A Family of Single Copy repABC-Type Shuttle Vectors Stably Maintained in the Alpha-Proteobacterium Sinorhizobium meliloti

Abstract

A considerable share of bacterial species maintains segmented genomes. Plant symbiotic α-proteobacterial rhizobia contain up to six repABC-type replicons in addition to the primary chromosome. These low or unit-copy replicons, classified as secondary chromosomes, chromids, or megaplasmids, are exclusively found in α-proteobacteria. Replication and faithful partitioning of these replicons to the daughter cells is mediated by the repABC region. The importance of α-rhizobial symbiotic nitrogen fixation for sustainable agriculture and Agrobacterium-mediated plant transformation as a tool in plant sciences has increasingly moved biological engineering of these organisms into focus. Plasmids are ideal DNA-carrying vectors for these engineering efforts. On the basis of repABC regions collected from α-rhizobial secondary replicons, and origins of replication derived from traditional cloning vectors, we devised the versatile family of pABC shuttle vectors propagating in Sinorhizobium meliloti, related members of the Rhizobiales, and Escherichia coli. A modular plasmid library providing the elemental parts for pABC vector assembly was founded. The standardized design of these vectors involves five basic modules: (1) repABC cassette, (2) plasmid-derived origin of replication, (3) RK2/RP4 mobilization site (optional), (4) antibiotic resistance gene, and (5) multiple cloning site flanked by transcription terminators. In S. meliloti, pABC vectors showed high propagation stability and unit-copy number. We demonstrated stable coexistence of three pABC vectors in addition to the two indigenous megaplasmids in S. meliloti, suggesting combinability of multiple compatible pABC plasmids. We further devised an in vivo cloning strategy involving Cre/lox-mediated translocation of large DNA fragments to an autonomously replicating repABC-based vector, followed by conjugation-mediated transfer either to compatible rhizobia or E. coli.

Keywords: Cre/loxP; Rhizobiales; Sinorhizobium; in vivo cloning; plasmid cloning vehicle; repABC.

Figure 1

pABC vector concept and assembly strategy. (a) A pABC is composed of up to five module library parts. Modules oriVSm and oriVEc mediate propagation in S. meliloti and E. coli, respectively. To ensure proper transcriptional control of the repABC operon, the multiple cloning site (yellow box) of the synTer-MCS module, serving as insertion site for the gene load, is flanked by transcriptional terminators (loop structures). A common RK2/RP4 mobilization site (module oriT) enables conjugal transfer of pABCs from E. coli to S. meliloti. For reliable selection in both organisms standardized antibiotic resistance cassettes were developed (module AR). Each module is flanked by linker sequences for standardized LCR-based assembly (black boxes). In the pABC design, order and orientation of modules is intended to minimize mutual influences of adjacent regions. (b) Detailed scheme of pABC design types A to C. The size of design-derived homologous regions potentially occurring in multi-pABC systems is denoted. Setups C1 and C2 differ in the number of transcription terminator sequences (loop structures) protecting the MCS after Cre-mediated deletion of modules oriVEc and oriT. (c) Module-specific linker sequences (gray and black lines) facilitate LCR-based assembly of module parts with standardized bridging oligonucleotides BO1 to BO4. Integration of the oriT module requires BO3a and BO3b.

Figure 2

Characterization of oriVSm module parts. (a) repABC cassettes analyzed in this study. Regions are derived from R. etli (Ret), M. loti (Mlo), and A. tumefaciens (Atu) plasmids. Since functional elements, such as partitioning sites (repS, yellow boxes) or the promoter of the antisense RNA incα potentially are located outside of the repABC operon, repABC cassettes were expanded several hundred base pairs up- and downstream of the coding regions. Although a suicide vector carrying the repABC operon of p42d plus 500 bp downstream of the repC stop codon was able to propagate in R. etli CFNX101, the downstream region was generously expanded in order to include a further predicted repS site. (b) Stability assay of pLoriVSm derivatives. S. meliloti Rm1021 carrying pLoriV-42b, -42d, 42e, -Mla, -Mlb, or -Ti, or the shorter constructs p42e_s or pMla_s was grown for 72 h in nonselective TY medium and diluted every 12 h to an OD₆₀₀ of ∼0.05. Single colonies were examined for antibiotic resistance (n = 100 for pLoriVSm derivatives; n = 50 for p42e_s and pMla_s). Error bars indicate the standard deviation calculated from three technical replicates. (c) Growth of S. meliloti Rm1021 carrying selected pLoriVSm plasmids compared to MWSm8 (wild type, Km^r). Cultures were adjusted to an OD₆₀₀ of 0.1 and grown for 12 h in selective TY medium. Error bars indicate the standard deviation calculated from four technical replicates. (d) qPCR-based copy number determination of pLoriVSm derivatives (-42b, -42d, -42e, -42f1, -42f2, -Mla, -Mlb, and -Ti) in exponentially growing S. meliloti. Measurements are based on eight technical replicates from two independent clones (see Table S6). SD: standard deviation.

Figure 3

Setup of pABC library modules. (a) The synTer-MCS module plasmids provide three ∼550 bp synTer regions composed of Rho-independent E. coli terminators rrnBT1 (90 bp) and rrnBT2 (60 bp) separated by unique synthetic sequences. Terminator sites (loop structures) are intended to protect the oriVSm module and the MCS, which provides unique recognition sites for rare cutters. pLsynTerX-lox plasmids are further equipped with a left arm mutated loxL site (arrows in brackets). (b) The oriT module comprises a 464 bp DNA fragment containing the RK2-based mob site (I). For Cre/lox applications the region is extended by sacB-loxR (II) and a further rrnBT2 transcription terminator (III). (c) Module AR contains the short Pmin2 fragment conferring resistance to kanamycin (Km), tetracycline (Tc), gentamicin (Gm), trimethoprim (Tmp), hygromycin (Hyg), or spectinomycin (Spec) in E. coli and S. meliloti.

Figure 4

Inheritance stability of pABCs in S. meliloti Rm1021. S. meliloti carrying pABC-egfp (-expR) derivatives were grown in nonselective TY medium at 30 °C for 96 h. During the time course, cultures were diluted every 12 h to an OD₆₀₀ of ∼0.05. An eGFP signal of 10.000 cells was measured by flow cytometry at time point 0 and after 1, 2, and 4 days. Additionally, after the initial 24 h, subcultures were incubated for 48 h until the stationary phase was reached, and reinoculated at day 3 (4 stat., separated by a line). Varying ordinate labeling should be noted. Positive: S. meliloti MWSm37 (genomically integrated egfp). Error bars indicate standard deviation calculated from three biological replicates. (a) Maintenance of single pABC-egfp derivatives. (i) Stable pABC-egfp constructs which did not deceed 97% propagation stability. (ii) Replicon stability of instable pABC-egfp derivatives and low copy plasmid pPHU231-egfp. Because of the heterogeneous behavior of pABC3-egfp, measurements from individual clones are shown (R1–3). (b) Replicon stability of pABCs in a triple pABC system. 3xpABC^a-egfp: pABCa-egfp, pABCb, pABCc-mob; 3xpABC^b-egfp: pABCa, pABCb-egfp, pABCc-mob; 3xpABC^c-egfp: pABCa, pABCb, pABCc-mob-egfp. (c) S. meliloti carrying pABC-egfp-expR derivatives.

Figure 5

Examination of pABCs in S. meliloti. (a) Fluorescence microscopy of S. meliloti JDSm106 carrying pABCa-tetO, pABCb, and pABCc-parS_Yp. Expression of reporters tetR-YFP (yellow) and mChr-parB_Yp (red) was induced with 15 mM taurine for 4 h. Snapshots show cells at different stages of the cell cycle. Scale bar: 1 μm. (b) eGFP fluorescence of S. meliloti Rm1021 carrying pABC1-egfp_f/r (synTer1), pABC1-ST2-egfp_f/r (synTer2), and pABC1-ST3-egfp_f/r (synTer3) was taken as a measure for the transcription activity in the MCS of library-derived pABCs. Promoter-less egfp_f/r cassettes were either forwardly or reversely (corresponding to up/down orientation) integrated into the MCS. Negative: S. meliloti/pABC1; positive: S. meliloti/pABC1-egfp. Error bars indicate standard deviation calculated from three technical replicates. FU: fluorescence units.

Figure 6

repABC mediated in vivo cloning in S. meliloti (ABC cloning). (a) The promoter region of repABC_pMLb was predicted according to the consensus motifs of S. meliloti σ⁷⁰ promoters (−35 and −10 elements are underlined). A single lacO box (red letters) was integrated at the predicted transcription start site (+1). (b) ABC cloning of the exo gene cluster in S. meliloti SmCre. Three consecutive arrow heads indicate lox sites with native (green) or mutated arms (red). (i) A gentamicin resistance cassette carrying a right arm-mutated loxR site (Pmin2-loxR-aacC1) was integrated downstream of the gene cluster via cloning-free genome editing (CFGE). Subsequently, pISO bearing repABC_pMlb-lacO (repABC*) was integrated via a ∼550 bp homologous region (HR) upstream of the gene cluster, resulting in S. meliloti SmCre_exo-IN. Taurine induction of Cre expression and simultaneous IPTG activation of oriV_{pMLb_lacO} gave rise to SmCre_exo-OUT which carries the relocated ∼35.5 kb region (pIso-exo) comprising the entire exo gene cluster. Both the deletion site on pSymB and the fusion site on pIso-exo were PCR-amplified with primers 94 + Rev (black arrows) and 680 + 128 (red arrows), respectively, and sequencing of PCR products confirmed proper Cre-mediated recombination. (ii) pISO-exo was transferred to E. coli via triparental mating, resulting in E. coli/pIso-exo. Purified plasmid DNA was digested with NheI, resulting in fragments of 2.03 kb, 13.8 kb, and 19.6 kb fragments (D). Sequencing of the fusion site comprising loxP and aadA1 with primers 680 and 128 (red arrows) further confirmed successful cloning. U: Undigested plasmid DNA. (iii) Calcofluor fluorescence assay. pISO-exo was transferred to the exopolysaccharide-deficient A. tumefaciens C58 exoB mutant (exoB^-) via triparental mating, giving rise to A. tumefaciens exoB/pIso-exo complemented for exopolysaccharide production (pIso-exo), and thus showing UV-induced fluorescence on Calcofluor-containing medium. Wild type: A. tumefaciens C58.