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. 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 nicR2Bs were deleted, the resulting mutant JY-Q ΔnicR2A ΔnicR2B1 ΔnicR2B2 (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, 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 hspB2, 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 QΔABs-ΔP_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.

Keywords: Pseudomonas; endogenous promoter; nicotine degradation; transcriptional repressors.

FIG 1

Nicotine metabolism modules (a), pyrrolidine pathway (b), and genetic organization of nicotine-degrading genes (c) in Pseudomonas sp. JY-Q. SP, 3-succinoyl-pyridine; HSP, 6-hydroxy-3-succinoyl pyridine.

FIG 2

RT-qPCR analysis of nicotine degradation gene transcripts produced in JY-Q and its mutants with a knockout of the transcriptional regulator gene grown in BSM containing 4 g/liter glucose. (a) PCR verified the deletion of nicR2A, nicR2B1, and nicR2B2 in mutants. A, the inner primer pair of nicR2A; B1, the primer pair outside nicR2B1; B2, the primer pair outside nicR2B2. (b) mRNA expression levels of spmA1, spmA2, hspB1, and hspB2. (c) mRNA expression levels of nox and nicA2. (d) mRNA expression levels of nicR2A and nicR2Bs. The data are the means of three independent experiments, and error bars indicate the standard deviations. t test was used for significance analysis; *, P < 0.05 (significant); **, P < 0.01 (extremely significant).

FIG 3

Growth (a); nicotine degradation (b); and expression levels of genes nox, nicA2, spmA1, spmA2, hspB1, and hspB2 (c) of WT strain JY-Q and its mutant strains QΔA, QΔnicR2AΔnicR2B1, and QΔABs in BSM containing 2 g/liter nicotine. The data are the means of three independent experiments, and error bars indicate the standard deviations. t test was used for significance analysis; *, P < 0.05 (significant); **, P < 0.01 (extremely significant).

FIG 4

Growth (a), nicotine degradation (b), and the broth color change (c) of the promoter replaced mutant and WT strains in BSM containing 2 g/liter nicotine. (d) Data regarding promoter replacement in the strains (+, original or inserted promoter replacing the original one; −, replaced).

FIG 5

mRNA expression levels of nox (a), spmA1 (b), hspB1 (c), and hspB2 (d) in seven mutants and the WT (Q) grown in BSM containing 2 g/liter nicotine. The data are the means of three independent experiments, and error bars indicate the standard deviations. t test was used for significance analysis between the mutant or/and the wild type JY-Q; *, P < 0.05 (significant); **, P < 0.01 (extremely significant). (e) Data regarding promoter replacement in the strains (+, original or inserted promoter replacing the original one; −, replaced).

FIG 6

Growth profile of WT and mutant strains after 40 h on solid BSM plate containing 15 g/liter nicotine (a), growth (b), and nicotine degradation curves (c) of the mutant QΔABs-28 and the control QΔABs in liquid BSM containing 2, 5, and 10 g/liter nicotine. It should be noted that, after 42 h, there was a large amount of green substance accumulation in 5 g/liter and 10 g/liter nicotine medium of strain QΔABs; thus, its OD₆₀₀ was not measured. The data are the means of three independent experiments, and error bars indicate the standard deviations. t test was used for significance analysis; *, P < 0.05 (significant); **, P < 0.01 (extremely significant). (d) Strain data.