Probing DNA segregation in archaea: molecular dissection of an atypical tricistronic partition system from Sulfolobus

The process of genome segregation is a fundamental stage of the life cycle of each cell: the genetic content is first duplicated, then separated and equally distributed into the two daughter cells. The mechanism whereby the genetic material (that is organized into linear DNA chromosomes) is separated in cells of higher organisms (like plants, animals, humans) has been extensively studied and is well understood. In these cells the molecular machine responsible for chromosome segregation is known as 'mitotic spindle': it consists of cables, called microtubules, which are anchored to chromosomes at a specific site, known as 'centromere'. The microtubules pull sister chromosomes to opposite poles of the spindle, before the cell divides. In bacteria the picture is more elusive. The genetic patrimony of bacteria consists of a single circular (more rarely linear) chromosome and sometimes smaller circles of DNA called plasmids. Historically, it was presumed that segregation of bacterial chromosomes and plasmids was a passive process, not requiring a dedicated apparatus and perhaps involving attachment of the newly-replicated genetic elements to the growing cell wall. However, in recent years evidence has been provided pointing to the existence of an active mechanism responsible for the segregation of chromosomes and plasmids, requiring the participation of dedicated factors. In bacteria, the most well-characterized DNA segregation systems are those specified by plasmids, which are present in the cell in low numbers. These plasmids harbour their own survival kit, a segregation cassette consisting of two genes, often termed parA and parB, and a centromere-like site. This cassette ensures an accurate segregation of the plasmids from one generation to the next at cell division. The molecular mechanisms underlying this process have not been thoroughly elucidated as yet; however, recent discoveries hint at the existence of mitotic spindle-like machineries. Archaea are the third domain of life: their discovery in 1977 represented a major biological milestone. They were initially identified as a different group of organisms on the basis of sequences contained in their RNA or ribonucleic acid. Further characterization of their physiology, biochemistry and genetics has provided unequivocal evidence that they are a distinct group of organisms (different from bacteria and from higher multicellular organisms). The first archaea to be studied were all from extreme environments, but they are now known to thrive in most of the earth's ecological niches and they constitute ~20% of the biosphere. Hyperthermophilic archaea grow at 80 C and above and exhibit unusual properties, which make these organisms a valuable resource for the development of novel biotechnological processes. Industrial applications include the production of archaea-derived enzymes, which are stable at high temperature, cellulose degrading enzymes, the use of their membranes as delivery systems for drugs and genes. Despite numerous studies on fundamental biological processes in archaea, to date no information is available on the mechanism of genome segregation in these organisms. We intend to analyze this process in an archaeon called Sulfolobus NOB8H2, which has been isolated from hot springs in the island of Hokkaido, Japan. This archaeon contains a plasmid, pNOB8, which harbours a putative DNA segregation cassette comprising three genes (designated as orf44, parB, parA). The project here proposed will focus on the characterization of the factors encoded by the genes above, their function and respective role in pNOB8 partitioning at cell division and their dynamic interactions. We also intend to identify the centromere of pNOB8 and dissect the interactions between this site and ParB, ParA and perhaps Orf44. Furthermore, investigations will be conducted to probe whether the ParA protein assembles into cable-like structures as observed for some bacterial ParA counterparts.

Genome segregation is a fundamental process in all organisms: it requires the concerted action of dedicated proteins and its timing and coordination with other cellular events, like DNA replication and cell division, is crucial to maintain euploidy. The molecular mechanisms promoting accurate chromosome partitioning in eukaryotes are well characterized. Bacterial DNA segregation has been explored mainly in model organisms like Escherichia coli, Bacillus subtilis and Caulobacter crescentus. Biochemical, structural and cell biology investigations have shed light on a growing number of cytoskeletal elements responsible for the segregation of chromosome and plamids in bacteria. In contrast with eukarya and bacteria, to date the mechanisms and dynamics of genome partitioning are entirely underexplored in archaea. The atypical orf44-parB-parA module of Sulfolobus NOB8H2 provides a particularly good platform to initiate a survey of DNA segregation in archaea.The three genes partially overlap, which suggests that they might be part of a single transcriptional unit. The products encoded by parB and parA genes exhibit homology, respectively, to the ParB and ParA superfamilies of bacterial DNA partition proteins, whereas Orf44 shares similarity with bacterial repressors of the ArsR family. The proteins encoded by this putative partition cassette are easily purified and amenable to molecular dissection. By using complementary approaches, we intend to elucidate the molecular mechanism underlying the segregational stability of pNOB8. The respective role played by ParB, ParA and Orf44 in plasmid partitioning at cell division and their interaction dynamics will be investigated. Parallel studies will focus on the identification of the centromere of pNOB8. A final objective is to investigate whether ParA is a motor protein capable of polymerization. These studies will provide valuable new perspectives on DNA segregation in an important and widespread group of organisms.