From the Cover: Montsechia,an ancient aquatic angiosperm

The early diversification of angiosperms in diverse ecological niches is poorly understood. Some have proposed an origin in a darkened forest habitat and others an open aquatic or near aquatic habitat. The research presented here centers on Montsechia vidalii, first recovered from lithographic limestone deposits in the Pyrenees of Spain more than 100 y ago. This fossil material has been poorly understood and misinterpreted in the past. Now, based upon the study of more than 1,000 carefully prepared specimens, a detailed analysis of Montsechia is presented. The morphology and anatomy of the plant, including aspects of its reproduction, suggest that Montsechia is sister to Ceratophyllum (whenever cladistic analyses are made with or without a backbone). Montsechia was an aquatic angiosperm living and reproducing below the surface of the water, similar to Ceratophyllum. Montsechia is Barremian in age, raising questions about the very early divergence of the Ceratophyllum clade compared with its position as sister to eudicots in many cladistic analyses. Lower Cretaceous aquatic angiosperms, such as Archaefructus and Montsechia, open the possibility that aquatic plants were locally common at a very early stage of angiosperm evolution and that aquatic habitats may have played a major role in the diversification of some early angiosperm lineages.When did early angiosperms begin to diversify ecologically? This question is currently unanswered. Age estimates of the divergence of crown-group angiosperms using molecular clock data vary considerably, although it is in the range of (max. 210–) often accepted, 150–140 (min. 130) million years (1–7). Parsimony reconstruction of early angiosperm habit suggests that they may have been shrubs living in “damp, dark, and disturbed” habitats (8). In contrast, many living aquatic angiosperms are basal in angiosperm phylogenies [e.g., Nymphaeales in Amborella, Nymphaeales and Illiciales, Trimeniaceae-Austrobaileya (ANITA) or Ceratophyllales with the eudicots as commonly understood]. In the fossil record, we have found an aquatic angiosperm, Montsechia vidalii (Zeiller) Teixeira, which is an atypical plant fossil found in the Barremian (130–125 million years ago) freshwater limestone in the Pyrenees and Iberian Range in Spain. Montsechia (Fig. 1) lacks roots (no proximal or adventitious roots were found in more than 1,000 shoots examined) and shows flexible axes and two types of phyllotaxy and leaf morphology. The cuticle is very thin with rare stomata. The fruit is closed with a pore near the distal tip, indehiscent, and contains one unitegmic seed developed from an orthotropous and pendent ovule (Figs. 2 and ​and3).3). Cladistic analysis of these characters places Montsechia on the stem lineage basal to extant Ceratophyllum or a clade formed by Ceratophyllum and Chloranthaceae (Fig. 4) suggesting that mesangiosperms (non-ANITA angiosperms) existed 125 million years ago, as indicated by the tricolpate pollen record. Montsechia is well-adapted to a submerged aquatic habit. Montsechia is contemporaneous with another aquatic plant fossil, Archaefructus, indicating that some of the earliest angiosperms were fully aquatic very early in their ecological diversification.Open in a separate windowFig. 1.Long- and short-leaved forms of Montsechia vidalii. (A) The long-leaved specimen shows very flexuous branches and opposite, long leaves. LH02556. (Scale bar, 10 mm.) (B) The short-leaved specimen shows regularly developed lateral branches and tiny leaf rosettes. LH07198. (Scale bar, 10 mm.)Open in a separate windowFig. 2.Fruit and seed of Montsechia vidalii. The fruit shows a small apical pore (po). The funicle (f) of the single, upside-down seed (orthotropous pendent) is attached from the hilum (h) to the placenta (pl). (Scale bar, 500 µm.)Open in a separate windowFig. 3.Reconstructions of Montsechia vidalii. (A) The long-leaved form shows the opposite leaves and branches. (B) The short-leaved form shows the alternate phyllotaxy of leaves and branches bearing pairs of ascidiate, nonornamented fruits. (C and D) The fruit shows a small apical pore and a single seed developed from an orthotropous pendent ovule. The funicle arises from the placenta (near the micropyle) to the hilum (near the pollination pore). (C) Lateral view. (D) Front view. Diagram by O. Sanisidro, B.G., and V.D.-G.Open in a separate windowFig. 4.Most parsimonious position of Montsechia in a simplified tree derived from the matrix by Endress and Doyle (26) using the J & M backbone. Taxa in blue are considered ancestrally water-related (27). Diagram by C.C. and B.G.     

Unilateral incompatibility gene ui1.1 encodes an S-locus F-box protein expressed in pollen of Solanum species

Unilateral interspecific incompatibility (UI) is a postpollination, prezygotic reproductive barrier that prevents hybridization between related species when the female parent is self-incompatible (SI) and the male parent is self-compatible (SC). In tomato and related Solanum species, two genes, ui1.1 and ui6.1, are required for pollen compatibility on pistils of SI species or hybrids. We previously showed that ui6.1 encodes a Cullin1 (CUL1) protein. Here we report that ui1.1 encodes an S-locus F-box (SLF) protein. The ui1.1 gene was mapped to a 0.43-cM, 43.2-Mbp interval at the S-locus on chromosome 1, but positional cloning was hampered by low recombination frequency. We hypothesized that ui1.1 encodes an SLF protein(s) that interacts with CUL1 and Skp1 proteins to form an SCF-type (Skp1, Cullin1, F-box) ubiquitin E3 ligase complex. We identified 23 SLF genes in the S. pennellii genome, of which 19 were also represented in cultivated tomato (S. lycopersicum). Data from recombination events, expression analysis, and sequence annotation highlighted 11 S. pennellii genes as candidates. Genetic transformations demonstrated that one of these, SpSLF-23, is sufficient for ui1.1 function. A survey of cultivated and wild tomato species identified SLF-23 orthologs in each of the SI species, but not in the SC species S. lycopersicum, S. cheesmaniae, and S. galapagense, pollen of which lacks ui1.1 function. These results demonstrate that pollen compatibility in UI is mediated by protein degradation through the ubiquitin–proteasome pathway, a mechanism related to that which controls pollen recognition in SI.Self-incompatibility (SI) in Solanum and other Solanaceae is the S-RNase–based, gametophytic type, in which S-specificity is determined by S-RNases in the pistil (1) and S-locus F-box proteins (SLFs) in pollen (2). F-box proteins, together with Skp1 and Cullin1 proteins, are components of SCF-type (Skp1, Cullin1, F-box) ubiquitin E3 ligases that mark target proteins for degradation by the 26S proteasome (3, 4). The ubiquitin–proteasome pathway controls the pollen compatibility phenotype in SI (5). In the “collaborative non–self-recognition” model (6), the S-locus encodes multiple SLF proteins that together recognize different sets of S-RNases. In a compatible pollination, the SLF/S-RNase interaction leads to protection of pollen tubes against cytotoxic S-RNase, whereas in incompatible pollinations, a failure to recognize self–S-RNase results in pollen tube arrest. In addition, modifier genes, such as those encoding HT-B, NaStEP, and 120-kDa proteins in the pistil, and CUL1 and SSK1 proteins in pollen, are required for SI function but do not confer S-specificity (7–11).Unilateral incompatibility (UI) is a reproductive barrier related to SI in which pollen from one species or population is rejected on pistils of a related species or population, whereas in the reciprocal crosses, no pollen rejection occurs. Pollen of SC species or populations is almost always rejected on pistils of related SI species or populations, whereas in the reciprocal crosses (SC pollinated by SI), pollen rejection rarely occurs. This unidirectional pattern of pollen rejection is referred to as the “SI × SC rule” (12). Although the mechanisms of pollen recognition and rejection by UI are complex (13), several SI factors, including S-RNase, CUL1, and HT, also function in UI (8, 14, 15).Cultivated and wild tomatoes provide a powerful model system to study the mechanisms of reproductive barriers in the Solanaceae (16). They display wide variation in mating systems, both between and within species (17). Cultivated tomato, S. lycopersicum, is SC and accepts pollen of its SI or SC wild relatives; conversely, pollen of S. lycopersicum is rejected by pistils of the SI species. Three other red- or orange-fruited species, S. cheesmaniae, S. galapagense, and S. pimpinellifolium, are bilaterally cross-compatible with each other and with S. lycopersicum. There are notable exceptions to SI × SC rule in the tomato clade (18). One is that pollen of all of the red/orange-fruited species (SC) are rejected on pistils of the SC green-fruited species. Another exception is that pollen of some SC biotypes of SI species are compatible on pistils of conspecific SI populations. Therefore, pollen rejection is complex and likely involves more than one mechanism (13). The ability to reject tomato pollen is dominant in interspecific F₁ hybrids between cultivated tomato and related wild SI species (i.e., SC × SI hybrids), although pollen tube arrest occurs lower in the style of hybrids, suggesting that expression of the pistil side barrier is weakened (19). Allotriploid hybrids comprised of two genomes from S. lycopersicum (SC) and one genome from S. lycopersicoides (SI) reject tomato pollen tubes lower in the style than corresponding diploid hybrids (19).We previously reported that two pollen factors from S. pennellii, ui1.1 and ui6.1, are required and sufficient to overcome the UI barrier on allotriploid S. lycopersicum × S. lycopersicoides hybrids (19, 20). These two factors are not sufficient for compatibility on diploid S. lycopersicum × S. lycopersicoides hybrids (19). The ui6.1 locus encodes a pollen specific Cullin1 (CUL1) protein (21) that functions in pollen recognition by UI and SI (8). Pollen lacking ui6.1 are incompatible on pistils expressing active S-RNases, but compatible on pistils expressing a mutant S-RNase lacking RNase activity (8). This observation suggested that ui6.1—and by extension, ui1.1—is required for pollen resistance to S-RNase–based rejection in the pistil. Interestingly, neither ui6.1 nor ui1.1 is required for resistance to S-RNase itself, because tomato pollen is fully compatible on pistils expressing active S-RNase in the absence of a functional HT protein (15, 22). Thus, both SI and UI require multiple pollen and pistil factors. The ui1.1 locus is located at or near the S locus region on the short arm of chromosome 1, suggesting it might encode one or more SLF proteins. The goal of the present research was to isolate ui1.1 from S. pennellii to elucidate the nature of pollen rejection by UI and its relationship to SI.     

Area burned in the western United States is unaffected by recent mountain pine beetle outbreaks

In the western United States, mountain pine beetles (MPBs) have killed pine trees across 71,000 km² of forest since the mid-1990s, leading to widespread concern that abundant dead fuels may increase area burned and exacerbate fire behavior. Although stand-level fire behavior models suggest that bark beetle-induced tree mortality increases flammability of stands by changing canopy and forest floor fuels, the actual effect of an MPB outbreak on subsequent wildfire activity remains widely debated. To address this knowledge gap, we superimposed areas burned on areas infested by MPBs for the three peak years of wildfire activity since 2002 across the western United States. Here, we show that the observed effect of MPB infestation on the area burned in years of extreme fire appears negligible at broad spatial extents. Contrary to the expectation of increased wildfire activity in recently infested red-stage stands, we found no difference between observed area and expected area burned in red-stage or subsequent gray-stage stands during three peak years of wildfire activity, which account for 46% of area burned during the 2002–2013 period. Although MPB infestation and fire activity both independently increased in conjunction with recent warming, our results demonstrate that the annual area burned in the western United States has not increased in direct response to bark beetle activity. Therefore, policy discussions should focus on societal adaptation to the effects of recent increases in wildfire activity related to increased drought severity.Predicting the consequences of climate change on ecosystems is one of the greatest challenges for contemporary ecology. The effects of climate change on disturbances, including fire and insect outbreaks, is expected to greatly exceed the effects of warming on more gradual forest change processes, such as tree physiology and tree reproductive success (1). Despite the potential for climate change to drive dramatic ecological change by altering rates, extents, and severities of disturbances, the effects of warming on forest disturbances, particularly how disturbances may interact under novel climate conditions, are poorly understood (2).During the early 21st century, warm and dry conditions, coupled with abundant susceptible tree hosts, have led to increased populations of mountain pine beetles (MPBs; Dendroctonus ponderosae Hopkins). Over the 2000–2013 period, the MPB has caused tree mortality over 71,000 km² of pine forest across the western United States (Fig. 1 A and B). Tree mortality caused by outbreaks of forest insects can dramatically alter ecosystems, leading to changes in timber resources; carbon sequestration (3); habitat quality (4); hydrology (5); and the likelihood or severity of subsequent disturbance, including wildfire (6, 7). To mediate the consequences of insect-induced tree mortality, the 2014 Farm Bill authorized $200 million to reduce the risk of insect outbreak, disease, and subsequent wildfire across 18 Mha of National Forest lands designated as landscape-scale insect and disease areas (8).Open in a separate windowFig. 1.Major wildland fires in 2006, 2007, and 2012 that intersect MPB hosts and cumulative MBP infestation in 2000–2013 across the western United States. (A) Extent of all pine hosts of MPBs (green), cumulative 2000–2013 MPB infestation (dark gray), and major wildland fires (>405 ha) that burned in 2006, 2007, or 2012 (red) and intersected MPB hosts (n = 546 fires). The fire area reflects the entire area burned, inclusive of MPB pine hosts and other cover types. Photographs of red-stage MPB infestation in ponderosa pine (B) and gray-stage infestation in lodgepole pine (C) are shown.The MPB is one of the most destructive forest insects in North America, and during outbreaks, high levels of tree mortality occur across large landscapes. The MPB inhabits the inner bark and feeds on the tree’s phloem tissues. Heavy colonization and reproduction within the inner bark interrupt the flow of water and nutrients throughout the tree and typically cause tree death. Endemic populations usually infest weakened trees. Initially, beetles selectively attack relatively large-diameter trees in dense stands, but as beetle populations increase, a wide range of tree sizes become susceptible to attack (9). Although the MPB feeds upon several pine species (10), most of the recent tree mortality has occurred in lodgepole pine and ponderosa pine forests (Fig. 2B). Outbreaks occur as beetle populations grow, typically in response to favorable effects of warmer temperatures on beetle population growth and drought stress in host trees (9, 11). Large and severe outbreaks of MPBs are also dependent on an abundance of susceptible host trees (9). The role of fire suppression in creating more homogeneous forest structures favorable to MPB outbreaks is debated and is likely to be different for forests types characterized by natural fire regimes of predominantly frequent surface fires vs. infrequent large stand-replacing fires (10, 12).Open in a separate windowFig. 2.Annual area burned by wildfires and cumulative area infested by MPBs (2002–2013) across the western United States in primarily ponderosa pine (light green), mixed ponderosa and lodgepole pine (gold), primarily lodgepole pine (dark green), other MPB hosts (dark brown), and non-MPB hosts (light gray). (A) Annual area burned, calculated from the MTBS fire perimeter dataset. (B) Cumulative area infested by MPBs, determined from US Forest Service ADS data. For each year, ADS polygon data were converted to grids of 990 × 990-m pixels and cross-validated with a corresponding map of the distribution of MPB hosts. (C) Annual area burned in the cumulative area infested by MPBs.MPB-induced tree mortality is hypothesized to affect fire behavior by altering the flammability, continuity, and structure of fuels (6, 7). The surface, ladder, and crown fuels (collectively, the fuel profile) are expected to change with time since outbreak, potentially altering fire behavior and fire risk. Initially following tree death, needles fade to red (red stage; Fig. 1B) and risks of ignition, torching, and canopy fire are expected to increase due to lower leaf moisture content and decreases in nonfiber carbohydrates and fats, which increase flammability (13, 14). About 3 y following attack, trees drop their needles and twigs and become exposed in the upper crown (6) (gray stage; Fig. 1C). At this time, forest floor fuels are expected to increase due to falling needles, branches, and/or the rapid growth of shrubs and understory trees into ladder fuels (6, 7). During this time period, many stand-scale, fuel-driven fire behavior models predict a decreased risk of crown fire due to lower canopy bulk density and decreased continuity of canopy fuels (6, 7). However, field-based studies suggest that understory trees may also serve as ladder fuels to carry fire into the crowns (15), leading to an expectation that surface fires will be more likely to spread into the canopy during the gray stage.Whereas MPB infestation alters fuels and, as suggested by stand-level fire behavior models (e.g., NEXUS, BehavePlus, FARSITE, FlamMAP), changes fire behavior (7, 16–19), it is unclear if these fuel alterations lead to observed increases in area burned across heterogeneous landscapes (6, 7). Retrospective field studies of the effect of prefire MPB outbreaks on fire severity have shown little to no effect (20–23). In addition, the observed effects of prefire outbreaks on fire occurrence appear to be varied or negligible (24, 25). Instead, retrospective field studies indicate fire occurrence and severity are more strongly associated with other drivers of fire behavior, including elevation, slope, cover type, and stand structure (19–21, 24).The apparent disagreement between stand-level fire behavior models and field case studies arises from (i) mechanistic and spatial limitations of the models and dependence on simulated weather scenarios and (ii) confounding factors in empirical case studies, where the effect of MPB infestation on fire covaries with initial forest conditions and weather (20, 22). Furthermore, at a landscape scale, both wildfire and the initiation of an MPB outbreak are associated with extreme drought, complicating detection of any potential synergy between these disturbances. These uncertainties highlight the need for a comprehensive broad-scale analysis of the observed effects of bark beetle outbreaks on subsequent wildfire.To determine if MPB infestation affects area burned at a broad spatial extent, we analyzed spatial data of MPB infestation and area burned across the western United States (exclusive of Alaska) (Materials and Methods). We examined the area burned in MPB host forests during the 3 y of greatest annual area burned between 2002 and 2013, which, combined, are responsible for 46% of the total area burned in the West during the 12-y period (Fig. 2). During these years (2006, 2007, and 2012), 546 fires (>405 ha each) burned in both infested and uninfested MPB host forests.     

Pigeons trade efficiency for stability in response to level of challenge during confined flight

Individuals traversing challenging obstacles are faced with a decision: they can adopt traversal strategies that minimally disrupt their normal locomotion patterns or they can adopt strategies that substantially alter their gait, conferring new advantages and disadvantages. We flew pigeons (Columba livia) through an array of vertical obstacles in a flight arena, presenting them with this choice. The pigeons selected either a strategy involving only a slight pause in the normal wing beat cycle, or a wings-folded posture granting reduced efficiency but greater stability should a misjudgment lead to collision. The more stable but less efficient flight strategy was not used to traverse easy obstacles with wide gaps for passage but came to dominate the postures used as obstacle challenge increased with narrower gaps and there was a greater chance of a collision. These results indicate that birds weigh potential obstacle negotiation strategies and estimate task difficulty during locomotor pattern selection.Locomotion in the real world is uncertain. Error is present in estimations of self-motion and external object location. In environments that present novel or unusually challenging obstacles, moving animals use different strategies to successfully navigate: either maintaining a consistent motor program and relying on passive properties of their neuromechanical systems to carry them through safely, or altering their motor program to detect and avoid or navigate the challenge. Cockroaches traversing substantial obstacles maintain a constant motor pattern (1), whereas, in contrast, blind Mexican cavefish halt their swimming and glide to avoid collision with solid obstacles (2). In avian flight, this choice has been subject to little study, despite the confined and cluttered environments in which nesting, social, foraging, and hunting flight behaviors occur. How are birds able to roost in trees and bushes, perch in the gaps of chain-link fences, and fly at speed through forests during hunting without collision? Is this managed through careful path planning while maintaining a constant gait or through altering wing beat patterns on encountering each new obstacle?Bird navigation has been studied extensively on the scale of migration with visual, olfactory, magnetic, and auditory map senses reported (3–5). However, local navigation through cluttered environments has received little attention (6), with most local bird flight studies addressing questions of takeoff or landing, visual perception, flight kinematics, or of energetics (7–10).Here, we challenge pigeons (Columba livia; n = 4) to traverse a linear array of vertical poles at a variety of spacings, with gaps ranging from as narrow as 0.2 wingspans to as wide as 0.4 wingspans. Although we expected to see a clear traversal strategy emerge as in the terrestrial and aquatic systems noted above, we instead saw the use of two distinct postures, each used by all individuals (Fig. 1). One posture, requiring less disruption of the wing beat flight pattern, was used predominately to pass although wider gaps, whereas use shifted to the more disruptive posture as gap size decreased (Fig. 2). We examine the advantages and disadvantages of each traversal strategy that cause them to be selected or discarded when facing a given locomotor challenge.Open in a separate windowFig. 1.Two traversal postures: wings paused in upstroke and wings folded. Two stereotyped groups of postures were adopted in traversing the gaps between obstacles: (A) the “wings-paused” case in which the wings were held at the top of upstroke, with downstroke resuming on the far side of the obstacle array, and (B) the “wings-folded” case in which the wing stroke was interrupted and the wings tucked along the body and unfolded after passing between obstacles. These postures were quantitatively classified into paused and folded clusters (C) by applying a normal mixtures model to wrist elevation and wing pitch. Each posture class exhibits a stereotyped or typical posture near the centroid of their respective clusters, within a distribution of similar poses. In D, wrist elevation, the height difference between the mean of the torso-mounted markers shown in Fig. S3A and the wrist marker, is directly compared between postures. Similarly, in E, wing pitch, the pitch angle between wrist–wingtip line and upper-lower torso markers line, is directly compared between postures. In both cases, one-way ANOVA tests show significant differences between each posture’s mean pitch and elevation. Additionally, standard least-squares linear models of pitch and elevation as they depend on individual pigeon, traversal method (pause or fold), gap size, and the interaction between traversal method and gap size show traversal method as having a significant effect (P_effect < 0.001 in both cases).Open in a separate windowFig. 2.Fraction of trials using each posture as gap size changes. As the openings narrow, the strategy used shifts from predominantly pausing the wings to folding the wings in a majority of trials. A least-squares linear fit to the fraction folded or fraction paused, means across individuals, shows a significant dependence on gap size (P = 0.035 in both cases, as each is the inverse of the other).     

Early guenon from the late Miocene Baynunah Formation,Abu Dhabi,with implications for cercopithecoid biogeography and evolution

A newly discovered fossil monkey (AUH 1321) from the Baynunah Formation, Emirate of Abu Dhabi, United Arab Emirates, is important in a number of distinct ways. At ∼6.5–8.0 Ma, it represents the earliest known member of the primate subfamily Cercopithecinae found outside of Africa, and it may also be the earliest cercopithecine in the fossil record. In addition, the fossil appears to represent the earliest member of the cercopithecine tribe Cercopithecini (guenons) to be found anywhere, adding between 2 and 3.5 million y (∼50–70%) to the previous first-appearance datum of the crown guenon clade. It is the only guenon—fossil or extant—known outside the continent of Africa, and it is only the second fossil monkey specimen so far found in the whole of Arabia. This discovery suggests that identifiable crown guenons extend back into the Miocene epoch, thereby refuting hypotheses that they are a recent radiation first appearing in the Pliocene or Pleistocene. Finally, the new monkey is a member of a unique fauna that had dispersed from Africa and southern Asia into Arabia by this time, suggesting that the Arabian Peninsula was a potential filter for cross-continental faunal exchange. Thus, the presence of early cercopithecines on the Arabian Peninsula during the late Miocene reinforces the probability of a cercopithecoid dispersal route out of Africa through southwest Asia before Messinian dispersal routes over the Mediterranean Basin or Straits of Gibraltar.Cercopithecine monkeys (Order Primates, Superfamily Cercopithecoidea, Family Cercopithecidae, Subfamily Cercopithecinae), also known as cheek-pouch monkeys, are the most speciose and widely distributed group of living Old World primates. Recent molecular estimates date the divergence of Cercopithecinae from Colobinae (leaf-eating monkeys) to between 17.6 Ma (range 21.5–13.9 Ma) and 14.5 Ma (range 16.2–12.8 Ma) and the origin of crown Cercopithecinae to around 11.5 Ma (range 13.9–9.2 Ma) (1, 2). However, the earliest known fossil cercopithecines only appear much later, around 7.4 Ma in the Turkana Basin of East Africa (3, 4).Cercopithecine monkeys are divided into two tribes: Cercopithecini, including African guenons (Allenopithecus, Miopithecus, Chlorocebus, Erythrocebus, Allochrocebus, Cercopithecus), and Papionini, which includes African and Eurasian macaques (Macaca) as well as African papionins (Papio, Lophocebus, Rungwecebus, Theropithecus, Mandrillus, Cercocebus). Of the living cercopithecines, only two genera are known outside of the African continent, both of them papionins: Papio (found on the Arabian Peninsula) and Macaca (found throughout Southern and Southeast Asia, and introduced in Gibraltar). The earliest fossil cercopithecines known outside of Africa are attributed to the genus Macaca and appear to be latest Miocene or early Pliocene in age (∼6.0–5.0 Ma) (Fig. 1) (5–9). Until now, no guenons, extant or extinct, have ever been known outside of the African continent.Open in a separate windowFig. 1.Hypothesized cercopithecoid dispersal routes out of Africa in relation to the known late Miocene fossil record. The oldest cercopithecine, Parapapio lothagamensis (light blue circles), is known from ∼7.4–6.1 Ma in the Turkana Basin and Tugen Hills, Kenya (3, 4, 41). An unnamed fossil papionin (purple circle) is known from the late Miocene of Ongoliba, Congo (5, 57). Macaca spp. (dark blue circles) are located throughout North Africa at sites ranging in age from ∼6.5–5.5 Ma (5, 8, 58, 59), and Macaca spp. first appear in Europe ∼6.0–5.3 Ma and in China in the early Pliocene (5–9). The oldest colobine outside of Africa, Mesopithecus (green circles), is known from a number of late Miocene sites securely dated between ∼8.5 and 5.3 Ma in Greece, Macedonia, Italy, Ukraine, Iran, Afghanistan, possibly Pakistan, and China (46–48). Three dispersal routes for cercopithecoids can be hypothesized: route 1 imagines a dispersal event over the Straits of Gibraltar or Mediterranean Basin into Europe and Asia; route 2 postulates a dispersal event through the Arabian Sinai Peninsula; and route 3 suggests a migration over the Arabian Straits of Bab el Mandeb. The discovery of AUH 1321 and AUH 35 in Abu Dhabi at >6.5–8 Ma (red circle), contemporaneous with the first appearance of Mesopithecus sp. in Eurasia and ∼1–2 million y earlier than the appearance of Macaca spp. in Eurasia, suggests scenarios 2 and 3 were possible before scenario 1. None of these scenarios is mutually exclusive and may have occurred in combination or succession.Three possible routes can be reasonably hypothesized for cercopithecine (and cercopithecoid) dispersal out of Africa and into Europe and Asia during the late Miocene: (i) over the Mediterranean Basin or Straits of Gibraltar to the north/northwest, (ii) across the Arabian Sinai Peninsula to the northeast, or (iii) across the Arabian Straits of Bab el Mandeb to the east (Fig. 1). Fossil Macaca specimens from the terminal Miocene of Spain and Italy have been suggested to provide evidence for the use of a route across the Mediterranean Basin or the Straits of Gibraltar via an ephemeral land bridge either immediately before—or perhaps associated with—the drop in Mediterranean sea levels during the Messinian (∼6.0–5.3 Ma) (8–19). Paleontological evidence for an Arabian route has been lacking, but paleogeographic and paleoenvironmental work on circum-Arabia suggests that the region did not present a persistent ecological barrier to some amount of intercontinental exchange during the late Miocene (20). In fact, an established land connection through Sinai was probably present during this time period, and oceanic spreading is not estimated to have begun in the southern Red Sea until around 5 Ma, with progressive development of open marine conditions throughout the Pliocene. Thus, before 6.5 Ma, a southern route in the region of the Straits of Bab el Mandeb was also possible (Fig. 1) (21).Although Arabia is a large area of the earth, fossil monkeys have so far been represented by only a single specimen, an isolated male lower canine (AUH 35), discovered in 1989 by A.H. and Peter Whybrow in the late Miocene Baynunah Formation, Abu Dhabi, United Arab Emirates (22–25). The specimen came from Jebel Dhanna, site JDH-3 (JD-3 in refs. 24 and 26) (Fig. 2), a locality now lost to industrial development. Because male cercopithecid lower canines are not metrically identifiable beyond the Family level of classification (23), AUH 35 was described as a cercopithecid with indeterminate affinities. Here we report the discovery of a second monkey specimen from the Baynunah Formation in Abu Dhabi (AUH 1321), found almost 20 y after the first. AUH 1321 clearly represents a cercopithecine and, because it is dated to between 6.5 and 8.0 Ma, it is the oldest cercopithecine yet known outside of Africa and possibly the oldest cercopithecine in the fossil record. Thus, the discovery of AUH 1321 provides the earliest paleontological evidence of cercopithecine dispersal out of continental Africa and possibly hints at an Arabian cercopithecoid dispersal route into Eurasia during the Late Miocene (Fig. 1). Furthermore, we believe AUH 1321 can be attributed to the Cercopithecini (guenons) and, therefore, it represents the only record of this tribe, living or fossil, yet known outside of Africa.Open in a separate windowFig. 2.Map illustrating the location of the two fossil sites in the Baynunah Formation that have produced fossil monkeys. Top Right Inset shows the location of the SHU 2–2 excavation (kite aerial photography by Nathan Craig).     

Internal and external components of the bacterial flagellar motor rotate as a unit

Most bacteria that swim, including Escherichia coli, are propelled by helical filaments, each driven at its base by a rotary motor powered by a proton or a sodium ion electrochemical gradient. Each motor contains a number of stator complexes, comprising 4MotA 2MotB or 4PomA 2PomB, proteins anchored to the rigid peptidoglycan layer of the cell wall. These proteins exert torque on a rotor that spans the inner membrane. A shaft connected to the rotor passes through the peptidoglycan and the outer membrane through bushings, the P and L rings, connecting to the filament by a flexible coupling known as the hook. Although the external components, the hook and the filament, are known to rotate, having been tethered to glass or marked by latex beads, the rotation of the internal components has remained only a reasonable assumption. Here, by using polarized light to bleach and probe an internal YFP-FliN fusion, we show that the innermost components of the cytoplasmic ring rotate at a rate similar to that of the hook.Bacterial flagella are driven at their base by rotary motors (1) as shown dramatically by the tethered cell technique (2). Most of what we know about the structure of the flagellar basal body, shown schematically in Fig. 1, has been found by EM studies of material attached to the base of flagellar filaments that survives weakening of cell walls by treatment with lysozyme-EDTA, solubilization with Triton X-100, and differential centrifugation (3, 4). DePamphilis and Adler (3) described four rings on a rod, the M and S rings and the P and L rings: M for membranous, S for supramembranous, P for peptidoglycan, and L for lipopolysaccharide. This arrangement led to the suggestion that torque is generated between the S and M rings, with the former attached to the peptidoglycan layer (5). This idea was abandoned when it was found that the M and S rings are made from multiple copies of a single protein, FliF (6). It was then realized that the stator complexes, linked to the peptidoglycan by the C terminus of MotB, were the membrane studs seen in freeze-fracture experiments (7, 8). Each stator complex is composed of (MotA)₄ (MotB)₂ and sports two ion channels (9). On the cytoplasmic side of the M ring is what is now called the C ring (C for cytoplasmic), which contains FliG, FliM, and FliN, components of the switch complex that control the direction of flagellar rotation, although FliG is considered by some as part of the M ring rather than the C ring. One model inspired by the symmetry mismatch between FliG (26 subunits) and FliM (34 subunits) has been proposed in which, when cells are tethered so that the filament is fixed and the cell body rotates, the C ring rotates 8/34 as fast as the cell body (10). However, most workers assume that the M ring and the C ring rotate as a unit. Recent work with cryo-EM tomography has embellished this picture (11, 12) but has not changed the basic story. A general review is in ref. 13.Open in a separate windowFig. 1.A scale drawing of the base of the Escherichia coli flagellum embedded in three layers of the cell wall. The outer and inner layers are fluid, but the intermediate layer, the peptidoglycan, is rigid, which gives the cell the shape of a rod with semispherical end caps. The external components of the flagellum (components that extend beyond the cell wall) include the hook (FlgE), the hook-associated proteins (FlgK, FlgL, FliD), and the filament (FliC). All the other components are internal. The internal components thought to rotate include the rod, the MS ring, and the C ring. The filament (a polymer of the protein FliC; also called flagellin) is shown broken, because it is several micrometers long. Polymerization occurs under the distal cap (FliD). Two adapter proteins (hook-associated proteins FlgK and FlgL) enable the FlgE to flex and the filament to rotate rigidly. The filament is a propeller that exhibits different polymorphic forms depending on the direction of rotation and torsional load, whereas the hook is a flexible coupling (or universal joint). A flexible coupling is required, because the hooks project from the sides of the cell, whereas the bundle of filaments (approximately four in number) that pushes the cell forward tends to align with the long axis of the cell. The rod (or drive shaft: FliE and FlgB, -C, -F, and -G) is connected to the hook at its distal end and the MS ring (the central part of the rotor) at its proximal end. The rod passes through the L and P rings (FlgH and FlgI), which are mounted in the outer membrane and peptidoglycan layers, respectively, and thought to serve as bushings. Torque is generated when protons flow from the outside to the inside of the cell through two channels in a stator complex bounded by 4MotA and 2MotB. Each stator complex—there are as few as 1 or as many as 11—is linked to the peptidoglycan by the C terminus of MotB and interacts electrostatically through a cytoplasmic domain of MotA with the end of FliG farthest from the axis of rotation. Other components of the C ring, FliM and FliN, interact with the signaling molecule of the chemotaxis network, CheY-P, to control the direction of rotation. At room temperature, the direction of rotation in the absence of CheY-P is counterclockwise (the direction of rotation of the rod when viewed from outside of the cell; i.e., from the top in this figure). Each motor comprises 26 copies of FliF and FliG, 34–45 copies of FliM, and 34–45 tetramers of FliN. The FliN tetramers appear as donuts in the cross-sectional view of the C ring shown here. The motor changes the number of stator complexes in response to viscous load and the number of FliM and FliN subunits in response to the ambient direction of rotation; the smaller number (34) is found in clockwise-spinning motors, and the larger number (45) is found in counterclockwise-spinning motors. The FliN tetramers are about 4 nm apart and separated by C-terminal domains of FliM. Not shown are CheY-P; FliH, an export component known to interact with FliN; FliL, a component that enhances torque and interacts with the stator complex and the MS ring; and the flagellar export apparatus that coordinates the export of axial flagellar components and is mounted at the center of the cytoplasmic face of the MS ring.To investigate the rotation of the C ring, we designed a polarized fluorescence bleaching experiment reminiscent of experiments used to study the orientation of single macromolecules (14, 15). We labeled the most abundant component of the C ring, FliN, with a YFP fluorophore and probed it with weak polarized light. Each YFP was linked to the C ring at both its N and C termini. Then, we applied an intense, short flash of light of the same polarization. Because the fluorophores with absorption transition moments oriented along the electric vector of the excitation light are preferentially excited (16) and thus, bleached by the flash, the surviving fluorophores are expected to be oriented mostly perpendicularly to the direction of polarization of the excitation light at the time of bleaching as shown schematically in Fig. 2.Open in a separate windowFig. 2.A schematic diagram of the experiment showing the polarization of the probe and bleaching beams (S), the ring of fluorophores before bleaching (a), the ring of fluorophores immediately after bleaching (b), the ring of fluorophores one-quarter and successive one-half turns later (c), and the fluorescence emission intensity expected as a function of time.Under the assumption that YFP and FliN are rigidly attached to each other and the ring, the only reason for a fluorophore to change its orientation is the rotation of the C ring itself. Thus, the fluorescence emission that abruptly decreased after bleaching is expected to rapidly increase as the surviving fluorophores rotate now toward a parallel orientation. For an expected rotation frequency near zero load of around 300 Hz (17), the maximum emission is expected to occur in about 830 μs (at one-quarter of a turn) and then, decrease to a minimum in another 830 μs (at one-half of a turn). Assuming that the transition moments of the fluorophores are roughly in the plane of the membrane, the fluorescence emission after bleaching is expected to ring at twice the frequency of rotation of the C ring, because each dipole aligns with the polarization of the probe beam twice during each revolution. If this experiment is done with a population of motors rotating at slightly different speeds (figure 2 a and b in ref. 17), then the fluorescence emission after bleaching will damp out as the different C rings get out of phase.     

Efficient plant male fertility depends on vegetative nuclear movement mediated by two families of plant outer nuclear membrane proteins

Increasing evidence suggests that nuclear migration is important for eukaryotic development. Although nuclear migration is conserved in plants, its importance for plant development has not yet been established. The most extraordinary plant nuclear migration events involve plant fertilization, which is starkly different from that of animals. Instead of evolving self-propelled sperm cells (SCs), plants use pollen tubes to deliver SCs, in which the pollen vegetative nucleus (VN) and the SCs migrate as a unit toward the ovules, a fundamental but barely understood process. Here, we report that WPP domain-interacting proteins (WIPs) and their binding partners the WPP domain-interacting tail-anchored proteins (WITs) are essential for pollen nuclear migration. Loss-of-function mutations in WIT and/or WIP gene families resulted in impaired VN movement, inefficient SC delivery, and defects in pollen tube reception. WIPs are Klarsicht/ANC-1/Syne-1 Homology (KASH) analogs in plants. KASH proteins are key players in animal nuclear migration. Thus, this study not only reveals an important nuclear migration mechanism in plant fertilization but also, suggests that similar nuclear migration machinery is conserved between plants and animals.Nuclear migration is essential for cell differentiation, polarization, and migration, which influence organism development (1–3). Examples range from Caenorhabditis elegans P-cell development to mammalian neural development (1–3). The key players in opisthokont nuclear migration are the inner nuclear membrane Sad1/UNC-84 (SUN) proteins and outer nuclear membrane Klarsicht/ANC-1/Syne-1 Homology (KASH) proteins. SUN and KASH proteins form the linkers of the nucleoskeleton and the cytoskeleton complexes at the nuclear envelope (NE) and transfer cytoplasmic forces to the nucleus (1–3). In plants, nuclear migration is associated with a number of developmental events and environmental responses, including fertilization, root and leaf hair formation, and plant–microbe interactions (4, 5). So far, little is known about the mechanism of plant nuclear migration. Although SUN proteins are conserved in plants (6, 7), absence of animal KASH homologs in plants suggests that plants may have evolved different molecular solutions to achieve nuclear migration. Recently, WPP domain-interacting proteins (WIPs) were identified as KASH proteins in plants (8), and their outer nuclear membrane binding partners WPP domain-interacting tail anchored proteins (WITs) were shown to interact with myosin XI-I (9). The WIT–myosin XI-I complexes regulate nuclear movement in root and mesophyll cells, but no developmental events have been linked to these nuclear movements (9).Essential for plant fertility, pollen tube growth harbors the most dramatic nuclear movement in plants. Unlike animals, which have sperm cells (SCs) that travel through self-propelled flagellum, flowering plants use pollen tubes to deliver SCs to ovules (10–13). In Arabidopsis, pollen tube growth is guided by chemical cues in carpel tissues and attracted by small peptides secreted by synergid cells in the vicinity of ovules (14–18). Pollen tube reception is completed by pollen tube burst, SC release, and degeneration of synergid cells (12). If this process fails, a second pollen tube can be attracted to the same ovule for a second attempt, resulting in polytubey (19). The SCs [or their progenitor the generative cell (GC)] are enclosed by an endocytic membrane tethered to the pollen vegetative nucleus (VN) (20). During pollen tube elongation, the VN and the SCs/GC are usually closely associated and move as a male germ unit (MGU) (13, 21). For decades, the movement of the MGU has been analyzed using cytoskeleton-depolymerizing reagents or heterogeneous antimyosin antibodies (22–27). However, no genes have been implicated in MGU movement, and the function of the joint migration of VN and GC/SC remains hypothetical.Here, we have identified the Arabidopsis WIT and WIP protein families as key players in VN movement. WIP1 and WIT1 are localized at the vegetative nuclear envelope (VNE). Loss of either WIT or WIP family proteins impaired VN movement, resulting in defective pollen tube reception and inefficient SC-to-ovule migration. This study has not only identified a molecular mechanism regulating the VN movement but also, revealed an important function of the VN in plant fertilization.     

Heterochrony underpins natural variation in Cardamine hirsuta leaf form

A key problem in biology is whether the same processes underlie morphological variation between and within species. Here, by using plant leaves as an example, we show that the causes of diversity at these two evolutionary scales can be divergent. Some species like the model plant Arabidopsis thaliana have simple leaves, whereas others like the A. thaliana relative Cardamine hirsuta bear complex leaves comprising leaflets. Previous work has shown that these interspecific differences result mostly from variation in local tissue growth and patterning. Now, by cloning and characterizing a quantitative trait locus (QTL) for C. hirsuta leaf shape, we find that a different process, age-dependent progression of leaf form, underlies variation in this trait within species. This QTL effect is caused by cis-regulatory variation in the floral repressor ChFLC, such that genotypes with low-expressing ChFLC alleles show both early flowering and accelerated age-dependent changes in leaf form, including faster leaflet production. We provide evidence that this mechanism coordinates leaf development with reproductive timing and may help to optimize resource allocation to the next generation.Leaves of seed plants present an attractive model to address the genetic basis for morphological diversity at different scales because they show considerable variation between and within species, and their morphology also differs according to developmental age in a phenomenon known as heteroblasty (1) (Fig. 1). Leaves can be classified into two broad morphological classes: simple, where the blade is entire, or dissected (also referred to as compound), where the blade comprises individual leaflets (Fig. 1A). Both simple and dissected leaves emerge from a pluripotent structure called the shoot apical meristem. Previous work has identified two processes that underlie such interspecies diversification of leaf shape. The first is the generation of lateral cell proliferation axes that give rise to leaflets. This process typically involves reactivation of meristem genes in leaves such as class I Knotted1-like homeobox (KNOX1) and CUP-SHAPED COTYLEDON (CUC) genes, which influence the patterning of peaks of auxin activity that are required for leaflet formation (2–6). The second is the action of local growth repressors at the flanks of emerging leaflet primordia that promote leaflet separation. This process involves the leaf-specific homeobox gene REDUCED COMPLEXITY (RCO) (7). Furthermore, in dissected leafed species, leaf complexity is regulated by the activity of TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR (TCP) genes, which modulate the competence of the leaf margin to respond to organogenic signals (8, 9).Open in a separate windowFig. 1.Morphological diversity of leaves across different scales. (A) Morphology of C. hirsuta (Ox) and A. thaliana (Col-0) rosette leaves; different parts of the leaf are indicated. (B, Upper) Silhouettes of the fifth rosette leaf illustrate diversity in leaf morphology of natural C. hirsuta strains. (B, Lower) Quantification of total leaflet number in the first eight rosette leaves is shown in the bar chart. Data are reported as means ± SD. (C) Silhouettes of the first nine rosette leaves from a typical heteroblastic series in C. hirsuta (Ox).     

Suppressed expression of RETROGRADE-REGULATED MALE STERILITY restores pollen fertility in cytoplasmic male sterile rice plants

Conflict/reconciliation between mitochondria and nuclei in plants is manifested by the fate of pollen (viable or nonviable) in the cytoplasmic male sterility (CMS)/fertility restoration (Rf) system. Through positional cloning, we identified a nuclear candidate gene, RETROGRADE-REGULATED MALE STERILITY (RMS) for Rf17, a fertility restorer gene for Chinese wild rice (CW)-type CMS in rice (Oryza sativa L.). RNA interference-mediated gene silencing of RMS restored fertility to a CMS plant, whereas its overexpression in the fertility restorer line induced pollen abortion. The mRNA expression level of RMS in mature anthers depended on cytoplasmic genotype, suggesting that RMS is a candidate gene to be regulated via retrograde signaling. We found that a reduced-expression allele of the RMS gene restored fertility in haploid pollen, whereas a normal-expression allele caused pollen to die in the CW-type CMS. RMS encodes a mitochondrial protein, 178 aa in length, of unknown function, unlike the majority of other Rf genes cloned thus far, which encode pentatricopeptide repeat proteins. The unique features of RMS provide novel insights into retrograde signaling and CMS.     

Metagenomic natural product discovery in lichen provides evidence for a family of biosynthetic pathways in diverse symbioses

Bacteria are a major source of natural products that provide rich opportunities for both chemical and biological investigation. Although the vast majority of known bacterial metabolites derive from free-living organisms, increasing evidence supports the widespread existence of chemically prolific bacteria living in symbioses. A strategy based on bioinformatic prediction, symbiont cultivation, isotopic enrichment, and advanced analytics was used to characterize a unique polyketide, nosperin, from a lichen-associated Nostoc sp. cyanobacterium. The biosynthetic gene cluster and the structure of nosperin, determined from 30 μg of compound, are related to those of the pederin group previously known only from nonphotosynthetic bacteria associated with beetles and marine sponges. The presence of this natural product family in such highly dissimilar associations suggests that some bacterial metabolites may be specific to symbioses with eukaryotes and encourages exploration of other symbioses for drug discovery and better understanding of ecological interactions mediated by complex bacterial metabolites.Symbiosis, defined by de Bary (1) as the “living together of two organisms,” includes a broad range of partnerships, from loose associations to obligate interdependencies and host–parasite interactions. Many involve microbes, with perhaps the most successful—between bacteria and early nucleated cells in the Precambrian—leading to mitochondria and chloroplasts in modern eukaryotes (2). Symbiotic interactions are being examined with increasing molecular detail, focusing not only on attributes that may be beneficial for each organism individually but also on what might be important for the association. It is increasingly being recognized that biosynthetic pathways leading to synthesis of specialized metabolites may play key roles in the biology of symbiosis (3).Lichens are ancient and physiologically highly integrated symbioses between heterotrophic filamentous fungi (mycobionts) and cyanobacteria or coccoidal green algae (photobionts) that may date as far back as 600 Mya (4). The morphology of the characteristic and stable macroscopic body of a lichen, the thallus, typically bears little resemblance to the individual organisms that form it and, in many cases, can be highly organized: fungal cells on the periphery for physical support and protection and photobiont cells inside, providing photosynthate or fixed nitrogen or both (5) (Fig. 1 A–C). Although the photobionts can often be isolated in pure culture (Fig. 1D), most mycobionts (almost exclusively from the Ascomycota) are refractory to propagation in vitro by standard methods, and intact lichens cannot be maintained artificially for long. Nevertheless, such limitations are gradually being overcome using advanced analytical platforms, e.g., metagenomics in the characterization of mycobiont lectin genes (6), and PCR-based phylogenetics in investigation of intrathalline bacterial diversity (7).Open in a separate windowFig. 1.The foliose lichen Peltigera membranacea and Nostoc symbiont. (A) Lichen in situ. (Scale bar, 5 cm.) (B) Rhizines (Rhi) on lower surface and apothecia (Apo) protruding from thallus edge. (C) Thallus cross section illustrating stratified internal structure including photosynthetic cyanobiont layer (shown with arrows) between cortical and medullary mycobiont layers (above and below, respectively). (Scale bar, 100 μm.) (D) Nostoc sp. N6 in culture. (Scale bar, 100 μm.) (photograph for Fig. 1C, courtesy of Martin Grube).In a number of bacterial–eukaryote symbioses, bacterial partners have been implicated in the production of complex molecules derived from polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) pathways (3, 8, 9). Examples include pederin, made by bacteria that live in rove beetles of the genus Paederus, and structurally related metabolites, the onnamides and psymberin, produced by bacteria that live in marine sponges (Fig. 2). In general, metabolites known or suspected to be of symbiont origin show remarkably low structural overlap with natural products discovered in screening programs from free-living bacteria (10). This phenomenon raises the intriguing question of whether symbiont chemistry might encompass structural scaffolds covering distinctive regions of chemical space.Open in a separate windowFig. 2.Pederin family compounds and symbioses. (Upper Left) Image of Paederus fuscipes courtesy of Christoph Benisch (www.kerbtier.de). (Upper Right) Image of Theonella swinhoei courtesy of Yoichi Nakao. (Lower Right) Image of Psammocinia aff. bulbosa adapted with permission from ref. 15. Copyright 2007 American Chemical Society. (Lower Left) Image of Mycale hentscheli courtesy of Mike Page.In this study, we applied a combination of metagenomic and natural product discovery methods to identify nosperin, the first member of the pederin family from a lichenized cyanobacterium and a further example toward the emerging concept of symbiosis-associated natural product pathways (10).     

Key driving forces in the biosynthesis of autoinducing peptides required for staphylococcal virulence

Staphylococci produce autoinducing peptides (AIPs) as quorum-sensing signals that regulate virulence. These AIPs feature a thiolactone macrocycle that connects the peptide C terminus to the side chain of an internal cysteine. AIPs are processed from ribosomally synthesized precursors [accessory gene regulator D (AgrD)] through two proteolytic events. Formation of the thiolactone is coupled to the first of these and involves the activity of the integral membrane protease AgrB. This step is expected to be thermodynamically unfavorable, and therefore, it is unclear how AIP-producing bacteria produce sufficient amounts of the thiolactone-containing intermediate to drive quorum sensing. Herein, we present the in vitro reconstitution of the AgrB-dependent proteolysis of an AgrD precursor from Staphylococcus aureus. Our data show that efficient thiolactone production is driven by two unanticipated features of the system: (i) membrane association of the thiolactone-containing intermediate, which stabilizes the macrocycle, and (ii) rapid degradation of the C-terminal proteolysis fragment AgrD^C, which affects the reaction equilibrium position. Cell-based studies confirm the intimate link between AIP production and intracellular AgrD^C levels. Thus, our studies explain the chemical principles that drive AIP production, including uncovering a hitherto unknown link between quorum sensing and peptide turnover.Quorum sensing (QS) is a process in which individual microbes produce and sense a diffusive signaling molecule (known as the autoinducer) to coordinate behaviors of the population (1). In the commensal pathogen Staphylococcus aureus, QS is required for the synchronized adjustment of gene expression patterns that ultimately enables the pathogen to establish an infection and endure the immune defense of the mammalian host (2, 3). The chromosomal locus that encodes this QS machinery is called the accessory gene regulator (agr). The autoinducer, autoinducing peptide (AIP), contains a thiolactone ring formed by condensation of the C-terminal carboxyl group and the sulfhydryl group of an internal cysteine (Fig. 1A, Inset). The resulting macrocycle is absolutely necessary for binding of the peptide to its receptor AgrC (2) and conserved in the autoinducer peptides produced by a plethora of low-GC, Gram-positive pathogens, with variations to oxolactones seen in a few cases (4, 5).Open in a separate windowFig. 1.AIP biosynthesis and models for free energy change estimation for the thiolactone formation. (A) Models of AIP production in S. aureus cells as exemplified by processing of AgrD-I. The generally accepted pathway and an alternative pathway are depicted using black and dashed arrows, respectively. (Inset) Sequences of AIPs produced by agr variants I and II. (B) A two-step model that recapitulates the thiolactone formation from a linear peptide. Estimated values of free energy change and/or equilibrium constant are shown for each step. Details are in the text.AIP biosynthesis is of particular interest to us, because formation of the high-energy thiolactone linkage is thought to not be coupled to ATP hydrolysis. It is unclear how the bacteria overcome the thermodynamic challenge to produce AIP at a rate sufficient for punctual QS induction. In the current AIP biosynthesis model (Fig. 1A, black arrows), the peptide is ribosomally translated as a precursor, AgrD, in which the mature AIP sequence is flanked by an N-terminal leader peptide and a C-terminal recognition sequence (6). The N-terminal leader forms an amphipathic helix, which attaches the precursor to the inner leaflet of the cell membrane (7). In the first step, AgrD is processed by a membrane-integrated peptidase, AgrB, such that the C-terminal recognition sequence (AgrD^C) is removed with concomitant installation of the thiolactone (Fig. 1A, step 1) (8, 9). This intermediate, herein referred to as AgrD(1–32)-thiolactone, is then translocated to the extracellular leaflet of the cell membrane, where the general signal peptidase SpsB is thought to clip off the N-terminal leader peptide (10), releasing AIP to the extracellular space (Fig. 1A, step 2). The first proteolytic event in this process is reminiscent of the ring closure step in the biosynthesis of a spectrum of lactam-containing ribosomal peptide natural products, including cyanobactins, amatoxins, cyclotides, and orbitides (11). However, although lactam formation through peptidyl transfer is roughly isoenthalpic (and hence, entropically driven overall), thiolactone formation through an analogous process is expected to be an enthalpically uphill process and thus, overall thermodynamically less favorable. We note that, although the pathway discussed above is generally accepted, the existence of the thiolactone intermediate has not been unambiguously proven (9, 12). Thus, an alternative and thermodynamically reasonable pathway could involve a process in which AgrD is first cleaved by AgrB to give a linear AgrD(1–32) peptide, which is then cyclized to give the AgrD(1–32)-thiolactone on, for example, ATP-dependent activation on the C-terminal carboxylate (Fig. 1A, dashed arrows). A full understanding of thiolactone formation in the AIP, therefore, necessitates precise characterization of the product of step 1 and if cyclization occurs, demonstration of its spontaneity under physiologically relevant conditions.Herein, we present the in vitro reconstitution of the first step in AIP biosynthesis using highly purified recombinant or synthetic reagents. AgrB from S. aureus forms stable dimers and is functional only when embedded in lipid bilayers. AgrB catalyzes the efficient, reversible cyclization of AgrD into the thiolactone intermediate alongside a slow, irreversible hydrolysis reaction that gives the linear AgrD(1–32) fragment. From a quasiequilibrium state involving AgrD, AgrD(1–32)-thiolactone, and the AgrD^C fragment, we determined the equilibrium constant of the cyclization. Our data suggest that the bacterium can maintain a reasonable intracellular level of thiolactone intermediate only when the turnover of AgrD^C through additional proteolysis is efficient. Membrane targeting of this intermediate by the N-terminal leader sequence induces lipid partitioning of the macrocycle and thereby, enhances its stability against ring-opening thiolysis. This effect partly ameliorates the enthalpic deficit of the thiolactone formation and renders the reaction more permissible than would otherwise be expected.     

Replication initiator DnaA binds at the Caulobacter centromere and enables chromosome segregation

During cell division, multiple processes are highly coordinated to faithfully generate genetically equivalent daughter cells. In bacteria, the mechanisms that underlie the coordination of chromosome replication and segregation are poorly understood. Here, we report that the conserved replication initiator, DnaA, can mediate chromosome segregation independent of replication initiation. It does so by binding directly to the parS centromere region of the chromosome, and mutations that alter this interaction result in cells that display aberrant centromere translocation and cell division. We propose that DnaA serves to coordinate bacterial DNA replication with the onset of chromosome segregation.Cell division requires the faithful transmission of genetic information to each daughter cell. Thus, in all forms of life, multiple mechanisms cooperate to ensure that DNA synthesis and chromosome segregation are temporally controlled and coordinated. Unlike eukaryotes, in which chromosomes are fully replicated and organized into higher order structures before segregation (1), most bacteria segregate their chromosomes progressively during replication (2). DnaA is a conserved bacterial protein responsible for the initiation of DNA synthesis at the chromosomal origin of replication (ori) (3, 4). The mechanism by which chromosome segregation is initiated in bacteria is less well understood.Although the factors responsible for DNA replication are highly conserved among bacterial species, multiple mechanisms have been proposed to account for chromosome segregation (5). In the G₁ phase of the Caulobacter crescentus cell cycle, the centromeric region of the chromosome (parS) is tethered to one pole of the cell (Fig. 1A). Upon the swarmer to stalked cell transition, replication initiates with replisome assembly at the origin of replication. The Par system in Caulobacter includes parS and two partitioning proteins, ParA and ParB. The ParB protein binds to parS (6, 7), which, in turn, interacts with the nucleoid-associated ParA ATPase to effect centromere movement (8–11).Open in a separate windowFig. 1.DnaA-dependent chromosome translocation. (A) Dynamics of Caulobacter chromosome segregation. (B–F) Time-lapse fluorescence micrographs of CFP-ParB in synchronized cells grown on M2G agarose pads (Left). Plots in Right depict fraction of synchronized cells with CFP-ParB translocated to the new pole (reported as % cells) grown in liquid media. Samples were taken every 30 min and imaged over a 3-h time-course. Data are represented as mean ± SD from two independent experiments with n equals on average 200 cells per time point per experiment. Caulobacter cells have a faster generation time in liquid media than on agarose pads. (B) Wild-type cells (parB::cfp-parB; MT190). Cells with dnaA expression regulated by P_xylX (parB::cfp-parB, dnaA::Ω, xylX::dnaA; LS5368) grown on M2G + 0.3% xylose (C) or M2G only (D). Cells with dnaA expression regulated by P_vanA (parB::cfp-parB, dnaA::Ω, vanA:dnaA; LS5369) grown on M2G + 250 μM vanillate (E) or M2G only (F); arrows indicate segregation of CFP-ParB/parS. (G) Vanillate promoter has leaky expression of DnaA. Relative levels of DnaA were followed by using Western blots with anti-DnaA antibodies (1 in 10,000 dilution) in mixed population of LS101 (Top), LS5368 (Middle), and LS5369 (Bottom) undergoing DnaA depletion. Cells were washed three times and grown on liquid M2G. Exposures of all samples were done simultaneously. (H) DnaA-dependent centromere translocation. Plotted are the percentages of cells (parB::cfp-parB, dnaA::Ω, xylX::dnaA; LS5368) with a single CFP-ParB focus translocated to the distant pole as a function of levels of xylose inducer added (i.e., varying subphysiological levels of DnaA). Localization of CFP-ParB was determined by fluorescence microscopy of cells grown in M2G liquid media with the respective xylose concentration.In Vibrio cholerae and Bacillus subtilis, the chromosome partitioning protein ParA (Soj) has been reported to regulate replication initiation by directly interacting with the DnaA replication initiator protein, suggesting a connection between segregation and the initiation of replication (12–14). However, the signals that trigger the Par system to initiate chromosome segregation are not known. By generating Caulobacter strains that express limited concentrations of DnaA, we sought to determine whether replication initiation is a prerequisite for the translocation of the centromere complex. Under these conditions, we were able to detect translocation of the chromosome in the absence of replication. We show that DnaA binds directly within the parS region and that altering binding of DnaA to parS leads to compromised chromosome segregation. These results suggest that, in Caulobacter, DnaA plays a direct role in the initiation of chromosome segregation.     

Peptidoglycan synthesis in Mycobacterium tuberculosis is organized into networks with varying drug susceptibility

Peptidoglycan (PG), a complex polymer composed of saccharide chains cross-linked by short peptides, is a critical component of the bacterial cell wall. PG synthesis has been extensively studied in model organisms but remains poorly understood in mycobacteria, a genus that includes the important human pathogen Mycobacterium tuberculosis (Mtb). The principle PG synthetic enzymes have similar and, at times, overlapping functions. To determine how these are functionally organized, we carried out whole-genome transposon mutagenesis screens in Mtb strains deleted for ponA1, ponA2, and ldtB, major PG synthetic enzymes. We identified distinct factors required to sustain bacterial growth in the absence of each of these enzymes. We find that even the homologs PonA1 and PonA2 have unique sets of genetic interactions, suggesting there are distinct PG synthesis pathways in Mtb. Either PonA1 or PonA2 is required for growth of Mtb, but both genetically interact with LdtB, which has its own distinct genetic network. We further provide evidence that each interaction network is differentially susceptible to antibiotics. Thus, Mtb uses alternative pathways to produce PG, each with its own biochemical characteristics and vulnerabilities.One of the leading causes of infectious disease deaths worldwide is tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb). One-third of the human population is thought to harbor Mtb and ∼1.5 million individuals died of TB last year (1). Mtb’s success as a pathogen is due in part to its unusual cell wall, which is notorious for its complexity and is implicated in Mtb’s innate resistance to many commonly used antibiotics (2). A critical component of the bacterial cell wall (including Mtb’s) is peptidoglycan (PG), a complex polymer that provides structural support and counteracts turgor pressure (3). PG is essential for cell survival, and its synthesis is targeted by many potent antibiotics (2).PG consists of long glycan chains composed of two different sugars (Fig. 1A) that are cross-linked via short peptide side chains that extend from the glycan chains. Notably, generation of mature PG occurs outside of the cell membrane and is mediated by enzymes that incorporate new PG subunits, which are formed in the cytoplasm, into the PG polymer. PonA1 and PonA2 are the two enzymes in Mtb that can both polymerize glycan strands and cross-link peptides [known as bifunctional penicillin binding proteins (PBPs), Fig. 1A]. The predominant peptide cross-links in mycobacteria join the third amino acids (3–3 link) of adjacent stem peptides (4, 5), which are synthesized by l,d-transpeptidases (Ldts) such as LdtB, one of the major Ldts in Mtb (Fig. 1A). The peptides can also be joined by cross-linking the fourth and third amino acids (4–3 link) (Fig. 1A) through the action of bifunctional or monofunctional (capable of only peptide cross-linking) PBPs. The activity of these distinct factors must be coordinated to ensure proper cell-wall synthesis. One method of coordination is the use of large protein complexes, the elongation complex and divisome, which mediate cell-wall biogenesis during cell elongation or division, respectively (2). The essential activity of these enzymes makes them prime drug targets; indeed, PBPs and Ldts are inhibited by carbapenems and penicillin (6, 7), which remains one of the most clinically important drugs in use.Open in a separate windowFig. 1.Deletion of PG synthases influences growth and morphology of Mtb. (A) Transglycosylation (TG) and transpeptidation (TP) reactions incorporate new PG subunits into the cell wall. PonA1 and PonA2 carry out both TG and 4–3 TP reactions. LdtB only mediates 3–3 TP reactions. M, N-acetylmuramic acid. G, N-acetylglucosamine. (B) Deletion of either ponA2 or ldtB does not greatly affect Mtb growth during log phase, although loss of ldtB reduces population density in stationary phase. Error bars are often too small to see. (C) ponA2 mutant cells (n = 179) have increased width compared with wild-type cells (n = 153) (approximate P value <0.0001 by the Kolmogorov–Smirnov test). (D) ldtB mutant cells (n = 193) have increased width and decreased length compared with wild-type cells (n = 153) (approximate P value <0.0001 by the Kolmogorov–Smirnov test for both length and width).Although the biosynthesis and structure of PG have been investigated for decades, predominantly in organisms such as Escherichia coli or Bacillus subtilis, the mechanisms that coordinate the biochemical activities required to polymerize and modify the cell wall remain incompletely understood. Moreover, much less is known about PG synthesis in many pathogenic organisms, including Mtb (2). However, previous studies in Mtb suggest that PG synthesis in this pathogen does not strictly conform to the E. coli paradigm. For example, E. coli has three bifunctional PBPs [PBP1A, PBP1B, and PBP1C (3)], whereas Mtb has just two [PonA1 and PonA2 (8)]. Additionally, PBP2 (known as PBPA in mycobacteria) is a monofunctional PBP and is required for cell elongation in E. coli, but instead seems to function in cell septation in mycobacteria (3, 9).The structure of PG is also different in Mtb than in E. coli: Mycobacterial PG has an unusual prevalence of 3–3 peptide linkages. The abundance of 3–3 cross-links in mycobacterial PG throughout different growth stages (5) suggests that Ldts are active during normal growth; however, their cellular roles or regulation during growth and PG biogenesis remain largely unknown. Whereas penicillins and cephalosporins target only enzymes that produce 4–3 cross-links, Ldts can be targeted by carbapenems (7). Recent work suggests that these agents might be far more efficacious against both dividing and nondividing bacteria (10). Although little is known about Mtb’s five encoded Ldts (11), one, LdtB, is implicated in antibiotic tolerance (11–13), is required for normal virulence in a mouse model of TB (12), and is important for normal cell shape (13).Previous studies have also revealed that PG biosynthesis differs between Mtb and the related saprophytic Mycobacterium smegmatis (Msm). As opposed to Mtb, Msm has three bifunctional PBPs: PonA1, PonA2, and PonA3 (14). PonA1 is required for Msm but not Mtb growth in culture (15, 16); however, PonA1 is required for robust growth of Mtb during infection (16). In contrast, Mtb and Msm ponA2 mutants do not have growth defects in culture (14, 17). However, Mtb strains with inactivated ponA1 or ponA2 exhibit similar survival defects during growth in a host (16, 18), suggesting that these two similar bifunctional enzymes have nonredundant and important contributions to PG synthesis during infection. Collectively, the differences in PG synthase functionality may imply that different PG synthetic pathways exist across species, which may have consequences for a pathogen’s virulence during infection.Here, we interrogated PG synthesis in Mtb by investigating the genetic interactions of ponA1, ponA2, and ldtB, which encode three PG synthases critical for Mtb’s growth during infection. To identify these interactions, we performed genome-wide transposon mutagenesis screens in Mtb mutant strains that lacked one of these enzymes. Advances in high-throughput sequencing technology coupled with the power of whole-genome studies provide unique insights into key bacterial processes, such as cell-wall biosynthesis. Such studies have been performed to a limited extent in bacteria, and further work would substantially expand our understanding of the organization of prokaryotic metabolic processes. In this study, we identified diverse genetic interaction networks for ponA1, ponA2, and ldtB, suggesting that these synthases are embedded within distinct cellular networks for assembling Mtb’s PG. We found that either ponA1 or ponA2 is required for cell growth, and that ldtB interacts with both ponA1 and ponA2. Moreover, mutants that lack these enzymes have differential susceptibility to agents that interfere with cell-wall biogenesis. Thus, the Mtb cell wall is synthesized using multiple interacting networks that are both overlapping and unique.     

Genome of Rhodnius prolixus,an insect vector of Chagas disease,reveals unique adaptations to hematophagy and parasite infection

Rhodnius prolixus not only has served as a model organism for the study of insect physiology, but also is a major vector of Chagas disease, an illness that affects approximately seven million people worldwide. We sequenced the genome of R. prolixus, generated assembled sequences covering 95% of the genome (∼702 Mb), including 15,456 putative protein-coding genes, and completed comprehensive genomic analyses of this obligate blood-feeding insect. Although immune-deficiency (IMD)-mediated immune responses were observed, R. prolixus putatively lacks key components of the IMD pathway, suggesting a reorganization of the canonical immune signaling network. Although both Toll and IMD effectors controlled intestinal microbiota, neither affected Trypanosoma cruzi, the causal agent of Chagas disease, implying the existence of evasion or tolerance mechanisms. R. prolixus has experienced an extensive loss of selenoprotein genes, with its repertoire reduced to only two proteins, one of which is a selenocysteine-based glutathione peroxidase, the first found in insects. The genome contained actively transcribed, horizontally transferred genes from Wolbachia sp., which showed evidence of codon use evolution toward the insect use pattern. Comparative protein analyses revealed many lineage-specific expansions and putative gene absences in R. prolixus, including tandem expansions of genes related to chemoreception, feeding, and digestion that possibly contributed to the evolution of a blood-feeding lifestyle. The genome assembly and these associated analyses provide critical information on the physiology and evolution of this important vector species and should be instrumental for the development of innovative disease control methods.Dating back to Wigglesworth’s pioneering work in the 1930s (1), Rhodnius prolixus (Fig. 1A) has served as a model organism for the study of cellular and physiological processes in insects, such as gametogenesis, the role of hemolymph proteins and lipids in oogenesis, and ion and water transport mechanisms. As an obligate blood-feeding hemipteran, this insect has adapted remarkably well to digesting and eliminating the potentially toxic by-products of blood digestion. R. prolixus is also a major vector of Trypanosoma cruzi, the parasitic protozoan that causes Chagas disease in humans. This disease, commonly considered a disease of the poor, causes premature heart failure in humans and is responsible for high economic and social costs. Approximately 10,000 people die from the disease annually and 100 million people are at risk for infection (2).Open in a separate windowFig. 1.Rhodnius genome. (A) Genome overview. All scaffolds are represented in layer I and are organized clockwise from the longest to the shortest, starting at the arrowhead. The genic (layer II, red) and TEs (layer III, blue) showed opposite densities until the asterisk (*) and were similarly low from this point until the end (shorter scaffolds). The Wolbachia sp. insertions (layer IV, orange) were observed throughout the genome without a trend. The kernel picture illustrates an adult R. prolixus. (B) Gene clustering. The Venn diagram partitions 15,439 OrthoMCL gene clusters according to their species compositions for R. prolixus and three other Hemimetabola (blue), four Diptera (yellow), four other Holometabola (green), and four noninsect outgroup species (pink). The 6,993 R. prolixus genes show widespread orthology (white circle, and bars, Bottom Left): these are part of the 7,115 clusters that have representatives from each of the four species sets, of which a conserved core of 2,253 clusters have orthologs in all 16 species. The 5,498 R. prolixus genes show no confident orthology (bars, Bottom Right), but most of these are homologous (e-value < 1e-05) to genes from other animals or to genes in its own genome. (C) TE distribution. The inner chart represents the three main classes of TEs (LTRs, non-LTRs, and class II), and the outer shows the distribution of TE superfamilies within each class. The charts are based on the total base pairs occupied by TE-related sequences longer than 0.5 Kb, as shown in SI Appendix, Table A1.     

Horizontal gene transfer and gene dosage drives adaptation to wood colonization in a tree pathogen

Some of the most damaging tree pathogens can attack woody stems, causing lesions (cankers) that may be lethal. To identify the genomic determinants of wood colonization leading to canker formation, we sequenced the genomes of the poplar canker pathogen, Mycosphaerella populorum, and the closely related poplar leaf pathogen, M. populicola. A secondary metabolite cluster unique to M. populorum is fully activated following induction by poplar wood and leaves. In addition, genes encoding hemicellulose-degrading enzymes, peptidases, and metabolite transporters were more abundant and were up-regulated in M. populorum growing on poplar wood-chip medium compared with M. populicola. The secondary gene cluster and several of the carbohydrate degradation genes have the signature of horizontal transfer from ascomycete fungi associated with wood decay and from prokaryotes. Acquisition and maintenance of the gene battery necessary for growth in woody tissues and gene dosage resulting in gene expression reconfiguration appear to be responsible for the adaptation of M. populorum to infect, colonize, and cause mortality on poplar woody stems.Domestication of forest trees, in contrast to agricultural crops, has become prevalent only during the last few centuries and often encompasses a transition from wild, complex ecosystems to homogeneous and intensively managed plantations that are frequently composed of a single tree species (1). The recent ability to use modern genetic and genomic techniques with conventional breeding promises to speed up tree domestication (2). Poplar has emerged as an extremely versatile tree with natural attributes favorable to its domestication. The ease of vegetative propagation and the breeding of interspecific hybrids with broad adaptability, improved growth, and disease resistance has contributed to the widespread use of poplar for a variety of commercial products, including lumber, paper, and bioenergy feedstock (3). One of the challenges of poplar domestication has been the emergence of pathogens that were innocuous in their natural pathosystems, but can cause severe losses in plantations. Native poplars still vastly outnumber planted trees, and this large reservoir of a naturally coevolved pathosystem in close proximity to intensively managed clonal plantations could destabilize the host–pathogen equilibrium, leading to new disease epidemics (4).A native endemic fungus on northeastern and north-central North American poplars, Mycosphaerella populorum (anamorph = Sphaerulina musiva; class Dothideomycetes) occurs in natural stands of native Populus deltoides, causing necrotic foliar lesions, but rarely resulting in early defoliation (5). With the introduction of exotic poplar species at the beginning of the 20th century and the intensification of hybrid poplar cultivation in North America, M. populorum has emerged as a stem-infecting pathogen, causing stem cankers that lead to weakening and breakage of the tree trunk, often resulting in plantation failure (Fig. 1A) (6, 7). The pathogen can attack a broad range of susceptible hybrid poplars and has also expanded its geographic range. It has recently been reported, to our knowledge, for the first time west of the Rocky Mountains and in Argentina and Brazil (6–8). This disease has become the most important factor limiting poplar plantations in eastern North America and could threaten the poplar industry worldwide (6, 9).Open in a separate windowFig. 1.M. populorum and M. populicola symptoms and divergence time estimate. (A, Top Left) M. populorum branch canker and leaf spots on a P. deltoides × P. trichocarpa clone. (Top Right) M. populicola leaf-spot symptoms on P. trichocarpa. (Bottom) M. populorum stem canker on P. deltoides × P. maximowiczii (Left) and P. deltoides × P. trichocarpa (Right) that caused the trunk to break at the canker. Photo provided by Harry Kope. (B) Maximum-likelihood phylogeny of M. populorum, M. populicola, and 16 other ascomycete fungi. All nodes received a bootstrap support of 100% except one, indicated with a 94% value. Colored stars: calibration points in million years (SI Appendix, Phylogenomic Analysis).A sister species to this pathogen, Mycosphaerella populicola (anamorph = S. populicola; class Dothideomycetes), is also endemic on native poplars, causing a leaf spot symptom on Populus balsamifera and Populus trichocarpa, but has a much broader geographical distribution than M. populorum (6, 10). This pathogen is considered a lower threat to poplar plantations because it does not cause stem cankers under natural conditions or in plantations and it has a narrow host range (11).To identify genetic factors underlying the canker symptom, we sequenced and compared the genomes of these two closely related pathogens with different natural host ranges and etiological characteristics. This provided a unique opportunity to contrast the evolutionary consequences of the adaptation of a tree pathogen to different hosts and the ability to gradually transition from natural to domesticated ecosystems. Our results show that the genome of M. populorum has evolved a broader battery of genes and has acquired genes through horizontal transfer that are absent in its sister species. These genes are enriched in functions that allow M. populorum to infect woody tissues.     

From the Cover: Experimental replacement of an obligate insect symbiont

Symbiosis, the close association of unrelated organisms, has been pivotal in biological diversification. In the obligate symbioses found in many insect hosts, organisms that were once independent are permanently and intimately associated, resulting in expanded ecological capabilities. The primary model for this kind of symbiosis is the association between the bacterium Buchnera and the pea aphid (Acyrthosiphon pisum). A longstanding obstacle to efforts to illuminate genetic changes underlying obligate symbioses has been the inability to experimentally disrupt and reconstitute symbiont–host partnerships. Our experiments show that Buchnera can be experimentally transferred between aphid matrilines and, furthermore, that Buchnera replacement has a massive effect on host fitness. Using a recipient pea aphid matriline containing Buchnera that are heat sensitive because of an allele eliminating the heat shock response of a small chaperone, we reduced native Buchnera through heat exposure and introduced a genetically distinct Buchnera from another matriline, achieving complete replacement and stable inheritance. This transfer disrupted 100 million years (∼1 billion generations) of continuous maternal transmission of Buchnera in its host aphids. Furthermore, aphids with the Buchnera replacement enjoyed a dramatic increase in heat tolerance, directly demonstrating a strong effect of symbiont genotype on host ecology.Symbiosis has been key in the ecological and evolutionary diversification of eukaryotes (1, 2). In many invertebrates, bacterial symbionts have been maternally transmitted for millions of years and are required for the growth and reproduction of hosts (3). These symbionts approach organelles in their degree of genetic and physiological integration with hosts and in their extreme genomic reduction. A model for obligate symbiosis is that of the pea aphid (Acyrthosiphon pisum) and its nutrient-provisioning bacterial symbiont, Buchnera aphidicola. Buchnera features a tiny genome (4), restriction to a small number of specialized host cells (bacteriocytes), host-controlled transmission (5), and regulated exchange of molecules with hosts (6, 7). This tight integration creates challenges for studies that aim to elucidate how symbiont variation affects host fitness and ecology.The ability to transfer obligate symbionts between host matrilines could provide a tool for teasing apart the separate contributions of symbiotic partners. Facultative symbionts, such as those conferring defense against pathogens, have been transferred experimentally into novel host matrilines, where they are typically stably inherited, enabling direct measures of symbionts on hosts (8). The success of these transfers is presumably related to the fact that facultative symbionts possess their own machinery for invading host cells (9, 10) and typically persist in several locations in the insect body. In contrast, obligate symbionts such as Buchnera are packaged into specialized host cells during early development and do not survive in the hemocoel or in other cell types.Buchnera colonizes developing aphids before birth, through a specialized transmission process that has been studied in detail in A. pisum (5, 11). Aphids are parthenogenetic for much of their life cycle, during which embryos develop within maternal ovarioles. Bacteriocytes and ovarioles containing developing embryos are located near one another within the mother’s abdomen, and transmission occurs when Buchnera cells are exocytosed from a maternal bacteriocyte in the vicinity of a blastula-stage embryo (5). The Buchnera cells become extracellular within the hemocoel, and some are endocytosed by the posterior syncytial cytoplasm of the embryo in which they are later packaged into the embryonic bacteriocytes. Buchnera cells released into the hemocoel quickly deteriorate if they do not enter an embryo. The molecular underpinnings of the transfer process are unknown. This specialized transmission process presents a challenge for the experimental transfer of symbionts between hosts.We developed a strategy aimed at replacing resident Buchnera with genetically distinct Buchnera from a different host matriline (Fig. 1), using parthenogenetic (all-female) lines of aphids. We used heat tolerance as a selectable phenotype. A previous study showed that a single nucleotide deletion in the promoter of a small heat shock protein [inclusion body-associated protein A (IbpA)] of Buchnera results in a reduction in Buchnera numbers of >100-fold after exposure to 4 h of 35 °C heat (12). Buchnera lacking this mutation undergo only modest declines after heat exposure. Our strategy was to reduce the native Buchnera in recipient aphids using heat, and then to inject homogenate containing heat-tolerant Buchnera from another A. pisum matriline. Because transmission to embryos includes an extracellular stage in which Buchnera is free in the hemocoel, the injected Buchnera might colonize embryos in lieu of maternal Buchnera, which have been eliminated or depleted. After successful replacement of native Buchnera, we demonstrated a large effect on aphid ability to withstand heat exposure.Open in a separate windowFig. 1.Experimental approach for replacement of the native Buchnera symbionts within an A. pisum matriline. The recipient line (LSR1) contains a heat-sensitive Buchnera genotype, and the donor (5AY) contains a heat-tolerant Buchnera genotype. Native Buchnera are depleted by heat in the recipient line, and microinjection is used to flood the hemocoel with donor Buchnera. Most embryos are successfully colonized by the donor symbionts. In some cases, complete replacement occurs in the progeny of injected females. In other cases, progeny have a mixed Buchnera population, which can be shifted completely to the donor type through further heat exposure.     

From the Cover: FtsK translocation permits discrimination between an endogenous and an imported Xer/dif recombination complex

In bacteria, the FtsK/Xer/dif (chromosome dimer resolution site) system is essential for faithful vertical genetic transmission, ensuring the resolution of chromosome dimers during their segregation to daughter cells. This system is also targeted by mobile genetic elements that integrate into chromosomal dif sites. A central question is thus how Xer/dif recombination is tuned to both act in chromosome segregation and stably maintain mobile elements. To explore this question, we focused on pathogenic Neisseria species harboring a genomic island in their dif sites. We show that the FtsK DNA translocase acts differentially at the recombination sites flanking the genomic island. It stops at one Xer/dif complex, activating recombination, but it does not stop on the other site, thus dismantling it. FtsK translocation thus permits cis discrimination between an endogenous and an imported Xer/dif recombination complex.In all organisms, the processing of chromosome ends or termini relies on specific activities for replication and segregation. In eukaryotes, telomeres are often targeted by mobile genetic elements, which may even substitute for telomeric functions (1). Circular chromosomes found in prokaryotes have no telomeres but harbor chromosome dimer resolution sites, called dif sites, on which dedicated Xer recombinases (XerC and XerD in most cases) act (2, 3). Besides their role in chromosome maintenance, dif sites are targeted by numerous mobile genetic elements, referred to as integrating mobile element exploiting Xer (IMEX) (4). How IMEXs integrate into dif without inactivating its cellular function and how they are stably maintained in their integrated state has remained unclear despite study over the past decade (4–7). Here we answer these questions by studying the gonococcal genomic island (GGI), an IMEX stably integrated into the dif site of pathogenic Neisseria species that encodes crucial functions for gene exchange and virulence (8, 9).In Escherichia coli, chromosome dimers form by homologous recombination during replication and are resolved by site-specific recombination between sister dif sites catalyzed by the XerC and XerD recombinases (Fig. 1) (3). The 28-bp dif site carries binding sites for each recombinase, separated by a 6-bp central region at the border of which strand exchanges are catalyzed. After assembly of the recombination complex (synapse), one pair of strands is exchanged by the XerD monomers, leading to a branched DNA intermediate (Holliday junction, HJ) subsequently resolved by XerC. Dimer resolution is integrated into the general processing of the terminal region of the chromosome (ter region) during cell division (10). FtsK, a DNA translocase associated with the division apparatus, segregates this region at the onset of cell division (10, 11). The translocation motor, FtsKαβ, is located in the C terminal of FtsK (12). Translocation is oriented toward the dif site located at the center of the ter region via a direct interaction between the extreme C-terminal subdomain of FtsK, FtsKγ, and the KOPS DNA motifs (13). Upon reaching the XerCD/dif complex, FtsK stops translocating and activates recombination via direct interaction with XerD (14, 15) (Fig. 1). The mechanisms of translocation arrest and of recombination activation are poorly understood but they both involve FtsKγ. However, these activities appear to be distinct from each other because FtsKγ can activate recombination in vivo and in vitro when isolated from the FtsKαβ motor or fused to XerC or XerD (16).Open in a separate windowFig. 1.The XerCD/dif recombination. (A) Chromosome dimer formation by homologous recombination (HR) during replication and resolution by site-specific recombination between the two dif sites. The dif site is represented as green and purple boxes for the XerC-binding and the XerD-binding sites, respectively. ori (black circle), some KOPS motifs (arrows), and the ter domain (thick line) are represented. The mechanism of XerCD/dif recombination is represented in the box. XerC (green circles) and XerD (purple circles) bind two distant dif sites to create a synapse. Hexamers of the FtsK C-terminal domain [FtsKC: FtsKαβ: (diamonds) + FtsKγ: (triangle) contacting XerD] translocate toward dif and contact XerD. This activates XerD (Y indicates the active recombinases), which catalyzes the first-strand exchange. This process leads to the formation of an HJ intermediate within which XerC is active and catalyzes the second-strand exchange (3). (B) Integration and excision of the GGI (dotted line) by XerCD catalysis. KOPS, dif_Ng, and dif_GGI sites are represented as in A. An alignment of dif_Ng, dif_GGI and consensus dif sequence (27, 28) is shown on the left. Substituted positions in dif_GGI are represented as lowercase characters and highlighted by stars.In numerous bacteria, the XerCD/dif system is hijacked by IMEXs, which integrate their host genome into dif sites by using XerCD-mediated catalysis (4). In all of the reported cases, integration of IMEXs recreates a bona fide dif site, thereby not interfering with chromosome dimer resolution, which would lead to their counter-selection. The best-described examples are Vibrio cholerae IMEXs, which carry crucial virulence determinants (5–7, 17). These IMEXs have developed different strategies to integrate and to remain stably integrated, although the mechanisms ensuring their stable maintenance are not fully understood. Neisseria species contain an unusually long IMEX called the gonococcal genomic island (GGI) (8). In Neisseria gonorrheae, the GGI is 57 kb long and encodes a type IV secretion system that exports the chromosomal DNA of its host, rendering it available to neighboring cells for gene exchange by genetic transformation (8, 18). The GGI carries a dif site, dif_GGI, consisting of a XerC-binding site, a central region homologous to the Neisseria dif site, dif_Ng, and a divergent XerD-binding site (Fig. 1B). Comparison of N. gonorrheae strains harboring or lacking the GGI, together with functional data, indicates that the GGI integrates by XerCD-dependent recombination (9). The nonreplicative excised circular form of the GGI can be detected and the GGI can also be lost, showing that excision occurs, although at low frequencies (9). Although the GGI was identified over a decade ago, it has remained unclear how DNA flanked by two Xer recombination sites is stably maintained at a chromosomal locus processed by FtsK during each cell cycle. In this study, we have combined in vitro and in vivo approaches to show that dif_GGI is indeed an active Xer recombination site at which the Neisseria Xer recombinases catalyze recombination when activated by FtsKγ. However, we find that recombination between dif_Ng and dif_GGI is inhibited by translocating FtsK. Inhibition is a result of the absence of translocation arrest at XerCD_Ng/dif_GGI complexes that most likely precludes recombination activation, an absence that causes the complex to dismantle. We conclude that, depending on the sequence of the recombination site, Xer recombination complexes have the intrinsic capacity to be activated or inhibited by FtsK.