Nucleotide sequence and expression in Escherichia coli of the Lactococcus lactis citrate permease gene.
The plasmid-encoded citrate determinant of the Lactococcus lactis subsp. lactis var. diacetylactis NCDO176 was cloned and functionally expressed in a Cit- Escherichia coli K-12 strain. From deletion derivative analysis, a 3.4-kilobase region was identified which encodes the ability to transport citrate. Analysis of proteins encoded by the cloned fragment in a T7 expression system revealed a 32,000-dalton protein band, which correlated with the ability of cells to transport citrate. Energy-dependent [1,5-14C]citrate transport was found with membrane vesicles prepared from E. coli cells harboring the citrate permease-expressing plasmid. The gene encoding citrate transport activity, citP, was located on the cloned fragment by introducing a site-specific mutation that abolished citrate transport and resulted in a truncated form of the 32,000-dalton expression product. The nucleotide sequence for a 2.2-kilobase fragment that includes the citP gene contained an open reading frame of 1,325 base pairs coding for a very hydrophobic protein of 442 amino acids, which shows no sequence homology with known citrate carriers.
David
S
S
Molecular Genetics Group, Netherlands Institute for Dairy Research, Ede.
van der Rest
M E
ME
Driessen
A J
AJ
Simons
G
G
de Vos
W M
WM
D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001483:Base Sequence; D002462:Cell Membrane / Q000201:enzymology; D002951:Citrates / Q000378:metabolism; D003001:Cloning, Molecular / Q000379:methods; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D007700:Kinetics; D013294:Lactococcus lactis / Q000201:enzymology / Q000235:genetics*; D026901:Membrane Transport Proteins / Q000235:genetics*; D008969:Molecular Sequence Data; D027361:Organic Anion Transporters; D011392:Proline / Q000378:metabolism; D015183:Restriction Mapping
D001426:Bacterial Proteins; D002951:Citrates; D026901:Membrane Transport Proteins; D027361:Organic Anion Transporters; C065431:citP protein, Lactococcus lactis; D011392:Proline