Genomes of Novel Myxococcota Reveal Severely Curtailed Machineries for Predation and Cellular Differentiation.

Cultured Myxococcota are predominantly aerobic soil inhabitants, characterized by their highly coordinated predation and cellular differentiation capacities. Little is currently known regarding yet-uncultured Myxococcota from anaerobic, nonsoil habitats. We analyzed genomes representing one novel order (o__JAFGXQ01) and one novel family (f__JAFGIB01) in the Myxococcota from an anoxic freshwater spring (Zodletone Spring) in Oklahoma, USA. Compared to their soil counterparts, anaerobic Myxococcota possess smaller genomes and a smaller number of genes encoding biosynthetic gene clusters (BGCs), peptidases, one- and two-component signal transduction systems, and transcriptional regulators. Detailed analysis of 13 distinct pathways/processes crucial to predation and cellular differentiation revealed severely curtailed machineries, with the notable absence of homologs for key transcription factors (e.g., FruA and MrpC), outer membrane exchange receptor (TraA), and the majority of sporulation-specific and A-motility-specific genes. Further, machine learning approaches based on a set of 634 genes informative of social lifestyle predicted a nonsocial behavior for Zodletone Myxococcota. Metabolically, Zodletone Myxococcota genomes lacked aerobic respiratory capacities but carried genes suggestive of fermentation, dissimilatory nitrite reduction, and dissimilatory sulfate-reduction (in f_JAFGIB01) for energy acquisition. We propose that predation and cellular differentiation represent a niche adaptation strategy that evolved circa 500 million years ago (Mya) in response to the rise of soil as a distinct habitat on Earth. IMPORTANCE The phylum Myxococcota is a phylogenetically coherent bacterial lineage that exhibits unique social traits. Cultured Myxococcota are predominantly aerobic soil-dwelling microorganisms that are capable of predation and fruiting body formation. However, multiple yet-uncultured lineages within the Myxococcota have been encountered in a wide range of nonsoil, predominantly anaerobic habitats, and the metabolic capabilities, physiological preferences, and capacity of social behavior of such lineages remain unclear. Here, we analyzed genomes recovered from a metagenomic analysis of an anoxic freshwater spring in Oklahoma, USA, that represent novel, yet-uncultured, orders and families in the Myxococcota. The genomes appear to lack the characteristic hallmarks for social behavior encountered in Myxococcota genomes and displayed a significantly smaller genome size and a smaller number of genes encoding biosynthetic gene clusters, peptidases, signal transduction systems, and transcriptional regulators. Such perceived lack of social capacity was confirmed through detailed comparative genomic analysis of 13 pathways associated with Myxococcota social behavior, as well as the implementation of machine learning approaches to predict social behavior based on genome composition. Metabolically, these novel Myxococcota are predicted to be strict anaerobes, utilizing fermentation, nitrate reduction, and dissimilarity sulfate reduction for energy acquisition. Our results highlight the broad patterns of metabolic diversity within the yet-uncultured Myxococcota and suggest that the evolution of predation and fruiting body formation in the Myxococcota has occurred in response to soil formation as a distinct habitat on Earth.

Long-latency "subthreshold" collicular responses to the constant-frequency components emitted by a bat.

A previously undescribed response pattern has been observed in certain single units in the posterior colliculus of Pteronotus suapurensis. These units, constituting about one-third of those tuned to the region of the dominant constant-frequency (CF) components of the orientation sounds, respond to a tone pip with a burst of spikes at a latency of 3 to 6 milliseconds, within the frequency-intensity domain of a normal V-shaped response area. In these units, however, as intensity is dropped below threshold for this response, a response of 5-to 10-milliseconds longer latency appears and persists throughout another 10 to 30 decibels of attenuation. These late responses can be very vigorous, are sharply tuned to frequencies at or just above the CF components of the signal, and are often strongest and of lowest threshold at stimulus durations of 1.5 to 3 milliseconds--approximately the duration of the CF component. These properties imply that the late responses are concerned with analysis of the CF components of echoes, apparently in ways not as prominent in other bats.

Integrins and modulation of transmitter release from motor nerve terminals by stretch.

The stretch of a frog muscle within the physiological range can more than double the spontaneous and evoked release of neurotransmitter from its motor nerve terminals. Here, stretch enhancement of release was suppressed by peptides containing the sequence arginine-glycine-aspartic acid (RGD), which blocks integrin binding. Integrin antibodies also inhibited the enhancement obtained by stretching. Stretch enhancement depended on intraterminal calcium derived both from external calcium and from internal stores. Muscle stretch thus might enhance the release of neurotransmitters either by elevating internal calcium concentrations or by increasing the sensitivity of transmitter release to calcium in the nerve terminal.

Tracking presynaptic Ca2+ dynamics during neurotransmitter release with Ca2+-activated K+ channels.

Neurotransmitter release during action potentials is thought to require transient, localized [Ca2+]i as high as hundreds of micromolar near presynaptic release sites. Most experimental attempts to characterize the magnitude and time course of these Ca2+ domains involve optical methods that sample large volumes, require washout of endogenous buffers and often affect Ca2+ kinetics and transmitter release. Endogenous calcium-activated potassium (KCa) channels colocalize with presynaptic Ca2+ channels in Xenopus nerve-muscle cultures. We used these channels to quantify the rapid, dynamic changes in [Ca2+]i at active zones during synaptic activity. Confirming Ca2+-domain predictions, these KCa channels revealed [Ca2+]i over 100 microM during synaptic activity and much faster buildup and decay of Ca2+ domains than shown using other techniques.

Effect of external and internal pH changes on K and Cl conductances in the muscle fiber membrane of a giant barnacle.

The membrane potential and conductance of the giant muscle fiber of a barnacle (Balanus nubilus Darwin) were analyzed in relation to changes in the external (3.5-10.0) and the internal (4.7-9.6) pH, under various experimental conditions. A sharp increase in membrane conductance, associated with a large increase in conductance to Cl ions, was observed when the external pH was lowered to values below 5.0. The ratio of Cl to K conductance in normal barnacle saline is between (1/6)-1/7 at pH 7.7, whereas at pH 4.0 the ratio is about 6-9. The behavior of the membrane in response to pH changes in a Cl-depleted muscle fiber shows that the K conductance decreases with decreasing external pH for the whole range of pH examined. A steep increase in Cl conductance is also observed when the internal pH of the fiber is lowered below 5.0. The K to Cl conductance ratio increases with increasing internal pH in a manner very similar to that found when the external pH is raised above 5.0. These facts suggest that the membrane is amphoteric with positive and negative fixed charge groups having dissociation constants such that at pH greater than 5, negative groups predominate and cations permeate more easily than anions, while at lower pH positive groups predominate, facilitating the passage of anions through the membrane.

Ultrastructural correlates of experimentally altered transmitter release efficacy in frog motor nerve terminals.

After experimentally inducing long term changes in transmitter release, a series of frog neuromuscular junctions were studied with intracellular recording and then semi-serially sectioned and examined in the electron microscope. Transmitter release per unit length of motor nerve terminal was well correlated with several measures of the length of individual presynaptic active zones and with the number of mitochondria per terminal. Total release from each terminal correlated with estimates of the total amount of active zone. This study of neuromuscular junctions in sartorius muscles of the frog Rana pipiens was undertaken to search for ultrastructural correlates of the increase in transmitter release efficacy that follows denervation of the contralateral sartorius. This treatment typically results in greatly enhanced release at some synapses while others appear unaffected. In the present study, nine identified junctions with known physiological properties were sectioned every 6 micron throughout much of their length to yield 40-105 cross-sectional profiles per junction. Overall, these 9 synapses showed a 33-fold range in quantal transmitter release and an 18-fold range in release per unit nerve terminal. Release correlated with estimates of active zone size. No correlations were found between release and the density of synaptic vesicles adjacent to active zones. Our results suggest that active zones in motor nerve terminals are plastic structures, and that changes in active zone size may be the structural basis of long term changes in transmitter release and synaptic efficacy.

Contribution of presynaptic calcium-activated potassium currents to transmitter release regulation in cultured Xenopus nerve-muscle synapses.

Using Xenopus nerve-muscle co-cultures, we have examined the contribution of calcium-activated potassium (K(Ca)) channels to the regulation of transmitter release evoked by single action potentials. The presynaptic varicosities that form on muscle cells in these cultures were studied directly using patch-clamp recording techniques. In these developing synapses, blockade of K(Ca) channels with iberiotoxin or charybdotoxin decreased transmitter release by an average of 35%. This effect would be expected to be caused by changes in the late phases of action potential repolarization. We hypothesize that these changes are due to a reduction in the driving force for calcium that is normally enhanced by the local hyperpolarization at the active zone caused by potassium current through the K(Ca) channels that co-localize with calcium channels. In support of this hypothesis, we have shown that when action potential waveforms were used as voltage-clamp commands to elicit calcium current in varicosities, peak calcium current was reduced only when these waveforms were broadened beginning when action potential repolarization was 20% complete. In contrast to peak calcium current, total calcium influx was consistently increased following action potential broadening. A model, based on previously reported properties of ion channels, faithfully reproduced predicted effects on action potential repolarization and calcium currents. From these data, we suggest that the large-conductance K(Ca) channels expressed at presynaptic varicosities regulate transmitter release magnitude during single action potentials by altering the rate of action potential repolarization, and thus the magnitude of peak calcium current.

Posttetanic potentiation in strong and weak neuromuscular junctions: physiological differences caused by a differential Ca2+-influx.

We have sought physiological explanations for large differences in synaptic strength of different frog sartorius neuromuscular junctions, using the time course of posttetanic potentiation (PTP) of mEPP frequency as an indicator of the kinetics of Ca2+-metabolism in junctions of varying strengths. Results obtained in Ca2+ vs Ca2+-free/EGTA Ringers suggest that differences in the Ca2+-influx contribute to the physiological differences between strong and weak junctions.

Specificity of innervation among Xenopus twitch muscle fibers.

More than 50% of the Xenopus pectoralis twitch muscle fibers with two distant endplates are innervated at both sites by the same neuron. This study indicates that there are three separable twitch motor unit types in this muscle which show very little overlap in innervation. This study also shows that each motor unit is topographically localized and that similar type units are shifted relative to one another. It is concluded that these two factors may contribute importantly to the observed high incidence of mononeuronal innervation.

Seasonal changes in the normal variability in release properties of motor nerve terminals in Rana pipiens.

During a 2 1/2 year period, we studied 298 identified endplates from 40 sartorius muscles, correlating their morphology with their synaptic release properties. There is a remarkably large range in evoked and spontaneous quantal release levels when junctions are studied in a low-Ca2+ Ringer. This diversity in release efficacy persists when release is normalized to identified nerve terminal length. We also found that release was significantly larger in the winter (Dec-Feb) than in the spring and summer (Mar-Aug), suggesting seasonal regulation of release properties.

Destabilization of junctional ACh receptors by a reinnervating frog motor nerve.

The distribution of ACh receptors (AChRs) in neuromuscular junctions of the frog cutaneous pectoris muscle has been investigated at various times after denervation, as the motor nerve regenerates to reinnervate the junctions. Original extent of synaptic gutter is judged by the distribution of ACh esterase, which is compared with the staining pattern of rhodamine-labelled alpha-bungarotoxin. It is found that within 8-10 days after the beginning of reinnervation, many junctions show loss of AChR from large stretches of original gutter, in contrast to other regions, which still have a high density of receptors. This loss of receptor does not happen if the muscle remains denervated. It is suggested that the innervated portions of the junctions retain AChR, while the receptors in uninnervated portions are destabilized and rapidly lost. These findings indicate a powerful role for the nerve in eliminating both extrajunctional receptors and junctional receptors that are not stabilized by the direct action of an overlying nerve. One can imagine that a similar removal of AChR or other important synaptic molecules from gutters that are innervated later or by a terminal of lower efficacy might account for the competitive elimination of certain inputs and retention of others during development of reinnervation.

Monitoring transient Ca2+ dynamics with large-conductance Ca2+-dependent K+ channels at active zones in frog saccular hair cells.

Neurotransmitter release from the basolateral surface of auditory and vestibular hair cells is mediated by Ca(2+) influx through voltage-gated Ca(2+) channels. Co-localization of large-conductance Ca(2+)-activated K(+) (BK) channels at the active zones of these cells affords them with an optimal location to act as reporters of the Ca(2+) concentration changes at active zones of transmitter release. In this report we use BK channels in frog (Rana pipiens) hair cells to monitor dynamic changes in intracellular Ca(2+) concentration during transient influxes of Ca(2+), showing that BK current magnitude and delay to onset are correlated with the rate and duration of Ca(2+) entry through Ca(2+) channels. We also show that BK channels exhibit a much higher Ca(2+) binding affinity in the open state than in the closed state.

Dynamics of nerve-muscle interaction in developing and mature neuromuscular junctions.

Neuromuscular connections have long served as models of synaptic structure and function. They also provide illuminating insights into the dynamic cell-cell interactions governing synaptogenesis, neuromuscular differentiation, and the maintenance of effective function. This paper reviews recent advances in our understanding of the regulatory and inductive interactions involved in motor axon pathfinding, target recognition, bidirectional control of gene expression during synapse formation, motoneuron cell death, terminal rearrangement, and the ongoing remodeling of synaptic number, structure, and function to adjust to growth and changes in use.

Electrical interaction between antidromically stimulated frog motoneurones and dorsal root afferents: enhancement by gallamine and TEA.

1. Electrical interactions have been studied in the isolated frog spinal cord preparation. It is found that gallamine and tetraethylammonium chloride (TEA) markedly enhance all non-cholinergic synaptic interactions, including the electrical interaction between motoneurones (VR-VRP). In addition, in the presence of either of these drugs, a short-latency interaction is seen to exist between antidromically stimulated motoneurones and dorsal root afferents (early VR-DRP). The early VR-DRP is rarely seen in the absence of gallamine or TEA.2. The early VR-DRP is of the same short latency as the VR-VRP and fulfils the same criteria for electrical interaction: it increases in amplitude with cooling from 17-10 degrees C, it is not blocked by a wide variety of pharmacological blocking agents, and it is suppressed by both Mg(2+) and Ca(2+), with no antagonism of action between the two.3. The early VR-DRP appears as a cluster of unitary events: all-or-none spikes conducted out the dorsal root fibres. No initial graded slow potentials are seen. Often there are two peaks in the response.4. The early VR-DRP is facilitated by a dorsal root volley, with a time course normally intermediate between that of the orthodromic ventral root potential (DR-VRP) and the dorsal root potential (DR-DRP). This orthodromic facilitation apparently is achieved by increasing invasion of motoneurone dendritic trees and depolarization of dorsal root afferents toward threshold.5. If the same ventral root is stimulated twice, or adjacent roots stimulated at different intervals, the second early VR-DRP, like the VR-VRP, is seen to be occluded for 10-20 msec, then facilitated to supranormal amplitudes. It is concluded that motoneurone dendrites are presynaptic to both interactions.6. Evidence is presented that gallamine and TEA act by increasing the duration of activity both in axon terminals and in antidromically invaded motoneurones. Often second or multiple spikes result. The increased duration of depolarization can increase transmitter release at terminals and increase coupling at electrical junctions.7. Possible morphological correlates for the two electrical interactions are discussed. It is speculated that the motoneurone interaction arises at numerous areas of close apposition between dendrites in dendritic ;thickets', and that the early VR-DRP is mediated by fewer, lower-resistance, electrical junctions between dendrites and afferent nerve terminals.

A study of the interaction between motoneurones in the frog spinal cord.

1. A short-latency interaction between motoneurones has been studied with intracellular and root potential recordings from the isolated spinal cord of the frog. Antidromic stimulation of one ventral root causes brief depolarization (VR-EPSP) of the motoneurones of adjacent, non-excited motoneurones. The summed activity of many such VR-EPSPs can be seen as a brief depolarization (VR-VRP) passing out an adjacent ventral root.2. Both intracellular and root-recorded signs of this interaction are graded in amplitude.3. It was found that this interaction decreased with increasing temperature. This is in contrast to the behaviour of the ventral root potential resulting from dorsal root stimulation (DR-VRP) or the dorsal root potentials resulting from either dorsal root (DR-DRP) or ventral root (VR-DRP) stimulation, all of which increased in amplitude from below 10 to about 17 degrees C.4. Pharmacological evidence suggests that the interaction between motoneurones is not chemically mediated. The VR-VRP was not affected by a large variety of transmitter blocking agents, including curare, dihydro-beta-erythroidine, atropine, succinylcholine, hexamethonium and DOPA, while the VR-DRP, which probably originates with the release of ACh from an axon collateral, was consistently blocked.5. Mg(2+) suppressed the VR-VRP more slowly than the other potentials, and this suppression was increased by adding Ca(2+), rather than reversed, as in the case of the other root potentials, which are presumably mediated by chemical transmission.6. The interaction between motoneurones is strongly facilitated by orthodromic depolarization of the motoneurones being antidromically stimulated. Extracellular recordings within the cord support the conclusion that this facilitation is a result of the enhancement of antidromic invasion, perhaps especially of the dendrites, by slight depolarization.7. One VR-VRP (or VR-EPSP) first suppresses response to another (for about 10 msec), then facilitates response to the second, with maximum effect around 20-40 msec. This is the case whether both stimuli go to the same or to different ventral roots, although occlusion is less and facilitation greater in the latter case. Occlusion of the VR-EPSP also results from full excitation of the cell in which recording is being done.8. The mechanism of this interaction remains uncertain, but it would seem likely that overlapping dendrites of adjacent motoneurones interact with each other electrically through close apposition or specialized contacts. Occlusion would result from the refractoriness of strongly depolarized dendrites, facilitation from the enhancement of invasion of antidromically stimulated motoneurones by the weaker (or residual) depolarization occurring after earlier activity of motoneurones or their dendrites.

The regulation of synaptic strength within motor units of the frog cutaneous pectoris muscle.

The physiological properties of frog neuromuscular junctions may vary widely in a single muscle. In order to understand the factors that contribute to this variation, we have studied populations of synapses belonging to individual motor units of the frog cutaneous pectoris muscle. Motor units in this muscle differ widely in twitch strength. A motor axon's synaptic contacts could be found throughout the muscle, at both singly and polyneuronally innervated endplates. Indeed, over 36% of the endplates contacted by each isolated motor axon were polyneuronally innervated. Comparisons of synapses on muscle fibers in large twitch motor units with those in small twitch motor units reveal that endplate potential amplitude, transmitter release, and muscle fiber diameter are positively correlated with the strength of the motor unit contraction. Large and small twitch motor units differ more in their transmitter release than in their nerve terminal length, indicating that larger twitch motor units have a higher release per unit length of terminal. Among motor units of roughly similar twitch tension, transmitter release at endplates receiving only one axonal input is remarkably constant, independent of postsynaptic muscle fiber input resistance, or, presumably, nerve terminal size. In cases where two different motor axons contribute to a single endplate, the synaptic strength of each input is again related to properties of the contributing motoneuron, although the individual synaptic inputs are markedly reduced in strength and size relative to singly innervated endplates. Additionally, the diameter of polyneuronally innervated muscle fibers appears related to properties of both innervating motoneurons. Thus, the pre- and postsynaptic characteristics of neuromuscular junctions may be determined both by the motoneuron and by peripheral interactions between motoneurons.

Effects of changes in motor unit size on transmitter release at the frog neuromuscular junction.

The dependence of transmitter release and synaptic effectiveness on the size of a neuron's peripheral field was studied using neuromuscular junctions in sartorius muscles of adult frogs (Rana pipiens). The size of the peripheral field (motor unit size) was reduced by crushing the sartorius nerve and surgically removing half of the muscle fibers. Synapses thus formed were compared with those formed when crushed nerves reinnervated intact whole muscles, as well as with synapses in normal unoperated muscles. Indirect observations suggested that all motor axons participated in reinnervation and that motor unit size was indeed smaller in half-muscles. Synaptic safety margins, as measured by the sensitivity of nerve stimulus-evoked twitching to low Ca2+, were substantially higher in muscles with reduced motor units. These higher safety margins were due to enhanced evoked transmitter release. In Ringer solution containing Mg2+ and lowered Ca2+, total evoked release and evoked release per unit nerve terminal length were approximately 2-fold higher in muscles with reduced motor units, when studied 7 to 18 weeks postoperatively. A similar difference was seen when unblocked release was measured in a normal physiological solution, after blocking excitation-contraction coupling and muscle fiber action potential generation with formamide. Miniature endplate potential frequency in half-muscles was 2 to 3 times higher than in controls when tested in normal physiological solution, but was not significantly different in low Ca2+, Mg2+-containing solution. By 34 weeks postoperatively, there was no longer a difference in evoked release, even though the difference in motor unit size persisted.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of synaptic position, size, and strength in anuran skeletal muscle.

An analysis of the physiology, morphology, and position of endplates on identified fibers in the Xenopus laevis pectoralis muscle has revealed the following. 1. The percentage of fibers with one endplate is lower in large muscles, and within the same muscle, singly innervated fibers are smaller than dually innervated fibers. 2. Single junctions tend to be stronger than junctions on dually innervated fibers. 3. Single junctions typically are located near the middle of their fibers, while the endplates on dually innervated fibers are located toward either end and usually are separated by at least 20% of the total fiber length. A significant proportion of dually innervated fibers appears to be innervated by the same axon at both junctions. 4. Junctions on the same dually innervated fiber tend to be more similar in length than do junctions on different fibers of the same input resistance. This observation is the same whether both junctions on a given fiber are formed by the same or different axons. There is no corresponding tendency for greater similarity in physiological strength of paired junctions, which frequently show large differences in endplate potential amplitude. 5. The total terminal length on dually innervated fibers of equivalent input resistance is inversely correlated with the mean release per unit length and total release of both junctions. There is no apparent correlation between the distance separating endplates and their strength or length. The data support a model of synaptic regulation in which nerve terminals are attracted, grow, and are maintained in proportion to the amount of a substance supplied by muscle fibers. Our findings suggest that such a substance is produced or distributed uniformly throughout each fiber in amounts proportional to the fiber size and inversely proportional to the total transmitter output of all junctions innervating the fiber. A form of competitive interaction between the terminals which helps to determine synaptic spacing may involve local depletion or inactivation of this substance.