Insights into functional and evolutionary analysis of carbaryl metabolic pathway from Pseudomonas sp. strain C5pp

Carbaryl (1-naphthyl N-methylcarbamate) is a most widely used carbamate pesticide in the agriculture field. Soil isolate, Pseudomonas sp. strain C5pp mineralizes carbaryl via 1-naphthol, salicylate and gentisate, however the genetic organization and evolutionary events of acquisition and assembly of pathway have not yet been studied. The draft genome analysis of strain C5pp reveals that the carbaryl catabolic genes are organized into three putative operons, ‘upper’, ‘middle’ and ‘lower’. The sequence and functional analysis led to identification of new genes encoding: i) hitherto unidentified 1-naphthol 2-hydroxylase, sharing a common ancestry with 2,4-dichlorophenol monooxygenase; ii) carbaryl hydrolase, a member of a new family of esterase; and iii) 1,2-dihydroxy naphthalene dioxygenase, uncharacterized type-II extradiol dioxygenase. The ‘upper’ pathway genes were present as a part of a integron while the ‘middle’ and ‘lower’ pathway genes were present as two distinct class-I composite transposons. These findings suggest the role of horizontal gene transfer event(s) in the acquisition and evolution of the carbaryl degradation pathway in strain C5pp. The study presents an example of assembly of degradation pathway for carbaryl.

Many microorganisms capable of utilizing carbaryl have been identified, but little or no information is available at the molecular level. This has limited our understanding about the genetic organization, regulation, possible route for acquisition of the genes involved in degradation and evolutionary history of the strain. The sequencing of Pseudomonas sp. strain C5pp genome will probably answer some of these questions. Here, in this section, we present an additional data to support the results presented and discussed in the main manuscript.
Though no t-RNA gene or tra-elements were identified at 3' or 5' of the 76.33 kb segment identified to be involved in carbaryl degradation, the segment was found to be interspersed with number of insertion sequences, repeats, duplication of ~2.6 kb region containing integrase and skewing of G+C content. This analysis suggests the 'patchwork' in the genetic organization of the pathway which indicates a very recent acquisition of the pathway in Pseudomonas sp. strain C5pp.

Analysis of G1 clone
The gentisate utilizing fosmid clone, designated as G1 obtained from genomic library constructed in E. coli was analyzed for restriction digestion pattern with BamHI. The restriction digestion of G1 with BamHI gave six fragments of ~12, 8, 5, 2.8, 2.5 and 1.8 kb size. These six fragments obtained after BamHI digestion were sub-cloned into pUC19 and partially sequenced.
The analysis and annotation of G1-DNA sequences by BLASTn analysis is given in Table S1.

Genome sequencing
The sequencing of strain C5pp genome generated 249174 raw reads and ~98 % of the total reads were assembled de novo giving 131 contigs totaling to 6161937 bases Figure S1A.
The large contigs (97) were used further for genome annotation and analyses. Genome features and the statistics of assembled genome of strain C5pp are summarized in and subsystem features in Figure S1B.

Gap filling of contigs involved in carbaryl degradation
The sequences obtained from six sub-clones of G1 were used as a query against strain C5pp draft genome which retrieved contig number 47 (32.72 kb), 62 (13.65 kb), 61 (14.04 kb) and 76 (2.64 kb). Based on the partial sequences obtained from G1 clone, the contig was expected to be in the following order 62-61-76-47. The contigs 68 (8.2 kb), 83 (1.47 kb) and 92 (0.87 kb) were also proposed to be a part of this assembly and expected to be present at 5' end of contig 62. The assembly of contigs was attempted by gap filling PCR reactions using contig specific primers ( Table S2). The PCR amplified fragments obtained using genomic DNA as template was sequenced to fill the gaps between the contigs. The contigs were also submitted to ORF finder to identify the probable proteins encoded in the sequence. The proteins were annotated based on blastp analysis. The annotation details for these contigs are summarized in Table S3. The order of the contig is 83-68-92-62-76-61-76-47 and the length of this assembled segment is 76334 bp. This is referred as Supercontig-A.

Functional analysis of carbaryl hydrolase
Carbaryl hydrolase (CH) catalyzes the conversion of carbaryl to 1-naphthol and Nmethylcarbamic acid by hydrolysis of either ester or amide linkage. Partial purification of CH was attempted from Pseudomonas sp. strain C5pp grown on carbaryl. The cells (14 g) were harvested at 12 h (OD 540 -~1) and cell-free extract was prepared as mentioned in Materials and Methods (see main MS). Lysate (64 ml) was loaded on to Q-Sepharose (40 ml) pre-equilibrated with potassium-phosphate buffer (50 mM, pH 7.5), the enzyme was obtained in the unbound (81 ml) fraction. The unbound pool was loaded onto Phenyl Sepharose (11 ml) pre-equilibrated with potassium-phosphate buffer (50 mM, pH 7.5). The enzyme activity was observed in the unbound (111 ml) fraction. The unbound enzyme was brought to 50 % ammonium sulphate saturation and loaded onto Phenyl Sepharose (10 ml) pre-equilibriated with potassium-phosphate buffer containing ammonium sulphate (30 %). The bound enzyme was eluted using increasing concentration gradient of ethylene glycol (0-80 %) and decreasing concentration gradient of ammonium sulphate (30-0 %). CH was eluted between 50-70 % of ethylene glycol and 10-5 % of ammonium sulphate. The active fractions (1.5 ml each) were pooled and loaded onto Sephacryl S200-HR which eluted at 49 ml. The enzyme was purified ~73 fold with yield of 4.3 % and specific activity of 11.3 µmole.min -1 .mg -1 . SDS-PAGE profile is shown in Fig. S2, CH was found to be a monomer with molecular weight ~80 kDa.
The molecular mass of McbA (annotated as hypothetical protein) was predicted to be 83 kDa (Expasy) which is similar to that observed for partially purified CH from strain C5pp (Fig.   S2). Thus, this hypothetical protein was speculated to be CH. McbA was cloned in pET28-a(+) at NheI and XhoI site and (Fig. S3AI  was performed using RP-C18 column (4.6×250 mm, particle size 5 µm, Eclipse plus C18, Agilent). The column was developed using solvent system methanol: water (60:40 vol/vol, flow rate 1 ml.min -1 ). The samples were detected using diode array with wavelength set at 280 nm and 322 nm for carbaryl and 1-naphthol, respectively. In HPLC analysis, besides carbaryl peak at RT 4.4 min, the reaction mixture at the end of 30 min revealed an additional peak with RT 5.2 min which corresponds to the standard 1-naphthol (RT 5.2 min) ( Fig. S3B-E). The lysate from E. coli transformed with vector pET-28a(+) failed to show any peak at RT 5.2 min. These results suggest that mcbA codes for CH which converts carbaryl to 1-naphthol.
Significant divergence at the functional (amidase or esterase) as well as at the sequence level has been observed for the CH. The phylogenetic analysis revealed the clustering of CH from strain C5pp with the protein sequences encoding esterases type of enzymes, suggesting the protein to be a member of esterase. The amino acid sequences of CH from strain C5pp, Rhizobium sp AC100 and Pseudomonas putida were compared with functionally characterized esterase belonging to fifteen different families (Fig. S4) Figure S4; and Figure S5).

Functional analysis of 1,2-dihydroxynaphthalene dioxygenase
The sequence alignment of the putative McbB with 12DHNDO from Burkholeria sp.

Functional analysis of 1-naphthol 2-hydroxylase
The gene for 1-naphthol 2-hydroxylase (1NH), mcbC was cloned into pET-28a(+) at EcoRI and NdeI site ( Fig. S7A-I  Alteromonas sp. SN2 and Marinomonas profundimaris (Fig. S8A). Similarly McbE was found to be closely related to the homologs from comamonas testosteroni and Xenophilus azovorans (Fig.   S8B). Whereas, McbF was found to closely related to PhnF from phenanthrene degrading Burkholderia sartisoli (Fig. S8C). This further supports the hypothesis that carbaryl degradation pathway is probably acquired from phenanthrene and naphthalene degrader Burkholderia. shows high similarity to dioxygenase type of enzymes, it remains to be seen whether S5H has evolved as a result of gene duplication from a dioxygenase.

Analysis of regulators present on Supercontig-A involved in carbaryl degradation
The