Cloning and Expression of Vitreoscilla Hemoglobin Gene in Burkholderia Sp

26 Biotechnol. Prog. 2000, 16, 26−30 Cloning and Expression of Vitreoscilla Hemoglobin Gene in Burkholderia sp. Strain DNT for Enhancement of 2,4-Dinitrotoluene Degradation Sangeeta M. Patel,† Benjamin C. Stark,† Kwang-Woo Hwang,† Kanak L. Dikshit,‡ and Dale A. Webster*,† Department of Biological, Chemical, and Physical Sciences, Illinois Institute of Technology, Chicago, Illinois 60616, and Institute of Microbial Technology, Sector 39, Chandigarh 160014, India The gene (vgb) encoding the hemoglobin (VHb) of Vitreoscilla sp. was cloned into a broad host range vector and stably transformed into Burkholderia (formerly Pseudomo- nas) sp. strain DNT, which is able to degrade and metabolize 2,4-dinitrotoluene (DNT). Vgb was stably maintained and expressed in functional form in this recombinant strain (YV1). When growth of YV1, in both tryptic soy broth and minimal salts broth containing DNT and yeast extract, was compared with that of the untransformed strain, YV1 grew significantly better on a cell mass basis (A600) and reached slightly higher maximum viable cell numbers. YV1 also had roughly twice the respiration as strain DNT on a cell mass basis, and in DNT-containing medium, YV1 degraded DNT faster than the untransformed strain. YV1 cells pregrown in medium containing DNT plus succinate showed the fastest degradation: 100% of the initial 200 ppm DNT was removed from the medium within 3 days. Introduction DNT degradation both indirectly, by enhancing respiration and thus growth, and directly, by enhancing the The prokaryotic hemoglobin (VHb) from the Gram- three oxygen-dependent oxidations in the DNT degrada- negative, aerobic bacterium Vitreoscilla is well-character- tion pathway. Thus, VHb technology may be useful for ized. Its in vivo role is suggested to be an oxygen trap to bioremediation in the field, especially when oxygen-poor allow Vitreoscilla to survive and grow in the microaerobic conditions exist in contaminated sites, for example, in environments that it normally inhabits (1, 2). The gene soil or water below the surface. (vgb) encoding VHb has been isolated, sequenced, and To this end we report here the engineering of Burkhold- characterized (3, 4). When this gene is transformed into eria strain DNT to contain vgb and produce VHb. We a heterologous host (e.g., Escherichia coli, fungi), it can show that the resulting strain (YV1) does, in fact, have increase the growth of and product production by the host enhanced growth and DNT-degrading abilities compared - (5 10). Presumably the VHb produced in these cells with strain DNT. enhances respiration at low oxygen concentrations as it is thought to do in Vitreoscilla. In Pseudomonads, it has Materials and Methods been shown to have potential for enhancing bioremediation as well (11). Bacterial Strains and Plasmids. Burkholderia (orig- inally Pseudomonas) sp. strain DNT (R34) was provided In the work reported here we have extended this by Jim C. Spain (Tyndall Air Force Base, FL). The technology to Burkholderia sp. strain DNT (formerly organism was maintained on a minimal salts medium Pseudomonas sp. strain DNT, also known as R34), which supplemented with 100 ppm DNT and 200 ppm yeast can degrade 2,4-dinitrotoluene (DNT). DNT, a byproduct extract (MSB-DNT-YE) at 35 °C (13). Plasmid pSC160 of trinitrotoluene production and usage, is considered (16.0 kb) was constructed as described previously (8)by toxic at levels greater than 0.13 ppm. Recent work from inserting plasmid pUC8:16 (3) into the EcoRI site of several laboratories suggests that nitrotoluenes can be autonomous vector pKT230 (11.9 kb) (15), which inacti- biodegraded by an oxidative pathway and that mineral- vated the Strr (streptomycin resistance) but left the Kmr ization of TNT and DNT by bacteria can occur (12, 13). (kanomycin resistance). Plasmid pUC8:16 (4.1 kb), in Suen and Spain (14), by biochemical and genetic dissec- turn, contains vgb, including its native promoter cloned tions, have characterized the pathway and genes of into vector pUC8 which confers ampicillin resistance (16). Burkholderia sp. strain DNT responsible for the complete Plasmid pSC160 thus confers resistance to both ampi- mineralization of DNT by an oxidative pathway. Because cillin and kanamycin, which was the initial method of oxygen is required at three early steps in this process, selection. engineering strain DNT so that it produces VHb may lead Transformation. Burkholderia sp. strain DNT was to improvements in its ability to bioremediate DNT; transformed with pSC160 by the method of Liu et al. (8). increased intracellular oxygen via VHb may enhance Plasmid pSC160 was isolated from P. aeruginosa (rather than E. coli) by a scale-up of the alkaline lysis method † Illinois Institute of Technology. described by Birnboim and Doly (17). This transformant ‡ Institute of Microbial Technology. is denoted as YV1. 10.1021/bp9901421 CCC: $19.00 © 2000 American Chemical Society and American Institute of Chemical Engineers Published on Web 01/20/2000 Biotechnol. Prog., 2000, Vol. 16, No. 1 27 Growth, Respiration, and Hemoglobin Measure- ment. Cells were inoculated from freezer stocks into 25 mL of fresh MSB-DNT-YE medium for the untransformed strain (DNT). For the transformed strain (YV1), 100 µg/mL ampicillin and 40 µg/mL kanamycin were added to the same medium. Cells were grown overnight at 35 °C and 150 rpm, harvested by centrifugation, washed with 0.01 M sodium phosphate buffer, pH 7.2, and inoculated into 200 mL of fresh MSB-DNT-YE medium without antibiotics for both strains DNT and YV1. Growth was continued at 35 °C and 150 rpm for 120 h. Inocula were prepared in the same way in MSB- DNT-YE, and cells were grown in the same way for growth comparisons (without antibiotics) in TSB (tryptic soy broth, Difco) or MSB-DNT-YE plus 0.02 M succinate for both strains. Samples were taken at intervals for determination of viable cell counts by plating on TSB agar. For determination of plasmid stability, 100 colonies were randomly transferred with sterile toothpicks from each plate to TSB agar plates containing 100 µg/mL ampicillin and 40 µg/mL kanamycin. Samples were also taken for determination of absorbance at 600 nm, diluted as necessary with the appropriate medium to keep the measured A600 below 0.3. Measurement of respiration rates (8) and hemoglobin levels by CO-difference spectra were as previously described (3, 18) using the extinction -1 -1 coefficient, E419 -436 nm ) 274 mM cm . Other Analytical Methods. Nitrite release was measured as described by Smibert and Krieg (19). DNT degradation and formation of intermediates in the degradation pathway were assayed in the cell-free culture medium using high performance liquid chromatography (HPLC) with a Spherosorb C18 column (Alltech, Deer- field, IL) and acetonitrile/13.5 mM trifluoroacetic acid in water, 1/1, (v/v) as the mobile phase. The flow rate was 1.5 mL/min, and the UV detector was set at 230 nm. Culture medium samples were clarified by centrifugation at 5000 × g for 15 min before chromatography. DNA Isolation and Southern and Western Blots. For the Southern blot, whole DNA was purified from the cells using a modification of the procedure of Marmur (20). The DNA samples were cut with EcoRI, and fragments were separated by electrophoresis on a 1% agarose gel, transferred to a Hybond nitrocellulose Figure 1. Presence of pSC160 and vgb in YV1. (A) Total DNA membrane, and processed using an ECL kit (Amersham) was isolated from strains DNT and YV1, digested with EcoRI, following the manufacturer’s instructions; plasmid pUC8: and separated on a 1% agarose gel as described in Materials 16 was used as the probe. For the Western blot analysis, and Methods. Lanes: 1, λ DNA digested with HindIII; 2, pUC8: 16 digested with EcoRI and HindIII; 3, pSC160 digested with purified VHb was used to immunize rabbits to generate EcoRI; 4 and 5, strain DNT DNA digested with EcoRI; 6-8, YV1 polyclonal VHb-specific antibodies. Burkholderia sp. DNA (from three separate isolates) digested with EcoRI. (B) strains DNT and YV1 were grown at 35 °C at 100 rpm Southern blot of gel in A. Transfer and hybridization procedures overnight, and total cellular proteins were separated on were as described in Materials and Methods. The probe used - an SDS-PAGE (15%) gel and transferred to a nitrocel- was pUC8:16, which contains the entire vgb gene. Lanes 1 8, same as for A. The position of the 4.1 kb pUC8:16 fragment lulose membrane (Hybond, Amersham) by the procedure produced by EcoRI digestion of pSC160 is indicated by the arrow of Towbin et al. (21) using a transblot cell (BioRad). The in the right margin. The 11.9 kb EcoRI fragment of pSC160 is membrane was treated with 2% skim milk for 2 h, obscured by the chromosomal DNA band. washed twice with PBS (140 mM NaCl, 10 mM Na2HPO4, 2.7 mM KCl, 1.8 mM KH2PO4, pH 7.4) containing 0.1% Results Tween 20, and incubated with polyclonal VHb antibodies Total DNA (chromosomal plus plasmid) isolated from for approximately 2 h. The membrane was washed twice strain YV1 and subjected to restriction analysis using with PBS and incubated with the secondary antibody EcoRI showed the presence in this strain of a DNA (peroxidase-conjugated goat anti-rabbit IgG) for another fragment of the same size (4.1 kb) as that produced from 2 h. Unreacted secondary antibodies were removed by pSC160; this band was absent from strain DNT DNA washing with PBS, and the membrane was treated with (Figure 1A). Southern blots using pUC8:16 as the probe 3,3-diaminobenzidineimidazole in 0.1 M phosphate buffer, confirmed that this 4.1 kb EcoRI fragment of pSC160 pH 7.0.

Load more