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Engineering of Nitrosomonas europaea to express Vitreoscilla hemoglobin enhances oxygen uptake and conversion of ammonia to nitrite.

Kunkel SA, Pagilla KR, Stark BC - AMB Express (2015)

Bottom Line: Vgb was maintained stably and appeared to be expressed in the transformants at VHb levels of about 0.75 nmol/g wet weight.Expression of VHb in the N. europaea transformants was correlated with an approximately 2 fold increase in oxygen uptake rate by whole cells at oxygen concentrations in the range of 75-100% saturation, but no change in oxygen uptake rate at oxygen concentrations below 25% saturation.VHb expression was also correlated with an increase of as much as about 30% in conversion of ammonia to nitrite by growing cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Illinois Institute of Technology, Chicago, IL, 60616, USA, skunkel@iit.edu.

ABSTRACT
Nitrosomonas europaea was transformed with a recombinant plasmid bearing the gene (vgb) encoding the hemoglobin (VHb) from the bacterium Vitreoscilla under control of the N. europaea amoC P1 promoter. Vgb was maintained stably and appeared to be expressed in the transformants at VHb levels of about 0.75 nmol/g wet weight. Expression of VHb in the N. europaea transformants was correlated with an approximately 2 fold increase in oxygen uptake rate by whole cells at oxygen concentrations in the range of 75-100% saturation, but no change in oxygen uptake rate at oxygen concentrations below 25% saturation. VHb expression was also correlated with an increase of as much as about 30% in conversion of ammonia to nitrite by growing cells. The results suggest that engineering of key aerobic wastewater bacteria to express bacterial hemoglobins may be a useful strategy to produce species with enhanced respiratory abilities.

No MeSH data available.


Related in: MedlinePlus

CO-difference spectral analysis. a CO-difference spectrum of cell extract of E. coli DH5α[pUC8:16] expressing VHb; peak and trough at 419 and 436 nm, respectively, characteristic of VHb are indicated. b CO-difference spectrum of cell extract of untransformed N. europaea; characteristic peak at 414 nm is indicated. c CO-difference spectra of cell extracts from three individual isolates of N. europaea transformed with vgb on plasmid pSK2; peak at 416 nm and shoulder at 423 nm are indicated.
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Fig3: CO-difference spectral analysis. a CO-difference spectrum of cell extract of E. coli DH5α[pUC8:16] expressing VHb; peak and trough at 419 and 436 nm, respectively, characteristic of VHb are indicated. b CO-difference spectrum of cell extract of untransformed N. europaea; characteristic peak at 414 nm is indicated. c CO-difference spectra of cell extracts from three individual isolates of N. europaea transformed with vgb on plasmid pSK2; peak at 416 nm and shoulder at 423 nm are indicated.

Mentions: VHb has a characteristic CO-difference spectrum with a peak at 419 nm and trough at 436 nm (Dikshit and Webster 1988; Fig. 3a). A CO-difference spectrum of a whole cell extract of untransformed N.europaea has several peaks, the most prominent of which is at 414 nm (Arnaldos et al. 2013; Fig. 3b). The identity of this peak is as yet unknown. CO-difference spectra of eight individual N.europaea/pSK2 transformants showed the 414 nm peak shifted from 414 to 416 nm with a shoulder at about 423 nm (three examples shown in Fig. 3c). The shift and shoulder are consistent with an overlap or combination of the 414 nm peak and the characteristic 419 nm VHb peak, the 423 shoulder presumably being part of the VHb signal. Although it is difficult to quantify the level of VHb from the height of the 423 shoulder, the average of calculations from three independent N. europaea[pSK2] spectra yielded a value of about 0.75 nmol/g wet weight of cells.Fig. 3


Engineering of Nitrosomonas europaea to express Vitreoscilla hemoglobin enhances oxygen uptake and conversion of ammonia to nitrite.

Kunkel SA, Pagilla KR, Stark BC - AMB Express (2015)

CO-difference spectral analysis. a CO-difference spectrum of cell extract of E. coli DH5α[pUC8:16] expressing VHb; peak and trough at 419 and 436 nm, respectively, characteristic of VHb are indicated. b CO-difference spectrum of cell extract of untransformed N. europaea; characteristic peak at 414 nm is indicated. c CO-difference spectra of cell extracts from three individual isolates of N. europaea transformed with vgb on plasmid pSK2; peak at 416 nm and shoulder at 423 nm are indicated.
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4522006&req=5

Fig3: CO-difference spectral analysis. a CO-difference spectrum of cell extract of E. coli DH5α[pUC8:16] expressing VHb; peak and trough at 419 and 436 nm, respectively, characteristic of VHb are indicated. b CO-difference spectrum of cell extract of untransformed N. europaea; characteristic peak at 414 nm is indicated. c CO-difference spectra of cell extracts from three individual isolates of N. europaea transformed with vgb on plasmid pSK2; peak at 416 nm and shoulder at 423 nm are indicated.
Mentions: VHb has a characteristic CO-difference spectrum with a peak at 419 nm and trough at 436 nm (Dikshit and Webster 1988; Fig. 3a). A CO-difference spectrum of a whole cell extract of untransformed N.europaea has several peaks, the most prominent of which is at 414 nm (Arnaldos et al. 2013; Fig. 3b). The identity of this peak is as yet unknown. CO-difference spectra of eight individual N.europaea/pSK2 transformants showed the 414 nm peak shifted from 414 to 416 nm with a shoulder at about 423 nm (three examples shown in Fig. 3c). The shift and shoulder are consistent with an overlap or combination of the 414 nm peak and the characteristic 419 nm VHb peak, the 423 shoulder presumably being part of the VHb signal. Although it is difficult to quantify the level of VHb from the height of the 423 shoulder, the average of calculations from three independent N. europaea[pSK2] spectra yielded a value of about 0.75 nmol/g wet weight of cells.Fig. 3

Bottom Line: Vgb was maintained stably and appeared to be expressed in the transformants at VHb levels of about 0.75 nmol/g wet weight.Expression of VHb in the N. europaea transformants was correlated with an approximately 2 fold increase in oxygen uptake rate by whole cells at oxygen concentrations in the range of 75-100% saturation, but no change in oxygen uptake rate at oxygen concentrations below 25% saturation.VHb expression was also correlated with an increase of as much as about 30% in conversion of ammonia to nitrite by growing cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Illinois Institute of Technology, Chicago, IL, 60616, USA, skunkel@iit.edu.

ABSTRACT
Nitrosomonas europaea was transformed with a recombinant plasmid bearing the gene (vgb) encoding the hemoglobin (VHb) from the bacterium Vitreoscilla under control of the N. europaea amoC P1 promoter. Vgb was maintained stably and appeared to be expressed in the transformants at VHb levels of about 0.75 nmol/g wet weight. Expression of VHb in the N. europaea transformants was correlated with an approximately 2 fold increase in oxygen uptake rate by whole cells at oxygen concentrations in the range of 75-100% saturation, but no change in oxygen uptake rate at oxygen concentrations below 25% saturation. VHb expression was also correlated with an increase of as much as about 30% in conversion of ammonia to nitrite by growing cells. The results suggest that engineering of key aerobic wastewater bacteria to express bacterial hemoglobins may be a useful strategy to produce species with enhanced respiratory abilities.

No MeSH data available.


Related in: MedlinePlus