Isolation And Characterization Of Malachite Green-Degrading Strains, Cloning And Expression Of The Tmr2 Gene

Two Malachite green degrdading strains designated as K4-W and K9 were isolated from the sludge sample collected from the wastewater treating system of a chemical plant. K4-W and K9 were preliminarily identified as Raoultella sp.(GenBank Accession No. EU876828) and Pseudomonas sp.(GenBank Accession No. EU855781) respectively according to the physiological & biochemical characteristics and 16S rRNA gene sequence analysis.Biological characteristics of strain K4-W and K9 were investigated respectively. Strain K4-W could grow well from pH5.0-10.0, the optimal pH for its growth was 7.0; The optimal temperature for its growth was 30℃; The optimal carbon source and nitrogen source of medium for its growth was xylitol and peptone; The optimal pH and temperature for the growth of strain K9 was 7.0 and 30℃respectively; Maltose and beef extract was the optimal carbon source and nitrogen source for its growth.The degradation rate against 20 mg·L-1 malachite green of strain K4-W and K9 was 82.5% and 88.3% respectively within 48h.30℃was the optimal degrading temperature for both of them. The increasing higher concentration of malachite green showed inhibitory effect on the degradation. Except the degradation promoting effect from the addition of Pb2+, other metal ions such as Al3+,Mn2+ and Zn2+ showed inhibitory effects to strain K4-W significantly; While all the tested metal ions could inhibit the degradation ability of K9. Strain K9 could also degrade other triphenylmethane dyes (Malachite Green, Crystal Violet, Basic Fuchsia), while strain K4-W could only degrade malachite green.The primers were designed according to the published sequences of the triphenylmethane reductase gene (tmr2). The tmr2 detection of strain K9 and K4-W was carried out by PCR and the tmr2 gene was obtained from strain K9. Its sequence had 100% similarity to tmr2 gene (GenBank accession No.EF463103),99% similarity to tmr (GenBank accession No.AY756172) and tmpD gene (GenBank accession No.EF010984). However, the tmr2 gene was not amplified successfully from strain K4-W. Therefore, whether there is a new tmr gene in this strain still needs further study. The tmr2 gene was overexpressed in E.coli BL21(DE3), the protein with the right molecular size (31KD) as the predicted was prouduced; It showed a NADH-dependency character and could degrde triphenylmethane dyes, which are the typical properties of triphenylmethane reductase.A rapid and efficient chromosome walking method (SEFA-PCR) was employed to clone the upstream and downstream fragments of the tmr2 gene. The obtaind sequences were spliced with the tmr2 gene, resulting a fragment (5946bp in length) including the tmr2 gene and the flankings. Its sequence analysis showed that tmr2 was associated with a typical mobile element ISPpu12 consisting of tnpA(encoding a transposase), lspA(encoding a lipoprotein signal peptidase), orf1(encoding a putative MerR family regulator) and orf2(encoding a CDF family heavy metal/H+ antiporter). We deduced that tmr2 gene was likely transferable among the malachite green-grading bacteria, which needs to be vertified further.