The expression of virulence factors essential for the invasion of host cells by Salmonella enterica is tightly controlled by a network of transcription regulators. The AraC/XylS transcription factor HilD is the main integration point of environmental signals into this regulatory network, with many factors affecting HilD activity. Long chain fatty acids (LCFAs), which are highly abundant throughout the host intestine directly bind to, and repress HilD, acting as environmental cues to coordinate virulence gene expression. The regulatory protein HilE also negatively regulates HilD activity, through a protein-protein interaction. Both of these regulators inhibit HilD dimerisation, preventing HilD from binding to target DNA. We investigated the structural basis of these mechanisms of HilD repression. LCFAs bind to a conserved pocket in HilD, in a comparable manner to that reported for other AraC/XylS regulators, whereas HilE forms a stable heterodimer with HilD by binding to the HilD dimerisation interface. Our results highlight two distinct mechanisms by which HilD activity is repressed, which could be exploited for the development of new antivirulence leads.

Histones are DNA-binding proteins that play a crucial role in DNA packaging and gene regulation in eukaryotes and archaea. In eukaryotes, histones form octamers that constitute the core of the nucleosome, the fundamental unit of chromatin. Archaeal histones, on the other hand, form tetramers that assemble into extended superhelices upon DNA binding. Although previously thought to occur only in archaea and eukaryotes, histone homologs have recently been discovered in bacteria. This work presents the dimeric crystal structure of the bacterial histone HBb from Bdellovibrio bacteriovorus determined at a resolution of 1.06 Angstrom, representing the first-ever structure of any histone protein determined at atomic resolution. Furthermore, this study shows that HBb binds DNA and is essential for bacterial viability, suggesting that bacterial histone homologs likely have a similar biological function as their eukaryotic and archaeal counterparts. These findings have important implications for our understanding of the fundamental processes of DNA organization and regulation in all domains of life.

The emergence of new proteins is a central question in biology. Most tertiary protein folds known to date appear to have an ancient origin, but it is clear from bioinformatic analyses that new proteins continuously emerge in all organismal groups. However, there is a paucity of experimental data on new proteins regarding their structure and biophysical properties. We performed a detailed phylogenetic analysis and identified 48 putative open reading frames in the honeybee-associated bacterium Apilactobacillus kunkeei for which no homologs could be identified in closely-related species, suggesting that they could be relatively new on an evolutionary time scale and represent recently evolved proteins. Using circular dichroism-, fluorescence- and nuclear magnetic resonance spectroscopy we investigated five of these proteins and show that they are not intrinsically disordered, but populate alpha-helical dominated folded states with relatively low thermodynamic stability (0-3 kcal/mol). The data demonstrate that small new proteins readily adopt simple folded conformations suggesting that more complex tertiary structures can be continuously re-invented during evolution by fusion of such simple secondary structure elements. These findings have implications for the general view on protein evolution, where de novo emergence of folded proteins may be a common event.