Overexpression, purification, and partial characterization of ADP-ribosyltransferases modA and modB of bacteriophage T4

Abstract

There is increasing experimental evidence that ADP-ribosylation of host proteins is an important means to regulate gene expression of bacteriophage T4. Surprisingly, this phage codes for three different ADP-ribosyltransferases, gene products Alt, ModA, and ModB, modifying partially overlapping sets of host proteins. While gene product Alt already has been isolated as a recombinant protein and its action on host RNA polymerases and transcription regulation have been studied, the nucleotide sequences of the two mod genes was published only recently. Their mode of action in the course of the infection cycle and the consequences of the ADP-ribosylations catalyzed by these enzymes remain to be investigated. Here we describe the cloning of the genes, the overexpression, purification, and partial characterization of ADP-ribosyltransferases ModA and ModB. Both proteins seem to act independently, and the ADP-ribosyl moieties are transferred to different sets of host proteins. While gene product ModA, similarly to the Alt protein, acts also on the alpha-subunit of host RNA polymerase, the ModB activity serves another set of proteins, one of which was identified as the S1 protein associated with the 30S subunit of the E. coli ribosomes.

FIG. 1

The arrangement of the reading frames modB and modA between the early promoters P13.149 and P11.815. The positions in the T4 genome and the sizes of the corresponding gene products are indicated. The positions of PCR primers for DNA amplification with restriction sites introduced to facilitate cloning are also shown.

FIG. 2

Solubility and prepurification of ModA. Polypeptides were separated on a 13% SDS-polyacrylamide gel and stained with Coomassie brilliant blue. M: marker proteins; C: total protein fraction of a ModA-expressing culture; S: soluble and P: insoluble protein fractions of this culture; PW: inclusion body wash; I: solubilized inclusion bodies; FT: column flow-through. For further information see text.

FIG. 3

Purified ModA, 1 and 3 μg protein per lane, respectively, separated on a 13% SDS-polyacrylamide gel. Proteins were detected by silver staining.

FIG. 4

Autoradiograph of soluble E. coli proteins incubated in the presence of T4 ADP-ribosyltransferases ModA and ModB, respectively. The crude cell extract was supplemented with 20 μg of RNAP. The in vitro labeling reaction followed the protocol as given in Materials and Methods. The radioactive substrate was [³²P]NAD⁺. The reaction products were separated by electrophoresis on a 13% SDS-polyacrylamide gel and stained with Coomassie brilliant blue (A). (B) The autoradiograph of (A). As a molecular weight standard a 10-kDa ladder was employed.

FIG. 5

The amino acid sequences of two independent isolates of the ADP-ribosylated 70-kDa protein, aligned with the sequence of the E. coli ribosomal protein S1.

FIG. 6

ADP-ribosylation of purified 70S ribosomes and of the overexpressed S1 protein. The in vitro labeling reaction with [³²P]NAD⁺ as a substrate followed the protocol given in Material and Methods. After ADP-ribosylation the products were seperated by 13% SDS-PAGE and stained with Coomassie brilliant blue. The band representing the S1 protein is marked by an arrow. M: 10-kDa protein ladder; 1a: purified 70S ribosomes; 1b: autoradiography of la; 2a: E. coli crude extract with the overexpressed SI protein; 2b: autoradiography of 2a.