Of Heavy Metals In Low-temperature Bacteria And Antibiotic Resistance Gene Cloning

Several cold-adapted bacteria were isolated from wastewater of a smeltery in Kunming, which showed resistance to many heavy metals. Based on morphological characteristics and 16S rDNA sequence analysis, four of them were identified as strains of Pseudomonas, Myroides, Chryseobacterium and Serratia, respectively. It was demostrated that Serratia sp. KMR3 showed universal resistance to many antibiotics and heavy metals. Morphological and physiological characteristics of this strain indicated that it belongs to the genus of Enterobacter, gram-negative, could produce red pigments and the pigments colors changed with culture temperatures and metal ions. KMR-3 strain could grow at 4℃-37℃, pH4 - 10, and the optimal temperature and pH were 28℃ and 5.0, respectively. The nucleotide sequence of KMR3 strain showed 99.9% similarity to Serratia marcescens, plus the results of morphological and physiological characteristics, the isolate was identified as Serratia marcescens KMR3, and Genbank accession number was DQ629026.Some genes involved in resistance to copper and chloramphenicol were cloned by constructing genomic DNA library of S. marcescens KMR3. It showed that cloned copper-resistant gene encoded a protein product of 194 amino acids that belongs to CutF protein and showed 70% similarity to uncharacterized lipoprotein NlpE involved in copper resistance of Yersinia mollaretii ATCC 43969. The cloned chloramphenicol-resistant gene encoded a protein product of 398 amino acids that belongs to AraJ protein and showed 85% similarity to drug resistance transporter, Bcr/CflA subfamily of Serratia proteamaculans 568. Regulation sequences including promoter, terminator, SD sequence and transcription start site of cloning genes were also identified. The predicted result showed the chloramphenicol-resistant protein located in cytoplasmic membrane with ten putative transmembrane domains. Although the isolate showed universal resistance to heavy metals and antibiotics, copper-resistant gene was not relative to chloramphenicol-resistant gene in the study. This is the first report of resisitant-gene cloning for Serratia strain in domestic.The study on the heavy metal and antibiotic resistant-genes of S. marcescens KMR3 will provide potential application on bioremediation of heavy metal polluted enviroment and prevention of S. marcescens KMR3 infection in hospital.