Differential Effects of Homologous Transcriptional Regulators NicR2A, NicR2B1, and NicR2B2 and Endogenous Ectopic Strong Promoters on Nicotine Metabolism in Pseudomonas sp. Strain JY-Q

• Published in: Applied and environmental microbiology Vol. 87; no. 3; pp. 1 - 16
• Main Authors: Huang, Chaochao, Shan, Lihui, Chen, Zeyu, He, Ziliang, Li, Jun, Yang, Yang, Shu, Ming, Pan, Fanda, Jiao, Yang, Zhang, Fuming, Linhardt, Robert J, Zhong, Weihong
• Format: Journal Article
• Language: English
• Published: United States American Society for Microbiology 15-01-2021
• Subjects: Bacterial Proteins - genetics; Biodegradation; Degradation; Efficiency; Environmental Microbiology; Gene clusters; Gene expression; Gene sequencing; Genes; Genomics; Homology; Hydroxylase; Metabolism; Mutants; Nicotine; Nicotine - metabolism; Pollutants; Promoter Regions, Genetic; Promoters; Pseudomonas; Pseudomonas - genetics; Pseudomonas - metabolism; Regulatory proteins; Ribonucleic acid; RNA; RNA-directed DNA polymerase; Tobacco; Transcription; Transcription Factors - genetics; Transcription, Genetic; Transcriptomes; Pseudomonas; transcriptional repressors; nicotine degradation; endogenous promoter
• ISSN: 0099-2240; 1098-5336
• DOI: 10.1128/AEM.02457-20

Nicotine is a toxic environmental pollutant that widely exists in tobacco wastes. As a natural nicotine-degrading strain,
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
and
were deleted, the resulting mutant JY-Q Δ
Δ
Δ
(QΔABs) 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,
,
, and
, 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
,
, and
were respectively 16.8, 2.6, and 1.6 times higher than that of
, encoding 6-hydroxy-3-succinylpyridine hydroxylase, which is involved in nicotine degradation. Thus, two strong endogenous promoters, namely,
and
, were selected to replace the original promoters of
gene clusters. The effect of the endogenous ectopic promoter was also related to the position of target gene clusters. When the promoter
replaced the promoter of
, the resultant mutant QΔABs-Δ
::
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.
This study evaluated the differential effects of homologous NicR2A and NicR2Bs and endogenous ectopic strong promoters on nicotine metabolism in
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.