Genomic and transcriptomic analyses of Agrobacterium tumefaciens S33 reveal the molecular mechanism of a novel hybrid nicotine-degrading pathway

Abstract

Agrobacterium tumefaciens S33 is able to degrade nicotine via a novel hybrid of the pyridine and pyrrolidine pathways. It can be utilized to remove nicotine from tobacco wastes and transform nicotine into important functionalized pyridine precursors for some valuable drugs and insecticides. However, the molecular mechanism of the hybrid pathway is still not completely clear. Here we report the genome analysis of strain S33 and its transcriptomes grown in glucose-ammonium medium and nicotine medium. The complete gene cluster involved in nicotine catabolism was found to be located on a genomic island composed of genes functionally similar but not in sequences to those of the pyridine and pyrrolidine pathways, as well as genes encoding plasmid partitioning and replication initiation proteins, conjugal transfer proteins and transposases. This suggests that the evolution of this hybrid pathway is not a simple fusion of the genes involved in the two pathways, but the result of a complicated lateral gene transfer. In addition, other genes potentially involved in the hybrid pathway could include those responsible for substrate sensing and transport, transcription regulation and electron transfer during nicotine degradation. This study provides new insights into the molecular mechanism of the novel hybrid pathway for nicotine degradation.

Figure 1

The pathways of nicotine and nicotinic acid degradation by some bacteria. (a) Pyridine pathway of nicotine degradation in Arthrobacter. Ndh, nicotine dehydrogenase; 6Hlno, 6-hydroxy-L-nicotine oxidase; Kdh, ketone dehydrogenase; Ponh, 2,6-dihydroxypseudooxynicotine hydrolase; Dhph, 2,6-dihydroxypyridine 3-hydroxylase; (b) A hybrid of pyridine and pyrrolidine pathways for nicotine degradation in A. tumefaciens S33 in this study. See Fig. 3 legend for enzyme abbreviations; (c) Pyrrolidine pathway of nicotine degradation in Pseudomonas. NicA, nicotine oxidoreductase; Pnao, pseudooxynicotine amidase; Sapd, 3-succinoylsemialdehyde-pyridne dehydrogenase; SpmABC, 3-succinoylpyridine monooxygenase; HspB, 6-hydroxy-3-succinoylpyridine hydroxylase; Hpo, 2,5-dihydroxypyridine dioxygenase; Nfo, N-formylmaleamate deformylase; Ami, maleamate amidohydrolase (amidase); Iso, maleate cis/trans-isomerase; (d) Nicotinic acid degradation pathway in Pseudomonas. NicAB, nicotinic acid hydroxylase; NicC, 6-hydroxynicotinate monooxygenase; NicX, 2,5-dihydroxypyridine dioxygenase; NicD, N-formylmaleamate deformylase; NicF, maleamate amidohydrolase (amidase); NicE, maleate cis/trans-isomerase. The same steps in the pyridine pathway and the hybrid pathway are shaded in red; the same steps in the hybrid pathway, the pyrrolidine pathway, and the nicotinic acid degradation pathway are shaded in blue.

Figure 2

Circular representation of the circular chromosome (left) and linear chromosome (right) of A. tumefaciens S33. Outer two rings indicated genes encoded on the forward and reverse strands of the chromosome, respectively, analysed using the COG database (colours were assigned according to the colour code of the COG functional classes). The third circle indicates the predicted GIs (red), prophages (green) and the nicotine-degrading genes (purple). On the fourth ring, the deviation from the average G + C content is plotted. The most inner ring shows the value of the GC skew (G − C/G + C).

Figure 3

The genetic organization of the gene cluster involved in the hybrid nicotine-degrading pathway in A. tumefaciens S33, Shinella sp. HZN7 and Ochrobactrum sp. SJY1. The gene modules of traGDCFBHR and trbIHGFLKJEDCB encode conjugal transfer proteins; repABC encode plasmid partitioning and replication initiation proteins. ndr, nicotine-degrading gene cluster; tnp, transposase; euo, electron transfer flavoprotein-ubiquinone oxidoreductase; etfAB, electron transfer flavoprotein subunit alpha and beta; mfs, major facilitator superfamily transporter; hsh (vppD), 6-hydroxy-3-succinoyl-pyridine hydroxylase; pno, 6-hydroxypseudooxynicotine oxidase; che, chemotaxis protein; abc, ABC transporter; tetR, TetR family transcriptional regulator; ami (vppG), maleamate amidohydrolase (amidase); hpo (vppE), 2,5-dihydroxypyridine dioxygenase; nfo (vppF), N-formylmaleamate deformylase; iso (vppH), maleate cis/trans-isomerase; ald, aldehyde dehydrogenase; hno (vppB), 6-hydroxynicotine oxidase; paz, pseudoazurin; ndhAB (vppA), nicotine dehydrogenase.

Figure 4

Density plot for genes expressed in A. tumefaciens S33 under two different nutrition conditions. S33Glu, cells grown in glucose-ammonium medium; S33Nic, cells grown in nicotine medium.

Figure 5

Scatter plot of FPKM values for genes expressed in A. tumefaciens S33 under two different nutrition conditions. S33Glu, cells grown in glucose-ammonium medium; S33Nic, cells grown in nicotine medium. The differentially-expressed genes were analysed based on S33Nic VS S33Glu.

Figure 6

Gene ontology (GO) annotation of the differentially-expressed genes in A. tumefaciens S33 in glucose-ammonium medium (S33Glu) and nicotine medium (S33Nic). The differentially-expressed genes were analysed based on S33Nic VS S33Glu.

Figure 7

qRT-PCR analysis of the expression levels of five genes involved in nicotine degradation in A. tumefaciens S33. S33Glu, cells grown in glucose-ammonium medium; S33Nic, cells grown in nicotine medium. The expression levels in glucose-ammonium medium were used as control and set as 1.