DEVELOPMENT OF CHIMERIC TYPE IV SECRETION SYSTEMS FOR TRANSFER OF HETEROLOGOUS SUBSTRATES ACROSS THE GRAM-NEGATIVE CELL ENVELOPE

Many bacteria use Type IV Secretion Systems (T4SSs) to aid in pathogenesis by translocating virulence factors across the cell envelope and into eukaryotic cells. These systems are structurally and functionally diverse, but are often compared to the archetypal VirB/VirD4 T4SS of Agrobacterium tumefaciens. This system is composed of the VirD4 type IV coupling protein (T4CP) and 11 VirB subunits (VirB1-11) that assemble as the secretion channel and an extracellular pilus. The T4CP is an inner membrane ATPase that interacts with T4SS substrates and the secretion channel, and is thought to link substrates with the secretion channel and possibly energize transfer through the channel lumen. In this thesis, I sought to adapt T4SSs in the surrogate hosts A. tumefaciens and Escherichia coli for use in identification of novel T4SS effector proteins from genetically-intractable Rickettsial species. I first constructed chimeric T4SSs in A. tumefaciens by substituting native VirD4 with Rickettsial VirD4 homologs. However, I was unable to demonstrate transfer of the promiscuous IncQ plasmid pML122 or known A. tumefaciens effector proteins. I next tested the E. coli pKM101-encoded T4SS, which is known to transfer DNA substrates, for the capacity to deliver heterologous protein substrates to E. coli recipients. Using the Cre-recombinase reporter assay for translocation (CRAfT), I showed that pKM101 translocates effector proteins from A. tumefaciens and two Rickettsial species, Anaplasma phagocytophilum and Wolbachia pipientis. I next created chimeric T4CPs by joining the transmembrane domain (TMD) of pKM101-encoded TraJ with the soluble domains (SDs) of VirD4 homologs from A. tumefaciens and the Rickettsial species. I showed that all of these chimeric systems translocate protein substrates, although less efficiently than the native pKM101 T4SS. Finally, I demonstrated that a variable C-terminal extension (CTE) that is present on the A. tumefaciens and Rickettsial T4CPs plays a modulatory role for secretion of different protein substrates. My findings showed for the first time that a T4SS encoded by an E. coli conjugative plasmid is capable of translocating a variety of protein substrates from phylogenetically diverse alphaproteobacterial species, including A. tumefaciens, A. phagocytophilum, and W. pipientis

Scientific access into Mercer Subglacial Lake: scientific objectives, drilling operations and initial observations

© The Author(s), 2021. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Priscu, J. C., Kalin, J., Winans, J., Campbell, T., Siegfried, M. R., Skidmore, M., Dore, J. E., Leventer, A., Harwood, D. M., Duling, D., Zook, R., Burnett, J., Gibson, D., Krula, E., Mironov, A., McManis, J., Roberts, G., Rosenheim, B. E., Christner, B. C., Kasic, K., Fricker, H. A., Lyons, W. B., Barker, J., Bowling, M., Collins, B., Davis, C., Gagnon, A., Gardner, C., Gustafson, C., Kim, O-S., Li, W., Michaud, A., Patterson, M. O., Tranter, M., Ryan Venturelli, R., Trista Vick-Majors, T., & Elsworth, C. Scientific access into Mercer Subglacial Lake: scientific objectives, drilling operations and initial observations. Annals of Glaciology, 62(85–86), (2021): 340–352, https://doi.org/10.1017/aog.2021.10.The Subglacial Antarctic Lakes Scientific Access (SALSA) Project accessed Mercer Subglacial Lake using environmentally clean hot-water drilling to examine interactions among ice, water, sediment, rock, microbes and carbon reservoirs within the lake water column and underlying sediments. A ~0.4 m diameter borehole was melted through 1087 m of ice and maintained over ~10 days, allowing observation of ice properties and collection of water and sediment with various tools. Over this period, SALSA collected: 60 L of lake water and 10 L of deep borehole water; microbes >0.2 μm in diameter from in situ filtration of ~100 L of lake water; 10 multicores 0.32–0.49 m long; 1.0 and 1.76 m long gravity cores; three conductivity–temperature–depth profiles of borehole and lake water; five discrete depth current meter measurements in the lake and images of ice, the lake water–ice interface and lake sediments. Temperature and conductivity data showed the hydrodynamic character of water mixing between the borehole and lake after entry. Models simulating melting of the ~6 m thick basal accreted ice layer imply that debris fall-out through the ~15 m water column to the lake sediments from borehole melting had little effect on the stratigraphy of surficial sediment cores.This material is based upon work supported by the US National Science Foundation, Section for Antarctic Sciences, Antarctic Integrated System Science program as part of the interdisciplinary (Subglacial Antarctic Lakes Scientific Access (SALSA): Integrated study of carbon cycling in hydrologically-active subglacial environments) project (NSF-OPP 1543537, 1543396, 1543405, 1543453 and 1543441). Ok-Sun Kim was funded by the Korean Polar Research Institute. We are particularly thankful to the SALSA traverse personnel for crucial technical and logistical support. The United States Antarctic Program enabled our fieldwork; the New York Air National Guard and Kenn Borek Air provided air support; UNAVCO provided geodetic instrument support. Hot water drilling activities, including repair and upgrade modifications of the WISSARD hot water drill system, for the SALSA project were supported by a subaward from the Ice Drilling Program of Dartmouth College (NSF-PLR 1327315) to the University of Nebraska-Lincoln. J. Lawrence assisted with manuscript preparation. Finally, we are grateful to C. Dean, the SALSA Project Manager, and R. Ricards, SALSA Project Coordinator at McMurdo Station, for their organizational skills, and B. Huber of Lamont-Doherty Earth Observatory for providing the SBE39 PT sensors and the Nortek Aquadopp current meter and assisting with interpretation of the data. B. Huber also provided helpful input on programing and calibrating the SBE19PlusV2 6112 CTD