Stephen D Bell

While there is a considerable body of knowledge regarding the molecular and structural biology and biochemistry of archaeal information processing machineries, far less is known about the nature of the substrate for these machineries—the archaeal nucleoid. In this article, we will describe recent advances in our understanding of the three‐dimensional organization of the chromosomes of model organisms in the crenarchaeal phylum.

Proteins in the structural maintenance of chromosomes (SMC) superfamily play key roles in chromosome organization and are ubiquitous across all domains of life. However, SMC proteins are notably absent in the Desulfurococcales of phylum Crenarchaeota. Intrigued by this observation, we performed chromosome conformation capture experiments in the model Desulfurococcales species Aeropyrum pernix. As in other archaea, we observe chromosomal interaction domains across the chromosome. The boundaries between chromosomal interaction domains show a dependence on transcription and translation for their definition. Importantly, however, we reveal an additional higher-order, bipartite organization of the chromosome—with a small high-gene-expression and self-interacting domain that is defined by transcriptional activity and loop structures. Viewing these data in the context of the distribution of SMC …

A key maxim in modernist architecture is that ‘form follows function'. While modernist buildings are hopefully the product of intelligent design, the architectures of chromosomes have been sculpted by the forces of evolution over many thousands of generations. In the following, I will describe recent advances in our understanding of chromosome architecture in the archaeal domain of life. Although much remains to be learned about the mechanistic details of archaeal chromosome organization, some general principles have emerged. At the 10–100 kb level, archaeal chromosomes have a conserved local organization reminiscent of bacterial genomes. In contrast, lineage-specific innovations appear to have imposed distinct large-scale architectural features. The ultimate functions of genomes are to store and to express genetic information. Gene expression profiles have been shown to influence chromosome …

In all organisms, the DNA sequence and the structural organization of chromosomes affect gene expression. The extremely thermophilic crenarchaeon Sulfolobus has one circular chromosome with three origins of replication. We previously revealed that this chromosome has defined A and B compartments that have high and low gene expression, respectively. As well as higher levels of gene expression, the A compartment contains the origins of replication. To evaluate the impact of three-dimensional organization on genome evolution, we characterized the effect of replication origins and compartmentalization on primary sequence evolution in eleven Sulfolobus species. Using single-nucleotide polymorphism analyses, we found that distance from an origin of replication was associated with increased mutation rates in the B but not in the A compartment. The enhanced polymorphisms distal to replication origins …

Replicative DNA polymerases cannot initiate DNA synthesis de novo and rely on dedicated RNA polymerases, primases, to generate a short primer. This primer is then extended by the DNA polymerase. In diverse archaeal species, the primase has long been known to have the ability to synthesize both RNA and DNA. However, the relevance of these dual nucleic acid synthetic modes for productive primer synthesis has remained enigmatic. In the current work, we reveal that the ability of primase to polymerize DNA serves dual roles in promoting the hand-off of the primer to the replicative DNA polymerase holoenzyme. First, it creates a 5′-RNA-DNA-3′ hybrid primer which serves as an optimal substrate for elongation by the replicative DNA polymerase. Second, it promotes primer release by primase. Furthermore, modeling and experimental data indicate that primase incorporates a deoxyribonucleotide …

Across the three domains of life, B-family DNA polymerases play a variety of roles in both DNA repair and DNA replication processes. We examine the phenotypic consequences of loss of the putative repair polymerases PolB2 and/or PolB3 in the crenarchaeon Sulfolobus islandicus. We detect a modest growth advantage when cells lacking the polymerase are grown in unperturbed conditions. Further, we observe a striking insensitivity of the mutant lines to acute treatment with the oxidizing agent, hydrogen peroxide. In addition, cells lacking PolB3 show enhanced sensitivity to the DNA damaging agent 4-NQO. Our data therefore suggest that these non-essential DNA polymerases may influence DNA repair pathway choice in these hyperthermophilic aerobes.

Chromosome conformation capture (3C) technologies have identified topologically associating domains (TADs) and larger A/B compartments as two salient structural features of eukaryotic chromosomes. These structures are sculpted by the combined actions of transcription and structural maintenance of chromosomes (SMC) superfamily proteins. Bacterial chromosomes fold into TAD-like chromosomal interaction domains (CIDs) but do not display A/B compartment-type organization. We reveal that chromosomes of Sulfolobus archaea are organized into CID-like topological domains in addition to previously described larger A/B compartment-type structures. We uncover local rules governing the identity of the topological domains and their boundaries. We also identify long-range loop structures and provide evidence of a hub-like structure that colocalizes genes involved in ribosome biogenesis. In addition to …

Chromosome organization in archaea has long been enigmatic due, in part, to the typically small cell size of archaea and the extremophilic nature of many of the model archaeal species studies, rendering live-cell imaging technically challenging. To circumvent these problems, we recently applied chromosome conformation capture combined with biotin enrichment and deep sequencing (Hi-C) to members of hyperthermophilic archaeal genus Sulfolobus. Our optimized Hi-C protocol described here permits delineation of how Sulfolobus species organize their chromosomes.For complete details on the use and execution of this protocol, please refer to Takemata et al. (2019).

Chromosome conformation capture (3C) techniques are emerging as promising approaches to study genome organization in Archaea, the least understood domain of life in terms of chromosome biology. Here, we describe a 3C technique combined with deep sequencing for the hyperthermophilic archaeal genus Sulfolobus. Instead of using restriction enzymes compatible with fill-in labeling, this protocol uses the 4-bp blunt cutter AluI to generate high-resolution (up to 2 kb) contact maps from Sulfolobus species.For complete details on the use and execution of this protocol, please refer to Takemata and Bell (2021).

It is now well recognized that the information processing machineries of archaea are far more closely related to those of eukaryotes than to those of their prokaryotic cousins, the bacteria. Extensive studies have been performed on the structure and function of the archaeal DNA replication origins, the proteins that define them, and the macromolecular assemblies that drive DNA unwinding and nascent strand synthesis. The results from various archaeal organisms across the archaeal domain of life show surprising levels of diversity at many levels—ranging from cell cycle organization to chromosome ploidy to replication mode and nature of the replicative polymerases. In the following, we describe recent advances in the field, highlighting conserved features and lineage-specific innovations.

Over the past decade, advances in methodologies for the determination of chromosome conformation have provided remarkable insight into the local and higher-order organization of bacterial and eukaryotic chromosomes. Locally folded domains are found in both bacterial and eukaryotic genomes, although they vary in size. Importantly, genomes of metazoans also possess higher-order organization into A- and B-type compartments, regions of transcriptionally active and inactive chromatin, respectively. Until recently, nothing was known about the organization of genomes of organisms in the third domain of life – the archaea. However, despite archaea possessing simple circular genomes that are morphologically reminiscent of those seen in many bacteria, a recent study of archaea of the genus Sulfolobus has revealed that it organizes its genome into large-scale domains. These domains further interact to …

The 822 eucalypt species (Angophora, Corymbia, Eucalyptus) within Australia were assessed using IUCN Red List Categories and Criteria. Overall, 193 (23%) eucalypts qualified as threatened and 36 were considered Data Deficient. One hundred and thirty-four threatened species qualified under criterion A2, representing a past and irreversible population decline of >30%. The remainder were narrow-range species with ongoing threats (mostly mining or urbanisation), or naturally rare. Habitat conversion to crops and pastures was the cause of decline for most threatened eucalypts. Threatened species were concentrated where deforestation and high eucalypt richness coincide, especially south-western Western Australia. Corymbia or Angophora species, and relatively few tropical eucalypts are threatened. Fire, timber harvesting and disease were rarely sufficient threats to eucalypts to warrant a threatened status …