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Functional Characterization of the FNT Family Nitrite Transporter of Marine Picocyanobacteria.

Maeda S, Murakami A, Ito H, Tanaka A, Omata T - Life (Basel) (2015)

Bottom Line: A strongly conserved hydrophilic amino acid sequence was found at the C-termini of the deduced NitM sequences from α-cyanobacteria, with a notable exception of the Synechococcus sp. strain CC9605 NitM protein, which entirely lacked the C-terminal amino acids.The C-terminal sequence was not conserved in the NitM proteins from β-cyanobacteria carrying the Type 1b RuBisCO, including the one from Synechococcus sp. strain PCC7002.Expression of the truncated nitM genes from Synechococcus sp. strain CC9311 and Prochlorococcus marinus strain MIT9313, encoding the proteins lacking the conserved C-terminal region, conferred nitrite uptake activity on the NA4 mutant, indicating that the C-terminal region of α-cyanobacterial NitM proteins inhibits the activity of the transporter.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan. maeda@agr.nagoya-u.ac.jp.

ABSTRACT
Many of the cyanobacterial species found in marine and saline environments have a gene encoding a putative nitrite transporter of the formate/nitrite transporter (FNT) family. The presumed function of the gene (designated nitM) was confirmed by functional expression of the gene from the coastal marine species Synechococcus sp. strain PCC7002 in the nitrite-transport-less mutant (NA4) of the freshwater cyanobacterium Synechococcus elongatus strain PCC7942. The NitM-mediated nitrite uptake showed an apparent Km (NO2-) of about 8 μM and was not inhibited by nitrate, cyanate or formate. Of the nitM orthologs from the three oceanic cyanobacterial species, which are classified as α-cyanobacteria on the basis of the occurrence of Type 1a RuBisCO, the one from Synechococcus sp. strain CC9605 conferred nitrite uptake activity on NA4, but those from Synechococcus sp. strain CC9311 and Prochlorococcus marinus strain MIT9313 did not. A strongly conserved hydrophilic amino acid sequence was found at the C-termini of the deduced NitM sequences from α-cyanobacteria, with a notable exception of the Synechococcus sp. strain CC9605 NitM protein, which entirely lacked the C-terminal amino acids. The C-terminal sequence was not conserved in the NitM proteins from β-cyanobacteria carrying the Type 1b RuBisCO, including the one from Synechococcus sp. strain PCC7002. Expression of the truncated nitM genes from Synechococcus sp. strain CC9311 and Prochlorococcus marinus strain MIT9313, encoding the proteins lacking the conserved C-terminal region, conferred nitrite uptake activity on the NA4 mutant, indicating that the C-terminal region of α-cyanobacterial NitM proteins inhibits the activity of the transporter.

No MeSH data available.


Related in: MedlinePlus

Uptake of nitrite from medium by cells of the NA4 derivatives expressing the NitM proteins and their derivatives from α-cyanobacteria. Changes in the concentration of nitrite in the medium after addition of nitrite to cell suspensions containing 5 μg of Chl/mL are shown. (A) NA402 (closed circles), NA403 (closed diamonds), and NA404 (closed squares) cells; (B) NA412 (open circles) and NA414 (open squares) cells. Cells grown in the presence of 1 mM isopropyl-β-d-thiogalactopyranoside were used for the measurements.
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life-05-00432-f004: Uptake of nitrite from medium by cells of the NA4 derivatives expressing the NitM proteins and their derivatives from α-cyanobacteria. Changes in the concentration of nitrite in the medium after addition of nitrite to cell suspensions containing 5 μg of Chl/mL are shown. (A) NA402 (closed circles), NA403 (closed diamonds), and NA404 (closed squares) cells; (B) NA412 (open circles) and NA414 (open squares) cells. Cells grown in the presence of 1 mM isopropyl-β-d-thiogalactopyranoside were used for the measurements.

Mentions: As Synechococcus sp. strain PCC 7002 belongs to β-cyanobacteria, we further characterized the nitM genes of the oceanic α-cyanobacterial strains. Transcriptional fusions of the trc promoter and the nitM genes of Synechococcus sp. strain CC9311 (sync_2899), Synechococcus sp. strain PCC9605 (Syncc9605_2657) and Prochlorococcus marinus strain MIT9313 (PMT2240) were introduced into the NA4 mutant of S. elongatus to construct the NA402, NA403 and NA404, respectively. On the medium containing 0.5 mM nitrite at pH 9.6, IPTG-induced cells of NA403 grew well (Figure 2d), while the NA402 and NA404 mutants failed to grow irrespective of the presence or absence of IPTG (Figure 2c,e). In accordance with the results of the growth experiment, IPTG-treated NA403 cells took up low concentrations of nitrite from the medium of pH 9.6 (Figure 4A), while the NA402 and NA404 strains failed to utilize low concentrations of nitrite. Thus the NitM protein of Synechococcus sp. strain PCC9605 was functional as a nitrite transporter when expressed in the S. elongatus NA4 mutant, but those from Synechococcus sp. strain CC9311 and Prochlorococcus marinus strain MIT9313 were not.


Functional Characterization of the FNT Family Nitrite Transporter of Marine Picocyanobacteria.

Maeda S, Murakami A, Ito H, Tanaka A, Omata T - Life (Basel) (2015)

Uptake of nitrite from medium by cells of the NA4 derivatives expressing the NitM proteins and their derivatives from α-cyanobacteria. Changes in the concentration of nitrite in the medium after addition of nitrite to cell suspensions containing 5 μg of Chl/mL are shown. (A) NA402 (closed circles), NA403 (closed diamonds), and NA404 (closed squares) cells; (B) NA412 (open circles) and NA414 (open squares) cells. Cells grown in the presence of 1 mM isopropyl-β-d-thiogalactopyranoside were used for the measurements.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4390861&req=5

life-05-00432-f004: Uptake of nitrite from medium by cells of the NA4 derivatives expressing the NitM proteins and their derivatives from α-cyanobacteria. Changes in the concentration of nitrite in the medium after addition of nitrite to cell suspensions containing 5 μg of Chl/mL are shown. (A) NA402 (closed circles), NA403 (closed diamonds), and NA404 (closed squares) cells; (B) NA412 (open circles) and NA414 (open squares) cells. Cells grown in the presence of 1 mM isopropyl-β-d-thiogalactopyranoside were used for the measurements.
Mentions: As Synechococcus sp. strain PCC 7002 belongs to β-cyanobacteria, we further characterized the nitM genes of the oceanic α-cyanobacterial strains. Transcriptional fusions of the trc promoter and the nitM genes of Synechococcus sp. strain CC9311 (sync_2899), Synechococcus sp. strain PCC9605 (Syncc9605_2657) and Prochlorococcus marinus strain MIT9313 (PMT2240) were introduced into the NA4 mutant of S. elongatus to construct the NA402, NA403 and NA404, respectively. On the medium containing 0.5 mM nitrite at pH 9.6, IPTG-induced cells of NA403 grew well (Figure 2d), while the NA402 and NA404 mutants failed to grow irrespective of the presence or absence of IPTG (Figure 2c,e). In accordance with the results of the growth experiment, IPTG-treated NA403 cells took up low concentrations of nitrite from the medium of pH 9.6 (Figure 4A), while the NA402 and NA404 strains failed to utilize low concentrations of nitrite. Thus the NitM protein of Synechococcus sp. strain PCC9605 was functional as a nitrite transporter when expressed in the S. elongatus NA4 mutant, but those from Synechococcus sp. strain CC9311 and Prochlorococcus marinus strain MIT9313 were not.

Bottom Line: A strongly conserved hydrophilic amino acid sequence was found at the C-termini of the deduced NitM sequences from α-cyanobacteria, with a notable exception of the Synechococcus sp. strain CC9605 NitM protein, which entirely lacked the C-terminal amino acids.The C-terminal sequence was not conserved in the NitM proteins from β-cyanobacteria carrying the Type 1b RuBisCO, including the one from Synechococcus sp. strain PCC7002.Expression of the truncated nitM genes from Synechococcus sp. strain CC9311 and Prochlorococcus marinus strain MIT9313, encoding the proteins lacking the conserved C-terminal region, conferred nitrite uptake activity on the NA4 mutant, indicating that the C-terminal region of α-cyanobacterial NitM proteins inhibits the activity of the transporter.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan. maeda@agr.nagoya-u.ac.jp.

ABSTRACT
Many of the cyanobacterial species found in marine and saline environments have a gene encoding a putative nitrite transporter of the formate/nitrite transporter (FNT) family. The presumed function of the gene (designated nitM) was confirmed by functional expression of the gene from the coastal marine species Synechococcus sp. strain PCC7002 in the nitrite-transport-less mutant (NA4) of the freshwater cyanobacterium Synechococcus elongatus strain PCC7942. The NitM-mediated nitrite uptake showed an apparent Km (NO2-) of about 8 μM and was not inhibited by nitrate, cyanate or formate. Of the nitM orthologs from the three oceanic cyanobacterial species, which are classified as α-cyanobacteria on the basis of the occurrence of Type 1a RuBisCO, the one from Synechococcus sp. strain CC9605 conferred nitrite uptake activity on NA4, but those from Synechococcus sp. strain CC9311 and Prochlorococcus marinus strain MIT9313 did not. A strongly conserved hydrophilic amino acid sequence was found at the C-termini of the deduced NitM sequences from α-cyanobacteria, with a notable exception of the Synechococcus sp. strain CC9605 NitM protein, which entirely lacked the C-terminal amino acids. The C-terminal sequence was not conserved in the NitM proteins from β-cyanobacteria carrying the Type 1b RuBisCO, including the one from Synechococcus sp. strain PCC7002. Expression of the truncated nitM genes from Synechococcus sp. strain CC9311 and Prochlorococcus marinus strain MIT9313, encoding the proteins lacking the conserved C-terminal region, conferred nitrite uptake activity on the NA4 mutant, indicating that the C-terminal region of α-cyanobacterial NitM proteins inhibits the activity of the transporter.

No MeSH data available.


Related in: MedlinePlus