Cloning and DNA sequence of a plasmid-determined citrate utilization system in Escherichia coli.
The citrate utilization determinant from a large 200-kilobase (kb) naturally occurring plasmid was previously cloned into the PstI site of plasmid vector pBR325 creating the Cit+ tetracycline resistance plasmid pWR61 (15 kb). Tn5 insertion mutagenesis analysis of plasmid pWR61 limited the segment responsible for citrate utilization to a 4.8-kb region bordered by EcoRI and PstI restriction nuclease sites. The 4.8-kb fragment was cloned into phage M13, and the DNA sequence was determined by the dideoxyribonucleotide method. Within this sequence was a 1,296-base-pair open reading frame with a preceding ribosomal binding site. The 431-amino-acid polypeptide that could be translated from this open reading frame would be highly hydrophobic. A second long open reading frame with the potential of encoding a 379-amino-acid polypeptide preceded the larger open reading frame. Portions of the 4.8-kb fragment were further subcloned with restriction endonucleases BglII and BamHI, reducing the minimum size needed for a citrate-positive phenotype to a 1.9-kb BamHI-BglII fragment (which includes the coding region for the 431-amino-acid polypeptide, but only the distal 2/3 of the reading frame for the 379-amino-acid polypeptide). Citrate utilization results from a citrate transport activity encoded by the plasmid. With the 4.8-kb fragment (as with larger fragments) the citrate transport activity was inducible by growth on citrate. On transfer from glucose, succinate, malate, or glycerol medium to citrate medium, the Cit+ Escherichia coli strains showed a delay of 36 to 48 h before growth.
D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001483:Base Sequence; D001693:Biological Transport, Active; D002951:Citrates / Q000378:metabolism*; D019343:Citric Acid; D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes / Q000378:metabolism; D004251:DNA Transposable Elements; D004269:DNA, Bacterial / Q000032:analysis; D015245:Deoxyribonuclease BamHI; D015252:Deoxyribonucleases, Type II Site-Specific; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D008970:Molecular Weight; D009154:Mutation; D010957:Plasmids
D001426:Bacterial Proteins; D002951:Citrates; D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D019343:Citric Acid; D004262:DNA Restriction Enzymes; D015245:Deoxyribonuclease BamHI; C024404:BglII endonuclease; C024388:CTGCAG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific