一株红球菌(Rhodococcus sp.)二噁英降解质粒的稳定性与接合转移特性

Maintenance and conjugative transfer of dioxin catabolic plasmids in a Rhodococcus sp.

【目的】考察一株红球菌Rhodococcus sp.strain p52中的二噁英降解质粒pDF01(170 kb)和pDF02(242 kb)的稳定性和接合转移特性。【方法】在无选择压力的条件下对菌株p52进行连续传代培养,考察质粒pDF01、pDF02的丢失;以菌株p52为供体菌,以不同种属的菌株作受体菌,通过平板接合实验探讨质粒pDF01、pDF02接合转移的受体菌范围以及接合转移频率,利用菌落杂交、Southern杂交对质粒转移结果进行确认,利用降解实验测试转移质粒降解基因的表达。【结果】质粒pDF01和pDF02在红球菌p52中均具有较高的稳定性,在LB培养基上连续传代少于47次时pDF02可保持,连续传代少于65次时pDF01可保持。质粒pDF01和pDF02具备在同属和属间接合转移的能力,可向受体菌——紫红红球菌(Rhodococcus rhodochrous)、红串红球菌(Rhodococcus erythropolis)、大地两面神菌(Terrabacter tumescens)和节杆菌(Arthrobacter sp.)转移,其中以节杆菌作受体菌时质粒pDF01和pDF02接合转移频率最高,达到3.5×10^(-6)(接合子/受体菌);对节杆菌接合子质粒进行Southern杂交进一步确认了质粒pDF01、pDF02的存在。另外获得质粒pDF01、pDF02后的节杆菌接合子可以对二苯并呋喃高效利用,且降解能力与红球菌供体菌株p52相当。【结论】红球菌菌株p52可通过降解质粒转移强化生物修复过程,在去除环境中二噁英污染中具有良好的应用前景。

[Objective] To examine the stable maintenance and conjugative transfer of the dioxin-catabolic plasmids pDF01 （170 kb） and pDF02 （242 kb） harbored by Rhodococcus sp. strain p52. [Methods] We monitored the presence of pDF01 and pDF02 in strain p52 during continuous transfer in LB medium. We also performed mating experiments using strain p52 as a donor and bacterial strains belonging to different genera as recipients, and confirmed the transconjugant by colony hybridization and Southem hybridization. Further, we examined the function of catabolic genes in a transconjugant by degrading test. [Results] Both pDF01 and pDF02 could maintain in strain p52 after continuous transfer in LB medium less than 47 times, but pDF01 was lost after 65 times transfer in LB medium. Plasmid pDF01 and pDF02 could transfer concomitantly from strain p52 to the recipient strains, such as Rhodococcus rhodochrous, Rhodococcus erythropolis, Janibacter terrae and Arthrobacter sp., while Arthrobacter sp. showed the highest transconjugation frequency of 3.5 × 10^-6 colonies per recipient. The results of Southem hybridization confirmed that A rthrobacter sp. transconjugant obtained pDF01 and pDF02. Furthermore, the catabolic plasmids could function in Arthrobacter sp. transconjugant, which could use dibenzofuran for growth comparing with the donor strain p52. [Conclusion] The dioxin-degrader Rhodococcus sp. strain p52 has potential for genetic bioaugmentation in contaminated environments.