MOE Laboratory of Biosystem Homeostasis and Protection, College of Life Sciences, Zhejiang University, Hangzhou, Zhejiang 310058, China.
Hangzhou Institute for Food and Drug Control, Hangzhou, Zhejiang 310018, China.
FEMS Microbiol Lett. 2021 May 17;368(9). doi: 10.1093/femsle/fnab049.
Rhodococcus spp. have broad potential applications related to the degradation of organic contaminants and the transformation or synthesis of useful compounds. However, some Gram-positive bacteria are difficult to manipulate genetically due to low transformation efficiency. In this study, we investigated the effects of chemicals including glycine, isonicotinic acid hydrazide (INH), Tween 80 and penicillin G, as well as cell growth status, competent cell concentration, electroporation field strength, electroporation time and heat shock time, on the electrotransformation efficiency of the tetrahydrofuran-degrading bacterium Rhodococcus ruber YYL with low transformation efficiency. The highest electrotransformation efficiency was 1.60 × 106 CFU/µg DNA after parameter optimization. GmhD (D-glycero-D-manno-heptose 1-phosphate guanosyltransferase) gene, which is important in the biosynthesis of lipopolysaccharide, was deleted via the optimized electrotransformation method. Compared with wild-type strain, YYL ΔgmhD showed extremely high electrotransformation efficiency because the surface of it had no mushroom-like extracellular polymeric substances (EPS). In addition, the results showed that cell wall-weakening reagents might cause some translucent substances like EPS, to detach from the cells, increasing the electrotransformation efficiency of strain YYL. We propose that these results could provide a new strategy for unique bacteria that are rich in EPS, for which genetic manipulation systems are difficult to establish.
红球菌属具有广泛的应用潜力，与有机污染物的降解、有用化合物的转化或合成有关。然而，由于转化效率低，一些革兰氏阳性菌难以进行基因操作。在这项研究中，我们研究了甘氨酸、异烟肼肼（INH）、吐温 80 和青霉素 G 等化学物质以及细胞生长状态、感受态细胞浓度、电穿孔场强、电穿孔时间和热休克时间对转化效率低的四氢呋喃降解菌红球菌 Ruber YYL 的电转化效率的影响。通过参数优化，最高电转化效率达到 1.60×106 CFU/μg DNA。通过优化的电转化方法，缺失了在脂多糖生物合成中起重要作用的 GmhD（D-甘油-D-甘露庚糖 1-磷酸鸟苷基转移酶）基因。与野生型菌株相比，YYL ΔgmhD 具有极高的电转化效率，因为其表面没有蘑菇状的胞外聚合物质（EPS）。此外，结果表明，细胞壁削弱试剂可能会使 EPS 等一些半透明物质从细胞上脱落，从而提高菌株 YYL 的电转化效率。我们提出，这些结果为富含 EPS 的独特细菌提供了一种新的策略，对于这些细菌，建立遗传操作系统较为困难。
