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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

Sequence of plasmid pSK2 in the region of vgb and the integrated amoC P1 promoter. The amoC promoter is highlighted in yellow, with the flanking HindIII sites highlighted in red. The native vgb promoter region is highlighted in green, and the vgb coding sequence, beginning with the ATG start codon and ending with the TAA stop codon, is highlighted in turquoise.
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Fig1: Sequence of plasmid pSK2 in the region of vgb and the integrated amoC P1 promoter. The amoC promoter is highlighted in yellow, with the flanking HindIII sites highlighted in red. The native vgb promoter region is highlighted in green, and the vgb coding sequence, beginning with the ATG start codon and ending with the TAA stop codon, is highlighted in turquoise.

Mentions: Plasmid pUC8:16 (vgb cloned into the HindIII-SalI sites of E. coli vector pUC8; Liu et al. 1994) was cleaved at its HindIII site. A synthetic sequence identical to the N. europaeaamoC P1 promoter (Hommes et al. 2001; Berube et al. 2007) was produced from two complementary oligonucleotides (Integrated DNA Technologies, Coralville, IA) that were 5′ phosphorylated by T4 polynucleotide kinase and annealed by heating to 95°C for 10 min followed by slow cooling to room temperature. Because of the design of the two oligonucleotides, the annealed product had 4 bp single stranded HindIII compatible overhangs at each end, which allowed sticky end ligation into the HindIII site in pUC8:16 to produce pSK2. This placed the amoC P1 promoter just upstream of the native vgb promoter (Fig. 1). The amoC P1 promoter was previously found to be active in the presence of ammonia (Hommes et al. 2001) and is thus a good candidate for expression of vgb for these studies. Because of its derivation from pUC8:16, pSK2 confers resistance to ampicillin.Fig. 1


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)

Sequence of plasmid pSK2 in the region of vgb and the integrated amoC P1 promoter. The amoC promoter is highlighted in yellow, with the flanking HindIII sites highlighted in red. The native vgb promoter region is highlighted in green, and the vgb coding sequence, beginning with the ATG start codon and ending with the TAA stop codon, is highlighted in turquoise.
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig1: Sequence of plasmid pSK2 in the region of vgb and the integrated amoC P1 promoter. The amoC promoter is highlighted in yellow, with the flanking HindIII sites highlighted in red. The native vgb promoter region is highlighted in green, and the vgb coding sequence, beginning with the ATG start codon and ending with the TAA stop codon, is highlighted in turquoise.
Mentions: Plasmid pUC8:16 (vgb cloned into the HindIII-SalI sites of E. coli vector pUC8; Liu et al. 1994) was cleaved at its HindIII site. A synthetic sequence identical to the N. europaeaamoC P1 promoter (Hommes et al. 2001; Berube et al. 2007) was produced from two complementary oligonucleotides (Integrated DNA Technologies, Coralville, IA) that were 5′ phosphorylated by T4 polynucleotide kinase and annealed by heating to 95°C for 10 min followed by slow cooling to room temperature. Because of the design of the two oligonucleotides, the annealed product had 4 bp single stranded HindIII compatible overhangs at each end, which allowed sticky end ligation into the HindIII site in pUC8:16 to produce pSK2. This placed the amoC P1 promoter just upstream of the native vgb promoter (Fig. 1). The amoC P1 promoter was previously found to be active in the presence of ammonia (Hommes et al. 2001) and is thus a good candidate for expression of vgb for these studies. Because of its derivation from pUC8:16, pSK2 confers resistance to ampicillin.Fig. 1

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