Genomic analysis of Pseudomonas putida: genes in a genome island are crucial for nicotine degradation

Abstract

Nicotine is an important chemical compound in nature that has been regarded as an environmental toxicant causing various preventable diseases. Several bacterial species are adapted to decompose this heterocyclic compound, including Pseudomonas and Arthrobacter. Pseudomonas putida S16 is a bacterium that degrades nicotine through the pyrrolidine pathway, similar to that present in animals. The corresponding late steps of the nicotine degradation pathway in P. putida S16 was first proposed and demonstrated to be from 2,5-dihydroxy-pyridine through the intermediates N-formylmaleamic acid, maleamic acid, maleic acid, and fumaric acid. Genomics of strain S16 revealed that genes located in the largest genome island play a major role in nicotine degradation and may originate from other strains, as suggested by the constructed phylogenetic tree and the results of comparative genomic analysis. The deletion of gene hpo showed that this gene is essential for nicotine degradation. This study defines the mechanism of nicotine degradation.

Figure 1. Chemical reactions and genes involved in the nicotine degradation pathway of P. putida S16.

(A) Pyrrolidine pathway of nicotine degradation. The reactions performed by the enzymes encoded by the nic2 gene cluster are inside the red-dashed box. (B) Genetic organization of the nic2 cluster of P. putida S16 as compared with similar gene clusters from other bacteria. HspB, HSP hydroxylase (black); Iso, maleate isomerase (red); Nfo, NFM deformylase (green); Hpo, DHP dioxygenase (orange); Ami, maleamate amidase (blue); Hna, 6-hydroxynicotinate 3-monooxygenase (dark cyan); Orfx, no predicted function (grey). The percentages represent amino acid homology of related enzymes.

Figure 2. Characterization of Hpo.

(A) SDS-PAGE. M, molecular size markers; lanes 1 and 2, cell extracts of expressed Hpo in E. coli BL21(DE3) expressing His₆-Hpo; lane 3, purified His₆-Hpo. (B) pH dependence of Hpo specific activity. (C) Effect of varying temperatures on specific Hpo activity. (D) Effect of the various metal salts on Hpo specific activity. CK, without metal salt; Ni, Ni²⁺; Co, Co²⁺; Ca, Ca²⁺; Cu, Cu²⁺; Mn, Mn²⁺; Zn, Zn²⁺; Mg, Mg²⁺; Mo, MoO₄²⁻; W, WO₄²⁻; Fe, Fe²⁺.

Figure 3. Characterization of Nfo, Ami, and Iso.

(A) SDS-PAGE of Nfo and Ami. M, molecular size markers; lanes 1 and 2, cell extracts of E. coli BL21(DE3) expressing His₆-Nfo; lane 3, purified His₆-Nfo; lanes 4 and 5, cell extracts of E. coli BL21(DE3) expressing His₆-Ami; 6, purified His₆-Ami. (B) Nfo activity, a, complete reaction: 695 μl buffer + 5 μl NAD + 50 μl Fdh + 50 μl Nfo + 200 μl NFM; b, positive control containing HCOOH (1.65 mM) instead of Nfo and NFM; c, negative control without NFM; d, negative control without Nfo; e, negative control without Nfo and NAD. (C) Enzymatic activity of Ami, a, negative control without Ami; b, negative control, without maleamic acid and Ami; c, negative control without maleamic acid; d, complete reaction containing 480 μl buffer (50 mM Tris-HCl + 0.001 mol l⁻¹ EDTA), 200 μl maleamic acid (0.034 mol l⁻¹), 10 μl α-ketoglutaric acid (0.5 mol l⁻¹), 10 μl NADPH (0.01 mol l⁻¹), 100 μl Gdh (9.5 unit ml⁻¹), and 200 μl amidase (0.106 mg protein ml⁻¹); e, positive control, containing ammonia (30 μM NH₄Cl) instead of maleamic acid and Ami. (D) SDS-PAGE of Iso. M, molecular size markers; lanes 1 and 2, cell extracts of expressed Iso in E. coli BL21(DE3); lanes 3 and 4, purified His₆-tagged Iso. (E) Transformation of maleic acid by Iso. (F) Determination of K_m of Iso.

Figure 4. Nicotine transformation by P. putida S16 and P. putida S16 hpo::pk18mob.

(A) The liquid culture of P. putida S16 and P. putida S16 hpo::pk18mob in nicotine medium. The medium was cultured at 30°C for 16 h. (B) The concentration of nicotine in P. putida S16 and P. putida S16 hpo::pk18mob resting cell systems. The resting cell systems were cultured at 30°C; sampling was conducted at 0, 3 and 5 h. (C) The concentration of 2,5-DHP in P. putida S16 and P. putida S16 hpo::pk18mob resting cell systems. The resting cell systems were cultured at 30°C sampling was conducted at 0, 3 and 5 h.

Figure 5. Phylogenetic trees of the nic gene cluster and 4 related enzymes in late pathway of nicotine degradation.

The phylogenetic trees were constructed with the neighbor joining method (NJ). (A) Phylogenetic tree of nic clusters of different strains constructed using MEGA 4.1. (B) Phylogenetic trees of Hpo, Nfo, Ami, and Iso of different strains constructed using MEGA 4.1.