Effective removal of a range of Ti/Ri plasmids using a pBBR1-type vector having a repABC operon and a lux reporter system

Yamamoto, Sachiko; Sakai, Ayako; Agustina, Vita; Moriguchi, Kazuki; Suzuki, K.

doi:10.1007/s00253-017-8721-7

Cited by 5 publications

(11 citation statements)

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“…This change in plasmid profile was observed for virtually all of the Ti plasmid-cured derivatives from multiple experiments. Interestingly, Eckhardt gel analysis of A. tumefaciens SA122, a 15955 derivative cured by a completely different approach using chemical and temperature stress in a different laboratory years earlier revealed the same pattern as the derivatives we had generated (Figure 1, and see Materials and Methods) (Uraji et al 2002; Yamamoto et al 2018).…”

Section: Resultssupporting

confidence: 57%

“…Several on-going studies rely on curing of the Ti plasmid from A. tumefaciens , using a plasmid incompatibility, eviction process (Uraji et al 2002; Platt et al 2012a; Yamamoto et al 2018). As a matter of practice, the plasmid profiles of putatively cured derivatives are analyzed by Eckhardt gel electrophoresis to visualize their large plasmid content (see Materials and Methods).…”

Section: Resultsmentioning

confidence: 99%

“…Curing of pTi15955 was performed as described by our lab previously (Morton and Fuqua 2012a) with slight modifications. The curing plasmid is similar to the pBBR1-type curing plasmids recently described (Yamamoto et al 2018), containing two origins capable of replication in Agrobacterium , a Km R cassette, and the counterselectable sacB marker for removal of the curing plasmid. Briefly, pSRKKm was digested with Bam HI (NEB) according with NEB guidelines, followed by purification using the E.Z.N.A.…”

Section: Methodsmentioning

confidence: 99%

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Destabilization of the Tumor-Inducing Plasmid from an Octopine-Type Agrobacterium tumefaciens Lineage Drives a Large Deletion in the Co-resident At Megaplasmid

Barton

Platt

Rusch

et al. 2019

G3 Genes|Genomes|Genetics

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Bacteria with multi-replicon genome organizations, including members of the family Rhizobiaceae, often carry a variety of niche-associated functions on large plasmids. While evidence exists for cross-replicon interactions and co-evolution between replicons in many of these systems, remarkable strain-to-strain variation is also observed for extrachromosomal elements, suggesting increased genetic plasticity. Here, we show that curing of the tumor-inducing virulence plasmid (pTi) of an octopine-type Agrobacterium tumefaciens lineage leads to a large deletion in the co-resident At megaplasmid (pAt). The deletion event is mediated by a repetitive IS-element, IS66, and results in a variety of environment-dependent fitness consequences, including loss of independent conjugal transfer of the plasmid. Interestingly, a related and otherwise wild-type A. tumefaciens strain is missing exactly the same large pAt segment as the pAt deletion derivatives, suggesting a similar event over its natural history. Overall, the findings presented here uncover a novel genetic interaction between the two large plasmids of A. tumefaciens and provide evidence for cross-replicon integration and co-evolution of these plasmids.

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Section: Resultssupporting

confidence: 57%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Destabilization of the Tumor-Inducing Plasmid from an Octopine-Type Agrobacterium tumefaciens Lineage Drives a Large Deletion in the Co-resident At Megaplasmid

Barton

Platt

Rusch

et al. 2019

G3 Genes|Genomes|Genetics

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“…167 pTi15955 curing and detection of pAt15955 segment loss 168 Curing of pTi15955 was performed as described by our lab previously (Morton and 169 Fuqua 2012a) with slight modifications. The curing plasmid is similar to the pBBR1-type 170 curing plasmids recently described (Yamamoto et al 2018), containing two origins 171 capable of replication in Agrobacterium, a Km R cassette, and the counterselectable 172 sacB marker for removal of the curing plasmid. Briefly, pSRKKm was digested with 173 BamHI (NEB) according with NEB guidelines, followed by purification using the E.Z.N.A.…”

Section: Introductionmentioning

confidence: 99%

“…To characterize the 317 connection between these events, the correlation between pTi15955 curing and 318 pAt15955 segment loss was determined under pTi15955 curing conditions. High 319 efficiency incompatibility-based curing strategies were used to determine the frequency 320 of both events Yamamoto et al 2018) gusA/Gm R , respectively, to generate A. tumefaciens 15955-IB118 to facilitate initial 326 screening for curing and deletion events (described in Materials and Methods).…”

mentioning

confidence: 99%

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

Barton

Platt

Rusch

et al. 2019

Preprint

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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 ...

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Systematic Part Transfer by Extending a Modular Toolkit to Diverse Bacteria

Keating

Young

2023

ACS Synth. Biol.

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It is impractical to develop a new parts collection for every potential host organism. It is well-established that gene expression parts, like genes, are qualitatively transferable, but there is little quantitative information defining transferability. Here, we systematically quantified the behavior of a parts set across multiple hosts. To do this, we developed a broad host range (BHR) plasmid system compatible with the large, modular CIDAR parts collection for E. coli, which we named openCIDAR. This enabled testing of a library of DNA constructs across the PseudomonadotaEscherichia coli, Pseudomonas putida, Cupriavidus necator, and Komagataeibacter nataicola. Part performance was evaluated with a standardized characterization procedure that quantified expression in terms of molecules of equivalent fluorescein (MEFL), an objective unit of measure. The results showed that the CIDAR parts enable graded gene expression across all organismsmeaning that the same parts can be used to program E. coli, P. putida, C. necator, and K. nataicola. Most parts had a similar expression trend across hosts, although each organism had a different average gene expression level. The variability is enough that to achieve the same MEFL in a different organism, a lookup table is required to translate a design from one host to another. To identify truly divergent parts, we applied linear regression to a combinatorial set of promoters and ribosome binding sites, finding that the promoter J23100 behaves very differently in K. nataicola than in the other hosts. Thus, it is now possible to evaluate any CIDAR compatible part in three other hosts of interest, and the diversity of these hosts implies that the collection will also be compatible with many other Proteobacteria (Pseudomonadota). Furthermore, this work defines an approach to generalize modular synthetic biology parts sets beyond a single host, implying that only a few parts sets may be needed to span the tree of life. This will accelerate current efforts to engineer diverse species for environmental, biotechnological, and health applications.

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Effective removal of a range of Ti/Ri plasmids using a pBBR1-type vector having a repABC operon and a lux reporter system

Cited by 5 publications

References 61 publications

Destabilization of the Tumor-Inducing Plasmid from an Octopine-Type Agrobacterium tumefaciens Lineage Drives a Large Deletion in the Co-resident At Megaplasmid

Destabilization of the Tumor-Inducing Plasmid from an Octopine-Type Agrobacterium tumefaciens Lineage Drives a Large Deletion in the Co-resident At Megaplasmid

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

Systematic Part Transfer by Extending a Modular Toolkit to Diverse Bacteria

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