Cloning and properties of the Salmonella typhimurium tricarboxylate transport operon in Escherichia coli.
The tricarboxylate transport operon (tctI) was cloned in Escherichia coli as a 12-kilobase (kb) fragment from an EcoRI library of the Salmonella typhimurium chromosome in lambda gtWES. It was further subcloned as a 12-kb fragment into pACYC184 and as an 8-kb fragment into pBR322. By insertional mutagenesis mediated by lambda Tn5, restriction mapping, and phenotypic testing, the tctI operon was localized to a 4.5-kb region. The tctC gene which encodes a periplasmic binding protein (C protein) was located near the center of the insert. E. coli/tctI clones on either multicopy or single-copy vectors grew on the same tricarboxylates as S. typhimurium, although unusually long growth lags were observed. E. coli/tctI clones exhibited similar [14C]fluorocitrate transport kinetics to those of S. typhimurium, whereas E. coli alone was virtually impermeable to [14C]fluorocitrate. The periplasmic C proteins (C1 and C2 isoelectric forms) were produced in prodigious quantities from the cloned strains. Motile E. coli/tctI clones were not chemotactic toward citrate, whereas tctI deletion mutants of S. typhimurium were. Taken together, these observations indicate that tctI is not an operon involved in chemotaxis.
Widenhorn
K A
KA
Department of Biochemistry and Microbiology, University of Victoria, British Columbia, Canada.
Boos
W
W
Somers
J M
JM
Kay
W W
WW
D001692:Biological Transport; D002874:Chromosome Mapping; D002951:Citrates / Q000378:metabolism; D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005786:Gene Expression Regulation; D005798:Genes, Bacterial; D009154:Mutation; D009876:Operon; D010641:Phenotype; D012486:Salmonella typhimurium / Q000235:genetics* / Q000378:metabolism; D014233:Tricarboxylic Acids / Q000378:metabolism*
D002951:Citrates; D014233:Tricarboxylic Acids; D004262:DNA Restriction Enzymes