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Title:Characterization of an agrobacterial plasmid inducible for transfer by mannopine and evolution of the core replication and transfer functions of repabc plasmids with class i quorum-sensing and transfer systems
Author(s):Wetzel, Margaret Elizabeth
Director of Research:Farrand, Stephen K.
Doctoral Committee Chair(s):Farrand, Stephen K.
Doctoral Committee Member(s):Imlay, James A.; Blanke, Steven R.; Orlean, Peter A. B.
Department / Program:Microbiology
Discipline:Microbiology
Degree Granting Institution:University of Illinois at Urbana-Champaign
Degree:Ph.D.
Genre:Dissertation
Subject(s):quorum-sensing
conjugative transfer
opines
mannopine
Alphaproteobacteria
RepABC
conjugative transfer genes
horizontal transfer
plasmid evolution
Abstract:repABC plasmids are ubiquitous in the α-proteobacteria and are important to the biology of the bacteria that harbor them for several reasons. First, they can carry large amounts of DNA, thereby conferring a wide variety of important characteristics. Some of these traits are important for the biology of the bacteria that harbor them. For example the repABC plasmids in species of Agrobacterium can encode virtually all of the genes responsible for inducing crown gall tumors and hairy roots on susceptible plant hosts. These plasmids also encode the genes required for production by the plant, and utilization by the bacteria of unique carbon conjugates called opines. Similarly, repABC plasmids in species of Rhizobium confer nodulation and nitrogen fixation when the bacteria are in symbiosis with a suitable plant host. Second, the repABC replicons have a broad host-range, and a subset of these plasmids encode a conjugative transfer system allowing these biologically relevant elements to transfer between and among species of bacteria. Perhaps the best-studied transfer system of the repABC plasmids is the Class I system composed of a type four secretion system encoded by the traI/trb operon and a DNA metabolism system encoded by the two tra operons. These operons are regulated by a quorum-sensing system involving three proteins: TraR, TraI, and TraM. TraR directly activates the transfer regulon but needs its ligand, an acyl-homoserine lacone quorum-sensing signal synthesized by TraI, to be active. One additional component, TraM, binds to TraR directly and inactivates the quorum-sensing protein when the signal for transfer is absent. The octopine-type Ti plasmids in A. tumefaciens strains 15955 and R10 are inducible for conjugative transfer by octopine because traR is the distal member of an operon inducible by the conjugative opine. However, a second non-functional allele of traR, called trlR, is present in the mannopine transport operon, an operon that is inducible by the opine mannopine. Based on the location and inducibility of trlR by mannopine, we hypothesized that there would be a functional allele of traR that is similarly located in a mannopine-inducible operon and that mannopine would induce transfer of a plasmid in a wild-type isolate of Agrobacterium. To this end we characterized and analyzed a collection of mannopine-utilizing field isolates for the ability of mannopine to induce transfer. We found five such isolates. Further characterization of the mannopine-utilizing plasmids in these strains indicated that these plasmids all are highly related. We analyzed and sequenced one such element, pAoF64/95. First, pAoF64/95 is not a virulence element; it does not contain the genes for virulence or a T-region. Instead pAoF64/95 is an opine-catabolic plasmid and encodes all of the genes for utilization of three of the four mannityl opines- mannopine, mannopinic acid and agropinic acid- as well as the agrocinopines. Indeed, strains harboring pAoF64/95 can utilize these three mannityl opines and are also sensitive to agrocin 84, an indication that the strain can utilize the agrocinopine opines. Second, an otherwise plasmid-less strain harboring pAoF64/95 transfers the mannopine-utilizing trait to a recipient when grown with mannopine. Moreover, mutational analysis of traR and traM encoded by pAoF64/95 suggests that the functions of TraR and TraM as activator and antiactivator are conserved. Finally, the genes involved in Class I transfer of pAoF64/95 are not organized as they are in Ti plasmids. For all repABC plasmids with Class I transfer systems, the traI/trb operon is always adjacent and divergently oriented to the repABC operon. In the Ti plasmids, the tra region along with traR and traM are located distantly from the trb-repABC region and more often are located near the genes for uptake and catabolism of the conjugative opine. Additionally, in the Ti plasmids known to be conjugative, traR is invariably located in an operon inducible by the conjugative opine. This organization of the genes for conjugative transfer we call Group I organization. However, in pAoF64/95, like the Ri plasmids of A. rhizogenes and many plasmids in species of Rhizobium, the location of the tra genes is contiguous with the trb-repABC region and traR is monocistronic, an organization we name Group II. Based upon these two modes of organization of plasmids with Class I transfer systems (Group I and Group II), we hypothesized that the component gene systems represent divergent evolutionary lineages. We assessed the evolution of the transfer, quorum-sensing, and replication and partition proteins and found that the quorum-sensing and transfer proteins form two clades that are consistent with the two modes of plasmid organization, indicating that the two organizational groups of plasmids are evolving divergently. Despite the obligatory linkage of the repABC operon with the traI/trb operon, the repABC proteins evolve independently of the transfer and quorum-sensing proteins. Moreover, while RepA and RepB coevolve, RepC evolves independently. Functional analysis indicates that TraR can dimerize and activate tra box-containing promoters of members within a clade, but not between clades. This is further evidence that proteins within, but not between clades are cross-functional. In contrast, the oriT regions are highly conserved and do not form two major clades. Consistent with the phylogeny, cloned oriT regions are processed and mobilized by members of either clade. We conclude that Group I and Group II plasmids diverged based upon where the cargo DNA is located and moreover that this divergence in organization extends to function.
Issue Date:2016-10-07
Type:Thesis
URI:http://hdl.handle.net/2142/95287
Rights Information:Copyright 2016 Margaret E. Wetzel
Date Available in IDEALS:2017-03-01
Date Deposited:2016-12


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