Atago Yuki, Shimodaira Jun, Araki Naoto, Bin Othman Nor'azizi, Zakaria Zuriati, Fukuda Masao, Futami Junichiro, Hara Hirofumi
a Division of Chemistry and Biochemistry, Department of Biotechnology , Graduate School of Natural Science and Technology, Okayama University , Okayama , Japan.
b Department of Bioengineering , Nagaoka University of Technology , Nagaoka , Japan.
Biosci Biotechnol Biochem. 2016 May;80(5):1012-9. doi: 10.1080/09168451.2015.1127134. Epub 2016 Feb 1.
Rhodococcus jostii RHA1 (RHA1) degrades polychlorinated biphenyl (PCB) via co-metabolism with biphenyl. To identify the novel open reading frames (ORFs) that contribute to PCB/biphenyl metabolism in RHA1, we compared chromatin immunoprecipitation chip and transcriptomic data. Six novel ORFs involved in PCB/biphenyl metabolism were identified. Gene deletion mutants of these 6 ORFs were made and were tested for their ability to grow on biphenyl. Interestingly, only the ro10225 deletion mutant showed deficient growth on biphenyl. Analysis of Ro10225 protein function showed that growth of the ro10225 deletion mutant on biphenyl was recovered when exogenous recombinant Ro10225 protein was added to the culture medium. Although Ro10225 protein has no putative secretion signal sequence, partially degraded Ro10225 protein was detected in conditioned medium from wild-type RHA1 grown on biphenyl. This Ro10225 fragment appeared to form a complex with another PCB/biphenyl oxidation enzyme. These results indicated that Ro10225 protein is essential for the formation of the PCB/biphenyl dioxygenase complex in RHA1.
约氏红球菌RHA1（RHA1）通过与联苯的共代谢作用降解多氯联苯（PCB）。为了鉴定RHA1中有助于PCB/联苯代谢的新开放阅读框（ORF），我们比较了染色质免疫沉淀芯片和转录组数据。鉴定出6个参与PCB/联苯代谢的新ORF。构建了这6个ORF的基因缺失突变体，并测试了它们在联苯上生长的能力。有趣的是，只有ro10225缺失突变体在联苯上生长存在缺陷。对Ro10225蛋白功能的分析表明，当向培养基中添加外源重组Ro10225蛋白时，ro10225缺失突变体在联苯上的生长得以恢复。尽管Ro10225蛋白没有假定的分泌信号序列，但在以联苯为碳源生长的野生型RHA1的条件培养基中检测到了部分降解的Ro10225蛋白。这个Ro10225片段似乎与另一种PCB/联苯氧化酶形成了复合物。这些结果表明，Ro10225蛋白对于RHA1中PCB/联苯双加氧酶复合物的形成至关重要。
