Overexpression of Escherichia coli udk mimics the absence of T7 Gp2 function and thereby abrogates successful infection by T7 phage

Andrey Shadrin,Carol Sheppard,Sivaramesh Wigneshweraraj,Dhruti Savalia,Konstantin Severinov

doi:10.1099/mic.0.064527-0

Andrey Shadrin, Carol Sheppard + Show 3 more

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https://doi.org/10.1099/mic.0.064527-0

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Abstract

Successful infection of Escherichia coli by bacteriophage T7 relies upon the transcription of the T7 genome by two different RNA polymerases (RNAps). The bacterial RNAp transcribes early T7 promoters, whereas middle and late T7 genes are transcribed by the T7 RNAp. Gp2, a T7-encoded transcription factor, is a 7 kDa product of an essential middle T7 gene 2, and is a potent inhibitor of the host RNAp. The essential biological role of Gp2 is to inhibit transcription of early T7 genes that fail to terminate efficiently in order to facilitate the coordinated usage of the T7 genome by both host and phage RNAps. Overexpression of the E. coli udk gene, which encodes a uridine/cytidine kinase, interferes with T7 infection. We demonstrate that overexpression of udk antagonizes Gp2 function in E. coli in the absence of T7 infection and thus independently of T7-encoded factors. It seems that overexpression of udk reduces Gp2 stability and functionality during T7 infection, which consequently results in inadequate inhibition of host RNAp and in the accumulation of early T7 transcripts. In other words, overexpression of udk mimics the absence of Gp2 during T7 infection. Our study suggests that the transcriptional regulation of the T7 genome is surprisingly complex and might potentially be affected at many levels by phage- and host-encoded factors.

Highlights

The Escherichia coli bacteriophage T7 relies upon the multisubunit RNA polymerase (RNAp) of its host and a single subunit RNA polymerases (RNAps) encoded by its own genome for transcription of its genes
Similar results were obtained when experiments were repeated in the context of the Gp2-like protein (CR44b_13) from Citrobacter rodentium infective phage CR44b which shares 47 % amino acid sequence identity with Gp2 that binds to the b9 jaw domain of the E. coli RNAp and inhibits
In this study we demonstrate that overexpression of E. coli udk allows transcription from early T7 promoters to continue late in infection, mimicking infections that occur in the absence of Gp2 function, i.e. when E. coli cells are infected by gene 2 mutant T7 phage or cells whose RNAp carries lesions in the Gp2-binding b9 jaw domain is infected by the wild-type T7 phage (Savalia et al, 2010)

Summary

Introduction

The Escherichia coli bacteriophage T7 relies upon the multisubunit RNA polymerase (RNAp) of its host and a single subunit RNAp encoded by its own genome for transcription of its genes. The host RNAp transcribes early T7 phage genes from the genetic left end of the viral genome, whereas the T7 RNAp transcribes the middle and late T7 phage genes. Whilst activities of both the host and T7 RNAp on the T7 phage genome are likely to be regulated and co-ordinated at multiple levels involving host and phage factors, it is generally accepted that the products of T7 phage early gene 0.7 and middle gene 2 are responsible for inhibition of host RNAp at later stages of infection such that host resources are shifted towards the production of viral transcripts synthesized by the T7 RNAp. T7 Gp0.7 is a serine/

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