The E. coli Global Regulator DksA Reduces Transcription during T4 Infection.

Jennifer Patterson-West,Deborah Hinton,Michael Cashel,Tamara James,Leslie Knipling,Kyung Moon,James Iben,Llorenç Fernández-Coll

doi:10.3390/v10060308

Abstract

Bacteriophage T4 relies on host RNA polymerase to transcribe three promoter classes: early (Pe, requires no viral factors), middle (Pm, requires early proteins MotA and AsiA), and late (Pl, requires middle proteins gp55, gp33, and gp45). Using primer extension, RNA-seq, RT-qPCR, single bursts, and a semi-automated method to document plaque size, we investigated how deletion of DksA or ppGpp, two E. coli global transcription regulators, affects T4 infection. Both ppGpp0 and ΔdksA increase T4 wild type (wt) plaque size. However, ppGpp0 does not significantly alter burst size or latent period, and only modestly affects T4 transcript abundance, while ΔdksA increases burst size (2-fold) without affecting latent period and increases the levels of several Pe transcripts at 5 min post-infection. In a T4motAam infection, ΔdksA increases plaque size and shortens latent period, and the levels of specific middle RNAs increase due to more transcription from Pe’s that extend into these middle genes. We conclude that DksA lowers T4 early gene expression. Consequently, ΔdksA results in a more productive wt infection and ameliorates the poor expression of middle genes in a T4motAam infection. As DksA does not inhibit Pe transcription in vitro, regulation may be indirect or perhaps requires additional factors.

Highlights

The integrity and regulation of gene expression is essential for proper cellular function and adaptation
We demonstrate that a deletion of either DksA or ppGpp results in significantly larger plaques for either T4 wt or T4motAam
These analyses provided a “proof of principle” that our RNA-seq analyses correctly extended previous work because one can investigate the effect of the motA knockdown on each reflected state of T4

Summary

Introduction

The integrity and regulation of gene expression is essential for proper cellular function and adaptation. Bacteria have evolved myriad mechanisms to tightly regulate gene expression to ensure that genes are expressed under the correct environmental conditions [1]. Bacteriophages have simultaneously evolved mechanisms for altering gene expression in their host to optimize the expression of genes required for viral proliferation [2,3,4]. In Escherichia coli (E. coli), the primary σ factor, σ70 , recognizes specific −10 and −35 elements within the promoter sequence and is essential for the transcription of genes needed for exponential growth under nutrient-rich conditions. Efficient transcription requires the interaction of RNAP with promoter DNA to create an “open complex” with a single-stranded region at the transcription start

Methods

Results

Conclusion