Summary We identified interactions between the conserved bacterial replication initiator and transcription factor DnaA and the nucleoid-associated protein Rok of Bacillus subtilis. DnaA binds directly to clusters of DnaA boxes at the origin of replication and elsewhere, including the promoters of several DnaA-regulated genes. Rok, an analog of H-NS from gamma-proteobacteria that affects chromosome architecture and Lsr2 from Mycobacteria, binds A+T-rich sequences throughout the genome and represses expression of many genes. Using crosslinking and immunoprecipitation followed by deep sequencing (ChIP-seq), we found that DnaA was associated with eight previously identified regions containing clusters of DnaA boxes, plus 36 additional regions that were also bound by Rok. Association of DnaA with these additional regions appeared to be indirect as it was dependent on Rok and independent of the DNA binding domain of DnaA. Gene expression and mutant analyses support a model in which DnaA and Rok cooperate to repress transcription of yxaJ, the yybNM operon, and the sunA-bdbB operon. Our results indicate that DnaA modulates the activity of Rok. We postulate that this interaction might affect nucleoid architecture. Furthermore, DnaA might interact similarly with Rok analogues in other organisms.

Methylation of cytosines is a prototypic epigenetic modification of the DNA. It has been implicated in various regulatory mechanisms across the animal kingdom and particularly in vertebrates. We mapped DNA methylation in 580 animal species (535 vertebrates, 45 invertebrates), resulting in 2443 genome-scale DNA methylation profiles of multiple organs. Bioinformatic analysis of this large dataset quantified the association of DNA methylation with the underlying genomic DNA sequence throughout vertebrate evolution. We observed a broadly conserved link with two major transitions—once in the first vertebrates and again with the emergence of reptiles. Cross-species comparisons focusing on individual organs supported a deeply conserved association of DNA methylation with tissue type, and cross-mapping analysis of DNA methylation at gene promoters revealed evolutionary changes for orthologous genes. In summary, this study establishes a large resource of vertebrate and invertebrate DNA methylomes, it showcases the power of reference-free epigenome analysis in species for which no reference genomes are available, and it contributes an epigenetic perspective to the study of vertebrate evolution.

SummarySignal transduction proteins are often multi-domain proteins that arose through the fusion of previously independent proteins. How such a change in the spatial arrangement of proteins impacts their evolution and the selective pressures acting on individual residues is largely unknown. We explored this problem in the context of bacterial two-component signalling pathways, which typically involve a sensor histidine kinase that specifically phosphorylates a single cognate response regulator. Although usually found as separate proteins, these proteins are sometimes fused into a so-called hybrid histidine kinase. Here, we demonstrate that the isolated kinase domains of hybrid kinases exhibit a dramatic reduction in phosphotransfer specificity in vitro relative to canonical histidine kinases. However, hybrid kinases phosphotransfer almost exclusively to their covalently attached response regulator domain, whose effective concentration exceeds that of all soluble response regulators. These findings indicate that the fused response regulator in a hybrid kinase normally prevents detrimental cross-talk between pathways. More generally, our results shed light on how the spatial properties of signalling pathways can significantly affect their evolution, with additional implications for the design of synthetic signalling systems.

The four named species of Branchipolynoe all live symbiotically in mytilid mussels (Bathymodiolus) that occur at hydrothermal vents or methane seeps. Analyses using mitochondrial (COI and 16S) and nuclear (ITS) genes, as well as morphology, were conducted on a collection of Branchipolynoe from Pacific Costa Rican methane seeps and West Pacific hydrothermal vents. This revealed five new species of Branchipolynoe, and these are formally described. The new species from Costa Rica live in three species of Bathymodiolus mussels (also new) at depths ranging from 1000 to 1800 m. Branchipolynoe kajsae n. sp. and Branchipolynoe halliseyae n. sp. were found in all three undescribed Bathymodiolus species, while Branchipolynoe eliseae n. sp. was found in Bathymodiolus spp. 1 and 2, and Branchipolynoe meridae n. sp. was found in Bathymodiolus spp. 1 and 3. Hence, Bathymodiolus sp. 1 hosted all four of the new species, while the other two Bathymodiolus hosted three each. Most mussels contained only one specimen of Branchipolynoe; where there was more than one, these were often a female and smaller male of the same species. The newly discovered species from the West Pacific, Branchipolynoe tjiasmantoi n. sp., lives in unidentified Bathymodiolus at depths ranging from 674 to 2657 m from hydrothermal vents in the North Fiji (Fiji) and Lau Basins (Tonga) and also from New Zealand, Vanuatu, and the Manus Basin (Papua New Guinea). The phylogenetic and biogeographical implications of this diversity of Branchipolynoe are discussed.