Chromosome and plasmid-borne PLacO3O1 promoters differ in sensitivity to critically low temperatures

Samuel M D Oliveira,Jason Lloyd-Price,Andre S Ribeiro,Jan Reyelt,Nadia S M Goncalves,Ramakanth Neeli-Venkata,Jose M Fonseca,Vinodh K Kandavalli,Leonardo Martins,Harald Kranz

doi:10.1038/s41598-019-39618-z

Abstract

Temperature shifts trigger genome-wide changes in Escherichia coli’s gene expression. We studied if chromosome integration impacts on a gene’s sensitivity to these shifts, by comparing the single-RNA production kinetics of a PLacO3O1 promoter, when chromosomally-integrated and when single-copy plasmid-borne. At suboptimal temperatures their induction range, fold change, and response to decreasing temperatures are similar. At critically low temperatures, the chromosome-integrated promoter becomes weaker and noisier. Dissection of its initiation kinetics reveals longer lasting states preceding open complex formation, suggesting enhanced supercoiling buildup. Measurements with Gyrase and Topoisomerase I inhibitors suggest hindrance to escape supercoiling buildup at low temperatures. Consistently, similar phenomena occur in energy-depleted cells by DNP at 30 °C. Transient, critically-low temperatures have no long-term consequences, as raising temperature quickly restores transcription rates. We conclude that the chromosomally-integrated PLacO3O1 has higher sensitivity to low temperatures, due to longer-lasting super-coiled states. A lesser active, chromosome-integrated native lac is shown to be insensitive to Gyrase overexpression, even at critically low temperatures, indicating that the rate of escaping positive supercoiling buildup is temperature and transcription rate dependent. A genome-wide analysis supports this, since cold-shock genes exhibit atypical supercoiling-sensitivities. This phenomenon might partially explain the temperature-sensitivity of some transcriptional programs of E. coli.

Highlights

Escherichia coli has evolved sophisticated regulatory programs to adapt to fluctuating environments that allow tuning gene expression so as to trigger appropriate responses[1,2]
We studied at the single-RNA level if the kinetics of RNA production under the control of PLacO3O1 differs in response to temperature changes when the gene is single-copy F-plasmid-borne and when it is chromosome-integrated
To explain the increased time-length of the events preceding the open complex formation in the chromosome-integrated construct at lower temperatures, we considered the model of transcription initiation (Supplementary Information, reactions 1–3)

Summary

Introduction

Escherichia coli has evolved sophisticated regulatory programs to adapt to fluctuating environments that allow tuning gene expression so as to trigger appropriate responses[1,2]. DNA compaction and supercoiling have distinct effects on plasmid-borne and chromosome integrated genes (see e.g.19). One reason for this is that the chromosome has topologically constrained segments that allow supercoiling buildup[12,20,21,22], as transcription occurs, since this process generates positive supercoiling ahead of the RNA polymerase (RNAP) and negative supercoiling behind it[23,24]. Www.nature.com/scientificreports carrying tandem copies of one or two DNA-binding sites[25,30] and plasmids carrying the T7 promoter, when expressed in topA mutant strains[31]. This may explain why temperature down-shifts affect the activity of most chromosomal genes in E. coli[37]

Methods

Results

Conclusion