Chaperon, David-Nicolas <jats:title>ABSTRACT</jats:title> <jats:p>This work reports the construction of <jats:italic>Escherichia coli</jats:italic> in-frame deletion strains of <jats:italic>tmk</jats:italic>, which encodes thymidylate kinase, Tmk. The <jats:italic>tmk</jats:italic> gene is located at the third position of a putative five-gene operon at 24.9 min on the <jats:italic>E. coli</jats:italic> chromosome, which comprises the genes <jats:italic>pabC</jats:italic>, <jats:italic>yceG</jats:italic>, <jats:italic>tmk</jats:italic>, <jats:italic>holB</jats:italic>, and <jats:italic>ycfH</jats:italic>. To avoid potential polar effects on downstream genes of the operon, as well as recombination with plasmid-encoded <jats:italic>tmk</jats:italic>, the <jats:italic>tmk</jats:italic> gene was replaced by the kanamycin resistance gene <jats:italic>kka1</jats:italic>, 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 <jats:italic>E. coli tmk</jats:italic> gene is essential under any conditions tested. To show functional complementation in bacteria, the <jats:italic>E. coli tmk</jats:italic> gene was replaced by thymidylate kinases of bacteriophage T4 gp<jats:italic>1</jats:italic>, <jats:italic>E. coli tmk</jats:italic>, <jats:italic>Saccharomyces cerevisiae cdc8</jats:italic>, or the <jats:italic>Homo sapiens</jats:italic> homologue, <jats:italic>dTYMK</jats:italic>. Growth of these transgenic <jats:italic>E. coli</jats:italic> 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 <jats:italic>holB</jats:italic> and <jats:italic>ycfH</jats:italic> 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.</jats:p> application/pdf https://sonar.ch/global/documents/293305 eng info:eu-repo/semantics/altIdentifier/doi/10.1128/aem.72.2.1288-1294.2006 info:eu-repo/semantics/altIdentifier/issn/0099-2240 info:eu-repo/semantics/openAccess Applied and Environmental Microbiology. - American Society for Microbiology. - 2006, vol. 72, no. 2, p. 1288-1294 Biotechnology Food Science Ecology Applied Microbiology and Biotechnology Construction and Complementation of In-Frame Deletions of the Essential Escherichia coli Thymidylate Kinase Gene http://purl.org/coar/resource_type/c_6501