Location and PCR analysis of catabolic genes in a novel Streptomyces sp.DUT AHX capable of degrading nitrobenzene
AI Haixin , ZHOU Jiti , LV Hong , WANG Jing , GUO Jianbo , LIU Guangfei , QU Yuanyuan
DOI:
Received September 25, 2007,Revised December 17, 2007, Accepted , Available online
Volume 20,2008,Pages 865-870
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A novel strain of Streptomyces sp. DUT AHX was isolated from sludge contaminated with nitrobenzene and identified on the basis
of physiological and biochemical tests and 16S ribosomal DNA (rDNA) sequence analysis. The optimal degradation conditions were
as follows: temperature 30°C, pH 7.0–8.0, shaking speed 150–180 r/min, and inocula 10% (V/V). The strain, which possessed a partial
reductive pathway with the release of ammonia, was also able to grow on mineral salts basal (MSB) medium plates with 2-aminophenol,
phenol, or toluene as the sole carbon source. Furthermore, the enzyme activity tests showed crude extracts of nitrobenzene-grown
DUT AHX contained 2-aminophenol 1,6-dioxygenase activity. The 17-kb plasmid was isolated by the modified alkaline lysis method
and was further cured by sodium dodecyl sulphate (SDS) together with 37°C. As a result, the cured derivative strain DUT AHX-4
lost the 2-aminophenol 1,6-dioxygenase activity. The results suggested that the catabolic genes encoding the nitrobenzene-degrading
enzymes were plasmid-associated. Moreover, the plasmid DNA was amplified with degenerate primers by touchdown PCR and an
expected size fragment (471 bp) was generated. The Blast results revealed that the gene encoding a 157 amino acid polypeptide
was 39%–76% identical to YHS domain protein. The further examination of the plasmid would demonstrate the molecular basis of
nitrobenzene catabolism in Streptomyces, such as regulation and genetic organization of the catabolic genes.
