Homologs of the Escherichia coli F Element Protein TraR, Including Phage Lambda Orf73, Directly Reprogram Host Transcription

mBio. 2022 May 18;e0095222. doi: 10.1128/mbio.00952-22. Online ahead of print.

Abstract

Bacterial cells and their associated plasmids and bacteriophages encode numerous small proteins of unknown function. One example, the 73-amino-acid protein TraR, is encoded by the transfer operon of the conjugative F plasmid of Escherichia coli. TraR is a distant homolog of DksA, a protein found in almost all proteobacterial species that is required for ppGpp to regulate transcription during the stringent response. TraR and DksA increase or decrease transcription initiation depending on the kinetic features of the promoter by binding directly to RNA polymerase without binding to DNA. Unlike DksA, whose full activity requires ppGpp as a cofactor, TraR is fully active by itself and unaffected by ppGpp. TraR belongs to a family of divergent proteins encoded by proteobacterial bacteriophages and other mobile elements. Here, we experimentally addressed whether other members of the TraR family function like the F element-encoded TraR. Purified TraR and all 5 homologs that were examined bound to RNA polymerase, functioned at lower concentrations than DksA, and complemented a dksA-null strain for growth on minimal medium. One of the homologs, λ Orf73, encoded by bacteriophage lambda, was examined in greater detail. λ Orf73 slowed host growth and increased phage burst size. Mutational analysis suggested that λ Orf73 and TraR have a similar mechanism for inhibiting rRNA and r-protein promoters. We suggest that TraR and its homologs regulate host transcription to divert cellular resources to phage propagation or conjugation without induction of ppGpp and a stringent response. IMPORTANCE TraR is a distant homolog of the transcription factor DksA and the founding member of a large family of small proteins encoded by proteobacterial phages and conjugative plasmids. Reprogramming transcription during the stringent response requires the interaction of DksA not only with RNA polymerase but also with the stress-induced regulatory nucleotide ppGpp. We show here that five phage TraR homologs by themselves, without ppGpp, regulate transcription of host promoters, mimicking the effects of DksA and ppGpp together. During a stringent response, ppGpp independently binds directly to, and inhibits the activities of, many proteins in addition to RNA polymerase, including translation factors, enzymes needed for ribonucleotide biosynthesis, and other metabolic enzymes. Here, we suggest a physiological role for TraR-like proteins: bacteriophages utilize TraR homologs to reprogram host transcription in the absence of ppGpp induction and thus without inhibiting host enzymes needed for phage development.

Keywords: DksA homologs; RNA polymerase; TraR homologs; bacteriophage lambda Orf73; regulation of transcription.