Construction and complementation of in-frame deletions of the essential Escherichia coli thymidylate kinase gene.
This work reports the construction of Escherichia coli in-frame deletion strains of tmk, which encodes thymidylate kinase, Tmk. The tmk gene is located at the third position of a putative five-gene operon at 24.9 min on the E. coli chromosome, which comprises the genes pabC, yceG, tmk, holB, and ycfH. To avoid potential polar effects on downstream genes of the operon, as well as recombination with plasmid-encoded tmk, the tmk gene was replaced by the kanamycin resistance gene kka1, encoding amino glycoside 3'-phosphotransferase kanamycin kinase. The kanamycin resistance gene is expressed under the control of the natural promoter(s) of the putative operon. The E. coli tmk gene is essential under any conditions tested. To show functional complementation in bacteria, the E. coli tmk gene was replaced by thymidylate kinases of bacteriophage T4 gp1, E. coli tmk, Saccharomyces cerevisiae cdc8, or the Homo sapiens homologue, dTYMK. Growth of these transgenic E. coli strains is completely dependent on thymidylate kinase activities of various origin expressed from plasmids. The substitution constructs show no polar effects on the downstream genes holB and ycfH with respect to cell viability. The presented transgenic bacteria could be of interest for testing of thymidylate kinase-specific phosphorylation of nucleoside analogues that are used in therapies against cancer and infectious diseases.
Chaperon
David-Nicolas
DN
Département de Biochimie Médicale, Centre Médical Universitaire, Université de Genève, CH-1211 Geneva, Switzerland. david.chaperon@medecine.unige.ch
D017122:Bacteriophage T4 / Q000235:genetics; D001483:Base Sequence; D002460:Cell Line; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D017353:Gene Deletion; D015870:Gene Expression; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D006801:Humans; D008969:Molecular Sequence Data; D009703:Nucleoside-Phosphate Kinase / Q000235:genetics*; D009876:Operon; D010957:Plasmids / Q000235:genetics; D013045:Species Specificity; D014161:Transduction, Genetic
D004269:DNA, Bacterial; D009703:Nucleoside-Phosphate Kinase; C020093:dTMP kinase