Pack it up, Pack it in: Unraveling H-NS Mediated Genome Packaging

How are bacterial genomes compacted? Nucleoid-associated proteins have been discovered that bind to the circular genome in either a sequence-dependent or sequence-independent manner, thereby resulting in a compact nucleoid that can be confined within the bacteria and be accessed by the cellular machinery required to transcribe the genome (1,2). Dame and co-workers performed a number of key experiments that elucidated the binding behavior of a particular nucleoid-associated protein, histone-like nucleoid structuring protein (H-NS), and its role in confining the genome. Their work provided a model for nucleoid condensation in which H-NS protein binds randomly to one strand and, upon meeting another strand of DNA, induces condensation through trans binding (3). Through optical-tweezer experiments, they were able to further characterize the kinetics and thermodynamics of this process (4). However, open questions remained as the work demonstrating condensation of DNA by H-NS was performed in two dimensions on a mica surface and neither the effect of this spatial confinement nor the nature of the DNA-surface interaction itself were fully addressed. Seeking to better understand the experimental findings of Dame et al., in their article Joyeux and Vreede (5) develop a representation of DNA and H-NS dimers consisting of bead-spring constructs. Through a careful choice of parameters, they closely reproduce the experimentally observed binding behavior of H-NS to DNA. They are able to then use this simple model to help elucidate the rich physics observed in experiments probing H-NS mediated genome condensation. In particular, their work gives insight into the nature of the H-NS dimer itself and its effect on genome condensation. In the work of Wiggins et al. (6), protein-mediated bridging is discussed in the context of two possible structural motifs, an H-NS linker domain (residues 65-89) that is either rigid or flexible. In the language of Joyeux and Vreede, this corresponds to an H-NS with a large or small value of G (the H-NS dimer bending rigidity), respectively. Coarse-grained simulations enable Joyeux and Vreede to test a hypothesis such as that of Wiggins et al. by simply assigning the dimer different physical parameters and observing the resulting change in behavior. They are able to demonstrate that a difference of ∼20% in binding affinity in the cis configuration (the result of a factor of two change in the H-NS bending rigidity) is sufficient to fundamentally change the dynamics of the condensed nucleoid, with the more flexible case resulting in a fluctuating, dynamic structure exhibiting open loops (more cis binding) whereas the less flexible case results in a more compact globular structure that changes little over time (increased trans binding). Joyeux and Vreede do not propose a precise value for the H-NS bending rigidity, as the experimental data to which they compare are difficult to interpret (primarily nucleoids condensed on a two-dimensional mica surface (3)); their work, however, is of fundamental significance in that it points the way toward experiments that might better elucidate the true nature of H-NS and its role in nucleoid condensation. Indeed the marked difference in the radius of gyration of three-dimensional condensed nucleoids reported in their work, driven by differences in cis binding affinity, represents a quantity that can be directly accessed in experiment to make progress in the problem of H-NS mediated genome condensation and the role played by H-NS flexibility and binding affinity therein. Looking forward in terms of developing molecular-level models to further understand protein-mediated genome compaction, the growing availability of advanced sampling techniques and reliable models provides the biophysics community with tools at every level of molecular description. The coarse-grained representation adopted by Joyeux and Vreede has provided valuable insights. Building on their results, future studies should aim to address potentially interesting details of the interaction between H-NS and DNA. Experimental data indicate that this interaction is not, in fact, entirely sequence-agnostic (as the Joyeux and Vreede representation assumes), but instead exhibits a preference for AT-rich genomic regions (7). Specifically, the minor groove width in these AT-rich regions is thought to be optimal for H-NS binding. Coarse-grain DNA (8,9) and protein (10) models have been developed that are capable of exploring such shape-dependent protein-nucleic acid interactions; approaching the problem at this scale may yield rich information regarding H-NS/DNA interactions. A particular detail that remains to be addressed is the effect of the ionic environment on the interaction of H-NS with the genome. In earlier work, Vreede and Dame used molecular simulations to demonstrate that the conformation of the dimerization domain of H-NS may indeed be sensitive to ionic conditions, with the parallel dimer increasing in stability with increasing salt (11). Experimental evidence also indicates that altering the ionic environment can fundamentally change the nature of the resulting H-NS/DNA complexes and promote either the cis or trans binding regime (12,13). Coarse-grained molecular models are uniquely suited to explore this wide range of environmental conditions and assess their impact on the mechanism of H-NS mediated nucleoid condensation. A particularly exciting idea that could now be addressed is that AT-rich sequences that promote H-NS bridging behavior act as domain barriers in bacterial genomes (1,14). Interestingly, the acquisition of genomic islands, generally AT-rich, is believed to potentially disrupt the three-dimensional structure of the nucleoid through the recruitment of H-NS proteins and decrease the fitness of the bacterial cell (14). This notion that stochastic addition of H-NS binding domains may drastically alter the organization of the genome could be directly probed in coarse-grained simulations in which sequence-specificity in H-NS binding is taken into account. In their work, Joyeux and Vreede provide a compelling example of how a simple and elegant molecular model can be used to provide profound insight into an otherwise complex biophysical problem. In addition to highlighting the large impact that small changes in DNA-protein interactions can have, their work suggests key experiments that, in due course, will provide a clearer picture of H-NS mediated genome condensation.