BRIEF REPORT A mutation in the gene for polynucleotide kinase of bacteriophage T4 K10 affects mRNA processing Z ˇ ivil _ e Strazdait _ e-Z ˇ ielien _ e • Aurelija Zajanc ˇkauskait _ e • Laura Kalinien _ e • Rolandas Mes ˇkys • Lidija Truncait _ e Received: 9 May 2013 / Accepted: 19 June 2013 Ó Springer-Verlag Wien 2013 Abstract The bacteriophage T4 insertion-substitution (I/S) vector system has become one of the most important tools for the introduction of site-directed mutations into the T4 genome. In this study, we show that the I/S phage T4 K10 carries two point mutations within the gene for polynucleotide kinase pseT, resulting in amino acid sub- stitutions G14D and R229H. The G14D mutation impairs 5 0 -kinase activity in vivo as well as in vitro and leads to diminished processing at secondary sites of several RegB- cleaved transcripts. Bacteriophage T4 is one of the best-characterized bacterial viruses that infect Escherichia coli [1, 2]. Extensive mutagenesis combined with biochemical and/or genetic screening has played key role in the study of phage biol- ogy. The availability of the complete genome sequence and the development of genetic engineering techniques have allowed the construction of site-directed T4 mutants with desired properties. Site-directed mutagenesis has been used to construct recombinant phages that display foreign pep- tides [3 and references therein] and to engineer phage particles that infect new bacterial hosts [4–7]. In addition, site-directed mutagenesis has been an invaluable technique for studying gene functions, protein structure-function relationships, and fundamental processes in cells. Genetic manipulation of the bacteriophage T4 genome has been facilitated by the development of an insertion/ substitution (I/S) system that allows direct introduction of in vitro-generated mutations into the phage chromosome [8]. The system has been widely used for construction of T4 mutants with mutations in essential or nonessential genes and regulatory sites [9–15]. The two basic compo- nents of the I/S system are the multiply mutant phage T4 I/S or its isogenic strain T4 K10 (38amB262 51amS29 denAnd28 denB-rIIBDrIIPT8) and a partner I/S plasmid vector, pBSPL0?, which contains a supF gene that is able to suppress amber mutations in the essential 38 and 51 genes of T4 K10. The mutation of interest is first con- structed as a cloned insert within the I/S plasmid. Homologous recombination between the cloned insert and the corresponding region of the T4 I/S genome results in plasmid integration and subsequent segregation. Integrant and segregant phage mutants are selected on the sup- pressing and nonsuppressing E. coli strains. Multiply mutant T4 I/S phage as well as its isogenic strain T4 K10 were constructed by genetic crosses between different T4 mutants obtained from several laboratories [8]. However, since generation of the parent strains was accomplished via general mutagenesis using UV irradia- tion or chemical compounds, these mutant phages may harbor additional unrecognized mutations. Here, we show that the pseT gene of T4 K10 carries two point mutations leading to amino acid substitutions in the encoded poly- nucleotide kinase (PNK). Phage T4 PNK catalyzes both the phosphorylation of 5 0 -hydroxyl termini and the hydrolysis of 3 0 -phosphomonoesters and 2 0 ,3 0 -cyclic phosphodiesters of polynucleotides [16]. In vivo, T4 PNK has the role of a healing enzyme involved in tRNA repair together with an Electronic supplementary material The online version of this article (doi:10.1007/s00705-013-1800-x) contains supplementary material, which is available to authorized users. Z ˇ . Strazdait _ e-Z ˇ ielien _ e Á A. Zajanc ˇkauskait _ e Á L. Kalinien _ e Á R. Mes ˇkys Á L. Truncait _ e(&) Department of Molecular Microbiology and Biotechnology, Institute of Biochemistry of Vilnius University, Mokslininku ˛ 12, 08662 Vilnius, Lithuania e-mail: lidija.truncaite@bchi.vu.lt 123 Arch Virol DOI 10.1007/s00705-013-1800-x