Characterization Of The Role Of The Pkm101-Encoded T4Ss Outer Membrane Core Complex In Substrate Transfer, Pilus Biogenesis, And Recipient Cell Contact

Author

Jay E. Gordon, The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical SciencesFollow

Author ORCID Identifier

http://www.orcid.org/0000-0001-7692-5569

Date of Graduation

5-2017

Document Type

Dissertation (PhD)

Program Affiliation

Microbiology and Molecular Genetics

Degree Name

Doctor of Philosophy (PhD)

Advisor/Committee Chair

Peter J. Christie, Ph.D.

Committee Member

Mikhail Bogdanov, Ph.D.

Committee Member

Heidi B. Kaplan, Ph.D.

Committee Member

Michael Lorenz, Ph.D.

Committee Member

William Margolin, Ph.D.

Abstract

The bacterial type IV secretion systems (T4SS) encoded on the Escherichia coli pKM101 and R388 conjugative plasmids and the Agrobacterium tumefaciens virB operon are composed of 12 subunits arranged in an architecturally similar fashion. Structural studies of the T4SS from R388 (Trw_R388) highlighted the presence of a distinct outer membrane core complex (OMCC) and inner membrane complex (IMC) joined together by a central stalk. This thesis is focused on the development of the pKM101 model system and the role of an OMCC cap region formed by alpha helical antenna projections (AP) found in VirB10 and TraF_pKM101. I introduced various internal deletions, truncations, and substitutions between TraF_pKM101 and VirB10 to study the role of the AP cap in (i) substrate transfer to recipient plant or bacterial cells, (ii) pilus biogenesis and donor-specific bacteriophage sensitivity, and (iii) recipient cell contact. My results demonstrate that the C-terminal tails of VirB10 and TraF_pKM101 are essential for DNA transfer and pilus biogenesis, whereas the AP domains are only important for pilus biogenesis. Neither of these domains is critical for recipient cell contact as monitored by sensitivity to Pseudomonas aeruginosa containing a functional type VI secretion system. Finally, I demonstrated that the IMC of pKM101 is able to functionally pair with the OMCCs from either VirB/VirD4, Trw_R388, or the Ptl T4SS of Bordetella pertussis restoring recipient cell contact and plasmid transfer. These results highlight the common ancestry between these functionally distinct machines and support a model where functionally independent IMCs and OMCCs have co-evolved to form the T4SS in Gram-negative bacteria.

Keywords

type IV secretion, conjugation, pKM101, pilus, Agrobacterium, TraF, VirB10, donor-specific bacteriophage, R388, Bordetella