medical School, Houston, Texas, USA Peter.J.Christie@uth.tmc.edu.
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Christie
mesh_terms: D003227:Conjugation, Genetic / Q000502:physiology*; D004269:DNA, Bacterial; D004926:Escherichia coli / Q000235:genetics / Q000502:physiology*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology; D000072417:Protein Domains; D011993:Recombinant Fusion Proteins; D000069319:Type IV Secretion Systems / Q000502:physiology*
chemical_list: D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D011993:Recombinant Fusion Proteins; D000069319:Type IV Secretion Systems