# Patent

## Title
Nitrate reductase fusion proteins and uses thereof

## Abstract

The present invention relates to a novel fusion protein comprising a nitrate reductase (NR) and a truncated hemoglobin N (trHbN) domain. The fusion protein may be used for bioremediation of nitric oxide.

## Background
CROSS-REFERENCE TO RELATED APPLICATION
This application claims the benefit of U.S. Provisional Application No. 61/364,583, filed Jul. 15, 2010, the content of which is incorporated herein by reference in its entirety for all purposes.


REFERENCE TO U.S. GOVERNMENT SUPPORT
This work is supported by a grant from the U.S. Environmental Protection Agency (Grant Nos. EPA STAR ECOHAB R83-3221 AND R83-1041) and a grant from the National Science Foundation (NSF) (Grant No. NA10NOS4780136). The United States has certain rights in the invention.


FIELD OF THE INVENTION
The invention relates generally to a novel fusion protein comprising a nitrate reductase (NR) and a truncated hemoglobin N (trHbN) domain, and the uses thereof for bioremediation of nitric oxide.
BACKGROUND OF THE INVENTION
Nitric oxide (NO) is a colorless and odorless gas. It is an important cell signaling molecule in mammals, including humans. It is also toxic as an air pollutant emitted mainly from combustion processes in, for example, fossil fuel power stations and automobile engines. Current methods for removing nitric oxide (NO) from flue gases rely on chemical processes with the disadvantages of expensive costs due to catalysts and chemicals, high energy requirements to sustain reaction temperatures, and negative environmental impacts from waste products such as urea and ammonia.
Alternative methods have been proposed to include complicated microbial bioreactors such as the BioDeNox process, which is a multiple step process that consumes ethanol, acetate, and/or Fe(II)EDTA2− as electron donors. This process is limited by the oxidation of Fe(II) to Fe(III) by oxygen, requires energy to sustain reaction temperatures of 140° F., and has a low NO removal efficiency of 40-70%.
Previous studies on the suitability of algal species for bioremediation of nitric oxide in flue gas are limited. The results of these studies have concluded that the species tested can only sustain removal of 40-65% nitric oxide for very short periods (10-15 days). The reported mechanism for nitric oxide removal in these studies relied on the pre-autooxidation of nitric oxide to nitrate and nitrite in the media in the presence of oxygen. The algal species tested then assimilated the nitrate/nitrite as nitrogen sources. Nitric oxide itself is highly toxic to cells, and the short durations of these previous experiments (10-15 days) suggest that those algal species did not have a method to metabolize nitric oxide directly.
There remains a need for an environmentally friendly and sustainable bioremediation of nitric oxide with great NO removal efficiencies.
SUMMARY OF THE INVENTION
The present invention relates to a novel fusion protein comprising a nitrate reductase (NR) and a truncated hemoglobin N (trHbN) domain, and uses thereof for bioremediation of nitric oxide.
An isolated fusion protein comprising a nitrate reductase (NR) and a truncated hemoglobin N (trHbN) domain is provided. The fusion protein may be capable of reducing nitric oxide. It may also be capable of converting nitric oxide to nitrate and nitrite.
The fusion protein may comprise a polypeptide having an amino acid sequence at least 90% identical to SEQ ID NO: 3 or 5, or a variant thereof. The fusion protein may be derived from a raphidophyte. The genus is selected from the group consisting of genus Heterosigma, Chattonella, Fibrocapsa, and Viridilobus. The raphidophyte may be Heterosigma akashiwo, Chattonella subsalsa, C. marina, C. antigua, Fibrocapsa japonica, or Viridilobus marinus. 
A composition for reducing nitric oxide is also provided. The composition comprises an effective amount of a fusion protein, wherein the fusion protein comprises a nitrate reductase (NR) and a truncated hemoglobin N (trHbN) domain. The fusion protein may be capable of converting nitric oxide to nitrate and nitrite. The fusion protein may comprise a polypeptide having an amino acid sequence at least 90% identical to SEQ ID NO: 3 or 5, or a variant thereof. The fusion protein may be derived from a raphidophyte. The raphidophyte is selected from the group consisting of genus Heterosigma, Chattonella, Fibrocapsa, and Viridilobus. The raphidophyte may be Heterosigma akashiwo, Chattonella subsalsa, C. marina, C. antigua, Fibrocapsa japonica, or Viridilobus marinus. 
A method for reducing nitric oxide is further provided. The method comprises applying an effective amount of a fusion protein, wherein the fusion protein comprises a nitrate reductase (NR) and a truncated hemoglobin N (trHbN) domain. The fusion protein may reduce nitric oxide by at least 50%. It may convert nitric oxide to nitrate and nitrite. The fusion protein may comprise a polypeptide having an amino acid sequence at least 90% identical to SEQ ID NO: 3 or 5, or a variant thereof. The fusion protein may be derived from a raphidophyte. The raphidophyte may be selected from the group consisting of genus Heterosigma, Chattonella, Fibrocapsa, and Viridilobus. The raphidophyte may be Heterosigma akashiwo, Chattonella subsalsa, C. marina, C. antigua, Fibrocapsa japonica, or Viridilobus marinus. 



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 illustrates a proposed mechanism for dual nitric oxide dioxygenase (NOD) and nitrate reductase activities by NR2-trHbN. (A) Mechanism of the NOD reaction known for mycobacterial group I truncated hemoglobins (trHbNs). Reductant is essential for the regeneration of active trHbN-Fe(II). (B) Proposed mechanism of NR2-trHbN coupling NOD and nitrate reductase activities. Electrons supplied by the FAD/NADH reductase domain (shaded arrows) may be used for nitrate reduction at the molybdenum-molybdopterin (MO-MPT) site and used for rapid regeneration of the active form of trHbN necessary for NO reactivity. Nitrate supplied by the detoxification of NO may be captured for reduction at the Mo-MPT site for incorporation as cellular nitrogen
FIG. 2 shows (A) the full length genomic DNA sequence (SEQ ID NO: 1), (B) the corresponding full length cDNA sequence (SEQ ID NO: 2), and (C) the corresponding translated amino acid sequence (SEQ ID NO: 3) for the NR2-trHbN fusion protein derived from H. askashiwo (HaNR2-trHbN). The underlined sequence reflects the trHbN domain.
FIG. 3 shows (A) a partial cDNA sequence (SEQ ID NO: 4) missing about 750-900 bases from the 5′ end of the sequence, and (B) the corresponding translated protein sequence (SEQ ID NO: 5) for the NR2-trHbN fusion protein derived from C. subsalsa (CsNR2-trHbN). The underlined sequence reflects the trHbN domain.
FIG. 4 illustrates a model of the domain architecture for the NR2-trHbN gene sequence. In NR2-trHbN, the truncated hemoglobin sequence (shown in grey) is inserted within the variable hinge 2 region of a traditional nitrate reductase (NR). NR is comprised of eight domains shown here, (i) an N-terminal domain, (ii) the molybdenum-molybdopterin cofactor (Mo-MPT) domain containing the catalytic active site for nitrate reduction, (iii) a dimer interface region (DI), (iv) a variable hinge 1 region, (v) a cyt b5-binding domain incorporating heme-Fe (Heme), (vi) a variable hinge 2 region, (vii) an FAD-binding domain, and (viii) an NADH-binding domain at the C-terminus (modified from Campbell 1999).
FIG. 5 shows translated amino acid sequence alignment of the trHbN domain of NR2-trHbN with mycobacterial trHbN. Features characteristic of trHb structure are indicated: boxed residues are essential to the structure of the trHb fold, shaded residues are important to the formation of a hydrophobic ligand access tunnel, key active site residues are marked in black (Vuletich and Lecomte 2006). Symbols directly above the sequences indicate strictly conserved residues (*) and two levels of highly conserved substitutions (: and •) according to ClustalW2 annotation (Chema 2003; Larkin et al. 2007). Helix designations are denoted above the alignment following notations previously described in Lama et al. (2009). The sequence of the H-helix included in the HaNR1 hinge 2 region is indicated by a horizontal box for the Heterosigma akashiwo sequence only.
FIG. 6 shows a minimum evolution tree of representative sequences from trHb groups I (N), II (O) & III (P). Bootstrap values above 50% for 500 replications are noted at the nodes. Sequences for Heterosigma akashiwo and Chattonella subsalsa are indicated (♦), and the node that indicates the separation of trHbN sequences into two groups based on amino acid composition at the E7 position (leucine versus glutamine) is circled.
FIG. 7 shows the effect of nitric oxide on the expression of HaNR2-trHbN by adding a chemical nitric oxide (NO) donor, sodium pentacyanonitrosylferrate (II) (aka: sodium nitroprusside; SNP), to Heterosigma akashiwo. FIG. 7A shows the concentration of the dissolved NO in controls (cell free medium+SNP) and treatment cultures (H. akashiwo culture+SNP) at 1 hour (T1) and 3 hours (T3) after the addition of SNP. FIG. 7B shows the relative expression of NR2-trHbN in H. akashiwo control (−SNP) and treatment (+SNP) cultures before the addition of SNP (T0) and at 1 hour (T1) and 3 hours (T3) after the addition of SNP. Transcript abundance was calculated relative to the average abundance of controls at each time point. FIG. 7C shows the cell numbers at T0, T1, and T3 for H. akashiwo control (−SNP) and treatment (+SNP) cultures. Data plotted are mean values+SD (n=3).
FIG. 8 shows (A) biomass and (B) Fv/Fm on day 6 of batch growth after treatment with 800 μM NaNO3/Ambient Air (800N-Air, control); 0 μM NaNO3/Ambient Air (0N-Air); 800 μM NaNO3/300 ppm NO (800N-NO); or 0 μM NaNO3/300 ppm NO (0N-NO). Error bars represent standard deviation of replicates (n=3).
FIG. 9 shows (A) relative gene expression of HaNR2-trHbN and (B) nitrate reducing activity in H. akashiwo cultures subjected to low light (60 μmol quanta m−2s−1) and high light (785 μmol quanta m−2s−1) with and without the addition of sodium tungstate. T=Sodium tungstate.
FIG. 10 shows (A) relative gene expression HaNR2-trHbN and (B) nitrate reducing activity in H. akashiwo cultures grown at each of the following four conditions: 25° C. and 375 ppm CO2, 25° C. and 750 ppm CO2, 30° C. and 375 ppm CO2, 30° C. and 750 ppm CO2.



DETAILED DESCRIPTION OF THE INVENTION
The present invention is based on the discovery that a novel fusion protein, comprising a nitrate reductase (NR) and a truncated hemoglobin N (trHbN) domain, is capable of reducing nitric oxide to nitrate and nitrite.
The terms “protein” and “polypeptide” are used herein interchangeably, and refer to a polymer of amino acid residues with no limitation with respect to the minimum length of the polymer. The definition includes both full-length proteins and fragments thereof, as well as modifications thereof (e.g., glycosylation, phosphorylation, deletions, additions and substitutions).
The term “derived from” used herein refers to the origin or source, and may include naturally occurring, recombinant, unpurified or purified molecules.
The term “variant” of a protein as used herein refers to a polypeptide having an amino acid sequence that is the same as the amino acid sequence of the protein except having at least one amino acid modified, for example, deleted, inserted, or replaced. The variant may have an amino acid sequence at least about 80%, 90%, 95%, or 99%, preferably at least about 90%, more preferably at least about 95%, identical to the amino acid sequence of the protein.
The term “about” as used herein when referring to a measurable value such as an amount, a percentage, and the like, is meant to encompass variations of +20% or +10%, more preferably +5%, even more preferably +1%, and still more preferably +0.1% from the specified value, as such variations are appropriate.
According to one aspect of the present invention, an isolated fusion protein is provided. The fusion protein comprises a nitrate reductase (NR) and a truncated hemoglobin (trHb). The fusion protein may be able to reduce or remove nitric oxide. It may also be able to convert nitric oxide to nitrate and nitrite.
The nitrate reductase (NR) is a protein or polypeptide that can catalyze the reduction of nitrate to nitrite. It may be derived from any eukaryotic nitrate reductase. For example, it may comprise eight sequence regions or domains: (i) an N-terminal sequence, (ii) a catalytic active site for nitrate reduction incorporating a molybdenum-molybdopterin cofactor (Mo-MPT), (iii) a dimer interface (DI) region, (iv) a variable hinge 1 region, (v) a cyt b5-binding domain incorporating heme-Fe, (vi) a variable hinge 2 region, (vii) an FAD-binding domain, and (viii) an NADH-binding domain at the C-terminus. (Campbell 2001).
The truncated hemoglobin (trHb, also known as 2/2Hbs) is a small hemeprotein found in bacteria, ciliates, algae or plants. The trHb comprises a 2-on-2 α-helical fold, and may catalyze the reduction of nitric oxide to nitrate. It may be any member of the three paralogous groups, group I (trHbN), group II (trHbO), and group III (trHbP), preferably a trHbN domain.
The fusion protein of the present invention may be derived from a raphidophyte. The raphidophyte may be in genus Heterosigma, Chattonella, Fibrocapsa, and Viridilobus. Examples of raphidophytes include species Heterosigma akashiwo, Chattonella subsalsa, C. marina, C. antigua, Fibrocapsa japonica, and Viridilobus marinus. 
Novel hybrid proteins (NR2-trHbN) in the raphidophytes, Heterosigma akashiwo and Chattonella subsalsa, have been discovered. In these hybrid proteins, a complete trHbN domain is inserted within the hinge 2 domain between the heme-Fe and FAD domains of a traditional NR sequence (FIGS. 2 and 3), resulting in a novel NR2-trHbN sequence. This novel NR2-trHbN sequence was found in multiple strains of H. akashiwo, including toxic and nontoxic strains isolated from the east and west coasts of the United States (e.g., CCMP2808, CCMP2393, CCMP1914 and C1 21 R2) and also in C. subsalsa (CCMP 2191) isolated from the Delaware Inland Bays. This NR2-trHbN sequence may also exist in other algal species within the raphidophyte group.
In one embodiment, the fusion protein of the present invention comprises 9 sequence regions or domains spanning over 900 amino acid residues, including a complete trHbN domain inserted within the hinge 2 domain of a traditional NR sequence. The trHbN domain within the trHbN-NR2 fusion protein may use the reductant supplied by the NR2 portion of the fusion protein such that the trHbN-NR2 fusion protein may exhibit coupled nitric oxide dioxygenase (NOD) and nitrate reductase activities (FIG. 1B), which is similar to the NOD reaction known for mycobacterial group I truncated hemoglobins (trHbNs) (FIG. 1A). For example, nitrate produced by trHbN NOD activity would then be captured for reduction at the Mo-MPT site of NR2-trHbN for incorporation into cellular nitrogen.
In another embodiment, a HaNR2-trHbN fusion protein derived from Heterosigma akashiwo, comprising a nitrate reductase NR2 and a trHbN domain, is provided. The HaNR2-trHbN fusion protein may comprise a polypeptide having an amino acid sequence of SEQ ID NO: 3, or a variant thereof. The variant may have an amino acid sequence at least about 80%, 85%, 90%, 95%, or 99%, preferably at least about 90%, identical to SEQ ID NO: 3.
In yet another embodiment, a CsNR2-trHbN protein derived from Chattonella subsalsa, comprising a nitrate reductase NR2 and a trHbN domain, is provided. The CsNR2-trHbN fusion protein may comprise a polypeptide having an amino acid sequence of SEQ ID NO: 5, or a variant thereof. The variant may have an amino acid sequence at least about 80%, 85%, 90%, 95%, or 99%, preferably at least about 90%, identical to SEQ ID NO: 5.
An isolated nucleic acid molecule encoding a fusion protein of the present invention is also provided. The fusion protein comprises a nitrate reductase (NR) and a truncated hemoglobin N (trHbN) domain. The fusion protein may be derived from a raphidophyte, preferably, the HaNR2-trHbN or CsNR2-trHbN fusion protein.
The nucleic acid molecule of the present invention may comprise a nucleotide sequence of SEQ ID NO: 2 or 4, or a variant thereof. The variant may have an amino acid sequence at least about 80%, 85%, 90%, 95%, or 99%, preferably at least about 90%, identical to SEQ ID NO: 2 or 4. The nucleic acid molecule may be operably linked to a promoter element suitable for expression of the gene in the nucleic acid molecule. It may also be in an expression vector.
For each fusion protein according to the present invention, a cell producing such a fusion protein is provided. Such a cell may be prepared using any conventional technique known in the art. A culture of cells producing a fusion protein of the present invention may also be prepared using standard methods known in the art. Such a cell culture may be used for purifying and isolating the fusion protein.
For each nucleic acid according to the present invention, a cell comprising such a nucleic acid molecule is provided. Such a cell may be prepared using any conventional technique known in the art. A culture of cells comprising a nucleic acid molecule of the present invention may also be prepared using standard methods known in the art. Such a cell culture may be used for producing the fusion protein.
Cells of the present invention may be grown under conditions suitable for the expression and/or production of a fusion protein according to the present invention. The cells are preferably grown under high light of at least about 50, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 mmol quanta m−2s−1, more preferably at least about 700 μmol quanta m−2s−1. The cells may be growth in the presence of an inhibitor (e.g., sodium tungstate (Na2WO4)) of the Mo-MPT nitrate reduction center of the nitrate reductase (NR) in the fusion protein. The cells may be grown at 25° C., 30° C., or 35° C., preferably at 25° C. The cells may be growth under 375 ppm, 750 ppm, or 15% CO2, preferably at 750 ppm CO2.
The present invention may be applied in several ways for the bioremediation of nitric oxide pollution. It is not limited to bioremediation of nitric oxide, but also allows for additional commercial applications (e.g., production of nitrate/nitrite as a nutrient source for biomass and/or directly reducing costs associated with algal biofuel production).
In one embodiment, whole cell algal suspensions of species expressing NR2 (such as H. akashiwo and C. subsalsa) may be grown on flue gas emissions for simultaneous removal of nitric oxide and carbon dioxide. H. akashiwo has been previously reported as an appropriate algal species for production of algal biofuels. Combining the bioremediation capabilities of H. akashiwo (and like species) with the production of algal biomass for biofuels will reduce the costs associated with algal biofuel production and provide a platform for sustainable and realistic commercialization of algae-derived biofuels. Further, the NR2 gene may be used to genetically modify existing algal species used in the biofuels industry, permitting them to survive in high nitric oxide conditions and allowing them to serve as bioremediators of nitric oxide.
In another embodiment, an engineered enzymatic reactor with a suspension of purified NR2 protein (supplied with NADH as a reductant) may be used to remove nitric oxide from flue gas. Nitric oxide-free flue gas still containing carbon dioxide can be used as a carbon source for nitric oxide-sensitive algal biomass growth. Additionally, the nitrate and nitrite produced by an enzymatic reaction can be sequestered, and supplied to the algal biomass as a free nitrogen source (fertilizer).
According to another aspect of the present invention, a composition for reducing nitric oxide is provided. The composition comprises an effective amount of a fusion protein of the present invention. The fusion protein comprises a nitrate reductase (NR) and a truncated hemoglobin N (trHbN) domain.
The term “reducing” used herein means “removing a portion of or all of”
In a composition of the present invention, the fusion protein is present in an amount effective, or sufficient, to reduce, or remove a portion or all of, nitric oxide such that the amount or concentration of the nitric oxide is lowered. The fusion protein may reduce nitric oxide by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%, preferably, at least about 20%, more preferably, at least about 50%.
In a composition according to the present invention, the fusion protein is capable of converting nitric oxide to nitrate and nitrite. The fusion protein may be derived from a raphidophyte. The raphidophyte may be in genus Heterosigma, Chattonella, Fibrocapsa, and Viridilobus. Examples of raphidophytes include species Heterosigma akashiwo, Chattonella subsalsa, C. marina, C. antigua, Fibrocapsa japonica, and Viridilobus marinus. The fusion protein may comprise a polypeptide having an amino acid sequence of SEQ ID NO: 3 or 5, or at least about 80%, 85%, 90%, 95%, or 99%, preferably at least about 90%, identical to SEQ ID NO: 3 or 5.
The composition may further comprise a culture of cells that produces a fusion protein of the present invention. The cells may be producing the fusion protein, or capable of producing the fusion protein. The cells may comprise a nucleic acid molecule encoding the fusion protein.
According yet another aspect of the present invention, a method for reducing nitric oxide is provided. The method comprises applying an effective amount of a fusion protein of the present invention. The fusion protein comprises a nitrate reductase (NR) and a truncated hemoglobin N (trHbN) domain.
In a method of the present invention, the fusion protein is present in an amount effective to reduce, or remove a portion or all of, nitric oxide such that the amount or concentration of the nitric oxide is lowered. The fusion protein may reduce nitric oxide by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%, preferably, at least about 20%, more preferably, at least about 50%. The reduction may be maintained for a period of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 days
In a method according to the present invention, the fusion protein is capable of converting nitric oxide to nitrate and nitrite. The fusion protein may be derived from a raphidophyte. The raphidophyte may be in genus Heterosigma, Chattonella, Fibrocapsa, and Viridilobus. Examples of raphidophytes include species Heterosigma akashiwo, Chattonella subsalsa, C. marina, C. antigua, Fibrocapsa japonica, and Viridilobus marinus. The fusion protein may comprise a polypeptide having an amino acid sequence of SEQ ID NO: 3 or 5, or at least about 80%, 85%, 90%, 95%, or 99%, preferably at least about 90%, identical to SEQ ID NO: 3 or 5.
The method may further comprise applying a culture of cells that produces a fusion protein of the present invention in an effective amount to reduce nitric oxide. The cells may be producing the fusion protein, or capable of producing the fusion protein. The cells may comprise a nucleic acid molecule encoding the fusion protein.
EXAMPLE 1
HaNR2-trHbN Fusion Protein
A NR2-trHbN fusion protein was purified from Heterosigma akashiwo, and its sequence was determined.
Heterosigma akashiwo (CCMP 2393) was obtained from the Provasoli-Guillard Center for the Culture of Marine Phytoplankton (CCMP; Boothbay Harbor, Me.). A stock culture was maintained in seawater diluted to a salinity of 20 and amended with f/2 nutrients (—Si) (Guillard 1975), grown at 25° C. and an irradiance of  ̃185 μmol quanta m−2s−1, and set to a 12:12 h light:dark cycle.
RNA was extracted from Heterosigma akashiwo (CCMP 2393) and reverse transcribed using oligo-dT-Heel primer (Coyne et al. 2004) as previously described in Coyne (2010). To obtain the 3′ end of the sequence, cDNA was then used as template in 20 μL PCR reactions containing 0.2 mM dNTPs, 0.25 μM Heel primer (Coyne et al. 2004), 0.25 μM HaNR—F primer (Coyne 2010), 2.5 mM MgCl2, 1× Taq polymerase buffer (Sigma, St. Louis, Ill. USA) and 0.5 units Jump-Start Taq Polymerase (Sigma). The reaction consisted of 35 cycles of 30 s at 94° C., 30 s at 56° C. and 2.5 min at 72° C., followed by a 5 min extension at 72° C. PCR products were cloned into pCR4 TOPO plasmid vector (Invitrogen, Carlsbad, Calif. USA) and sequenced using Big Dye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Foster City, Calif. USA).
The 5′ end of the HaNR2-trHbN cDNA sequence was obtained as described in Coyne (2010) using the GeneRacer RACE Ready cDNA Kit (Invitrogen) and primer HaNR-954R (AAGCCCAGAACATGCTCCG) (SEQ ID NO: 6) for the initial PCR reaction. The GeneRacer 5′ Nested Primer included in the kit and HaNRGlob-312R primer (GCTGGTATCCTTCAGCACCT) (SEQ ID NO: 7) were used in a nested PCR reaction as described in Coyne (2010) followed by cloning and sequencing as above.
To obtain the genomic DNA sequence, DNA was extracted from a stock culture of H. akashiwo as previously described in Coyne et al. (2001). The full length NR gene was amplified by PCR using the following primers:







HaNRFull-F (SEQ ID NO: 8):


AAGCTTAGAAGGAGATATACATATGGCCCCTCCTTCTACGATCAAGATTG


 


HaNRFull-R (SEQ ID NO: 9):


AAGCTTGCTCGAATTCACTAAAACTGAAAAATCTGCTCCTTCTTG






These primers were designed for recombinant protein expression, so the specific sequence that targets HaNR2-trHbN is underlined for clarity. PCR reactions consisted of 20 μA reactions containing 0.2 mM dNTPs, 0.5 μM each primer, 2.5 mM MgCl2, 1× Taq polymerase buffer (Sigma) and 0.5 units Jump-Start Taq Polymerase (Sigma). The PCR cycle consisted of 40 cycles of 30 s at 94° C., 30 s at 60° C. and 3 min at 72° C., followed by a 5 min extension at 72° C. PCR products were cloned and sequenced as described above.

The full-length nucleotide sequence for HaNR1 (GenBank accession #GQ149451) was previously described as a traditional nitrate reductase (Coyne 2010). The full-length nucleotide sequence of HaNR2-trHbN (SEQ ID NO: 1) (FIG. 2A) derived from cDNA (SEQ ID NO: 2) (FIG. 2B) is 2,869 bases in length with a translated sequence of 936 amino acids (SEQ ID NO: 3) (FIG. 2C). The transcript included the domain regions as previously annotated in Coyne (2010) for HaNR1 with the addition of a 321-base nucleotide insertion (following nucleotide 1635 in HaNR1) in the hinge 2 region separating the cyt b5-binding domain from the FAD-binding domain (FIG. 4). The insertion in HaNR2-trHbN encodes a translated amino acid sequence of 107 amino acids. A direct repeat of 9 nucleotide bases (GAGCTGGGT) was found to flank the 321-base insertion in HaNR2-trHbN, whereas a single copy of this sequence was observed in HaNR1 at the insertion site (nucleotides 1627-1635 in HaNR1). Excluding the insert, the full-length cDNA sequence of HaNR2-trHbN is identical to HaNR1. The full-length gene sequence of HaNR2-trHbN was also obtained from genomic DNA. The gene sequence was found to contain the 321-base nucleotide insertion in the hinge 2 region and a single intron of 182 bases located within the Mo-MPT domain (following nucleotide 621 in HaNR1).
EXAMPLE 2
CsNR2-trHbN Fusion Protein
A CsNR2-trHbN fusion protein was purified from Chattonella subsalsa, and its sequence was determined.
Chattonella subsalsa (CCMP 2191) was obtained from the Provasoli-Guillard Center for the Culture of Marine Phytoplankton (CCMP; Boothbay Harbor, Me.). A stock culture was maintained in seawater diluted to a salinity of 20 and amended with f/2 nutrients (—Si) (Guillard 1975), grown at 25° C. and an irradiance of  ̃185 μmol quanta m−2s−1, and set to a 12:12 h light:dark cycle.
Chattonella subsalsa (CCMP2191) was cultured in f/2 medium and DNA was extracted as described in Coyne et al. (2001). A fragment of the NR gene was amplified by PCR using degenerate primers designed from conserved regions of NR. PCR reactions consisted of 20 μL reactions containing 0.2 mM dNTPs, 0.5 μM each primer NR231F (ATHGGNGGNMGNATGATHAARTGG) (SEQ ID NO: 10) and NR394R (RTTRTTCATCATNCCCAT) (SEQ ID NO: 11), 2.5 mM MgCl2, 1× Taq polymerase buffer (Sigma) and 0.5 units Jump-Start Taq Polymerase (Sigma). The PCR cycle consisted of 40 cycles of 30 s at 94° C., 30 s at 54° C. and 1 min at 72° C., followed by a 5 min extension at 72° C. PCR products were cloned and sequenced as described above.
To obtain the 3′ end of the CsNR2-trHbN cDNA sequence, total RNA was reverse transcribed as described for H. akashiwo above. cDNA was then used as template in 20 μL PCR reactions containing 0.2 mM dNTPs, 0.25 μM Heel primer (Coyne et al. 2004), 0.25 μM CsNR-F2 primer (TTCCGAACAGTCCTCGAATC) (SEQ ID NO: 12), 2.5 mM MgCl2, 1× Taq polymerase buffer (Sigma) and 0.5 units Jump-Start Taq Polymerase (Sigma). The reaction consisted of 35 cycles of 30 s at 94° C., 30 s at 56° C. and 2 min at 72° C., followed by a 5 min extension at 72° C. PCR products were cloned and sequenced as described above.
A partial sequence of 2,248 bases was obtained for the CsNR2-trHbN cDNA sequence (SEQ ID NO: 4) (FIG. 3A) from Chattonella subsalsa. This sequence spans the DI domain through the C-terminus, and was also found to include a 342-base nucleotide sequence in the hinge 2 region with similarity to the insert described for HaNR2-trHbN. A partial sequence of 611 bases was also obtained from genomic DNA, which contains a single intron of 95 bases located within the DI domain following nucleotide 218.
EXAMPLE 3
Phylogenetic Analysis and Alignment of trHbN Region of HaNR2
The boundaries for the amino acid sequence of the trHbN domain in HaNR2-trHbN were initially defined to include the 107 amino acids inserted in HaNR2-trHbN that were excluded from HaNR1. However, alignment with other trHb sequences (FIG. 5) revealed that the H-helix of the trHbN domain was also included in the HaNR1 sequence (Coyne 2010). Therefore, a total of 137 amino acids including the H-helix were used for phylogenetic and sequence analyses of the trHbN domain in HaNR2-trHbN. An NR1 gene was not found for C. subsalsa. Consequently, the boundary of the trHbN domain in CsNR2-trHbN was defined by alignment with HaNR2-trHbN and a total of 148 amino acids were included in the analysis.
The nucleotide sequences for trHbN domains in both NR2-trHbNs were compared to sequences in the NCBI protein database using the BLASTX algorithm (Altschul et al. 1997). Phylogenetic analyses were conducted in MEGA4 (Tamura et al. 2007). Database locations for sequences used in this analysis are listed in Online Resource 1 in Table Si. The evolutionary history for the translated amino acid sequences of the trHbN domains in HaNR2-trHbN and CsNR2-trHbN was inferred using the Minimum Evolution (ME) method (Rzhetsky and Nei 1992). The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (500 replicates) is shown next to each node (Felsenstein 1985). The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The ME tree was searched using the Close-Neighbor-Interchange (CNI) algorithm at a search level of 1. The Neighbor-joining algorithm (Saitou and Nei 1987) was used to generate the initial tree. All positions containing gaps and missing data were eliminated from the dataset. There were a total of 101 positions in the final dataset.
A subset of seven mycobacterial sequences identified in the phylogenetic analysis were aligned with the translated amino acid sequence of the trHbN domains in HaNR2-trHbN and CsNR2-trHbN by ClustalW2 (Chema 2003; Larkin et al. 2007) and annotated following helix designations previously given (Lama et al. 2009).
The similarity of FAD/NADH reductase domains of NR2-trHbN sequences to other enzymes was evaluated by comparison of the translated amino acid sequences to sequences in the NCBI protein database using the BLASTP algorithm (Altschul et al. 1997).
The translated amino acid sequence of the trHbN domains in HaNR2-trHbN and CsNR2-trHbN were shown to be homologous to trHbs by BLASTX (Altschul et al. 1997). Phylogenetic analysis of the amino acid sequences with 50 known trHb sequences representative of trHbN, trHbO and trHbP subtypes generated a robust tree with significant bootstrap values and placed the sequences within the group I (N-type) truncated hemoglobins (FIG. 6). The trHbN sequences in HaNR2-trHbN and CsNR2-trHbN were distinct from trHbN sequences of other algal and eukaryotic species and instead grouped strongly with trHbN sequences from the actinobacteria, Mycobacterium spp. Alignment of the trHbN domain from HaNR2-trHbN and CsNR2-trHbN with mycobacterial trHbN showed conserved residues that are involved in active site reactions as well as residues that are essential to the trHb 2-on-2 α-helical fold and residues that form a characteristic hydrophobic ligand access tunnel (FIG. 5; Vuletich and Lecomte 2006; Lama et al. 2009).
Notably, in NR2-trHbN and the actinobacteria sequences, leucine occupies the E7 residue in the active site, whereas glutamine is located at E7 in all other trHbN sequences analyzed. The node circled in FIG. 6 reflects this difference in active site composition between the two groups defined by the phylogenetic analysis. The alignment of HaNR2-trHbN with HaNR1 showed that the trHbN H-helix is part of the hinge 2 region of HaNR1 following the 9-nucleotide direct repeat (described above).
Amino acid sequences for the FAD/NADH reductase domains in HaNR2-trHbN and CsNR2-trHbN were also analyzed independently and found to be homologous to eukaryotic NR sequences.
EXAMPLE 4
Effect of Nitric Oxide on Expression of HaNR2-trHbN
H. akashiwo was grown semi-continuously in f/2 medium to maintain steady state growth for 8 days prior to the experiment. To examine the effect of NO on the expression of HaNR2-trHbN, triplicate cultures of H. akashiwo were treated with the chemical NO donor, sodium pentacyanonitrosylferrate (II) (SNP), at a final concentration of 0.4 mM SNP. Two sets of controls were included in this experiment: triplicate cultures not treated with SNP (for comparison of transcript abundance) and triplicate aliquots of cell-free f/2 medium treated with 0.4 mM SNP (for comparison of NO concentrations). Cell counts were determined by microscopy using a Neubauer Hemocytometer for control and SNP-treated cultures before the addition of SNP (T0), at 1 hour (T1) and 3 hours (T3) after the addition of SNP. NO generated by the chemical decomposition of SNP was measured electrochemically in treatment cultures and cell-free medium at T1 and T3 as previously described (Sakihama et al. 2002; Xing et al. 2005). Briefly, a NO-specific microsensor (ISO-NOP, World Precision Instruments, Sarasota, Fla.), capable of detecting NO in the range of 0.3 nM to 100 μM, was used to selectively measured NO in algal culture media. The method for sensor calibration according to the manufacturer's instructions for the chemical generation of NO was modified by diluting the calibration solution in 20 psu seawater. NO concentrations in treatment cultures (+SNP) and controls (cell-free medium+SNP) were then determined by linear regression analysis.
For transcript analysis, 50 mL of culture were collected from treatment (+SNP) and control (−SNP) cultures at T0, T1 and T3 and filtered onto 3.0 μm polycarbonate filters. The filters were submerged into buffer RLT (RNEasy Plant Mini Kit, Qiagen) and heated at 56° C. before freezing at −80° C. RNA was extracted using the RNEasy Plant Mini Kit (Qiagen) and resuspended in RNase-free water. The purity of total RNA was analyzed spectroscopically and RNA was treated with DNase I (Invitrogen) as previously described (Coyne and Cary 2005). Approximately 500 ng of DNase-treated total RNA was reverse transcribed with random hexamers using the Superscript III First Strand Synthesis System (Invitrogen). Duplicate reactions for each DNase-treated RNA sample without reverse transcriptase were also evaluated by PCR. Transcript abundances for HaNR2-trHbN and glyceraldehyde 3-phosphate dehydrogenase (HaGAP) as a reference gene were determined by quantitative real time-PCR using the Stratagene MX3005P Sequence Detection System (Agilent Technologies, Santa Clara, Calif.). cDNA was diluted 1:20 in LoTE [3 mM Tris-HCl (pH 7.5), 0.2 mM EDTA] and used as template in triplicate 10 μL reactions. Each reaction consisted of 14 diluted cDNA, 5 μL of SYBR Green Master Mix (Applied Biosystems), and either 0.9 μM HaNRGlob-239F primer (CTGTGAGCCTGTTTGAGAAG) (SEQ ID NO: 13) and 0.3 μM HaNRGlob-312R primer (GCTGGTATCCTTCAGCACCT) (SEQ ID NO: 7) for HaNR2-trHbN analysis or 0.9 μM HaGAP-448F primer (Coyne 2010) and 0.3 μM HaGAP-638R primer (Coyne 2010) for HaGAP analysis. Cycling parameters were as follows: 10 min at 50° C., 2 min at 95° C., followed by 40 cycles of 15 s at 95° C., 30 s at 56° C., and 1 min at 60° C. The dissociation of duplex PCR products was monitored during a stepwise increase in temperature from 60° C. to 95° C. to evaluate reaction specificity. Average values of transcript abundance were determined by linear regression analysis of triplicate reactions. HaNR2-trHbN transcript abundances were calculated from a standard curve prepared from 1×10−2 ng to 1×10−6 ng of HaNR2-trHbN plasmid. NR transcript abundances were then normalized to HaGAP expression as described in Coyne (2010). Relative expression was then calculated as the ratio of the normalized transcript levels in each treatment to the average normalized transcript levels of controls.
For statistical analyses, standard deviations were calculated from the average of replicates (n=3), and means were compared using a one-way ANOVA followed by Tukey HSD post hoc testing using PAST v2.05 software (Hammer et al. 2001). Differences were determined to be statistically significant when p<0.05. Prior to analysis by ANOVA, data were assessed for normality and equality of variance. Raw data for relative gene expression did not meet assumptions of equal variance and were log transformed prior to statistical analysis.
Measurements of NO in cell-free medium (+SNP) showed that cells were exposed to nanomolar concentrations of NO in this experiment, with the highest NO concentration (277 nM) occurring at T1. NO concentrations were significantly lower in H. akashiwo cultures (+SNP) compared to cell-free medium (+SNP) (FIG. 7A), with a 64% reduction in NO at T1 (p<0.001) and a 51% reduction at T3 (p<0.007). HaNR2-trHbN transcript abundance in treatment cultures (+SNP) was significantly higher than control cultures (−SNP) at T1 and T3 (p<0.05; FIG. 7B). Additionally, cell numbers remained constant over the course of the experiment for cultures with and without SNP addition (FIG. 7C).
EXAMPLE 5
Biomass and Photosynthetic Efficiency after Treatment of H. akashiwo Cultures with NO Gas
Goals of this experiment were to (i) show that H. akashiwo can initiate batch growth after exposure to 300 ppm NO and (ii) evaluate the potential of cells to utilize NO as a sole nitrogen source. Cultures were inoculated at 30,000 cells/mL in modified f/2 medium with or without nitrate supplied as a nitrogen source. After inoculation, cultures were treated with either ambient air (control) or 300 ppm NO gas balanced in N2 for 16 hours at a flow rate of 100 mL/min. Growth was monitored over the following batch cycle for triplicate cultures in four treatments: 800 μM NaNO3/Ambient Air (control); 0 μM NaNO3/Ambient Air; 800 μM NaNO3/300 ppm NO; or 0 μM NaNO3/300 ppm NO.
On day 6 of batch growth, biomass and photosynthetic efficiency were compared (FIG. 8). Chlorophyll fluorescence of whole cells was read on a fluorescence induction and relaxation fluorometer (FIRe) to measure the ratio of variable fluorescence (Fv) to maximal fluorescence (Fm). Fv/Fm measurements quantify the efficiency of photosystem II and are used as an indicator of photosynthetic efficiency and general cell health. The interpretation of Fv/Fm values is species specific. As a general rule for H. akashiwo, an Fv/Fm of 0.6-0.7 indicates high efficiency, 0.5-0.6 is average and often observed in healthy laboratory cultures, 0.4-0.5 indicates a decline in photosystem II efficiency, and below 0.4 indicates severe stress.
800 μM NaNO3/Ambient Air control cultures had the highest average biomass and Fv/Fm (0.64), while 0 μM NaNO3/Ambient Air had the lowest average biomass and Fv/Fm (0.26), indicating that these cells were severely stressed under nitrogen starvation. 800 μM NaNO3/300 ppm NO cultures maintained moderate Fv/Fm values (0.54) indicating that photosynthetic activity remained normal after exposure to NO gas. The average Fv/Fm for 0 μM NaNO3/300 ppm NO (0.44) was above the 0.4 minimum cutoff and higher than air treated cells without nitrogen. It was also observed that 0 μM NaNO3/Ambient Air cultures did not grow and instead formed non-motile spherical cysts, however, 0 μM NaNO3/300 ppm NO did not encyst (data not shown). These results suggest that H. akashiwo is able to use NO as a nitrogen source for growth, but the mechanism of NO utilization was not determined. The observation that cells remained healthy throughout the growth cycle and can use NO gas as a source of nitrogen is promising, but data for long-term acclimation under constant NO supply is needed to truly assess the impact of NO on the growth and physiology of H. akashiwo. 
EXAMPLE 6
Effect of Light on the Expression of HaNR2-trHbN
Cultures of H. akashiwo were subjected to shifts in irradiance from low light (60 μmol quanta m−2s−1) to high light (785 μmol quanta m−2s−1) with and without the addition of sodium tungstate (Na2WO4, an inhibitor of the Mo-MPT nitrate reduction center in NR). Samples were taken for gene expression analysis and nitrate reductase activity four hours after the shift to highlight. Data (FIG. 9) shows that expression of HaNR2 and nitrate reducing activity both increase in response to high light. In the presence of Na2WO4, nitrate reducing activity is abolished and the expression of HaNR2 increases greater than 60 times that of controls (FIG. 9). Na2WO4 blocks nitrate reduction at the Mo-MPT center but does not interfere with the flow of electrons to the trHbN domain in HaNR2 (FIG. 9).
EXAMPLE 7
Effect of Temperature and Carbon Dioxide on the Expression of HaNR2-trHbN
Cultures of H. akashiwo were grown at each of the following four conditions: 25° C. and 375 ppm CO2, 25° C. and 750 ppm CO2, 30° C. and 375 ppm CO2, 30° C. and 750 ppm CO2. Cultures were grown under these conditions for three 7-day batch cycles followed by 10 days of semi-continuous growth before sampling for gene expression analysis and nitrate reducing activity. The expression of HaNR2 was the lowest in 30° C. and 375 ppm CO2, which was the condition that showed the highest nitrate reducing activity (FIG. 10). Furthermore, HaNR2 expression was the highest in the combined elevated temperature and CO2 condition (30° C. and 750 ppm CO2) (FIG. 10).
All documents, books, manuals, papers, patents, published patent applications, guides, abstracts, and/or other references cited herein are incorporated by reference in their entirety. Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with the true scope and spirit of the invention being indicated by the following claims.
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## Claims

1. An isolated fusion protein comprising a nitrate reductase (NR) and a truncated hemoglobin N (trHbN) domain, wherein the fusion protein comprises a polypeptide having an amino acid sequence at least 90% identical to SEQ ID NO: 3 or 5.

2. The isolated fusion protein of claim 1, wherein the fusion protein is capable of reducing nitric oxide.

3. The isolated fusion protein of claim 1, wherein the fusion protein is capable of converting nitric oxide to nitrate and nitrite.

4. The isolated fusion protein of claim 1, wherein the fusion protein comprises a polypeptide having the amino acid sequence of SEQ ID NO: 3 or 5.

5. The isolated fusion protein of claim 1, wherein the fusion protein is derived from a raphidophyte.

6. The isolated fusion protein of claim 5, wherein the raphidophyte is selected from the group consisting of genus Heterosigma, and Chattonella.

7. The isolated fusion protein of claim 5, wherein the raphidophyte is selected from the group consisting of species Heterosigma akashiwo, and Chattonella subsalsa.

8. A composition for reducing nitric oxide, comprising an effective amount of an isolated fusion protein, wherein the fusion protein comprises a nitrate reductase (NR) and a truncated hemoglobin N (trHbN) domain, wherein the fusion protein comprises a polypeptide having an amino acid sequence at least 90% identical to SEQ ID NO: 3 or 5.

9. The composition of claim 8, wherein the fusion protein is capable of converting nitric oxide to nitrate and nitrite.

10. The composition of claim 8, wherein the fusion protein comprises a polypeptide having the amino acid sequence of SEQ ID NO: 3 or 5.

11. The composition of claim 8, wherein the fusion protein is derived from a raphidophyte.

12. The composition of claim 11, wherein the raphidophyte is selected from the group consisting of genus Heterosigma, and Chattonella.

13. The composition of claim 11, wherein the raphidophyte is selected from the group consisting of species Heterosigma akashiwo, and Chattonella subsalsa.

14. A method for reducing nitric oxide, comprising applying an effective amount of a fusion protein, wherein the fusion protein comprises a nitrate reductase (NR) and a truncated hemoglobin N (trHbN) domain, and wherein the fusion protein comprises a polypeptide having an amino acid sequence at least 90% identical to SEQ ID NO: 3 or 5.

15. The method of claim 14, wherein the fusion protein converts nitric oxide to nitrate and nitrite.

16. The method of claim 14, wherein the fusion protein comprises a polypeptide having the amino acid sequence of SEQ ID NO: 3 or 5.

17. The method of claim 14, wherein the fusion protein is derived from a raphidophyte.

18. The method of claim 17, wherein the raphidophyte is selected from the group consisting of genus Heterosigma, and Chattonella.

19. The method of claim 17, wherein the raphidophyte is selected from the group consisting of species Heterosigma akashiwo, and Chattonella subsalsa.

20. The method of claim 14, wherein the fusion protein reduces nitric oxide by at least 50%.