| Chlorpyrifos is a broad spectrum and moderately toxic organophosphorus insecticide. Its widely used has promoted a wide contamination to the environment and has aroused much public concern. In this work, we isolated a chlorpyrifos degrading bacterium and found that chlorpyrifos stress also could trigger a classic stringent response in this strain. We cloned the (p)ppGpp synthetase gene and found that it plays an important role in growth under several stress conditions and may be a key factor regulating chlorpyrifos biodegradation.The main results are as follows:Strain CPK, which could utilize chlorpyrifos as the sole carbon for growth and transform chlorpyrifos to 3,5,6-trichloro-2-pyridinol, was isolated from activated sludge by enrichment culture technique. Based on the results of phenotypic features, physiological-biochemical properties, and phylogenetic similarity of 16S rRNA gene sequences, this strain was identified as Klebsiella sp. CPK. The Genebank accession number is GU301269. The degradation patterns of strain CPK was studied in MSM with chlorpyrifos as the sole carbon source and was determined by GC. With total biomass amount of 0.2 g dry wt/L, Klebsiella sp. CPK nearly degraded 80% chlorpyrifos within 11d and degradation of chlorpyrifos was accompanied by bacterial growth. The result of HPLC showed that 3,5,6-trichloro-2-pyridinol was the only major metabolite of chlorpyrifos degradation, and it accumulated in the medium without further metabolism. Therefore, like most other aerobic degradation bacteria, CPK could only transform chlorpyrifos to 3,5,6-trichloro-2-pyridinol, but it could not further degrade 3,5,6-trichloro-2-pyridinol.A specific protein was obtained through whole cell protein native-PAGE, which was from the Klebsiella sp. CPK treated by chlorpyrifos in the nutrient rich medium TYC. The specific protein was identified as a (p)ppGpp synthetase (RelA) through TOF-MS sequencing. A 2238 bp complete (p)ppGpp synthetase coding region was cloned and identified by ORF Finder (NCBI), showorf (EMBOSS explorer) bioinformatics software etc. Sequence and phylogenetic analysis of Klebsiella sp. CPK RelA showed that it shared 92% identity to E. coli RelA, but it showed low identity to bifunctional Rel-SpoT protein. These results indicated that Klebsiella sp. CPK RelA may only have (p)ppGpp synthetase activity. In addition, the detection of (p)ppGpp accumulation and complementation test of Klebsiella sp. CPK relA in E. coli also proved that the RelA has only (p)ppGpp synthetase activity.A Klebsiella sp. CPKâ–³relA strain was obtained by homologous recombination and it was verified by PCR and RT-PCR amplification. Compared to its parent, theâ–³relA strain showed weaker degradation ability to chlorpyrifos and it was more sensitive to several stress conditions, such as high salt, low pH values and high concentration of chlorpyrifos. Interestingly, growth curves and semi-quantitative RT-PCR indicated that chlorpyrifos stress would affect the growth and relA expression. In addition, biodegradation study showed that relA expression may play an important role in the degradation of chlorpyrifos by Klebsiella sp. CPK.The results of this work indicated that chlorpyrifos stress could trigger a classic stringent response in Klebsiella sp. CPK and it provided an evidence of the role of (p)ppGpp in biodegradation chlorpyrifos. More specifically, the stringent response is triggered not in nutrient limitation conditions such as amino acids starvation, carbon starvation, etc, but in nutrient rich medium containing chlorpyrifos. Significantly, it reveals that (p)ppGpp modulate environmental stress response, chlorpyrifos tolerance and chlorpyrifos biodegradation in Klebsiella sp. CPK. This regulation may also involve in the detoxification and degradation of others insecticides, xenobiotic or environmental pollutants. These results lead to a better understanding of molecular regulatory mechanisms of bioremediation by microbes.
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