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Nucleoid associated proteins (NAPs) maintain the architecture of bacterial chromosomes and regulate gene expression. Thus, their role as transcription factors may involve three-dimensional chromosome re-organisation. While this model is supported by in vitro studies, direct in vivo evidence is lacking. Here, we use RT-qPCR and 3C-qPCR to study the transcriptional and architectural profiles of the H-NS (histone-like nucleoid structuring protein)-regulated, osmoresponsive proVWX operon of Escherichia coli at different osmolarities and provide in vivo evidence for transcription regulation by NAP-mediated chromosome re-modelling in bacteria. By consolidating our in vivo investigations with earlier in vitro and in silico studies that provide mechanistic details of how H-NS re-models DNA in response to osmolarity, we report that activation of proVWX in response to a hyperosmotic shock involves the destabilization of H-NS-mediated bridges anchored between the proVWX downstream and upstream regulatory elements (DRE and URE), and between the DRE and ygaY that lies immediately downstream of proVWX . The re-establishment of these bridges upon adaptation to hyperosmolarity represses the operon. Our results also reveal additional structural features associated with changes in proVWX transcript levels such as the decompaction of local chromatin upstream of the operon, highlighting that further complexity underlies the regulation of this model operon. H-NS and H-NS-like proteins are wide-spread amongst bacteria, suggesting that chromosome re-modelling may be a typical feature of transcriptional control in bacteria. Here, the authors use the proVWX operon of Escherichia coli as a model system to show how the nucleoid associated protein H-NS regulates gene expression in vivo by local chromatin remodelling.

Bacterial chromosomes are folded to compact DNA and facilitate cellular processes. Studying model bacteria has revealed aspects of chromosome folding that are applicable to many species. Primarily controlled by nucleoid-associated proteins, chromosome folding is hierarchical, from large-scale macrodomains to smaller-scale structures that influence DNA transactions, including replication and transcription. Here we review the environmentally regulated, architectural and regulatory roles of nucleoid-associated proteins and the implications for bacterial cell biology. We also highlight similarities and differences in the chromosome folding mechanisms of bacteria and eukaryotes.Advances in sequencing- and imaging-based techniques for chromosome structure analysis have led to a mature understanding of bacterial chromosome structure and dynamics. In this Review, Dame, Rashid and Grainger discuss the hierarchical nature of bacterial chromosome structure and how it is influenced by diverse types of nucleoid-associated proteins. Furthermore, they describe roles for nucleoid-associated proteins and chromosome structure, including in gene expression, chromosome segregation and cell cycle regulation.

Transcription is a vital cellular process in which RNA polymerase produces messenger RNAs (mRNA) from a DNA template. Many transcription systems have been developed to reproduce this process in vitro and in the confined environment of giant unilamellar vesicles (GUVs). However, these systems and the studies employing them often use DNA concentrations significantly higher than those found in natural cells, which typically contain a single DNA copy. In this work, we introduce single-molecule in vitro transcription (smIVT) that overcomes this limitation, enabling the visualization and tracking of individual mRNA molecules both in solution and within GUVs. We achieved this by employing a molecular beacon-a quenched RNA or DNA hairpin probe that becomes fluorescent after binding to the transcript mRNA-and template DNA encoding 32 repeats of the molecular beacon binding sequence. With these, we ensured a signal-to-noise ratio sufficient to detect single mRNAs. We use smIVT to compare the performance of various commercially-available in vitro transcription kits, and to quantitatively describe single molecule transcription inside GUVs. We establish smIVT as a remarkable technique for scrutinizing in vitro transcription with single-molecule resolution.

Nucleoid associated proteins (NAPs) maintain the architecture of bacterial chromosomes and regulate gene expression. Thus, their role as transcription factors may involve three-dimensional chromosome re-organisation. While this model is supported by in vitro studies, direct in vivo evidence is lacking. Here, we use RT-qPCR and 3C-qPCR to study the transcriptional and architectural profiles of the H-NS-regulated, osmoresponsive proVWX operon of Escherichia coli at different osmolarities and provide in vivo evidence for transcription regulation by NAP-mediated chromosome re-modelling in bacteria. We show that activation of proVWX in response to a hyperosmotic shock involves the destabilization of H-NS-mediated bridges anchored between the proVWX downstream and upstream regulatory elements (DRE and URE), and between the DRE and ygaY that lies immediately downstream of proVWX. The re-establishment of these bridges upon adaptation to hyperosmolarity represses the operon. H-NS and H-NS-like proteins are wide-spread amongst bacteria, suggesting that chromosome re-modelling may be a typical feature of transcriptional control in bacteria.