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Journal of Bacteriology, November 2004, p. 7262-7272, Vol. 186, No. 21
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.21.7262-7272.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

ModA and ModB, Two ADP-Ribosyltransferases Encoded by Bacteriophage T4: Catalytic Properties and Mutation Analysis

Bernd Tiemann,^1, Reinhard Depping,^1, Egle Gineikiene,² Laura Kaliniene,² Rimas Nivinskas,² and Wolfgang Rüger^1*

Arbeitsgruppe Molekulare Genetik, Fakultät für Biologie, Ruhr-Universität Bochum, Bochum, Germany,¹ Department of Gene Engineering, Institute of Biochemistry, Vilnius, Lithuania²

Received 8 May 2004/ Accepted 27 July 2004

Bacteriophage T4 encodes three ADP-ribosyltransferases, Alt, ModA, and ModB. These enzymes participate in the regulation of the T4 replication cycle by ADP-ribosylating a defined set of host proteins. In order to obtain a better understanding of the phage-host interactions and their consequences for regulating the T4 replication cycle, we studied cloning, overexpression, and characterization of purified ModA and ModB enzymes. Site-directed mutagenesis confirmed that amino acids, as deduced from secondary structure alignments, are indeed decisive for the activity of the enzymes, implying that the transfer reaction follows the Sn1-type reaction scheme proposed for this class of enzymes. In vitro transcription assays performed with Alt- and ModA-modified RNA polymerases demonstrated that the Alt-ribosylated polymerase enhances transcription from T4 early promoters on a T4 DNA template, whereas the transcriptional activity of ModA-modified polymerase, without the participation of T4-encoded auxiliary proteins for middle mode or late transcription, is reduced. The results presented here support the conclusion that ADP-ribosylation of RNA polymerase and of other host proteins allows initial phage-directed mRNA synthesis reactions to escape from host control. In contrast, subsequent modification of the other cellular target proteins limits transcription from phage early genes and participates in redirecting transcription to phage middle and late genes.

Present address: Klinik für Nephrologie and Rheumatologie, Universitätsklinikum Düsseldorf, 40225 Düsseldorf, Germany.

Present address: Institut für Physiologie, Universität zu Lübeck, 23538 Lübeck, Germany.

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