The Assembly of Bacteria Living in Natural Environments Shapes Neuronal Integrity and Behavioral Outputs in Caenorhabditis elegans.

Bacterivore nematodes are the most abundant animals in the biosphere, largely contributing to global biogeochemistry. Thus, the effects of environmental microbes on the nematodes' life-history traits are likely to contribute to the general health of the biosphere. Caenorhabditis elegans is an excellent model to study the behavioral and physiological outputs of microbial diets. However, the effects of complex natural bacterial assemblies have only recently been reported, as most studies have been carried out with monoxenic cultures of laboratory-reared bacteria. Here, we quantified the physiological, phenotypic, and behavioral traits of C. elegans feeding on two bacteria that were coisolated with wild nematodes from a soil sample. These bacteria were identified as a putative novel species of Stenotrophomonas named Stenotrophomonas sp. strain Iso1 and a strain of Bacillus pumilus designated Iso2. The distinctive behaviors and developmental patterns observed in animals fed with individual isolates changed when bacteria were mixed. We studied in more depth the degeneration rate of the touch circuit of C. elegans and show that B. pumilus alone is protective, while the mix with Stenotrophomonas sp. is degenerative. The analysis of the metabolite contents of each isolate and their combination identified NAD+ as being potentially neuroprotective. In vivo supplementation shows that NAD+ restores neuroprotection to the mixes and also to individual nonprotective bacteria. Our results highlight the distinctive physiological effects of bacteria resembling native diets in a multicomponent scenario rather than using single isolates on nematodes. IMPORTANCE Do behavioral choices depend on animals' microbiota? To answer this question, we studied how different bacterial assemblies impact the life-history traits of the bacterivore nematode C. elegans using isolated bacteria found in association with wild nematodes in Chilean soil. We identified the first isolate, Iso1, as a novel species of Stenotrophomonas and isolate Iso2 as Bacillus pumilus. We find that worm traits such as food choice, pharyngeal pumping, and neuroprotection, among others, are dependent on the biota composition. For example, the neurodegeneration of the touch circuit needed to sense and escape from predators in the wild decreases when nematodes are fed on B. pumilus, while its coculture with Stenotrophomonas sp. eliminates neuroprotection. Using metabolomics analysis, we identify metabolites such as NAD+, present in B. pumilus yet lost in the mix, as being neuroprotective and validated their protective effects using in vivo experiments.

Assignment of structural transitions during mechanical unwrapping of nucleosomes and their disassembly products.

Nucleosome DNA unwrapping and its disassembly into hexasomes and tetrasomes is necessary for genomic access and plays an important role in transcription regulation. Previous single-molecule mechanical nucleosome unwrapping revealed a low- and a high-force transitions, and force-FRET pulling experiments showed that DNA unwrapping is asymmetric, occurring always first from one side before the other. However, the assignment of DNA segments involved in these transitions remains controversial. Here, using high-resolution optical tweezers with simultaneous single-molecule FRET detection, we show that the low-force transition corresponds to the undoing of the outer wrap of one side of the nucleosome (∼27 bp), a process that can occur either cooperatively or noncooperatively, whereas the high-force transition corresponds to the simultaneous unwrapping of ∼76 bp from both sides. This process may give rise stochastically to the disassembly of nucleosomes into hexasomes and tetrasomes whose unwrapping/rewrapping trajectories we establish. In contrast, nucleosome rewrapping does not exhibit asymmetry. To rationalize all previous nucleosome unwrapping experiments, it is necessary to invoke that mechanical unwrapping involves two nucleosome reorientations: one that contributes to the change in extension at the low-force transition and another that coincides but does not contribute to the high-force transition.

Friction-driven membrane scission by the human ESCRT-III proteins CHMP1B and IST1.

The endosomal sorting complexes required for transport (ESCRT) system is an ancient and ubiquitous membrane scission machinery that catalyzes the budding and scission of membranes. ESCRT-mediated scission events, exemplified by those involved in the budding of HIV-1, are usually directed away from the cytosol ("reverse topology"), but they can also be directed toward the cytosol ("normal topology"). The ESCRT-III subunits CHMP1B and IST1 can coat and constrict positively curved membrane tubes, suggesting that these subunits could catalyze normal topology membrane severing. CHMP1B and IST1 bind and recruit the microtubule-severing AAA+ ATPase spastin, a close relative of VPS4, suggesting that spastin could have a VPS4-like role in normal-topology membrane scission. Here, we reconstituted the process in vitro using membrane nanotubes pulled from giant unilamellar vesicles using an optical trap in order to determine whether CHMP1B and IST1 are capable of membrane severing on their own or in concert with VPS4 or spastin. CHMP1B and IST1 copolymerize on membrane nanotubes, forming stable scaffolds that constrict the tubes, but do not, on their own, lead to scission. However, CHMP1B-IST1 scaffolded tubes were severed when an additional extensional force was applied, consistent with a friction-driven scission mechanism. We found that spastin colocalized with CHMP1B-enriched sites but did not disassemble the CHMP1B-IST1 coat from the membrane. VPS4 resolubilized CHMP1B and IST1 without leading to scission. These observations show that the CHMP1B-IST1 ESCRT-III combination is capable of severing membranes by a friction-driven mechanism that is independent of VPS4 and spastin.

A DNA packaging motor inchworms along one strand allowing it to adapt to alternative double-helical structures.

Ring ATPases that translocate disordered polymers possess lock-washer architectures that they impose on their substrates during transport via a hand-over-hand mechanism. Here, we investigate the operation of ring motors that transport ordered, helical substrates, such as the bacteriophage ϕ29 dsDNA packaging motor. This pentameric motor alternates between an ATP loading dwell and a hydrolysis burst wherein it packages one turn of DNA in four steps. When challenged with DNA-RNA hybrids and dsRNA, the motor matches its burst to the shorter helical pitches, keeping three power strokes invariant while shortening the fourth. Intermittently, the motor loses grip on the RNA-containing substrates, indicating that it makes optimal load-bearing contacts with dsDNA. To rationalize these observations, we propose a helical inchworm translocation mechanism in which, during each cycle, the motor increasingly adopts a lock-washer structure during the ATP loading dwell and successively regains its planar form with each power stroke during the burst.

Molecular switch-like regulation enables global subunit coordination in a viral ring ATPase.

Subunits in multimeric ring-shaped motors must coordinate their activities to ensure correct and efficient performance of their mechanical tasks. Here, we study WT and arginine finger mutants of the pentameric bacteriophage φ29 DNA packaging motor. Our results reveal the molecular interactions necessary for the coordination of ADP-ATP exchange and ATP hydrolysis of the motor's biphasic mechanochemical cycle. We show that two distinct regulatory mechanisms determine this coordination. In the first mechanism, the DNA up-regulates a single subunit's catalytic activity, transforming it into a global regulator that initiates the nucleotide exchange phase and the hydrolysis phase. In the second, an arginine finger in each subunit promotes ADP-ATP exchange and ATP hydrolysis of its neighbor. Accordingly, we suggest that the subunits perform the roles described for GDP exchange factors and GTPase-activating proteins observed in small GTPases. We propose that these mechanisms are fundamental to intersubunit coordination and are likely present in other ring ATPases.

ß1-subunit-induced structural rearrangements of the Ca2+- and voltage-activated K+ (BK) channel.

Large-conductance Ca(2+)- and voltage-activated K(+) (BK) channels are involved in a large variety of physiological processes. Regulatory ß-subunits are one of the mechanisms responsible for creating BK channel diversity fundamental to the adequate function of many tissues. However, little is known about the structure of its voltage sensor domain. Here, we present the external architectural details of BK channels using lanthanide-based resonance energy transfer (LRET). We used a genetically encoded lanthanide-binding tag (LBT) to bind terbium as a LRET donor and a fluorophore-labeled iberiotoxin as the LRET acceptor for measurements of distances within the BK channel structure in a living cell. By introducing LBTs in the extracellular region of the α- or ß1-subunit, we determined (i) a basic extracellular map of the BK channel, (ii) ß1-subunit-induced rearrangements of the voltage sensor in α-subunits, and (iii) the relative position of the ß1-subunit within the α/ß1-subunit complex.

Mechanism of potassium ion uptake by the Na(+)/K(+)-ATPase.

The Na(+)/K(+)-ATPase restores sodium (Na(+)) and potassium (K(+)) electrochemical gradients dissipated by action potentials and ion-coupled transport processes. As ions are transported, they become transiently trapped between intracellular and extracellular gates. Once the external gate opens, three Na(+) ions are released, followed by the binding and occlusion of two K(+) ions. While the mechanisms of Na(+) release have been well characterized by the study of transient Na(+) currents, smaller and faster transient currents mediated by external K(+) have been more difficult to study. Here we show that external K(+) ions travelling to their binding sites sense only a small fraction of the electric field as they rapidly and simultaneously become occluded. Consistent with these results, molecular dynamics simulations of a pump model show a wide water-filled access channel connecting the binding site to the external solution. These results suggest a mechanism of K(+) gating different from that of Na(+) occlusion.

Temperature and voltage coupling to channel opening in transient receptor potential melastatin 8 (TRPM8).

Expressed in somatosensory neurons of the dorsal root and trigeminal ganglion, the transient receptor potential melastatin 8 (TRPM8) channel is a Ca(2+)-permeable cation channel activated by cold, voltage, phosphatidylinositol 4,5-bisphosphate, and menthol. Although TRPM8 channel gating has been characterized at the single channel and macroscopic current levels, there is currently no consensus regarding the extent to which temperature and voltage sensors couple to the conduction gate. In this study, we extended the range of voltages where TRPM8-induced ionic currents were measured and made careful measurements of the maximum open probability the channel can attain at different temperatures by means of fluctuation analysis. The first direct measurements of TRPM8 channel temperature-driven conformational rearrangements provided here suggest that temperature alone is able to open the channel and that the opening reaction is voltage-independent. Voltage is a partial activator of TRPM8 channels, because absolute open probability values measured with fully activated voltage sensors are less than 1, and they decrease as temperature rises. By unveiling the fast temperature-dependent deactivation process, we show that TRPM8 channel deactivation is well described by a double exponential time course. The fast and slow deactivation processes are temperature-dependent with enthalpy changes of 27.2 and 30.8 kcal mol(-1). The overall Q10 for the closing reaction is about 33. A three-tiered allosteric model containing four voltage sensors and four temperature sensors can account for the complex deactivation kinetics and coupling between voltage and temperature sensor activation and channel opening.

Energy landscape of the reactions governing the Na+ deeply occluded state of the Na+/K+-ATPase in the giant axon of the Humboldt squid.

The Na(+)/K(+) pump is a nearly ubiquitous membrane protein in animal cells that uses the free energy of ATP hydrolysis to alternatively export 3Na(+) from the cell and import 2K(+) per cycle. This exchange of ions produces a steady-state outwardly directed current, which is proportional in magnitude to the turnover rate. Under certain ionic conditions, a sudden voltage jump generates temporally distinct transient currents mediated by the Na(+)/K(+) pump that represent the kinetics of extracellular Na(+) binding/release and Na(+) occlusion/deocclusion transitions. For many years, these events have escaped a proper thermodynamic treatment due to the relatively small electrical signal. Here, taking the advantages offered by the large diameter of the axons from the squid Dosidicus gigas, we have been able to separate the kinetic components of the transient currents in an extended temperature range and thus characterize the energetic landscape of the pump cycle and those transitions associated with the extracellular release of the first Na(+) from the deeply occluded state. Occlusion/deocclusion transition involves large changes in enthalpy and entropy as the ion is exposed to the external milieu for release. Binding/unbinding is substantially less costly, yet larger than predicted for the energetic cost of an ion diffusing through a permeation pathway, which suggests that ion binding/unbinding must involve amino acid side-chain rearrangements at the site.