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). TnS 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 epdonucleases BglII and BamHI, reducing the minimum size needed for a citrate-positive phenotype to a 1.9-kb BamHI-BgIII 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.The inability of Escherichia coli to utilize citrate as the sole carbon and energy source has been recognized for 60 years (22) and provides the basis for an often-used distinction between Cit-E. coli and many similar but Cit+ bacterial species. The recent discovery of naturally occurring plasmids that confer a Cit+ phenotype on E. coli strains (14,15,35) has provided an explanation for the periodic isolation of bacteria that would have been called E. coli except for their ability to utilize citrate. A wide range of Cit+ plasmids have been found from animal and human sources (10-17, 34, 35), and the existence of such determinants requires answers as to how they have arisen and spread and what the biochemical basis for the Cit+ phenotype might be. Ishiguro et al. (19) found one such Cit+ determinant on a transposon. Reynolds and Silver (32) demonstrated that the Cit+ plasmid confers the ability to transport citrate by a system that was partially inducible. The substrate range of tricarboxylic acids for the Cit+ plasmid-containing E. coli and the requirement for H+ but neither K+, Na+, nor Mg2+ (32) distinguished this plasmid-determined system from those known in other organisms, including Salmonella typhimurium (9, 20, 21), and also from the unique chromosomal rearrangement of E. coli that led to a Cit+ phenotype (6,32). Recently, one Cit+ plasmid determinant has been cloned and analyzed by restriction nuclease mapping and a polypeptide product has ...

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