Complete nucleotide sequence and organization of the naphthalene catabolic plasmid pND6-1 from Pseudomonas sp. strain ND6

doi:10.1016/j.gene.2004.03.027

Gene

Volume 336, Issue 2, 21 July 2004, Pages 231-240

https://doi.org/10.1016/j.gene.2004.03.027 Get rights and content

Abstract

Pseudomonas sp. strain ND6, which was isolated from industrial wastewater in Tianjin, China, was capable of dissimilating naphthalene as sole carbon and energy sources. We identified one plasmid, pND6-1, which was associated with the metabolism of naphthalene and determined the complete nucleotide sequence of pND6-1 (101,858 bp) using a whole-genome-shotgun approach. Computational analyses indicated that the naphthalene metabolism of the strain ND6 is associated with this plasmid. This is the first report of a complete sequence of naphthalene catabolic plasmid. pND6-1 encodes 102 putative coding sequences (CDSs). Among them, 23 CDSs were predicted to be involved in naphthalene catabolism, 14 were predicted to be involved in transposition and integration, 2 encoded putative transporters, 3 were putative transcriptional regulators, and 9 were proteins necessary for plasmid replication and partitioning. Most of the naphthalene catabolic genes of pND6-1 have 99–100% identity in amino acid sequences homologous to their nearest counterparts found in plasmid pDTG1, NAH7 and in a chromosome region in Pseudomonas stutzeri AN10 except for two duplicated genes (ND013 and ND016). Results of this study indicated that globally distributed naphthalene catabolic genes are highly conserved among different bacterial species.

Introduction

Many bacterial strains have been shown to degrade environmental contaminants and their degradation genes can often be located on catabolic plasmids (Sayler et al., 1990). For example, plasmids encoding the catabolism of toluene, camphor, naphthalene, 2,4-dichlorophenoxyacetate, biphenyl, and herbicide atrazine have been reported Sayler et al., 1990, Romine et al., 1999, Martinez et al., 2001, Greated et al., 2002. Many of the genes for these catabolic pathways have been cloned and sequenced, but to date only four catabolic plasmids, pNL1 from Sphingomonas aromaticivorans strain F199, pADP-1 from Pseudomonas sp. strain ADP, pWWO from Pseudomonas putida PaW1, pCAR1 from Pseudomonas resinovorans strain CA10, have been completely sequenced and their gene organizations were published. pNL1 contains the genes encoding enzymes for the metabolism of biphenyl, naphthalene, m-xylene, and p-cresol (Romine et al., 1999). The six genes (atzA, B, C, D, E, F) of pADP-1 encode the atrazine degradation pathway (Martinez et al., 2001). pWWO encodes catabolic pathways of toluene and xylene (Greated et al., 2002). pCAR1 contains the genes encoding the carbazole/dioxin-degrading enzymes and anthranilate 1,2-dioxygenase, respectively (Maeda et al., 2003).

The aromatic hydrocarbon naphthalene has been identified as a priority pollutant. The bacterial oxidation of naphthalene has been extensively investigated, especially in Pseudomonas strains. Naphthalene-degrading genes have been localized on catabolic plasmids or are present in the chromosomes of bacteria. The plasmid NAH7 from P. putida G7 and pDTG1 from P. putida NCIB 9816 are the two best-characterized naphthalene catabolic plasmids (Yen and Serdar, 1988). Incomplete sets of naphthalene-degrading genes from plasmid NAH7 were cloned and sequenced Eaton, 1994, Grimm and Harwood, 1999. Recently, after pND6-1 sequence was submitted to GenBank (Dec. 28, 2002), another naphthalene catabolic plasmid, pDTG1 from P. putida NCIB 9816-4 (gi: 28976042, unpublished), was released on May 17, 2003. On the other hand, about 20 chromosomally located naphthalene-degrading genes from Pseudomonas stutzeri AN10 were sequenced and analyzed Bosch et al., 1999a, Bosch et al., 2000.

Pseudomonas sp. strain ND6 was isolated from industrial wastewater in Tianjin, China by selection for its ability to use naphthalene as sole carbon and energy sources (Zhang et al., 2000). One plasmid, designated pND6-1, was identified to be associated with these features. We undertook the whole-genome-shotgun sequencing of this plasmid to probe the catabolic functions and accessory genes in it. Computational analyses indicated that the naphthalene catabolism of the strain ND6 is associated with this plasmid, pND6-1. The complete nucleotide sequence and annotation of pND6-1 are reported here.

Section snippets

Bacterial strains, plasmids and growth conditions

Pseudomonas sp. strain ND6 was grown with shaking at 30 °C in LB medium for isolation of plasmids. The plasmid, named pND6-1, was isolated for the study. Plasmid pUC18 was used as vector for the construction of the plasmid shotgun library. Escherichia coli DH5 α was used as recipient strain for shotgun library and grown at 37 °C in LB medium.

Isolation and purification of plasmid DNA

A 100 ml culture of strain ND6 was grown in LB medium and incubated overnight at 30 °C with shaking. Cells were harvested by centrifugation at 7000 rpm for

Nucleotide sequence and genetic organization of pND6-1

DNA sequencing indicated that the size of pND6-1 was 101,858 bp in length. In silicon analyses indicated that naphthalene metabolism of the strain ND6 was associated with this plasmid. pND6-1 has an overall G+C content of 57%. This plasmid contains an origin of replication (oriV), a region involved in plasmid replication and stable inheritance, and a region involved in the naphthalene catabolism (Fig. 1). This is the first report of a complete nucleotide sequence of naphthalene catabolic

Acknowledgements

This work is supported by Grant 30270274 from the National Natural Science Foundation of China.

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¹: Present address: Department of Biostatistics, Harvard School of Public Health, Dana-Farber Cancer Institute, Boston, MA 02115, USA

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Complete nucleotide sequence and organization of the naphthalene catabolic plasmid pND6-1 from Pseudomonas sp. strain ND6

Abstract

Introduction

Section snippets

Bacterial strains, plasmids and growth conditions

Isolation and purification of plasmid DNA

Nucleotide sequence and genetic organization of pND6-1

Acknowledgements

Gene

Gene

J. Mol. Biol.

FEMS Microbiol. Ecol.

Gapped BLAST and PSI-BLAST: a new generation of protein database search programs

Nucleic Acids Res.

The InterPro database, an integrated documentation resource for protein families, domains and functional sites

Nucleic Acids Res.

Cloning and functional analysis of the pbr lead resistance determinant of Ralstonia metallidurans CH34

J. Bacteriol.

NahW, a novel, inducible salicylate hydroxylase involved in mineralization of naphthalene by Pseudomonas stutzeri AN10

J. Bacteriol.

Cloning and nucleotide sequence analysis of naphthalene chemotaxis gene nahY in naphthalene-degrading plasmid pND6-1

Chin. J. Appl. Environ. Biol.

Improved microbial gene identification with GLIMMER

Nucleic Acids Res.

Metabolism of dibenzothiophene and naphthalene in Pseudomonas strains: complete DNA sequence of an upper naphthalene catabolic pathway

J. Bacteriol.

Organization and evolution of naphthalene catabolic pathways: sequence of the DNA encoding 2-hydroxychromene-2-carboxylate isomerase and trans-o-hydroxybenzylidenepyruvate hydratase-aldolase from the NAH7 plasmid

J. Bacteriol.

Base-calling of automated sequencer traces using phred: II. Error probabilities

Genome Res.

Base-calling of automated sequencer traces using phred: I. Accuracy assessment

Genome Res.

Consed: a graphical tool for sequence finishing

Genome Res.

Complete sequence of the IncP-9 TOL plasmid pWW0 from Pseudomonas putida

Environ. Microbiol.

NahY, a catabolic plasmid-encoded receptor required for chemotaxis of Pseudomonas putida to the aromatic hydrocarbon naphthalene

J. Bacteriol.