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IPOD-HR, ChIP-seq, RNA-seq of E. coli nucleoid associated protein deletions

ABSTRACT: There is increasing evidence that microbes maintain a structured chromosome, which in turn impacts gene expression. However tools to profile the genome landscape and binding of key architectural proteins have been limited. We recently discovered densely occupied, multi-kilobase regions in E. coli that are transcriptionally silent, similar to eukaryotic heterochromatin. These regions, termed EPODs, overlap metabolic pathways and parasitic elements such as prophages. Here, we investigate the contributions of nucleoid associated proteins (NAPs) to these domains by examining the impacts of deleting NAPs on EPODs genome-wide in E. coli and an evolutionarily distant species, Bacillus subtilis. We identify key NAPs contributing to the silencing of specific EPODs, where deletion of a particular NAPs opens a chromosomal region to RNA polymerase binding. In E. coli, we distinguish novel xenogeneic silencing NAPs, Fis and Hfq, that are essential for cell viability in the presence of domesticated prophages. Furthermore, we show that changes in EPODs facilitate an extra layer of transcriptional regulation to prepare cells for exposure to exotic carbon sources. Our findings demonstrate a new suite of mechanisms through which genomic architecture primes bacteria for changing metabolic environments and silences harmful genomic elements

ORGANISM(S): Escherichia coli Escherichia coli str. K-12 substr. MG1655

PROVIDER: GSE164796 | GEO | 2021/10/01

REPOSITORIES: GEO

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Project description:Nucleoid remodeling facilitated by DNA supercoiling results in changes in nucleoid configuration and involves nucleoid-associated proteins (NAPs) and structural maintenance of chromosomes (SMC) proteins among others. Changes in nucleoid configurations regulated by NAPs are synchronized with cellular adaptation and influence the simultaneous expression of several genes. HU, a ubiquitous bacterial histone-like protein, is among the most conserved and abundant NAPs in eubacteria. In Escherichia coli, HU forms dimers by HUα self-association (HUαα) or by HUα-HUβ interactions (HUαβ). HUα is mostly expressed during lag and early exponential growth phase and HUβ is expressed only during the later exponential and stationary phase, pointing to distinct HUαα/DNA and HUαβ/DNA packaging of the nucleoid in regulating expression patterns during growth and stasis. Mutations or the deletion of HU transform the E. coli nucleoid to a different form and alter overall transcription program; thus, HU interactions with DNA directly affect global gene regulation. Recently, analysis of high-resolution contact maps of the E. coli nucleoid revealed an important role of HU to promote long-range DNA-DNA contacts within the nucleoid. Yet, the molecular connections between HU-DNA interactions and changes in nucleoid architecture that regulate gene expression globally remain unknown. Here, we explored the higher-order E. coli nucleoid organization by soft x-ray tomography (SXT) and revealed an effect of HU surface charges in overall nucleoid organization and rearrangements. We also studied global transcription by next-generation RNA sequencing (RNA-Seq) and found a link between nucleoid rearrangement and changes in global transcription. To determine the functional relationships of observed nucleoid rearrangement and HU-DNA interactions, we also characterized the overall organization of HU nucleoprotein complexes in solution by small angle x-ray scattering (SAXS). We found that HUαα-mediated DNA networks are different at different ionic strengths and pHs. By means of macromolecular crystallography, we additionally elucidate HUαα dependent molecular switches that modulate DNA networking. This integrative structural study explains how HUαα regulates dynamic transformations of the nucleoid by DNA bridging to control nucleoid rearrangement and global gene regulation.

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IPOD-HR, ChIP-seq, RNA-seq of E. coli nucleoid associated protein deletions

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