A sirA-like gene, sirA2, is essential for 3-succinoyl-pyridine metabolism in the newly isolated nicotine-degrading Pseudomonas sp. HZN6 strain

Abstract

A novel nicotine-degrading Pseudomonas sp. strain, HZN6, was isolated from a pesticide-wastewater treatment facility in Hangzhou. The strain could grow on nicotine as its sole source of carbon, nitrogen, and energy. The strain's main intermediate metabolites were determined to be pseudooxynicotine, 3-succinoyl-pyridine (SP), and 6-hydroxy-3-succinoyl-pyridine (HSP). A Tn5 transposon mutant was generated in which the degradation pathway was blocked at the SP. A 4,583-bp DNA fragment flanking the transposon insertion site was obtained through self-formed adaptor PCR and analyzed. The mutant gene orfC displays 89% deduced amino acid sequence identity with the sirA-like gene (sirA2, a sulfurtransferase homologue gene) of Pseudomonas stutzeri A1501. The orfC-disrupted strain lost the ability to degrade SP, and the complementation strains with the orfC from the Pseudomonas sp. HZN6 and the sirA2 (PP_1233) from Pseudomonas putida KT2440 recovered the degradation ability. Though the orfC-disrupted strain also lost the xanthine dehydrogenase activity, the effects of tungsten on the degradation of SP and hypoxanthine revealed that the hydroxylation of SP to HSP was not a xanthine dehydrogenase type. These results demonstrated that the orfC gene was essential for the SP metabolism involved in the nicotine metabolic pathway in the Pseudomonas sp. HZN6 strain. This study might advance the understanding of the nicotine metabolic mechanism in Pseudomonas.

Functional Identification of Two Novel Genes from Pseudomonas sp. Strain HZN6 Involved in the Catabolism of Nicotine

“…The degradation mechanism of nicotine in Pseudomonas has been studied for more than 50 years (21,28). However, there have been few studies at the genetic level reported so far.…”

“…The in-frame disruption of the pao and sap genes in Pseudomonas sp. strain HZN6 was performed using suicide plasmid pJQ200SK and a twostep homologous recombination method, as described previously (21). Briefly, to disrupt the pao gene, a 1.749-kb fusion product was constructed by PCR, which consisted of an upstream sequence of the pao gene, kanamycin resistance gene, and downstream sequence using primers paoUFBamHI/paoUR, KmF/KmR, and paoDF/paoDR-SpeI (see Table S1 in the supplemental material), respectively.…”

“…Pseudomonas sp. strain HZN6 was isolated and identified as a nicotinedegrading bacterium and was deposited in the China Center for Type Culture Collection (CCTCC 2010196) (21). The Pseudomonas strains and their derivatives were grown aerobically at 30°C in LB or mineral salts medium (MSM) as previously described (21).…”

“…The generation and selection of nicotine growth-deficient mutants of Pseudomonas sp. HZN6 and the cloning of mutant genes were performed according to methods described previously (21). The mutant gene was amplified by self-formed adaptor PCR (SEFA-PCR) (30) and assembled using the Omega 2.0 software.…”

“…strain HZN6 with the pyrrolidine pathway (21). The sirA2 gene of strain HZN6 was disrupted by the Tn5 transposon and identified as essential for SP hydroxylation.…”