Bacterial chromatin organization by H-NS protein unravelled using dual DNA manipulation

Abstract

Both prokaryotic and eukaryotic organisms contain DNA bridging proteins, which can have regulatory or architectural functions. The molecular and mechanical details of such proteins are hard to obtain, in particular if they involve non-specific interactions. The bacterial nucleoid consists of hundreds of DNA loops, shaped in part by non-specific DNA bridging proteins such as histone-like nucleoid structuring protein (H-NS), leucine-responsive regulatory protein (Lrp) and SMC (structural maintenance of chromosomes) proteins. We have developed an optical tweezers instrument that can independently handle two DNA molecules, which allows the systematic investigation of protein-mediated DNA-DNA interactions. Here we use this technique to investigate the abundant non-specific nucleoid-associated protein H-NS, and show that H-NS is dynamically organized between two DNA molecules in register with their helical pitch. Our optical tweezers also allow us to carry out dynamic force spectroscopy on non-specific DNA binding proteins and thereby to determine an energy landscape for the H-NS-DNA interaction. Our results explain how the bacterial nucleoid can be effectively compacted and organized, but be dynamic in nature and accessible to DNA-tracking motor enzymes. Finally, our experimental approach is widely applicable to other DNA bridging proteins, as well as to complex DNA interactions involving multiple DNA molecules.

Lsr2 is a nucleoid-associated protein that targets AT-rich sequences and virulence genes inMycobacterium tuberculosis

“…H-NS is responsible for binding and repressing >400 genes in Salmonella (4, 7) and in Escherichia coli (8,9), many of which are DNA sequences obtained through horizontal gene transfer and involved in adaptive stress responses and virulence (10). Numerous phenotypes associated with hns mutations have been described, and the effects of H-NS on gene expression are largely inhibitory (2,11), which is partially explained by the ability of H-NS to bridge adjacent helices of DNA (12,13), causing either the trapping or the occlusion of RNA polymerase in the promoter regions (2,14). H-NS homologs are widespread in the Gram-negative α-, β-, and γ-proteobacteria but have not been identified in Gram-positive bacteria or in any other groups of bacteria, leaving it unclear as to how these bacterial species regulate the genes that they obtain through genetic exchange.…”

The DNA static curvature has a role in the regulation of the ompS1 porin gene in Salmonella enterica serovar Typhi

“…7A), where the role of the curvature would be to facilitate the formation of DNA-H-NS-DNA bridges between downstream and upstream sites. Chromatin organization by loop domain formation conducive to DNA bridging has been discussed previously in detail (Dame et al, 2005(Dame et al, , 2006Noom et al, 2007;Dorman & Kane, 2009). In the Fmt fragments, where the curvature has been lowered by two point mutations, H-NS would still be binding, albeit at lower affinity, and could still cause some repression of expression by the formation of a nucleofilament-type structure (Figs 5B, C and 7B).…”

Anti-silencing: overcoming H-NS-mediated repression of transcription in Gram-negative enteric bacteria

“…H-NS forms at least dimers in solution and these have the ability to create DNA-protein-DNA bridges both between separate DNA molecules and between different portions of the same DNA molecule (Fig. 1a) (Dame et al, 2005(Dame et al, , 2006Dorman, 2007a;Noom et al, 2007). H-NS has also been implicated in closing the looped domains that form an important part of the higher organization of the bacterial chromosome (Noom et al, 2007).…”

“…The result has been described as H-NSmediated transcriptional silencing (Bouffartigues et al, 2007;Fang & Rimsky, 2008;Göransson et al, 1990;Lang et al, 2007;Lucchini et al, 2006;Madhusudan et al, 2005;McGovern et al, 1994;Murphree et al, 1997;Navarre et al, 2006;Nye et al, 2000;Petersen et al, 2002;Westermark et al, 2000;Will et al, 2004). It has been estimated from single-molecule studies using optical tweezers that the force required to disrupt an H-NS-DNA bridge is 7 pN at an unzipping rate of 70 bp s 21 , which is the speed of RNA polymerase; RNA polymerase can exert a force of up to 25 pN (Dame et al, 2006). Perhaps the H-NS-DNA bridge is strong enough to contain a stationary RNA polymerase (Fig.…”

“…It seems likely that this sequence, or related sequences, can form nucleation sites from which the H-NS protein can spread laterally along DNA, forming H-NS filaments and possibly DNA-H-NS-DNA bridges (Lang et al, 2007;Rimsky et al, 2001). Currently our impressions of H-NS behaviour following binding at nucleation sites remain somewhat speculative as they are derived from data obtained in highly artificial in vitro single-molecule studies carried out in flow chambers using optical tweezers (Dame et al, 2006). ChIP-on-chip studies have shown that H-NS binds to the A+T-rich portions of the genomes of Salmonella Typhimurium (Lucchini et al, 2006;Navarre et al, 2006) and E. coli (Grainger et al, 2006;Oshima et al, 2006).…”