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

Abstract

Nicotine is a toxic environmental pollutant that widely exists in tobacco wastes. As a natural nicotine-degrading strain, Pseudomonas sp. JY-Q still has difficulties to degrade 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 NicR2A and NicR2Bs can repress nicotine-degrading genes expression. When both of nicR2A and nicR2Bs were deleted, the resulting mutant QΔnicR2AΔnicR2B1ΔnicR2B2 exhibit 17% higher nicotine degradation efficiency than wide type JY-Q. The RNA-seq analysis showed that the transcription level (FPKM value) of six genes was particularly higher than the other genes in JY-Q. Based on the genetic organization of these genes, three putative promoters, PRS28250, PRS09985 and PRS24685, were identified. Their promoter activities were evaluated by comparing their expression levels using RT-qPCR. We found that the transcription levels of RS28250, RS09985 and RS24685 were 16.8-, 2.6-, and 1.6-times higher than that of hspB2, encoding 6-hydroxy-3-succinylpyridine hydroxylase involved in nicotine degradation. Thus, two strong endogenous promoters PRS28250 and PRS09985 were selected to replace the original promoters of Nic2 gene clusters. The effect of endogenous ectopic promoter was also related to the replaced position of target gene clusters. When the promoter PRS28250 replaced the promoter of hspB2, the resultant mutant, QΔABs-ΔPhspB2::PRS28250, exhibited 69% higher nicotine degrading efficiency than the JY-Q. This research suggests a feasible strategy to enhance strain ability by removal of repressing regulatory proteins and replacing target promoter with strong endogenous ectopic promoters. IMPORTANCE This study evaluated the differential effects of homologous NicR2A/NicR2Bs and endogenous ectopic strong promoters on nicotine metabolism in Pseudomonas sp. JY-Q. Based on our differential analysis, a feasible strategy is presented to modify wild type strain JY-Q by removing repressing regulatory proteins, NicR2A/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 pollutant-degrading capacity of naturally strains through genomic modification.

Combined Transcriptomics and Proteomics Analysis to Reveal the Mechanism underlying the Degradation of TSNAs by Bacillus pumilus 05–5402

“…For instance, according to the KEGG analysis conducted in a previous study, ABC transporter could have a key role in the degradation of nicotine by Pseudomonas sp. JY-Q [27], and this ABC transporter was also enriched in the present study (Fig. 4).…”

“…A combined transcriptomics and proteomics analysis is, consequently, a popular strategy in systems research to obtain information on both genes and proteins, along with gaining insights into the expressions, regulations, and interactions of these biomolecules in an organism [25,26]. Both transcriptomics and proteomics have been applied widely for studying the degradation of harmful substances in tobacco and revealing the underlying mechanisms [27][28][29].…”

Bacillus sp. YC7 from intestines of Lasioderma serricorne degrades nicotine due to nicotine dehydrogenase

“…Differential effects of the homologous transcriptional regulators, NicR2A, NicR2B1, and NicR2B2, and endogenous ectopic strong promoters on nicotine metabolism in Pseudomonas sp. strain JY-Q have been found (Huang et al 2021 ). In addition, functional enhancement of a metabolic module via endogenous promoter replacement for Pseudomonas sp.…”

Neonicotinoid Insecticide-Degrading Bacteria and Their Application Potential in Contaminated Agricultural Soil Remediation

“…However, in a complex of tobacco waste water extract, the effect of JY-Q on nicotine degradation was poor due to the lack of bacterial tolerance and nicotine conversion ability. To break through this bottleneck, researchers first identified the regulatory factors and promoters of the three modules (upper, middle, and downstream) of the nicotine metabolism in JY-Q and then applied metabolic engineering strategies for combined replacement of the endogenous promoters, and the nicotinic degradation rate of the modified bacterial strains increased by 67% and 69%, respectively, compared with the wild type [82,83].…”