Destabilization of the Tumor-Inducing Plasmid from an Octopine-TypeAgrobacterium tumefaciensLineage Drives a Large Deletion in the Co-Resident At Megaplasmid

Abstract

1Bacteria with multi-replicon genome organizations, including members of the family 2 Rhizobiaceae, often carry a variety of niche-associated functions on large plasmids. 3 While evidence exists for cross-replicon interactions and co-evolution between replicons 4 in many of these systems, remarkable strain-to-strain variation is also observed for 5 extrachromosomal elements, suggesting increased genetic plasticity. Here, we show 6 that curing of the tumor-inducing virulence plasmid (pTi) of an octopine-type 7 Agrobacterium tumefaciens lineage leads to a large deletion in the co-resident At 8 megaplasmid (pAt). The deletion event is mediated by a repetitive IS-element, IS66, 9 and results in a variety of environment-dependent fitness consequences, including loss 10 of independent conjugal transfer of the plasmid. Interestingly, a related and otherwise 11 wild-type A. tumefaciens strain is missing exactly the same large pAt segment as the 12 pAt deletion derivatives, suggesting a similar event over its natural history. Overall, the 13 findings presented here uncover a novel genetic interaction between the two large 14 plasmids of A. tumefaciens and provide evidence for cross-replicon integration and co-15 evolution of these plasmids. 16 Bacteria exhibit a diversity of genomic architectures and contain replicons that range 18 from small, transient plasmids and megaplasmids, to chromids, to secondary and 19 primary chromosomes (diCenzo and Finan 2017). Primary chromosomes are the largest 20 replicons encoding core functions, whereas any replicating elements in addition to the 21 primary chromosome are defined as secondary replicons. Secondary chromosomes are 48 limitation to the coexistence of multiple replicons and have been observed in diverse 49 bacterial species, including species of Vibrio (Heidelberg et al. 2000; Ramachandran et 50 al. 2017) and Sinorhizobium (Ronson et al. 1987; Barnett et al. 2004; Bobik et al. 2006; 51 Galardini et al. 2015; Pini et al. 2015; diCenzo et al. 2018). In most cases, chromosomal 52 factors influence the regulation of secondary replicon factors, with limited regulation in 53 the opposite direction (Ronson et al. 1987; Barnett et al. 2004; Bobik et al. 2006; Agnoli 54 et al. 2012; Galardini et al. 2015; Pini et al. 2015; diCenzo et al. 2018), likely due to 55 intolerance of integral pathway perturbation and the suppression of costly accessory 56 functions required to stabilize secondary replicons. However, metabolic redundancy has 57 been observed between rhizobial replicons (González et al. 2005; diCenzo and Finan 58 2017), suggesting that favorable interaction and pathway integration between primary 59 and secondary replicons can occur. However, because the majority of organisms 60 possessing multi-partite genomes exist and transition between multiple environmental 61 reservoirs, and are often host-associated, the extent to which cross-replicon interactions 62 occur and their consequences are often not determined.63Here, we characterize a cross-replicon genetic interaction in ...

Co-Dependent and Interdigitated: Dual Quorum Sensing Systems Regulate Conjugative Transfer of the Ti Plasmid and the At Megaplasmid in Agrobacterium tumefaciens 15955

“…Taken together with our prior observation of pTi15955-dependent octopine-induction of pAt15955 conjugation (Barton, et al 2019), this data suggests that pTi15955 is directly involved in activation of pAt15955 tra gene expression in response to octopine Winans 1994). Additionally, octopine-induction had differential effects on pAt15955 and pTi15955 gene activation, where octopine addition stimulated comparable modest expression from both traA genes (Figure 1A), but had a more dramatic effect on traI Ti expression than that of traI At (Figure 1B), suggesting complex cross-regulation between both plasmids and supporting our previous observations for pAt15955 conjugation control, as well as previous work on the activation of octopine-type Ti plasmid conjugation Winans 1994).…”

“…The basal expression of the traI Ti gene is in fact modestly elevated in the traR At mutant raising the possibility that TraR At plays a mildly inhibitory role in the absence of octopine. In contrast to pTi15955, low level conjugation of pAt15955 is detectable in the absence of octopine (Barton et al 2019). The observation that even this minimal conjugation is largely abolished in the traI Ti traI At double mutants suggests that quorum sensing is functioning to regulate the process even at its lowest levels.…”

“…We have demonstrated that this second At-encoded system controls conjugative transfer through regulation of tra genes on the At plasmid, but that it can also have a profound impact on Ti plasmid functions. Likewise, the Ti-encoded system can impact At plasmid conjugative transfer, most notably imparting octopine-inducible control over pAt15955 conjugation (Barton, et al 2019). These co-resident quorum sensing systems interdigitate at multiple levels to regulate horizontal transfer of both plasmids (Fig.…”

“…We previously showed that pAt15955 encodes a functional conjugation system and observed that conjugation of pAt15955∆270 (a derivative with a 270 kb deletion on pAt) which lacks cisencoded trb conjugation functions (Figure S1A), could be stimulated by octopine-induction in a pTi15955-dependent manner (Barton et al 2019) suggesting a direct interaction between the conjugation systems of both plasmids.…”

“…The pAt15955 plasmid has two annotated traI homologs, one of which is encoded as the predicted first gene of a traI-trb operon (traI At_1 , At15955_49060) in a position similar to pTiencoded systems, and another ~20 Kbp distant from trb in an uncharacterized locus (traI At_2 , At15955_RS25205) (Figure S1A and S1C). Both are within the large segment of pAt15955 that is deleted upon curing of pTi15955 (purple bar in Figure S1), and a pAt15955 derivative with this deletion is unable to conjugate independently (Barton et al 2019). The annotated coding sequence of traI At_2 specifies a protein that has a amino acid C-terminal extension relative to TraI Ti and TraI At_1 (Figure S2).…”