A novel and complete gene cluster involved in the degradation of anilineby Delftia sp. AN3
ZHANG Tao , ZHANG Jinglei , LIU Shuangjiang , LIU Zhipei
DOI:
Received August 01, 2007,Revised September 18, 2007, Accepted , Available online
Volume 20,2008,Pages 717-724
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A recombinant strain, Escherichia coli JM109-AN1, was obtained by constructing of a genomic library of the total DNA of Delftia
sp. AN3 in E. coli JM109 and screening for catechol 2,3-dioxygenase activity. This recombinant strain could grow on aniline as sole
carbon, nitrogen and energy source. Enzymatic assays revealed that the exogenous genes including aniline dioxygenase (AD) and
catechol 2,3-dioxygenase (C23O) genes could well express in the recombinant strain with the activities of AD and C23O up to 0.31
U/mg wet cell and 1.92 U/mg crude proteins, respectively. The AD or C23O of strain AN3 could only catalyze aniline or catechol
but not any other substituted substrates. This recombinant strain contained a recombinant plasmid, pKC505-AN1, in which a 29.7-kb
DNA fragment from Delftia sp. AN3 was inserted. Sequencing and open reading frame (orfs) analysis of this 29.7 kb fragment revealed
that it contained at least 27 orfs, among them a gene cluster (consisting of at least 16 genes, named danQTA1A2BRDCEFG1HIJKG2)
was responsible for the complete metabolism of aniline to TCA-cycle intermediates. This gene cluster could be divided into two main
parts, the upper sequences consisted of 7 genes (danQTA1A2BRD) were predicted to encode a multi-component aniline dioxygenase
and a LysR-type regulator, and the central genes (danCEFG1HIJKG2) were expected to encode meta-cleavage pathway enzymes
for catechol degradation to TCA-cycle intermediates. Unlike clusters tad from Delftia tsuruhatensis AD9 and tdn from Pseudomonas
putida UCC22, in this gene cluster, all the genes were in the same transcriptional direction. There was only one set of C23O gene (danC)
and ferredoxin-like protein gene (danD). The presence of only one set of these two genes and specificity of AD and C23O might be the
reason for strain AN3 could only degrade aniline. The products of danQTA1A2BRDC showed 99%–100% identity to those from Delftia
acidovorans 7N, and 50%–85% identity to those of tad cluster from D. tsuruhatensis AD9 in amino acid residues. Besides this dan
cluster, the 29.7 kb fragment also contained genes encoding the trans-membrane transporter and transposases which might be needed
for transposition of the gene cluster. Pulsed-field gel electrophoresis (PFGE) and plasmid curing experiments suggested that the dan
cluster might be encoded on the chromosome of strain AN3. The GenBank accession number for the dan cluster of Delftia sp. AN3 is
DQ661649.
