Cloning, sequencing, and characterization of a gene (narT) encoding a transport protein involved in dissimilatory nitrate reduction in Staphylococcus carnosus.
A Tn917 mutant of Staphylococcus carnosus TM300, nrIII, was isolated and characterized. Mutant nrIII did not take up nitrate or accumulate nitrite when grown in B-medium supplemented with up to 10 mM nitrate under anoxic conditions; however, it displayed wild-type levels of benzyl Delta viologen-linked nitrate reductase activity. Cultivated in B-medium with nitrate under oxic conditions, mutant nrIII accumulated fivefold less nitrite than the wild-type. The mutation in S. carnosus nrIII could be complemented with a 2-kb chromosomal EcoRI-HpaI fragment from the wild-type. The gene affected by transposon insertion in mutant nrIII was cloned and sequenced. Analysis of the deduced amino acid sequence revealed that this gene, designated narT, encodes a highly hydrophobic 42-kDa transmembrane protein of 388 amino acids and shows similarities to transport proteins that play a role in nitrate import or nitrite export. The inability of nrIII to take up nitrate under anoxic conditions and its ability to take up and accumulate nitrite in the presence of benzyl viologen, a nitrate ionophore, under the same conditions suggest that NarT represents a transport protein required for nitrate uptake under anoxic conditions in S. carnosus.
Fast
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B
Mikrobielle Genetik, Universität Tübingen, Waldhäuserstrasse 70/8, D-72076 Tübingen, Germany.
Lindgren
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P
Götz
F
F
D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D002352:Carrier Proteins / Q000235:genetics*; D003001:Cloning, Molecular; D004251:DNA Transposable Elements; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D008565:Membrane Proteins / Q000235:genetics*; D026901:Membrane Transport Proteins; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D050901:Nitrate Reductase; D009565:Nitrate Reductases / Q000378:metabolism; D009566:Nitrates / Q000378:metabolism*; D009573:Nitrites / Q000378:metabolism; D010957:Plasmids; D011995:Recombination, Genetic; D015183:Restriction Mapping; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013210:Staphylococcus / Q000235:genetics*; D014170:Transformation, Genetic
D001426:Bacterial Proteins; D002352:Carrier Proteins; D004251:DNA Transposable Elements; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; C104761:NarT protein, Staphylococcus carnosus; D009566:Nitrates; D009573:Nitrites; D009565:Nitrate Reductases; D050901:Nitrate Reductase