卤代芳烃代谢质粒pBHB与宿主Comamonas sp. 7D-2的补偿性进化机制

The evolution of catabolic plasmid is an important mechanism for bacteria to adapt to organic pollutant contaminated-environment. However, the plasmid replication, conjugative mobilization and gene expression will also bring corresponding physiological burden to its host. Therefore, plasmid tends to reduce its host burden, improve self-stability and mobilization in host populations through compensatory evolution with its host. However, few studies on the co-evolution of catabolic plasmids with their hosts have been carried out, hindering the application of plasmid-mediated bioaugmentation for the bioremediation of organic pollutant contaminated-sites. In this project, we will carry out the co-evolution research of the halogenated aromatic-catabolic plasmid pBHB (belongs to IncP-1β subgroup) with its host Comamonas sp. 7D-2 under the selective pressure of 3,5-dibromo-4-hydroxybenzoate, assess the adaptability of co-evolutionary plasmid to its host, reveal the roles of plasmid gene loss, constrictive regulation of the expression of the catabolic gene cluster and compensatory mutation of core genes of plasmid and host chromosome on the adaptability of plasmid to its host, and finally elucidate the mechanisms involved in the compensatory evolution of pBHB with its host strain 7D-2. The expected results will enrich the theory of compensatory evolution of catabolic plasmids with their hosts, and provide theoretical guidance for the application of plasmid-mediated bioaugmentation for bioremediation.

代谢型质粒进化是细菌适应污染环境的重要机制，但是质粒复制、接合转移以及基因表达等生理活动会给宿主带来相应的生理负担。因此，质粒往往通过与宿主的补偿性进化来降低宿主负担，提高质粒在宿主菌群中的稳定性和传播性。然而，代谢型质粒与宿主的补偿性进化机制研究十分匮乏，制约了质粒介导的有机污染物微生物强化修复技术的应用。本项目以IncP-1β亚群的卤代芳烃代谢质粒pBHB和宿主Comamonas sp. 7D-2为研究对象，以3,5-二溴-4-羟基苯甲酸为选择压力，开展质粒与宿主的共进化研究，评估质粒与宿主的适应性，揭示质粒基因丢失、代谢基因簇表达的紧缩调控、质粒和宿主染色体核心基因的补偿性突变对质粒与宿主适应性改善的作用，最终阐明pBHB与宿主7D-2的补偿性进化机制。预期成果将丰富代谢型质粒与宿主补偿性进化的理论，也为质粒介导的生物强化修复技术的应用提供理论指导。

细菌种群中接合质粒的维持是一个令人困惑的进化问题。近年来在解决“质粒悖论”方面取得了重大进展，但主要集中在耐药质粒与临床病原体的协同进化上。代谢型接合质粒通过在种群内或跨物种传播其编码的代谢功能，对污染物降解以及宿主适应污染环境起着重要作用。然而，关于代谢型质粒与宿主的共进化研究十分匮乏。本项目以3,5-二溴-4-羟基苯甲酸（DBHB）代谢质粒pBHB与宿主Comamonas sp. 7D-2为研究对象，开展了DBHB持续选择下质粒pBHB与宿主7D-2的共进化，发现共进化群体的适应性（生长速率和质粒维持率）得到显著改善，并从中筛选出两株适应性明显增强的共进化菌株7D-2evo1 pBHBevo1和7D-2evo2 pBHBevo2。通过比较基因组学分析，共进化菌株的染色体上存在结构变异、SNP/Indel突变和转座子随机插入突变，共进化质粒pBHBevo1和pBHBevo2附属区发生了大片段丢失。尽管染色体变异和质粒变异都提高了质粒与宿主的适应性，但后者起主要作用。此外，发现质粒编码的LysR型转录调控因子调控DBHB代谢基因簇bhbA2B2的转录，质粒附属基因表达的紧缩调控可以降低宿主的成本。值得注意的是，携带质粒导致宿主染色体中与tRNA合成相关的基因转录下调。从机制上讲，质粒上附属区大规模基因丢失和代谢基因簇的紧缩调控，以及宿主染色体基因表达的收紧改善了质粒携带和维持成本。本项目研究丰富了代谢型质粒与宿主的共进化理论，揭示了代谢型接合质粒在环境微生物种群中维持的进化策略。