古菌ESCRT系统研究进展

内体分拣转运复合体（ESCRT,endosomal sorting complex required for transport）曾被认为是真核生物特有的系统,涉及膜重塑、泛素化蛋白质分拣等重要细胞生命过程。近年的研究显示,TACK（包括Thaumarchaeota、Aigarchaeota、Crenarchaeota和Korarchaeota门）古菌超门中存在着一类与分泌膜囊泡、古菌病毒出胞以及细胞分裂过程等膜重塑过程相关的细胞分裂（Cdv,cell division）系统,该系统中的CdvB和CdvC是真核生物ESCRT-III和Vps4的同源蛋白,提示真核生物ESCRT系统可能起源自古菌。然而,由于TACK古菌中缺少真核生物ESCRT系统的其他关键成分,这一假设仍有争议。最近发现的阿斯加德（Asgard）古菌是一类被认为与真核生物最近缘的古菌,其基因组具有较完整的ESCRT相关蛋白的编码基因,提示真核生物的ESCRT很可能起源于阿斯加德古菌。本文首先简要介绍真核生物ESCRT系统的组成及生物学功能,然后分别总结TACK古菌的Cdv系统和阿斯加德古菌的ESCRT系统的研究进展,重点讨论它们的组成及生物学功能,为进一步了解古菌ESCRT系统与真核生物起源的关系提供参考。     

Crenarchaeal CdvA forms double-helical filaments containing DNA and interacts with ESCRT-III-like CdvB

Background

The phylum Crenarchaeota lacks the FtsZ cell division hallmark of bacteria and employs instead Cdv proteins. While CdvB and CdvC are homologues of the eukaryotic ESCRT-III and Vps4 proteins, implicated in membrane fission processes during multivesicular body biogenesis, cytokinesis and budding of some enveloped viruses, little is known about the structure and function of CdvA. Here, we report the biochemical and biophysical characterization of the three Cdv proteins from the hyperthermophilic archaeon Metallospherae sedula.

Methodology/Principal Findings

Using sucrose density gradient ultracentrifugation and negative staining electron microscopy, we evidenced for the first time that CdvA forms polymers in association with DNA, similar to known bacterial DNA partitioning proteins. We also observed that, in contrast to full-lengh CdvB that was purified as a monodisperse protein, the C-terminally deleted CdvB construct forms filamentous polymers, a phenomenon previously observed with eukaryotic ESCRT-III proteins. Based on size exclusion chromatography data combined with detection by multi-angle laser light scattering analysis, we demonstrated that CdvC assembles, in a nucleotide-independent way, as homopolymers resembling dodecamers and endowed with ATPase activity in vitro. The interactions between these putative cell division partners were further explored. Thus, besides confirming the previous observations that CdvB interacts with both CdvA and CdvC, our data demonstrate that CdvA/CdvB and CdvC/CdvB interactions are not mutually exclusive.

Conclusions/Significance

Our data reinforce the concept that Cdv proteins are closely related to the eukaryotic ESCRT-III counterparts and suggest that the organization of the ESCRT-III machinery at the Crenarchaeal cell division septum is organized by CdvA an ancient cytoskeleton protein that might help to coordinate genome segregation.     

FtsZ-less cell division in archaea and bacteria

A dedicated cell division machinery is needed for efficient proliferation of an organism. The eukaryotic actin-myosin based mechanism and the bacterial FtsZ-dependent machinery have both been characterized in detail, and a third division mechanism, the Cdv system, was recently discovered in archaea from the Crenarchaeota phylum. Despite these findings, division mechanisms remain to be identified in, for example, organisms belonging to the bacterial PVC superphylum, bacteria with extremely reduced genomes, wall-less archaea and bacteria, and in archaea that carry out the division process without cell constriction. Cytokinesis mechanisms in these clades and individual taxa are likely to include adaptation of host functions to division of bacterial symbionts, transfer of bacterial division genes into the host genome, vesicle formation without a dedicated constriction machinery, cross-wall formation without invagination, as well as entirely novel division mechanisms.     

Split decision: a thaumarchaeon encoding both FtsZ and Cdv cell division proteins chooses Cdv for cytokinesis

Cytoskeletal proteins play a pivotal role in cytokinesis in prokaryotes and eukaryotes. Most bacteria and a major branch of the archaea called the Euryarchaeota harbour a tubulin homologue, FtsZ, which assembles into a dynamic polymeric ring structure required for cytokinesis. However, Crenarchaeota, another branch of the archaea, lack FtsZ and instead use Cdv proteins, which are homologues of the ESCRT-III-like system involved in vesicular sorting and cytokinesis in eukaryotes, for cell division. Recently, a group of Crenarchaeota that grow in non-extreme environments was found to be sufficiently divergent to warrant its own branch of the archaea called the Thaumarchaeota. Notably, Thaumarchaeota have both Cdv and FtsZ homologues, which begs the question of which system is used for cell division. In this issue of Molecular Microbiology,Pelve et al. (2011) Pelve and colleagues tackle this question. They found that cells of the thaumarchaeon Nitrosopumilus maritimus likely divide using the Cdv system and not FtsZ, based on localization of Cdv proteins but not FtsZ to division sites. The authors also provide evidence that the cell cycle during growth of N. maritimus differs significantly from those of other archaea.     

Deletion of cdvB paralogous genes of Sulfolobus acidocaldarius impairs cell division

The majority of Crenarchaeota utilize the cell division system (Cdv) to divide. This system consists of three highly conserved genes, cdvA, cdvB and cdvC that are organized in an operon. CdvC is homologous to the AAA-type ATPase Vps4, involved in multivesicular body biogenesis in eukaryotes. CdvA is a unique archaeal protein that interacts with the membrane, while CdvB is homologous to the eukaryal Vps24 and forms helical filaments. Most Crenarcheota contain additional CdvB paralogs. In Sulfolobus acidocaldarius these are termed CdvB1–3. We have used a gene inactivation approach to determine the impact of these additional cdvB genes on cell division. Independent deletion mutants of these genes were analyzed for growth and protein localization. One of the deletion strains (ΔcdvB3) showed a severe growth defect on plates and delayed growth on liquid medium. It showed the formation of enlarged cells and a defect in DNA segregation. Since these defects are accompanied with an aberrant localization of CdvA and CdvB, we conclude that CdvB3 fulfills an important accessory role in cell division.     

Cdv-based cell division and cell cycle organization in the thaumarchaeon Nitrosopumilus maritimus

Cell division is mediated by different mechanisms in different evolutionary lineages. While bacteria and euryarchaea utilize an FtsZ-based mechanism, most crenarchaea divide using the Cdv system, related to the eukaryotic ESCRT-III machinery. Intriguingly, thaumarchaeal genomes encode both FtsZ and Cdv protein homologues, raising the question of their division mode. Here, we provide evidence indicating that Cdv is the primary division system in the thaumarchaeon Nitrosopumilus maritimus. We also show that the cell cycle is differently organized as compared to hyperthermophilic crenarchaea, with a longer pre-replication phase and a shorter post-replication stage. In particular, the time required for chromosome replication is remarkably extensive, 15-18 h, indicating a low replication rate. Further, replication did not continue to termination in a significant fraction of N. maritimus cell populations following substrate depletion. Both the low replication speed and the propensity for replication arrest are likely to represent adaptations to extremely oligotrophic environments. The results demonstrate that thaumarchaea, crenarchaea and euryarchaea display differences not only regarding phylogenetic affiliations and gene content, but also in fundamental cellular and physiological characteristics. The findings also have implications for evolutionary issues concerning the last archaeal common ancestor and the relationship between archaea and eukaryotes.     

ZipN, an FtsA-like orchestrator of divisome assembly in the model cyanobacterium Synechocystis PCC6803

We pursued the characterization of the divisome of the spherical-celled cyanobacterium Synechocystis PCC6803, through deletion, site-directed mutagenesis, GFP tagging, two-hybrid and co-immunoprecipitation assays. We presently report that the DivIVA-like protein Cdv3 is essential to both cell growth and division, whereas the AmiC, AmpH, FtsE, FtsN, SpoIID, YlmD, YlmE and YlmG proteins are dispensable. With the exception of the self-interacting protein YlmD, none of these dispensable factors appeared to interact with ZipN, the crucial cytokinetic factor we previously characterized. By contrast, we found that ZipN interacts with itself and the self-interacting protein Cdv3, as well as with all other crucial cytokinetic factors we previously characterized, namely: FtsZ, FtsI, FtsQ, SepF and ZipS. We also identified ZipN amino acids selectively involved in ZipN interaction with one of its following partners, Cdv3, FtsQ or SepF. Finally, we found no direct interaction between Cdv3, SepF and ZipS. Collectively, these results indicate that ZipN is a central player of divisome assembly in cyanobacteria, similarly to the FtsA protein of E. coli that is absent in cyanobacteria and chloroplast.     

Division plane placement in pleomorphic archaea is dynamically coupled to cell shape

One mechanism for achieving accurate placement of the cell division machinery is via Turing patterns, where nonlinear molecular interactions spontaneously produce spatiotemporal concentration gradients. The resulting patterns are dictated by cell shape. For example, the Min system of Escherichia coli shows spatiotemporal oscillation between cell poles, leaving a mid‐cell zone for division. The universality of pattern‐forming mechanisms in divisome placement is currently unclear. We examined the location of the division plane in two pleomorphic archaea, Haloferax volcanii and Haloarcula japonica, and showed that it correlates with the predictions of Turing patterning. Time‐lapse analysis of H. volcanii shows that divisome locations after successive rounds of division are dynamically determined by daughter cell shape. For H. volcanii, we show that the location of DNA does not influence division plane location, ruling out nucleoid occlusion. Triangular cells provide a stringent test for Turing patterning, where there is a bifurcation in division plane orientation. For the two archaea examined, most triangular cells divide as predicted by a Turing mechanism; however, in some cases multiple division planes are observed resulting in cells dividing into three viable progeny. Our results suggest that the division site placement is consistent with a Turing patterning system in these archaea.     

Starch division and partitioning. A mechanism for granule propagation and maintenance in the picophytoplanktonic green alga Ostreococcus tauri

Whereas Glc is stored in small-sized hydrosoluble glycogen particles in archaea, eubacteria, fungi, and animal cells, photosynthetic eukaryotes have resorted to building starch, which is composed of several distinct polysaccharide fractions packed into a highly organized semicrystalline granule. In plants, both the initiation of polysaccharide synthesis and the nucleation mechanism leading to formation of new starch granules are currently not understood. Ostreococcus tauri, a unicellular green alga of the Prasinophyceae family, defines the tiniest eukaryote with one of the smallest genomes. We show that it accumulates a single starch granule at the chloroplast center by using the same pathway as higher plants. At the time of plastid division, we observe elongation of the starch and division into two daughter structures that are partitioned in each newly formed chloroplast. These observations suggest that in this system the information required to initiate crystalline polysaccharide growth of a new granule is contained within the preexisting polysaccharide structure and the design of the plastid division machinery.     

Coordination of cell division and chromosome segregation by a nucleoid occlusion protein in Bacillus subtilis

A range of genetical and physiological experiments have established that diverse bacterial cells possess a function called nucleoid occlusion, which acts to prevent cell division in the vicinity of the nucleoid. We have identified a specific effector of nucleoid occlusion in Bacillus subtilis, Noc (YyaA), as an inhibitor of division that is also a nonspecific DNA binding protein. Under various conditions in which the cell cycle is perturbed, Noc prevents the division machinery from assembling in the vicinity of the nucleoid. Unexpectedly, cells lacking both Noc and the Min system (which prevents division close to the cell poles) are blocked for division, apparently because they establish multiple nonproductive accumulations of division proteins. The results help to explain how B. subtilis specifies the division site under a range of conditions and how it avoids catastrophic breakage of the chromosome by division through the nucleoid.     

The Structure, Function, and Regulation of Mycobacterium FtsZ

FtsZ is a widely distributed major cytoskeletal protein involved in the archaea and bacteria cell division. It is the most critical component in the division machinery and similar to tubulin in structure and function. Four major roles of FtsZ have been characterized: cell elongation, GTPase, cell division, and bacterial cytoskeleton. FtsZ subunits can be assembled into protofilaments. Mycobacteria consist of a large family of medical and environmental important bacteria, such as M. leprae, M. tuberculosis, the pathogen of leprosy, and tuberculosis. Structure, function, and regulation of mycobacteria FtsZ are summarized here, together with the implication of FtsZ as potential novel drug target for anti-tuberculosis therapeutics.     

Insights into archaeal chaperone machinery: a network-based approach

Molecular chaperones are a diverse group of proteins that ensure proteome integrity by helping the proteins fold correctly and maintain their native state, thus preventing their misfolding and subsequent aggregation. The chaperone machinery of archaeal organisms has been thought to closely resemble that found in humans, at least in terms of constituent players. Very few studies have been ventured into system-level analysis of chaperones and their functioning in archaeal cells. In this study, we attempted such an analysis of chaperone-assisted protein folding in archaeal organisms through network approach using Picrophilus torridus as model system. The study revealed that DnaK protein of Hsp70 system acts as hub in protein-protein interaction network. However, DnaK protein was present only in a subset of archaeal organisms and absent from many archaea, especially members of Crenarchaeota phylum. Therefore, a similar network was created for another archaeal organism, Sulfolobus solfataricus, a member of Crenarchaeota. The chaperone network of S. solfataricus suggested that thermosomes played an integral part of hub proteins in archaeal organisms, where DnaK was absent. We further compared the chaperone network of archaea with that found in eukaryotic systems, by creating a similar network for Homo sapiens. In the human chaperone network, the UBC protein, a part of ubiquitination system, was the most important module, and interestingly, this system is known to be absent in archaeal organisms. Comprehensive comparison of these networks leads to several interesting conclusions regarding similarities and differences within archaeal chaperone machinery in comparison to humans.     

Cryo-EM structure of the archaeal 50S ribosomal subunit in complex with initiation factor 6 and implications for ribosome evolution

Translation of mRNA into proteins by the ribosome is universally conserved in all cellular life. The composition and complexity of the translation machinery differ markedly between the three domains of life. Organisms from the domain Archaea show an intermediate level of complexity, sharing several additional components of the translation machinery with eukaryotes that are absent in bacteria. One of these translation factors is initiation factor 6 (IF6), which associates with the large ribosomal subunit. We have reconstructed the 50S ribosomal subunit from the archaeon Methanothermobacter thermautotrophicus in complex with archaeal IF6 at 6.6?? resolution using cryo-electron microscopy (EM). The structure provides detailed architectural insights into the 50S ribosomal subunit from a methanogenic archaeon through identification of the rRNA expansion segments and ribosomal proteins that are shared between this archaeal ribosome and eukaryotic ribosomes but are mostly absent in bacteria and in some archaeal lineages. Furthermore, the structure reveals that, in spite of highly divergent evolutionary trajectories of the ribosomal particle and the acquisition of novel functions of IF6 in eukaryotes, the molecular binding of IF6 on the ribosome is conserved between eukaryotes and archaea. The structure also provides a snapshot of the reductive evolution of the archaeal ribosome and offers new insights into the evolution of the translation system in archaea.     

A novel component of the division‐site selection system of Bacillus subtilis and a new mode of action for the division inhibitor MinCD

Cell division in bacteria is governed by a complex cytokinetic machinery in which the key player is a tubulin homologue, FtsZ. Most rod‐shaped bacteria divide precisely at mid‐cell between segregated sister chromosomes. Selection of the correct site for cell division is thought to be determined by two negative regulatory systems: the nucleoid occlusion system, which prevents division in the vicinity of the chromosomes, and the Min system, which prevents inappropriate division at the cell poles. In Bacillus subtilis recruitment of the division inhibitor MinCD to cell poles depends on DivIVA, and these proteins were thought to be sufficient for Min function. We have now identified a novel component of the division‐site selection system, MinJ, which bridges DivIVA and MinD. minJ mutants are impaired in division because MinCD activity is no longer restricted to cell poles. Although MinCD was thought to act specifically on FtsZ assembly, analysis of minJ and divIVA mutants showed that their block in division occurs downstream of FtsZ. The results support a model in which the main function of the Min system lies in allowing only a single round of division per cell cycle, and that MinCD acts at multiple levels to prevent inappropriate division.     

Homologous recombinational repair of DNA ensures mammalian chromosome stability

The process of homologous recombinational repair (HRR) is a major DNA repair pathway that acts on double-strand breaks and interstrand crosslinks, and probably to a lesser extent on other kinds of DNA damage. HRR provides a mechanism for the error-free removal of damage present in DNA that has replicated (S and G2 phases). Thus, HRR acts in a critical way, in coordination with the S and G2 checkpoint machinery, to eliminate chromosomal breaks before the cell division occurs. Many of the human HRR genes, including five Rad51 paralogs, have been identified, and knockout mutants for most of these genes are available in chicken DT40 cells. In the mouse, most of the knockout mutations cause embryonic lethality. The Brca1 and Brca2 breast cancer susceptibility genes appear to be intimately involved in HRR, but the mechanistic basis is unknown. Biochemical studies with purified proteins and cell extracts, combined with cytological studies of nuclear foci, have begun to establish an outline of the steps in mammalian HRR. This pathway is subject to complex regulatory controls from the checkpoint machinery and other processes, and there is increasing evidence that loss of HRR gene function can contribute to tumor development. This review article is meant to be an update of our previous review [Biochimie 81 (1999) 87].     

CRISPR-Cas systems restrict horizontal gene transfer in Pseudomonas aeruginosa

CRISPR-Cas systems provide bacteria and archaea with an adaptive immune system that targets foreign DNA. However, the xenogenic nature of immunity provided by CRISPR-Cas raises the possibility that these systems may constrain horizontal gene transfer. Here we test this hypothesis in the opportunistic pathogen Pseudomonas aeruginosa, which has emerged as an important model system for understanding CRISPR-Cas function. Across the diversity of P. aeruginosa, active CRISPR-Cas systems are associated with smaller genomes and higher GC content, suggesting that CRISPR-Cas inhibits the acquisition of foreign DNA. Although phage is the major target of CRISPR-Cas spacers, more than 80% of isolates with an active CRISPR-Cas system have spacers that target integrative conjugative elements (ICE) or the conserved conjugative transfer machinery used by plasmids and ICE. Consistent with these results, genomes containing active CRISPR-Cas systems harbour a lower abundance of both prophage and ICE. Crucially, spacers in genomes with active CRISPR-Cas systems map to ICE and phage that are integrated into the chromosomes of closely related genomes lacking CRISPR-Cas immunity. We propose that CRISPR-Cas acts as an important constraint to horizontal gene transfer, and the evolutionary mechanisms that ensure its maintenance or drive its loss are key to the ability of this pathogen to adapt to new niches and stressors.Subject terms: Microbiology, Microbial genetics, Evolution