NasFED proteins mediate assimilatory nitrate and nitrite transport in Klebsiella oxytoca (pneumoniae) M5al.
Klebsiella oxytoca can use nitrate and nitrite as sole nitrogen sources. The enzymes required for nitrate and nitrite assimilation are encoded by the nasFEDCBA operon. We report here the complete nasFED sequence. Sequence comparisons indicate that the nasFED genes encode components of a conventional periplasmic binding protein-dependent transport system consisting of a periplasmic binding protein (NasF), a homodimeric intrinsic membrane protein (NasE), and a homodimeric ATP-binding cassette (ABC) protein (NasD). The NasF protein and the related NrtA and CmpA proteins of cyanobacteria contain leader (signal) sequences with the double-arginine motif that is hypothesized to direct prefolded proteins to an alternate protein export pathway. The NasE protein and the related NrtB and CmpB proteins of cyanobacteria contain unusual variants of the EAA loop sequence that defines membrane-intrinsic proteins of ABC transporters. To characterize nitrate and nitrite transport, we constructed in-frame nonpolar deletions of the chromosomal nasFED genes. Growth tests coupled with nitrate and nitrite uptake assays revealed that the nasFED genes are essential for nitrate transport and participate in nitrite transport as well. Interestingly, the delta nasF strain exhibited leaky phenotypes, particularly at elevated nitrate concentrations, suggesting that the NasED proteins are not fully dependent on the NasF protein.
Wu
Q
Q
Section of Microbiology, Cornell University, Ithaca, New York 14853-8101, USA.
Stewart
V
V
D018528:ATP-Binding Cassette Transporters / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D027321:Anion Transport Proteins; D001426:Bacterial Proteins / Q000235:genetics; D001483:Base Sequence; D001692:Biological Transport; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003470:Culture Media; D005798:Genes, Bacterial; D006863:Hydrogen-Ion Concentration; D007709:Klebsiella / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D026901:Membrane Transport Proteins; D008969:Molecular Sequence Data; D016296:Mutagenesis; D050901:Nitrate Reductase; D009565:Nitrate Reductases / Q000235:genetics; D000091285:Nitrate Transporters; D009566:Nitrates / Q000378:metabolism*; D009573:Nitrites / Q000378:metabolism*; D009876:Operon; D033902:Periplasmic Binding Proteins; D000644:Quaternary Ammonium Compounds / Q000494:pharmacology; D017422:Sequence Analysis, DNA; D012965:Sodium Chloride / Q000494:pharmacology; D013696:Temperature
D018528:ATP-Binding Cassette Transporters; D027321:Anion Transport Proteins; D001426:Bacterial Proteins; D002352:Carrier Proteins; D003470:Culture Media; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; C110971:NasD protein, Klebsiella oxytoca; C110970:NasE protein, Klebsiella oxytoca; C110969:NasF protein, Klebsiella oxytoca; D000091285:Nitrate Transporters; D009566:Nitrates; D009573:Nitrites; D033902:Periplasmic Binding Proteins; D000644:Quaternary Ammonium Compounds; D012965:Sodium Chloride; D009565:Nitrate Reductases; D050901:Nitrate Reductase