Hydrolase CehA and Monooxygenase CfdC Are Responsible for Carbofuran Degradation in Sphingomonas sp. Strain CDS-1

Abstract

Carbofuran, a broad-spectrum systemic insecticide, has been extensively used for approximately 50 years. Diverse carbofuran-degrading bacteria have been described, among which sphingomonads have exhibited an extraordinary ability to catabolize carbofuran; other bacteria can only convert carbofuran to carbofuran phenol, while all carbofuran-degrading sphingomonads can degrade both carbofuran and carbofuran phenol. However, the genetic basis of carbofuran catabolism in sphingomonads has not been well elucidated. In this work, we sequenced the draft genome of Sphingomonas sp. strain CDS-1 that can transform both carbofuran and carbofuran phenol but fails to grow on them. On the basis of the hypothesis that the genes involved in carbofuran catabolism are highly conserved among carbofuran-degrading sphingomonads, two such genes, cehA_CDS-1 and cfdC_CDS-1, were predicted from the 84 open reading frames (ORFs) that share ≥95% nucleic acid similarities between strain CDS-1 and another sphingomonad Novosphingobium sp. strain KN65.2 that is able to mineralize the benzene ring of carbofuran. The results of the gene knockout, genetic complementation, heterologous expression, and enzymatic experiments reveal that cehA_CDS-1 and cfdC_CDS-1 are responsible for the conversion of carbofuran and carbofuran phenol, respectively, in strain CDS-1. CehA_CDS-1 hydrolyzes carbofuran to carbofuran phenol. CfdC_CDS-1, a reduced flavin mononucleotide (FMNH₂)- or reduced flavin adenine dinucleotide (FADH₂)-dependent monooxygenase, hydroxylates carbofuran phenol at the benzene ring in the presence of NADH, FMN/FAD, and the reductase CfdX. It is worth noting that we found that carbaryl hydrolase CehA_AC100, which was previously demonstrated to have no activity toward carbofuran, can actually convert carbofuran to carbofuran phenol, albeit with very low activity.IMPORTANCE Due to the extensive use of carbofuran over the past 50 years, bacteria have evolved catabolic pathways to mineralize this insecticide, which plays an important role in eliminating carbofuran residue in the environment. This study revealed the genetic determinants of carbofuran degradation in Sphingomonas sp. strain CDS-1. We speculate that the close homologues cehA and cfdC are highly conserved among other carbofuran-degrading sphingomonads and play the same roles as those described here. These findings deepen our understanding of the microbial degradation mechanism of carbofuran and lay a foundation for the better use of microbes to remediate carbofuran contamination.

Keywords: carbofuran; hydrolase CehA; monooxygenase CfdC; sphingomonads; upstream catabolic pathway.

FIG 1

Catabolism of carbofuran in strains Novosphingobium sp. KN65.2 and Sphingomonas sp. CDS-1 and genomic context of cehA and cfdC in different hosts. (A) Tentative catabolic pathway of carbofuran in strains Novosphingobium sp. KN65.2 (black arrows) and Sphingomonas sp. CDS-1 (red arrows). Strains KN65.2 and CDS-1 are able to transform both carbofuran and carbofuran phenol (11, 13). Strain KN65.2 can also cleave the aromatic moiety of carbofuran and further degrade the cleaved aromatic ring. Strain CDS-1 failed to break the aromatic moiety and cannot grow on carbofuran. CehA and CfdC are responsible for the initial two steps of carbofuran catabolism. CfdE_KN65.2 supposedly catalyzes the cleavage of the benzene ring of carbofuran in strain KN65.2 (13). Notably, the proposed products of carbofuran phenol catalyzed by CfdC are different in strains KN65.2 and CDS-1. (B) Genomic context of cehA in strains Sphingomonas sp. CDS-1, Rhizobium sp. AC100 (20), and Novosphingobium sp. KN65.2 (21). (C) Genomic context of cfdC in strains Sphingomonas sp. CDS-1 and Novosphingobium sp. KN65.2 (13). The nearly identical ORFs are drawn in the same color.

FIG 2

Transformation of carbofuran or carbofuran phenol by tested strains. WT, Sphingomonas sp. strain CDS-1; CDS-1-cehA, cehA_CDS-1-inactivated mutant of strain CDS-1; CDS-1-cfdC, cfdC-inactivated mutant of strain CDS-1. Plasmid pBBR-A contains cehA_CDS-1 and its putative promoter region. Plasmid pBBR-C carries cfdC and its putative promoter region. Plasmid pET-X harbors cfdX under the control of the T7 promoter. (A) HPLC analysis of the products of carbofuran degraded by strains CDS-1-cehA and CDS-1-cfdC. The degradation test is described in Materials and Methods. (B) Degradation of carbofuran and carbofuran phenol by the tested strains. The strains that can functionally express CfdC are able to transform carbofuran phenol and generate red compounds.

FIG 3

Phylogenetic analysis of CfdC_CDS-1 and the five flavin reductases predicted in Sphingomonas sp. strain CDS-1. (A) Phylogenetic comparison of CfdC_CDS-1 with selected group D flavin-dependent monooxygenases. (B) Phylogenetic tree of the five flavin reductases predicted in Sphingomonas sp. strain CDS-1 with selected flavin reductases. The multiple alignment analysis of the amino acid sequences was performed with Clustal X 2.1. The neighbor-joining method was used to construct the phylogenetic unrooted tree with MEGA 5.0. Protein identifiers (IDs) are shown at the end of each protein.

FIG 4

HPLC and MS/MS analyses of the products of carbofuran phenol treated with purified 6His-CfdC and CfdX-6His. (A) HPLC analysis of the products. The peak on the far left was also present in the control, which may be the peak of NAD⁺ or proteins added. The peaks of carbofuran phenol and two metabolites were further analyzed by tandem mass spectrometry (MS/MS) (B, C, and D). The mass spectra were collected using a TripleTOF 5600 (AB SCIEX) mass spectrometer. The metabolites were ionized by electrospray with positive polarity, and characteristic fragment ions were detected using MS/MS. The main fragment peaks are displayed with their chemical structures.

FIG 5

Relationship between the carbofuran phenol monooxygenase activity and various concentrations of CfdX-6His while the concentration of 6His-CfdC was kept constant. The concentration of CfdX-6His was increased from 0 to 0.06 μM, while the concentration of 6His-CfdC was kept constant at 1 μM. The 6His-CfdC specific activity against carbofuran phenol was defined as 100% when 0.04 μM CfdX-6His was added. Data were calculated from three independent replicates, and the error bars indicate standard deviations.