Fig 1.
Growth curves of A. caldus strains grown in liquid Starkey-S0 media with different antibiotics.
(A) A. caldus MTH-04 wild type in media with kanamycin, streptomycin, ampicillin and chloramphenicol, respectively. (B) A. caldus MTH-04 wild type in media with different concentrations of chloramphenicol. (C) A. caldus MTH-04 carrying pSDU1 in various concentrations of chloramphenicol.
Fig 2.
Construction of pJRD215-derived plasmids.
(A) Construction processes of pSDU1, pSDU1Δmob, and pSDU1ΔmobΔorfEF. A P2-cat cassette was constructed by the fusion of the promoter P2 from the plasmid pBR322 and the cat gene from plasmid pACBSR. The generated cassette and the backbone amplified from plasmid pJRD215 were ligated to generate plasmid pSDU. After that, the multiple cloning sites originated from pUC19 were introduced into pSDU to construct the plasmid pSDU1. To construct pSDU1Δmob, the mobB gene and a part of mobA gene on pSDU1 was removed. Finally, the orfEF region was further deleted from pSDU1Δmob, resulting in plasmid pSDU1ΔmobΔorfEF. (B) The detail diagram of the new pJRD215-derived plasmids. (C) Electrophoretic analysis of plasmids. Lane 1, 5: pJRD215; lane 2, 6: pSDU1; lane 3, 7: pSDU1Δmob; lane 4, 8: pSDU1ΔmobΔorfEF; lane 5–8, plasmids digested with Hind Ⅲ.
Table 1.
Transformation efficiency and frequency of plasmids in electroporation of A. caldus MTH-04.
Table 2.
Transfer frequencies of plasmids between A. caldus MTH-04 and E. coli C600.
Fig 3.
Real-time qPCR experiments to determine the copy numbers of plasmids in A. caldus MTH-04.
(A) Electrophoretic analysis of the total DNA from A. caldus MTH-04 carrying pJRD215 (lane 1), A. caldus MTH-04 carrying pSDU1 (lane 2), A. caldus MTH-04 carrying pSDU1Δmob (lane 3), A. caldus MTH-04 carrying pSDU1ΔmobΔorfEF (lane 4), respectively. Confirmation of PCR amplification specificities of tetH-set (B) and repA-set (C) primers. Standard curves and amplification efficiencies of tetH-set (D) and the repA-set (E) primers.
Table 3.
Estimated plasmid copy number (PCN) by absolute quantification.
Table 4.
Estimated plasmid copy number (PCN) by relative quantification method.
Fig 4.
Maintenance of the plasmids in A. caldus MTH-04 cultivated in liquid Starkey-S0 media during serial passages.
(A) without antibiotics; (B) with antibiotics, streptomycin 200 μg/ml, chloramphenicol 60μg/ml.
Table 5.
The bacterial strains and plasmids used in this work.
Table 6.
Primers used in this work.