Diving into the complexity of the spirochetal endoflagellum.

Spirochaetes, a phylum that includes medically important pathogens such as the causative agents of Lyme disease, syphilis, and leptospirosis, are in many ways highly unique bacteria. Their cell morphology, subcellular organization, and metabolism reveal atypical features. Spirochetal motility is also singular, dependent on the presence of periplasmic flagella or endoflagella, inserted subterminally at cell poles and not penetrating the outer membrane and elongating outside the cell as in enterobacteria. In this review we present a comprehensive comparative genomics analysis of endoflagellar systems in spirochetes, highlighting recent findings on the flagellar basal body and filament. Continued progress in understanding the function and architecture of spirochetal flagella is uncovering paradigm-shifting mechanisms of bacterial motility.

3D cryo-EM imaging of bacterial flagella: Novel structural and mechanistic insights into cell motility.

Bacterial flagella are nanomachines that enable cells to move at high speeds. Comprising 25 and more different types of proteins, the flagellum is a large supramolecular assembly organized into three widely conserved substructures: a basal body including the rotary motor, a connecting hook, and a long filament. The whole flagellum from Escherichia coli weighs ∼20 MDa, without considering its filament portion, which is by itself a ∼1.6 GDa structure arranged as a multimer of ∼30,000 flagellin protomers. Breakthroughs regarding flagellar structure and function have been achieved in the last few years, mainly because of the revolutionary improvements in 3D cryo-EM methods. This review discusses novel structures and mechanistic insights derived from such high-resolution studies, advancing our understanding of each one of the three major flagellar segments. The rotation mechanism of the motor has been unveiled with unprecedented detail, showing a two-cogwheel machine propelled by a Brownian ratchet device. In addition, by imaging the flagellin-like protomers that make up the hook in its native bent configuration, their unexpected conformational plasticity challenges the paradigm of a two-state conformational rearrangement mechanism for flagellin-fold proteins. Finally, imaging of the filaments of periplasmic flagella, which endow Spirochete bacteria with their singular motility style, uncovered a strikingly asymmetric protein sheath that coats the flagellin core, challenging the view of filaments as simple homopolymeric structures that work as freely whirling whips. Further research will shed more light on the functional details of this amazing nanomachine, but our current understanding has definitely come a long way.

An asymmetric sheath controls flagellar supercoiling and motility in the leptospira spirochete.

Spirochete bacteria, including important pathogens, exhibit a distinctive means of swimming via undulations of the entire cell. Motility is powered by the rotation of supercoiled 'endoflagella' that wrap around the cell body, confined within the periplasmic space. To investigate the structural basis of flagellar supercoiling, which is critical for motility, we determined the structure of native flagellar filaments from the spirochete Leptospira by integrating high-resolution cryo-electron tomography and X-ray crystallography. We show that these filaments are coated by a highly asymmetric, multi-component sheath layer, contrasting with flagellin-only homopolymers previously observed in exoflagellated bacteria. Distinct sheath proteins localize to the filament inner and outer curvatures to define the supercoiling geometry, explaining a key functional attribute of this spirochete flagellum.

Palaeoproteomics resolves sloth relationships.

The living tree sloths Choloepus and Bradypus are the only remaining members of Folivora, a major xenarthran radiation that occupied a wide range of habitats in many parts of the western hemisphere during the Cenozoic, including both continents and the West Indies. Ancient DNA evidence has played only a minor role in folivoran systematics, as most sloths lived in places not conducive to genomic preservation. Here we utilize collagen sequence information, both separately and in combination with published mitochondrial DNA evidence, to assess the relationships of tree sloths and their extinct relatives. Results from phylogenetic analysis of these datasets differ substantially from morphology-based concepts: Choloepus groups with Mylodontidae, not Megalonychidae; Bradypus and Megalonyx pair together as megatherioids, while monophyletic Antillean sloths may be sister to all other folivorans. Divergence estimates are consistent with fossil evidence for mid-Cenozoic presence of sloths in the West Indies and an early Miocene radiation in South America.

Crystallization of FcpA from Leptospira, a novel flagellar protein that is essential for pathogenesis.

The protein FcpA is a unique component of the flagellar filament of spirochete bacteria belonging to the genus Leptospira. Although it plays an essential role in translational motility and pathogenicity, no structures of FcpA homologues are currently available in the PDB. Its three-dimensional structure will unveil the novel motility mechanisms that render pathogenic Leptospira particularly efficient at invading and disseminating within their hosts, causing leptospirosis in humans and animals. FcpA from L. interrogans was purified and crystallized, but despite laborious attempts no useful X ray diffraction data could be obtained. This challenge was solved by expressing a close orthologue from the related saprophytic species L. biflexa. Three different crystal forms were obtained: a primitive and a centred monoclinic form, as well as a hexagonal variant. All forms diffracted X-rays to suitable resolutions for crystallographic analyses, with the hexagonal type typically reaching the highest limits of 2.0 Šand better. A variation of the quick-soaking procedure resulted in an iodide derivative that was instrumental for single-wavelength anomalous diffraction methods.

Synergistic roles of climate warming and human occupation in Patagonian megafaunal extinctions during the Last Deglaciation.

The causes of Late Pleistocene megafaunal extinctions (60,000 to 11,650 years ago, hereafter 60 to 11.65 ka) remain contentious, with major phases coinciding with both human arrival and climate change around the world. The Americas provide a unique opportunity to disentangle these factors as human colonization took place over a narrow time frame (~15 to 14.6 ka) but during contrasting temperature trends across each continent. Unfortunately, limited data sets in South America have so far precluded detailed comparison. We analyze genetic and radiocarbon data from 89 and 71 Patagonian megafaunal bones, respectively, more than doubling the high-quality Pleistocene megafaunal radiocarbon data sets from the region. We identify a narrow megafaunal extinction phase 12,280 ± 110 years ago, some 1 to 3 thousand years after initial human presence in the area. Although humans arrived immediately prior to a cold phase, the Antarctic Cold Reversal stadial, megafaunal extinctions did not occur until the stadial finished and the subsequent warming phase commenced some 1 to 3 thousand years later. The increased resolution provided by the Patagonian material reveals that the sequence of climate and extinction events in North and South America were temporally inverted, but in both cases, megafaunal extinctions did not occur until human presence and climate warming coincided. Overall, metapopulation processes involving subpopulation connectivity on a continental scale appear to have been critical for megafaunal species survival of both climate change and human impacts.

Ancient mitochondrial DNA reveals convergent evolution of giant short-faced bears (Tremarctinae) in North and South America.

The Tremarctinae are a subfamily of bears endemic to the New World, including two of the largest terrestrial mammalian carnivores that have ever lived: the giant, short-faced bears Arctodus simus from North America and Arctotherium angustidens from South America (greater than or equal to 1000 kg). Arctotherium angustidens became extinct during the Early Pleistocene, whereas Arctodus simus went extinct at the very end of the Pleistocene. The only living tremarctine is the spectacled bear (Tremarctos ornatus), a largely herbivorous bear that is today only found in South America. The relationships among the spectacled bears (Tremarctos), South American short-faced bears (Arctotherium) and North American short-faced bears (Arctodus) remain uncertain. In this study, we sequenced a mitochondrial genome from an Arctotherium femur preserved in a Chilean cave. Our molecular phylogenetic analyses revealed that the South American short-faced bears were more closely related to the extant South American spectacled bear than to the North American short-faced bears. This result suggests striking convergent evolution of giant forms in the two groups of short-faced bears (Arctodus and Arctotherium), potentially as an adaptation to dominate competition for megafaunal carcasses.

Mitochondrial genomes reveal the extinct Hippidion as an outgroup to all living equids.

Hippidions were equids with very distinctive anatomical features. They lived in South America 2.5 million years ago (Ma) until their extinction approximately 10 000 years ago. The evolutionary origin of the three known Hippidion morphospecies is still disputed. Based on palaeontological data, Hippidion could have diverged from the lineage leading to modern equids before 10 Ma. In contrast, a much later divergence date, with Hippidion nesting within modern equids, was indicated by partial ancient mitochondrial DNA sequences. Here, we characterized eight Hippidion complete mitochondrial genomes at 3.4-386.3-fold coverage using target-enrichment capture and next-generation sequencing. Our dataset reveals that the two morphospecies sequenced (H. saldiasi and H. principale) formed a monophyletic clade, basal to extant and extinct Equus lineages. This contrasts with previous genetic analyses and supports Hippidion as a distinct genus, in agreement with palaeontological models. We date the Hippidion split from Equus at 5.6-6.5 Ma, suggesting an early divergence in North America prior to the colonization of South America, after the formation of the Panamanian Isthmus 3.5 Ma and the Great American Biotic Interchange.

Revising the recent evolutionary history of equids using ancient DNA.

The rich fossil record of the family Equidae (Mammalia: Perissodactyla) over the past 55 MY has made it an icon for the patterns and processes of macroevolution. Despite this, many aspects of equid phylogenetic relationships and taxonomy remain unresolved. Recent genetic analyses of extinct equids have revealed unexpected evolutionary patterns and a need for major revisions at the generic, subgeneric, and species levels. To investigate this issue we examine 35 ancient equid specimens from four geographic regions (South America, Europe, Southwest Asia, and South Africa), of which 22 delivered 87-688 bp of reproducible aDNA mitochondrial sequence. Phylogenetic analyses support a major revision of the recent evolutionary history of equids and reveal two new species, a South American hippidion and a descendant of a basal lineage potentially related to Middle Pleistocene equids. Sequences from specimens assigned to the giant extinct Cape zebra, Equus capensis, formed a separate clade within the modern plain zebra species, a phenotypicically plastic group that also included the extinct quagga. In addition, we revise the currently recognized extinction times for two hemione-related equid groups. However, it is apparent that the current dataset cannot solve all of the taxonomic and phylogenetic questions relevant to the evolution of Equus. In light of these findings, we propose a rapid DNA barcoding approach to evaluate the taxonomic status of the many Late Pleistocene fossil Equidae species that have been described from purely morphological analyses.