The Characteristic Analysis And Partial Gene Clone Of Novel Salt-tolerant Bacteria Capable Of Degrading Nitrobenzene

The purpose of this dissertation is to investigate the characteristics and the partial gene of novel salt-tolerant bacteria capable of utilizing nitrobenzene (NB) as the sole carbon, nitrogen and energy sources. The exploitation of salt-tolerant bacteria would be a remarkable improvement in NB bioremediation and wastewater treatment in high salinity. The physiological and biochemical tests and 16S ribosomal DNA (rDNA) sequence analysis were carried out. The characteristics of tolerating salt and degrading NB of Streptomyces sp. DUT_AHX and Micrococcus sp. DUT_AHX were also investigated. Meanwhile, catabolic genes of degrading NB were localized. The partial gene was cloned by modern molecular biological techniques and then was analyzed by bioinformatics techniques.Two novel salt-tolerant bacteria Streptomyces sp. DUT_AHX and Micrococcus sp. DUT_AHX, which were isolated from the sludge and could utilize NB as the sole carbon, nitrogen and energy sources, were identified on the basis of physiological and biochemical tests and 16S rDNA sequence analysis. The 16S rDNA sequences of Streptomyces sp. DUT_AHX and Micrococcus sp. DUT_AHX were submitted to GenBank with the accession number DQ409080 and DQ409081, respectively.The optimal degradation and growth conditions of Streptomyces sp. DUT_AHX are as follows: temperature 30℃, pH 7.0 and shaking speed 150 r/min. It can grow in the presence of NB up to 900 mg/L in mineral salts basal (MSB) medium. The optimal degradation and growth conditions of Micrococcus sp. DUT_AHX are as follows: temperature 37℃, pH 7.0 and shaking speed 150 r/min. It can grow in the presence of NB up to 600 mg/L in MSB medium. Besides, NB-grown cells in MSB medium can degrade NB with the concomitant release of ammonia. The NB degradation rate of strain 1# and strain 2# were 98.8% and 97.6% in 80 h and TOC removing rate were 99.2% and 98.1%. Thus, NB is mineralized to CO₂ and H₂O. The enzyme activity tests show that crude extracts mainly contain 2-aminophenol 1,6-dioxygenase activity. Thus, they may involve a partial reductive pathway of degrading nitrobenzene.Streptomyces sp. DUT_AHX can tolerate moderately NaCl but does not require NaCl for growth. Hence, it is not halophilic but halotolerant. The exogenously added osmoprotectants such as glycin, glutamic acid, proline, betaine and ectoine can improve growth of Streptomyces sp. DUT_AHX in the presence of 10% (w/v) NaCl. The proteins induced by salinity stress or NB were analyzed by native-gradient polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate (SDS)-PAGE. There were three prominent types of modifications, which several proteins were declined, certain proteins were enhanced and some proteins were induced de novo. In NB-induced proteins de novo, 141 kDa protein on the native-gradient PAGE gel was excised and electroeluted. Furthermore, enzyme tests exhibit the 2-aminophenol 1,6-dioxygenase activity of purified 141 kDa protein is 5.825μmol/min/mg protein, which is 11-fold that of the cell-free extracts. Thus, 141 kDa protein is the 2-aminophenol 1,6-dioxygenase of Streptomyces sp. DUT_AHX.A 17-kb plasmid was detected in Streptomyces sp. DUT_AHX and designated pSNB1. The use of elevating growth temperature together with SDS was effective in curing the plasmid pSNB1. A cured derivative designated AHX-4 lost NB degradability and ampicillin resistance. Moreover, pSNB1 was successfully transformed into E. coli JM109 competent cells by calcium chloride method and one transformant AHX-JM109 capable of degrading NB was obtained. As a result, some NB catabolic genes and antibiotic resistance genes are plasmid-mediated in Streptomyces sp. DUT_AHX.Moreover, the plasmid DNA was amplified with degenerate primers by touchdown polymerase chain reaction (PCR) and an expected size fragment (465 bp) was generated. The Blast results reveal that the gene encoding a 155 amino acid polypeptide is 33% to 76% identical to YHS domain protein. The sequences amplified by cassette-ligation-mediated PCR were spliced and the sequence is 2026 bp. The open reading frames (ORFs) in the sequence were analyzed by ORF Finder program. The ORF encoding 126 amino acid polypeptide is identical to YHS domain protein. The ORF designated pPH_AHX may be the gene encoding NB nitroreductase of Streptomyces sp. DUT_AHX. The sequence of pPH_AHX gene was submitted to GenBank with the accession number EF998874. The pPH_AHX gene was analyzed by bioinformatics techniques. It shows that the isoelectric point of the protein encoded by pPH_AHX gene is 4.8 and the theoretic molecular weight is 13.49089 kDa. The protein is acidic, soluble and spherical in cytoplasm. Furthermore, the partial second and tertiary structure of the protein were predicated and three-dimensional structure homologous model was constructed.