Limits...
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

Growth test on nitrite-containing media showing the effects of expression of the genes encoding the FNT family proteins of marine cyanobacteria in the NA4 mutant lacking the ABC-transporters capable of nitrite transport. Synechococcus elongatus strain PCC 7942 cells (n = 106) were spotted onto solid medium containing 7.5 mM ammonium or 0.5 mM nitrite and incubated under illumination for four days. The medium containing ammonium was buffered at pH 8.2 and the medium containing nitrite was buffered at pH 9.6. Where indicated, isopropyl-β-d-thiogalactopyranoside (IPTG; 1 mM) was added to induce the expression of the plasmid-borne genes.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4390861&req=5

life-05-00432-f002: Growth test on nitrite-containing media showing the effects of expression of the genes encoding the FNT family proteins of marine cyanobacteria in the NA4 mutant lacking the ABC-transporters capable of nitrite transport. Synechococcus elongatus strain PCC 7942 cells (n = 106) were spotted onto solid medium containing 7.5 mM ammonium or 0.5 mM nitrite and incubated under illumination for four days. The medium containing ammonium was buffered at pH 8.2 and the medium containing nitrite was buffered at pH 9.6. Where indicated, isopropyl-β-d-thiogalactopyranoside (IPTG; 1 mM) was added to induce the expression of the plasmid-borne genes.

Mentions: In our study on the putative nitrite transporter genes of marine cyanobacteria, we first examined the function of the gene from the β-cyanobacterium Synechococcus sp. strain PCC 7002 (synPCC7002_A0125). A transcriptional fusion of the trc promoter and synPCC7002_A0125 was introduced into the NA4 mutant to construct the NA401 strain by using the pSE1 shuttle expression vector [23]. The pSE1 vector was also introduced into NA4 to construct the reference strain NA41. Both strains grew well on the medium containing ammonium (Figure 2a,b). Whereas NA41 failed to grow on the nitrite (0.5 mM)-containing medium of pH 9.6 irrespective of the presence of IPTG (Figure 2a), NA401 grew well on the medium in the absence of IPTG (Figure 2b). In the presence of IPTG, NA401 died on the nitrite-containing medium but grew well on the ammonium-containing medium (Figure 2b). These results suggested that the basal-level expression of synPCC7002_A0125 from the trc promoter was sufficient to support nitrite uptake from the medium containing low concentrations of nitrite, but overexpression of the gene resulted in uptake of excessive nitrite from the medium to kill the cells. To further analyze the role of the SynPCC7002_A0125 in nitrite transport, nitrite uptake activity of these mutants was determined by measuring consumption of nitrite in liquid medium (Figure 3). Whereas NA41 failed to utilize low concentrations of nitrite, the non-induced cells of NA401 without IPTG treatment took up low concentrations of nitrite (Figure 3A). Nitrite uptake rate of the cells of NA401 was calculated to be 80 µmol (mg·Chl)−1·h−1 in the extracellular nitrite concentration range of 20–100 µM. These results confirmed that the FNT family protein of Synechococcus sp. strain PCC 7002 has nitrite uptake activity. We therefore named the gene as nitM for nitrite transporter of marine cyanobacteria. When grown under the same conditions, the wild-type cells of S. elongatus strain PCC 7942, expressing the ABC-type bispecific nitrate/nitrite transporter, showed a rate of about 40 µmol (mg Chl)−1·h−1 for the uptake of nitrate or nitrite [23]. Thus, the nitrite uptake rate of the non-induced NA401 cells was two fold of that of the wild-type S. elongatus strain PCC 7942 cells.


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

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

Growth test on nitrite-containing media showing the effects of expression of the genes encoding the FNT family proteins of marine cyanobacteria in the NA4 mutant lacking the ABC-transporters capable of nitrite transport. Synechococcus elongatus strain PCC 7942 cells (n = 106) were spotted onto solid medium containing 7.5 mM ammonium or 0.5 mM nitrite and incubated under illumination for four days. The medium containing ammonium was buffered at pH 8.2 and the medium containing nitrite was buffered at pH 9.6. Where indicated, isopropyl-β-d-thiogalactopyranoside (IPTG; 1 mM) was added to induce the expression of the plasmid-borne genes.
© Copyright Policy
Related In: Results  -  Collection

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

life-05-00432-f002: Growth test on nitrite-containing media showing the effects of expression of the genes encoding the FNT family proteins of marine cyanobacteria in the NA4 mutant lacking the ABC-transporters capable of nitrite transport. Synechococcus elongatus strain PCC 7942 cells (n = 106) were spotted onto solid medium containing 7.5 mM ammonium or 0.5 mM nitrite and incubated under illumination for four days. The medium containing ammonium was buffered at pH 8.2 and the medium containing nitrite was buffered at pH 9.6. Where indicated, isopropyl-β-d-thiogalactopyranoside (IPTG; 1 mM) was added to induce the expression of the plasmid-borne genes.
Mentions: In our study on the putative nitrite transporter genes of marine cyanobacteria, we first examined the function of the gene from the β-cyanobacterium Synechococcus sp. strain PCC 7002 (synPCC7002_A0125). A transcriptional fusion of the trc promoter and synPCC7002_A0125 was introduced into the NA4 mutant to construct the NA401 strain by using the pSE1 shuttle expression vector [23]. The pSE1 vector was also introduced into NA4 to construct the reference strain NA41. Both strains grew well on the medium containing ammonium (Figure 2a,b). Whereas NA41 failed to grow on the nitrite (0.5 mM)-containing medium of pH 9.6 irrespective of the presence of IPTG (Figure 2a), NA401 grew well on the medium in the absence of IPTG (Figure 2b). In the presence of IPTG, NA401 died on the nitrite-containing medium but grew well on the ammonium-containing medium (Figure 2b). These results suggested that the basal-level expression of synPCC7002_A0125 from the trc promoter was sufficient to support nitrite uptake from the medium containing low concentrations of nitrite, but overexpression of the gene resulted in uptake of excessive nitrite from the medium to kill the cells. To further analyze the role of the SynPCC7002_A0125 in nitrite transport, nitrite uptake activity of these mutants was determined by measuring consumption of nitrite in liquid medium (Figure 3). Whereas NA41 failed to utilize low concentrations of nitrite, the non-induced cells of NA401 without IPTG treatment took up low concentrations of nitrite (Figure 3A). Nitrite uptake rate of the cells of NA401 was calculated to be 80 µmol (mg·Chl)−1·h−1 in the extracellular nitrite concentration range of 20–100 µM. These results confirmed that the FNT family protein of Synechococcus sp. strain PCC 7002 has nitrite uptake activity. We therefore named the gene as nitM for nitrite transporter of marine cyanobacteria. When grown under the same conditions, the wild-type cells of S. elongatus strain PCC 7942, expressing the ABC-type bispecific nitrate/nitrite transporter, showed a rate of about 40 µmol (mg Chl)−1·h−1 for the uptake of nitrate or nitrite [23]. Thus, the nitrite uptake rate of the non-induced NA401 cells was two fold of that of the wild-type S. elongatus strain PCC 7942 cells.

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