阿拉伯海假交替单胞菌N1230-9几丁质裂解酶的功能

Functions of five chitin lyases from Pseudoalteromonas arabiensis N1230-9

摘要:

目的 几丁质裂解酶是细菌有效降解几丁质的关键酶。阿拉伯海假交替单胞菌(Pseudoalteromonas arabiensis) N1230-9拥有5个几丁质裂解酶编码基因(woc28159、woc28160、woc28161、woc27404和woc27232)，对这5个基因的功能进行鉴定是解析该菌株具有高效几丁质降解能力的关键。方法 通过生物信息学方法分析菌株N1230-9中的5个几丁质裂解酶在假交替单胞菌44个模式菌株中的分布规律；采用实时荧光定量PCR技术(RT-qPCR)检测菌株N1230-9中5个几丁质裂解酶编码基因在以几丁质为唯一碳源培养基中的转录水平；利用同源重组策略构建菌株N1230-9中5个几丁质裂解酶编码基因的单基因缺失突变体，并分析突变体的几丁质降解能力。结果 有33株假交替单胞菌模式菌株拥有与菌株N1230-9几丁质裂解酶同源的蛋白；几丁质可以强烈诱导woc28159、woc28160、woc28161和woc27404的转录水平上调，且这4个基因的单基因缺失均在不同程度上削弱了菌株的几丁质降解能力。然而woc27232的转录水平不受几丁质的诱导，且该基因的缺失对菌株的几丁质降解能力无影响。结论 几丁质酶WOC28159和WOC28161是菌株N1230-9降解几丁质所必需的酶，WOC28160和WOC27404增强了菌株的几丁质降解效率，而WOC27232并不参与菌株N1230-9的几丁质降解过程。

关键词:假交替单胞菌;阿拉伯海假交替单胞菌;几丁质裂解酶;几丁质酶

Abstract:

Objective Chitin lyases are the key enzymes for bacteria to degrade chitin. Pseudoalteromonas arabiensis N1230-9 possesses five chitin lyase-encoding genes (woc28159, woc28160, woc28161, woc27404, and woc27232). Functional identification of these five genes is crucial for determining the ability of strain to degrade chitin.Methods The distribution patterns of five chitin lyases from strain N1230-9 in 44 model strains of Pseudomonas were analyzed by bioinformatics methods. The transcription levels of five chitin lyase-encoding genes of strain N1230-9 cultured in the medium with chitin as the sole carbon source were analyzed by RT-qPCR. The mutants with deletion of chitin lyase-encoding genes were constructed by homologous recombination, and their abilities to degrade chitin were evaluated.Results Thirty-three Pseudoalteromonas strains possessed chitin lyase homologous proteins of strain N1230-9. Chitin significantly upregulated the transcription levels of woc28159, woc28160, woc28161, and woc27404, and the deletion of each of the four genes weakened the chitin-degradation ability. The transcription level of woc27232 was not affected by chitin, and the deletion of this gene did not affect the chitin-degradation ability of the strain.Conclusion WOC28159 and WOC28161 are necessary for the degradation of chitin by strain N1230-9. WOC28160 and WOC27404 endow strain N1230-9 with efficient chitin-degrading ability, while WOC27232 does not participate in the chitin degradation process.

Key words:Pseudoalteromonas;Pseudoalteromonas arabiensis;chitin lyase;chitinase