Differential Effects of Homologous Transcriptional Regulators NicR2A, NicR2B1, and NicR2B2 and Endogenous Ectopic Strong Promoters on Nicotine Metabolism in Pseudomonas sp. Strain JY-Q
Differential Effects of Homologous Transcriptional Regulators NicR2A, NicR2B1, and NicR2B2 and Endogenous Ectopic Strong Promoters on Nicotine Metabolism in Pseudomonas sp. Strain JY-Q
复制标题
同源 NicR2A/NicR2B 和内源异位强启动子对假单胞菌尼古丁代谢的差异作用。
DOI：
10.1128/aem.02457-20
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发表时间：
2021-02-01
影响因子：
4.4
通讯作者：
Zhong, Weihong
中科院分区：
文献类型：
--
作者：
Huang, Chaochao;Shan, Lihui;Zhong, Weihong
尼古丁是一种有毒的环境污染物，广泛存在于烟草废弃物中。假单胞菌JY-Q作为一株天然烟碱降解菌，在高浓度烟碱降解方面仍存在困难。在这项研究中，我们研究了两个同源的转录调控因子和内源性异位强启动子对尼古丁降解效率的影响。比较基因组学分析表明，两个同源的转录调控因子，即NicR 2A和NicR 2Bs（NicR 2B 1加NicR 2B 2），可以抑制尼古丁降解基因的表达。当nicR 2A和nicR 28两者缺失时，所得突变体JY-Q Delta nicR 2A Delta nicR 2B 1 Delta nicR 282（Q Delta AB）表现出比野生型JY-Q高17%的烟碱降解效率。转录组测序（RNA-seq）分析表明，JY-Q中有6个基因的转录水平（每百万个酶的片段数[FPKM]值）高于其他基因。基于这些基因的遗传组织，鉴定了三个推定的启动子，P-RS 28250、P-RS 09985和P（RS 24685）。通过使用逆转录酶定量PCR（RT-qPCR）比较它们的表达水平来评估它们的启动子活性。我们发现RS 28250、RS 09985和RS 24685的转录水平分别是hsp 82的16.8、2.6和1.6倍，编码6-羟基-3-琥珀酰吡啶羟化酶，该酶参与尼古丁降解。因此，选择了两个强内源启动子，即P（RS 28250）和P（RS 09985），以替换nic 2基因簇的原始启动子。内源性异位启动子的作用还与目的基因簇的位置有关。当启动子P（RS 28250）取代hspB 2的启动子时，所得到的突变体Q Δ ABs-Delta P-hspB 2：：P（RS 28250）表现出比JY-Q高69%的烟碱降解效率。本研究提出了一种可行的策略，以提高菌株的尼古丁降解的能力，通过去除阻遏调节蛋白和替换强内源性异位promoters. IMPORTION目标promoter。本研究评估了同源NicR 2A和NicR 2Bs和内源性异位强启动子对假单胞菌菌株JY-Q尼古丁代谢的差异影响。基于我们的差异分析，提出了一个可行的策略，以修改野生型（WT）菌株JY-Q通过删除阻遏调控蛋白NicR 2A和NicR 2Bs和替换的目标启动子与强大的内源性异位启动子。所得突变体表现出较高的尼古丁耐受性和降解性。这些发现将有助于通过基因组改造提高天然菌株的污染物降解能力。
Nicotine is a toxic environmental pollutant that widely exists in tobacco wastes. As a natural nicotine-degrading strain, Pseudomonas sp. strain JY-Q still has difficulties degrading high concentrations of nicotine. In this study, we investigated the effect of two homologous transcriptional regulators and endogenous ectopic strong promoters on the efficiency of nicotine degradation. Comparative genomics analysis showed that two homologous transcriptional regulators, namely, NicR2A and NicR2Bs (NicR2B1 plus NicR2B2), can repress nicotine degradation gene expression. When both nicR2A and nicR28s were deleted, the resulting mutant JY-Q Delta nicR2A Delta nicR2B1 Delta nicR282 (Q Delta ABs) exhibits a 17% higher nicotine degradation efficiency than wild-type JY-Q. Transcriptome sequencing (RNA-seq) analysis showed that the transcription levels (fragments per kilobase per million [FPKM] value) of six genes were higher than those of the other genes in JY-Q. Based on the genetic organization of these genes, three putative promoters, P- RS28250, P- RS09985, and P (RS24685), were identified. Their promoter activities were evaluated by comparing their expression levels using reverse transcriptase quantitative PCR (RT-qPCR). We found that the transcription levels of RS28250, RS09985, and RS24685 were respectively 16.8, 2.6, and 1.6 times higher than that of hsp82, encoding 6-hydroxy-3-succinylpyridine hydroxylase, which is involved in nicotine degradation. Thus, two strong endogenous promoters, namely, P (RS28250) and P (RS09985), were selected to replace the original promoters of nic2 gene clusters. The effect of the endogenous ectopic promoter was also related to the position of target gene clusters. When the promoter P (RS28250) replaced the promoter of hspB2, the resultant mutant Q Delta ABs-Delta P-hspB2::P (RS28250) exhibited nicotine-degrading efficiency 69% higher than that of JY-Q. This research suggests a feasible strategy to enhance strains' capacity for nicotine degradation by removal of repressing regulatory proteins and replacing the target promoter with strong endogenous ectopic promoters.IMPORTANCE This study evaluated the differential effects of homologous NicR2A and NicR2Bs and endogenous ectopic strong promoters on nicotine metabolism in Pseudomonas sp. strain JY-Q. Based on our differential analysis, a feasible strategy is presented to modify wild-type (WT) strain JY-Q by removing repressing regulatory proteins NicR2A and NicR2Bs and replacing the target promoter with strong endogenous ectopic promoters. The resulting mutants exhibited high tolerance and degradation of nicotine. These findings should be beneficial for improving the pollutant-degrading capacity of natural strains through genomic modification.