Probing the physical properties of intact bacterial chromosomes

Biophysical tools are increasingly providing key insights into understanding genome organisation in bacterial cells. We demonstrated the use of two complementary techniques, using manipulation by optical tweezers and high-throughput resistive pulse based sensing in micro-capillaries, to size individual bacterial genomes in a label-free manner.

HFSP Young Investigator Grant holders Marco Cosentino Lagomarsino, Pietro Cicuta, and Bianca Sclavi and colleagues
authored on Thu, 15 May 2014

One of the fundamental facts of biology is that prokaryotes such as bacteria differ from their eukaryotic cousins due to the lack of sub-cellular compartmentalisation. Prokaryotes do not organise their genome into a nucleus-like organelle. However, the lack of compartmentalisation does not automatically imply a lack of organisation. The spatial organisation of the bacterial genome is arguably (at least in some aspects) more complex than in eukaryotes. It is now clear that an important class of proteins contribute to organising the bacterial genome in a structured complex known as the nucleoid. These proteins are known as Nucleoid Associated Proteins (NAPs). The structural organization of the nucleoid is thought to play a role in mediating the cellular response to changing environmental cues. The important role of NAPs in maintaining this organization can be gauged from the fact that when the cells are lysed and nucleoids released, the NAPs dissociate from the nucleoid very slowly and the nucleoids retain part of their compactness over a timescale of tens of minutes.

In our HFSP project, we seek to approach the physical organization of the nucleoid and its link to physiological changes with new methods. Although the role of NAPs in structuring the bacterial nucleoid has been studied using biophysical methods, most of these are unsuited to high-throughput lab-on-chip approaches. In this study, we demonstrate a novel approach that combines a steerable optical trap with micro-capillary coulter counter experiments. Intact nucleoids are extracted from bacterial cells at different growth stages. A characteristic ionic current signature detects the passage of many hundreds of individual nucleoids into a glass capillary with a micron-sized pore. The characteristics of this signature can also be used to infer nucleoid size. Using optical tweezers, individual nucleoids are dragged through a fluid of known viscosity to determine their hydrodynamic radii and calibrate the size measurement. Using these complementary techniques, we are now able to study changes in global nucleoid organisation at both the population and the single nucleoid level.

The technique can be applied to study any perturbation of the genome. As a first test, we chose to consider the Histone-like Nucleoid Structuring (H-NS) protein, which has been demonstrated both in vivo and in vitro to affect nucleoid organization. We studied the growth phase dependent differences in size between nucleoids extracted from wild type (WT) and a strain of E. coli cells lacking the H-NS protein (Δhns). We effectively capture the increase in nucleoid size as a result of deletion of the NAP H-NS on E. coli cells entering stationary phase – a result consistent with the known biological role of H-NS. The microcapillary experiments also reveal the relevant biophysical feature that nucleoids behave as solid colloids and are impermeable to ionic flow.

Reference

Bacterial nucleoid structure probed by active drag and resistive pulse sensing of purified genomes. V. V. Thacker*, K. Bromek*, B. Meijer, J. Kotar, B. Sclavi, M. Cosentino Lagomarsino, U. F. Keyser and P. Cicuta.  (*equal contribution). Integrative Biology, 6, 184-191, 2014.

Pubmed link

Link to abstract and paper