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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

pSK2 is stably maintained in N. europaea[pSK2]. Lane 1 pSK2 plasmid prep from N. europaea [pSK2]; lane 2 positive control PCR of vgb amplicon (from pUC8:16 from E. coli); lane 3 negative control PCR of vgb (plasmid prep from untransformed N. europaea); lane 4 PCR amplicon of vgb from N. europaea [pSK2]; lane 5 2-log ladder (New England Biolabs; position of 500 bp fragment is noted). The amplicons in lanes 2 and 4 are of the expected size (about 450 bp).
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Fig2: pSK2 is stably maintained in N. europaea[pSK2]. Lane 1 pSK2 plasmid prep from N. europaea [pSK2]; lane 2 positive control PCR of vgb amplicon (from pUC8:16 from E. coli); lane 3 negative control PCR of vgb (plasmid prep from untransformed N. europaea); lane 4 PCR amplicon of vgb from N. europaea [pSK2]; lane 5 2-log ladder (New England Biolabs; position of 500 bp fragment is noted). The amplicons in lanes 2 and 4 are of the expected size (about 450 bp).

Mentions: Transformation of N.europaea with pSK2 was successful and stable with antibiotic pressure (25 μg/mL ampicillin), as proven by miniprep analysis and PCR amplification of vgb repeated at 2 week intervals (Fig. 2). In addition, this construct allowed production of VHb in N.europaea, as demonstrated by CO-difference spectral analysis.Fig. 2


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)

pSK2 is stably maintained in N. europaea[pSK2]. Lane 1 pSK2 plasmid prep from N. europaea [pSK2]; lane 2 positive control PCR of vgb amplicon (from pUC8:16 from E. coli); lane 3 negative control PCR of vgb (plasmid prep from untransformed N. europaea); lane 4 PCR amplicon of vgb from N. europaea [pSK2]; lane 5 2-log ladder (New England Biolabs; position of 500 bp fragment is noted). The amplicons in lanes 2 and 4 are of the expected size (about 450 bp).
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4522006&req=5

Fig2: pSK2 is stably maintained in N. europaea[pSK2]. Lane 1 pSK2 plasmid prep from N. europaea [pSK2]; lane 2 positive control PCR of vgb amplicon (from pUC8:16 from E. coli); lane 3 negative control PCR of vgb (plasmid prep from untransformed N. europaea); lane 4 PCR amplicon of vgb from N. europaea [pSK2]; lane 5 2-log ladder (New England Biolabs; position of 500 bp fragment is noted). The amplicons in lanes 2 and 4 are of the expected size (about 450 bp).
Mentions: Transformation of N.europaea with pSK2 was successful and stable with antibiotic pressure (25 μg/mL ampicillin), as proven by miniprep analysis and PCR amplification of vgb repeated at 2 week intervals (Fig. 2). In addition, this construct allowed production of VHb in N.europaea, as demonstrated by CO-difference spectral analysis.Fig. 2

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