Abstract
Bacteria respond to their environment by regulating mRNA synthesis, often by altering the genomic sites at which RNA polymerase initiates transcription. Here, we investigate genome-wide changes in transcription start site (TSS) usage by Clostridium phytofermentans, a model bacterium for fermentation of lignocellulosic biomass. We quantify expression of nearly 10,000 TSS at single base resolution by Capp-Switch sequencing, which combines capture of synthetically capped 5′ mRNA fragments with template-switching reverse transcription. We find the locations and expression levels of TSS for hundreds of genes change during metabolism of different plant substrates. We show that TSS reveals riboswitches, non-coding RNA and novel transcription units. We identify sequence motifs associated with carbon source-specific TSS and use them for regulon discovery, implicating a LacI/GalR protein in control of pectin metabolism. We discuss how the high resolution and specificity of Capp-Switch enables study of condition-specific changes in transcription initiation in bacteria.
Highlights
Bacteria respond to their environment by regulating messenger RNA (mRNA) synthesis, often by altering the genomic sites at which RNA polymerase initiates transcription
The initiating nucleotide of nascent mRNA is distinguished by a 50 triphosphate (50-PPP), which has been exploited for genomewide transcription start site (TSS) identification with dRNA-seq[8] by depleting rRNA and other monophosphorylated transcripts using terminal exonuclease (TEX). dRNA-seq has been applied to diverse bacteria[9,10,11,12,13], but incomplete and non-specific degradation of processed RNA requires TSS identification to be based on statistical comparison of read coverage in þ TEX and À TEX samples
We identified 9,457 TSS across treatments (Supplementary Data 1), one-third of which were expressed in both sugar and polysaccharide cultures (Fig. 2c)
Summary
Bacteria respond to their environment by regulating mRNA synthesis, often by altering the genomic sites at which RNA polymerase initiates transcription. Correspondence and requests for materials should be addressed to Bacteria translate environmental signals into cellular responses using a network of regulatory RNA and proteins that control genome-wide transcription patterns Many of these regulators affect where RNA polymerase initiates messenger RNA (mRNA) synthesis at transcription start sites (TSS). We apply Capp-Switch sequencing to define a genome-wide map of 9,457 TSS during C. phytofermentans growth on raw biomass, heterogeneous polysaccharides (cellulose, hemicellulose and pectin) and their constituent sugars. We use this TSS map to investigate features controlling gene regulation, such as RNA polymerase binding sites, 50 untranslated region (UTR) structure, alternative promoters, operons and non-standard (leaderless and antisense) transcription. We discuss how Capp-Switch sequencing can be applied as a general approach to explore transcription regulation in prokaryotes
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