Cloning and DNA sequence of a plasmid-determined citrate utilization system in Escherichia coli

Abstract

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 ...

Nucleotide sequence of the gene determining plasmid-mediated citrate utilization

“…2) corresponds to the 431 amino acids shown in Fig. 4 or to the 379-aminoacid polypeptides reported by Sasatsu et al (19) to be encoded by a preceding open reading frame. The 379-aminoacid product is not needed for the Cit+ phenotype.…”

“…Since the single long open reading frame occupied 1,293 bp within the 2,047-bp BglII fragment of Tn3411, there may be present DNA sequences influencing the regulation of the cit gene. Sasatsu et al (19) determined the nucleotide sequence of an EcoRI-PstI fragment ( Fig. 4.…”

Molecular Biology of Sugar Transporters in Plants

“…In a recent review article Marger and Saier Jr. (1993) examined, for the first time, the amino acid sequences of more than 50 uni-, sym-, and antiporters specific for sugars, organic acids, and drugs, using statistical methods. According to this work a superfamily of transport proteins exists which can be divided into five clusters of more closely related transporters: cluster 1 covers the drug-resistance proteins, which extrude drugs from bacterial and yeast cells (Lomovskaya, 1992), in cluster 2 are included W Isugar symporters and uniporters from pro-and eucaryotes (Mueckler et al, 1985;Davis and Henderson, 1987;Sauer and Tanner, 1989;Sauer et al, 1990), cluster 3 contains uptake systems for organic acids (Sasatsu et al, 1985), cluster 4 antiporters for organic phosphate esters and inorganic phosphate (Friedrich and Kadner, 1987), and cluster 5 the W I symporters catalyzing the transport of oligosaccharides such as lactose or sucrose in enteric bacteria (Btichel et al, 1980;Bockmann et al, 1992). This tells us that, for example, the Arabidopsis STP1 W Iglucose symporter (cluster 2), the GLUTl glucose uniporter (cluster 2), and the E. coli LacYW /lactose symporter (cluster 5) are all derived N. Sauer and W Tanner from a common ancestral transporter gene and that the development of LacY, however, has been separated very early in evolution from STP1 and GLUTl.…”

Cloning and characterization of a Campylobacter jejuni 72Dz/92 gene encoding a 30 kDa immunopositive protein, component of the ABC transport system; expression of the gene in avirulent Salmonella typhimurium

“…The product of the truncated ORF3 sequence showed extensive homology to Gram‐negative bacterial proteins, members of the sugar transporter family which catalyze ion‐linked transport (symport) of organic solutes. The highest scores observed were to ProP protein of E. coli (37.3% identity) [27], to citrate carriers from E. coli (35.6%) and Klebsiella pneumoniae (37.1% identity) [28, 29]and finally to α‐ketoglutarate permease of E. coli (31.6% identity) [30].…”