Evidence for Cyclic Di-GMP-Mediated Signaling in Bacillus subtilis

Cyclic di-GMP (c-di-GMP) is a second messenger that regulates diverse cellular processes in bacteria, including motility, biofilm formation, cell-cell signaling, and host colonization. Studies of c-di-GMP signaling have chiefly focused on Gram-negative bacteria. Here, we investigated c-di-GMP signaling in the Gram-positive bacterium Bacillus subtilis by constructing deletion mutations in genes predicted to be involved in the synthesis, breakdown, or response to the second messenger. We found that a putative c-di-GMP-degrading phosphodiesterase, YuxH, and a putative c-di-GMP receptor, YpfA, had strong influences on motility and that these effects depended on sequences similar to canonical EAL and RxxxR-D/NxSxxG motifs, respectively. Evidence indicates that YpfA inhibits motility by interacting with the flagellar motor protein MotA and that yuxH is under the negative control of the master regulator Spo0A～P. Based on these findings, we propose that YpfA inhibits motility in response to rising levels of c-di-GMP during entry into stationary phase due to the downregulation of yuxH by Spo0A～P. We also present evidence that YpfA has a mild influence on biofilm formation. In toto, our results demonstrate the existence of a functional c-di-GMP signaling system in B. subtilis that directly inhibits motility and directly or indirectly influences biofilm formation.     

Monkey Abundance and Social Structure in Two High-Elevation Forest Reserves in the Udzungwa Mountains of Tanzania

The effects of human activity on population and social structure are a pantropical concern for primate conservation. We compare census data and social group counts from two forests in the Udzungwa Mountains, Tanzania. The main aim is to relate differences within and between the forests to current theory on the effect of human disturbance on primate abundance and group size. The survey reveals the presence of the restricted-range red colobus, Procolobus gordonorum, in New Dabaga/Ulangambi Forest Reserve (NDUFR). The primate community of NDUFR is impoverished compared to that in Ndundulu forest. Red colobus and black-and-white colobus (Colobus angolensis palliatus) abundance and group size are lowest in NDUFR. Fission-fusion of red colobus social groups may be occurring in previously logged areas of both forests. Our observations are consistent with current theory on the effect of habitat degradation and hunting on primates, but the relative effects of the 2 factors could not be differentiated. We pooled the results with previous data to show that abundance of red colobus in the Udzungwa Mountains is lowest at high elevations. Low red colobus group sizes appear to be related to human activity rather than elevation. Black-and-white colobus and Sykes monkeys (Cercopithecus mitis) show no relationship with elevation. Future studies will require more detailed information on vegetation, diet and ranging patterns to interpret fully intraspecific variation in population demography and social structure in the Udzungwa Mountains.     

Multifactorial Competition and Resistance in a Two-Species Bacterial System

Microorganisms exist almost exclusively in interactive multispecies communities, but genetic determinants of the fitness of interacting bacteria, and accessible adaptive pathways, remain uncharacterized. Here, using a two-species system, we studied the antagonism of Pseudomonas aeruginosa against Escherichia coli. Our unbiased genome-scale approach enabled us to identify multiple factors that explained the entire antagonism observed. We discovered both forms of ecological competition–sequestration of iron led to exploitative competition, while phenazine exposure engendered interference competition. We used laboratory evolution to discover adaptive evolutionary trajectories in our system. In the presence of P. aeruginosa toxins, E. coli populations showed parallel molecular evolution and adaptive convergence at the gene-level. The multiple resistance pathways discovered provide novel insights into mechanisms of toxin entry and activity. Our study reveals the molecular complexity of a simple two-species interaction, an important first-step in the application of systems biology to detailed molecular dissection of interactions within native microbiomes.     

Metabolic Commensalism and Competition in a Two-Species Microbial Consortium

We analyzed metabolic interactions and the importance of specific structural relationships in a benzyl alcohol-degrading microbial consortium comprising two species, Pseudomonas putida strain R1 and Acinetobacter strain C6, both of which are able to utilize benzyl alcohol as their sole carbon and energy source. The organisms were grown either as surface-attached organisms (biofilms) in flow chambers or as suspended cultures in chemostats. The numbers of CFU of P. putida R1 and Acinetobacter strain C6 were determined in chemostats and from the effluents of the flow chambers. When the two species were grown together in chemostats with limiting concentrations of benzyl alcohol, Acinetobacter strain C6 outnumbered P. putida R1 (500:1), whereas under similar growth conditions in biofilms, P. putida R1 was present in higher numbers than Acinetobacter strain C6 (5:1). In order to explain this difference, investigations of microbial activities and structural relationships were carried out in the biofilms. Insertion into P. putida R1 of a fusion between the growth rate-regulated rRNA promoter rrnBP1 and a gfp gene encoding an unstable variant of the green fluorescent protein made it possible to monitor the physiological activity of P. putida R1 cells at different positions in the biofilms. Combining this with fluorescent in situ hybridization and scanning confocal laser microscopy showed that the two organisms compete or display commensal interactions depending on their relative physical positioning in the biofilm. In the initial phase of biofilm development, the growth activity of P. putida R1 was shown to be higher near microcolonies of Acinetobacter strain C6. High-pressure liquid chromatography analysis showed that in the effluent of the Acinetobacter strain C6 monoculture biofilm the metabolic intermediate benzoate accumulated, whereas in the biculture biofilms this was not the case, suggesting that in these biofilms the excess benzoate produced by Acinetobacter strain C6 leaks into the surrounding environment, from where it is metabolized by P. putida R1. After a few days, Acinetobacter strain C6 colonies were overgrown by P. putida R1 cells and new structures developed, in which microcolonies of Acinetobacter strain C6 cells were established in the upper layer of the biofilm. In this way the two organisms developed structural relationships allowing Acinetobacter strain C6 to be close to the bulk liquid with high concentrations of benzyl alcohol and allowing P. putida R1 to benefit from the benzoate leaking from Acinetobacter strain C6. We conclude that in chemostats, where the organisms cannot establish in fixed positions, the two strains will compete for the primary carbon source, benzyl alcohol, which apparently gives Acinetobacter strain C6 a growth advantage, probably because it converts benzyl alcohol to benzoate with a higher yield per time unit than P. putida R1. In biofilms, however, the organisms establish structured, surface-attached consortia, in which heterogeneous ecological niches develop, and under these conditions competition for the primary carbon source is not the only determinant of biomass and population structure.     

Revegetation Methods for High-Elevation Roadsides at Bryce Canyon National Park, Utah

Establishment of native plant populations on disturbed roadsides was investigated at Bryce Canyon National Park (BCNP) in relation to several revegetation and seedbed preparation techniques. In 1994, the BCNP Rim Road (2,683–2,770 m elevation) was reconstructed resulting in a 23.8‐ha roadside disturbance. Revegetation comparisons included the influence of fertilizer on plant establishment and development, the success of indigenous versus commercial seed, seedling response to microsites, methods of erosion control, and shrub transplant growth and survival. Plant density, cover, and biomass were measured 1, 2, and 4 years after revegetation implementation (1995–1998). Seeded native grass cover and density were the highest on plots fertilized with nitrogen and phosphorus, but by the fourth growing season, differences between fertilized and unfertilized plots were minimal. Fertilizers may facilitate more rapid establishment of seeded grasses following disturbance, increasing soil cover and soil stability on steep and unstable slopes. However the benefit of increased soil nutrients favored few of the desired species resulting in lower species richness over time compared to unfertilized sites. Elymus trachycaulus (slender wheatgrass) plants raised from indigenous seed had higher density and cover than those from a commercial seed source 2 and 4 years after sowing. Indigenous materials may exhibit slow establishment immediately following seeding, but they will likely persist during extreme climatic conditions such as cold temperatures and relatively short growing seasons. Seeded grasses established better near stones and logs than on adjacent open microsites, suggesting that a roughened seedbed created before seeding can significantly enhance plant establishment. After two growing seasons, total grass cover between various erosion‐control treatments was similar indicating that a variety of erosion reduction techniques can be utilized to reduce erosion. Finally shrub transplants showed minimal differential response to fertilizers, water‐absorbing gels, and soil type. Simply planting and watering transplants was sufficient for the greatest plant survival and growth.     

具有阶段结构的两种群竞争系统的周期解

本文研究了具有阶段结构的两种群竞争系统的渐近行为.我们得到了系统持续生存的条件.由Brouwer不动点定理和李亚普诺夫函数,我们证明相应的周期系统在满足一定的条件下,存在一个唯一的全局渐近稳定的正周期解.最后我们把没有阶段结构的系统与有阶段结构的系统进行了比较.     

Evidence for Cyclic Electron Flow around Photosystem II in Chlorella pyrenoidosa

Electron flow around photosystem II was investigated in Chlorella pyrenoidosa. Using a bare platinum O₂ electrode, simultaneous measuremnts were made of steady-state photosynthesis in continuous light, the yield of oxygen (Y_o2) produced by a superimposed saturating xenon flash, and the change in fluorescence yield of a weak flash triggered before and 70 microseconds after the saturating flash. Throughout most of the continuous photosynthesis-irradiance curve, normalized O₂ flash yields (Y_o2/Y_o2max) and normalized variable fluorescence yields (Δ/Δ′) were linearly correlated with a slope of 1.0. As photosynthetic rates reached light saturation, however, the variable fluorescence yields remained relatively constant while O₂ flash yields decreased. These results strongly suggest that there is a cyclic electron flow around photosystem II in unpoisoned intact cells at light saturation and supraoptimal light intensities.     

Fumarole-Supported Islands of Biodiversity within a Hyperarid, High-Elevation Landscape on Socompa Volcano, Puna de Atacama, Andes

Fumarolic activity supports the growth of mat-like photoautotrophic communities near the summit (at 6,051 m) of Socompa Volcano in the arid core of the Andes mountains. These communities are isolated within a barren, high-elevation landscape where sparse vascular plants extend to only 4,600 m. Here, we combine biogeochemical and molecular-phylogenetic approaches to characterize the bacterial and eucaryotic assemblages associated with fumarolic and nonfumarolic grounds on Socompa. Small-subunit rRNA genes were PCR amplified, cloned, and sequenced from two fumarolic soil samples and two reference soil samples, including the volcanic debris that covers most of the mountain. The nonfumarolic, dry, volcanic soil was similar in nutrient status to the most extreme Antarctic Dry Valley or Atacama Desert soils, hosted relatively limited microbial communities dominated by Actinobacteria and Fungi, and contained no photoautotrophs. In contrast, modest fumarolic inputs were associated with elevated soil moisture and nutrient levels, the presence of chlorophyll a, and ¹³C-rich soil organic carbon. Moreover, this soil hosted diverse photoautotroph-dominated assemblages that contained novel lineages and exhibited structure and composition comparable to those of a wetland near the base of Socompa (3,661-m elevation). Fumarole-associated eucaryotes were particularly diverse, with an abundance of green algal lineages and a novel clade of microarthropods. Our data suggest that volcanic degassing of water and ¹³C-rich CO₂ sustains fumarole-associated primary producers, leading to a complex microbial ecosystem within this otherwise barren landscape. Finally, we found that human activities have likely impacted the fumarolic soils and that fumarole-supported photoautotrophic communities may be exceptionally sensitive to anthropogenic disturbance.     

Evidence for Seed Dispersal by Rodents in Tropical Montane Forest in Africa

Seed dispersal by rodents has been understudied in Africa. Based on seed‐removal experiments, the presence of seeds in burrows and caches, cotyledon burial of seedlings, and images from camera traps, we provide evidence that rodents (Cricetomys kivuensis) remove and hoard large seeds of Carapa grandiflora in Nyungwe National Park, Rwanda.     

Complex Effects of Prescribed Fire on Restoring the Soil Water Content in a High-Elevation Riparian Meadow, Arizona

One of the largest and rarest Bebb willow (Salix bebbiana) communities in the United States occurs at Hart Prairie, Arizona. Low recruitment of the willow over the past several decades has been linked to inadequate soil water content for seed germination and seedling establishment. We tested a hypothesis that a prescribed burn would reduce biomass of and evapotranspiration by herbaceous plants, thereby increasing soil water content. Three treatments (unburned control, early‐growing season burned, late‐growing season burned) were applied in year 2001 to replicated plots in fern‐ and grass‐dominated herbaceous communities. Soil water content (0–30 cm) was measured weekly in plots during the 2001, 2002, and 2003 growing seasons. Both early‐ and late‐season burning reduced herbaceous biomass in the fern‐dominated community in 2002 and 2003 and reduced biomass in the grass‐dominated community in 2002 but not in 2003. Soil water content increased for approximately four weeks in 2001 following the early‐season burn, but the early‐season and late‐season burns reduced soil water content in both communities over much of the 2002 and 2003 growing seasons. Thus, early‐season burning may benefit willow seed germination by increasing soil water content immediately following burning but be detrimental to germination in the second and third growing seasons after burning because of drier soil. Large temporal variation in the effect of prescribed burning on soil water content will complicate the use of fire as a restoration tool to manage soil water available for threatened plants such as Bebb willow and for recharge of groundwater.     

The Principle of Competitive Exception in a Two-Species Community with One Metabolic Regulation Factor

The validity of the competitive exclusion principle (the Gause’s principle) at one metabolic regulation factor is demonstrated for a general model of two-species community. The competitive exclusion principle postulates that a long-term coexistence of species is impossible if their number exceeds the number of density-dependent growth-regulating factors. Previously, this principle was proved for the stationary states in a general model of a community with any number of factors. In the dynamic modes, the number of species in a community may exceed the number of regulating factors. However, under the influence of one factor, only one species survives.     

Cyclic GMP phosphodiesterase from bovine retina. Evidence for interspecies conservation.

A light-activated phosphodiesterase (PDE) from retinal rod outer segments (ROS) has been strongly implicated as a possible mediator in the propagation of the visual response. In view of the probable importance of this enzyme in the visual system, a comparison of the PDE proteins from ROS of different evolutionary species was made. Partial purification of the PDE, as measured by enzymic activity, followed by resolution of the protein components on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, indicated that the major Coomassie Blue-staining species in the ROS of all species studied is a doublet of 84000-88000 Da. After radioiodination, this doublet was converted in all but the frog proteins into a single band of 85000 Da. Two-dimensional tryptic peptide mapping of the radioiodinated peptides indicated that at least six major peptides of the putative PDE have been conserved in all of the species studies. If this protein is indeed associated with PDE activity, such conserved peptides may play an important role in the catalytic and/or regulatory functions of the enzyme.     

Evidence from water chemistry as a criterion of ombrotrophy in the mire complexes of Abernethy Forest,Scotland

Question: Can cation analyses of water samples within a peatland site be used to infer solute sources to the samples, and so provide an objective criterion of ombrotrophy? Location: Mire complexes within native Pinus sylvestris forest, in Abernethy Forest, north of the Cairngorm Mountains, Scotland, UK. Methods: Chemical analyses of major cations in 200 water samples, and χ² analysis of contingency tables relating species occurrence in the corresponding quadrat samples to the ombrotrophic‐telluric division. Results and conclusions: The chemical data (especially the ratios Ca²⁺/Mg²⁺ and Na⁺/Mg²⁺) indicated a separation of about 140 essentially ombrotrophic samples from about 60 showing clear telluric influence; these conclusions are consistent with published rainwater analyses. For hydrological and meteorological reasons, a sharp separation cannot be expected, so the limit adopted here (Ca/Mg=1.0) is to some extent arbitrary, but the methods described provide a more objective criterion of ombrotrophy than any other. Contingency tables showed highly significant associations between species occurrence and the ombrotrophic‐telluric division. The strongest associations included: (ombrotrophic) Eriophorum vaginatum, Odontoschisma sphagni and Sphagnum cuspidatum; (telluric) Carex panicea, Potentilla erecta, Carex echinata, Narthecium ossifragum, Sphagnum auriculatum s.l., Agrostis canina, Molinia caerulea, Eriophorum angustifolium, and Sphagnum recurvum s.l. The Abernethy mires are arguably more comparable with mire complexes on base‐poor rocks in southern Scandinavia than with most ombrotrophic sites further south and west in Britain. As in Sweden, Narthecium ossifragum and Eriophorum angustifolium, generally ubiquitous on British and Irish ombrotrophic bogs, are “fen” plants at Abernethy.     

Conservation of Forest Birds: Evidence of a Shifting Baseline in Community Structure

Background

Quantifying changes in forest bird diversity is an essential task for developing effective conservation actions. When subtle changes in diversity accumulate over time, annual comparisons may offer an incomplete perspective of changes in diversity. In this case, progressive change, the comparison of changes in diversity from a baseline condition, may offer greater insight because changes in diversity are assessed over longer periods of times. Our objectives were to determine how forest bird diversity has changed over time and whether those changes were associated with forest disturbance.

Methodology/Principal Findings

We used North American Breeding Bird Survey data, a time series of Landsat images classified with respect to land cover change, and mixed-effects models to associate changes in forest bird community structure with forest disturbance, latitude, and longitude in the conterminous United States for the years 1985 to 2006. We document a significant divergence from the baseline structure for all birds of similar migratory habit and nest location, and all forest birds as a group from 1985 to 2006. Unexpectedly, decreases in progressive similarity resulted from small changes in richness (<1 species per route for the 22-year study period) and modest losses in abundance (−28.7–−10.2 individuals per route) that varied by migratory habit and nest location. Forest disturbance increased progressive similarity for Neotropical migrants, permanent residents, ground nesting, and cavity nesting species. We also documented highest progressive similarity in the eastern United States.

Conclusions/Significance

Contemporary forest bird community structure is changing rapidly over a relatively short period of time (e.g., ∼22 years). Forest disturbance and forest regeneration are primary factors associated with contemporary forest bird community structure, longitude and latitude are secondary factors, and forest loss is a tertiary factor. Importantly, these findings suggest some regions of the United States may already fall below the habitat amount threshold where fragmentation effects become important predictors of forest bird community structure.     

Seed Banks in Savanna,Forest Fragments,and Continuous Forest Edges Differ in a Tropical Dry Forest in Madagascar

Rapid deforestation has fragmented habitat across the landscape of Madagascar. To determine the effect of fragmentation on seed banks and the potential for forest regeneration, we sampled seed viability, density and diversity in 40 plots of 1 m² in three habitat types: forest fragments, the near edge of continuous forest, and deforested savanna in a highly fragmented dry deciduous forest landscape in northwestern Madagascar. While seed species diversity was not different between forest fragments and continuous forest edge, the number of animal‐dispersed seeds was significantly higher in forest fragments than in continuous forest edge, and this pattern was driven by a single, small‐seeded species. In the savanna, seeds were absent from all but three of the 40 plots, indicating that regeneration potential is low in these areas. Several pre‐ and post‐dispersal biotic and abiotic factors, including variation in the seed predator communities and edge effects could explain these findings. Understanding the extent to which seed dispersal and seed banks influence the regeneration potential of fragmented landscapes is critical as these fragments are the potential sources of forest expansion and re‐connectivity.     

Cyclic nucleotide phosphodiesterase from a particulate fraction of rat brain. Evidence for an activator deficient form.

A cyclic nucleotide phosphodiesterase from a particulate fraction of rat brain was partially purified after solubilization with a non-ionic detergent. Influence of divalent ions, of phosphodiesterase inhibitors and of the activator protein from different sources were tested. Determination of K_m-values shows two enzymes with different values, one at 7.3 μM and the other at 15 mM. Two distinct activity peaks were determined after resolution by isoelectric focusing. It was concluded that this particulate enzyme is regulated in a way opposite to that of the soluble enzyme and is independent from calcium and the activator protein.     

Permanence and Global Stability for Two-Species Cooperative System with Time Delays in Two-Patch Environment

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Tracking Seed Fates of Tropical Tree Species: Evidence for Seed Caching in a Tropical Forest in North-East India

Rodents affect the post-dispersal fate of seeds by acting either as on-site seed predators or as secondary dispersers when they scatter-hoard seeds. The tropical forests of north-east India harbour a high diversity of little-studied terrestrial murid and hystricid rodents. We examined the role played by these rodents in determining the seed fates of tropical evergreen tree species in a forest site in north-east India. We selected ten tree species (3 mammal-dispersed and 7 bird-dispersed) that varied in seed size and followed the fates of 10,777 tagged seeds. We used camera traps to determine the identity of rodent visitors, visitation rates and their seed-handling behavior. Seeds of all tree species were handled by at least one rodent taxon. Overall rates of seed removal (44.5%) were much higher than direct on-site seed predation (9.9%), but seed-handling behavior differed between the terrestrial rodent groups: two species of murid rodents removed and cached seeds, and two species of porcupines were on-site seed predators. In addition, a true cricket, Brachytrupes sp., cached seeds of three species underground. We found 309 caches formed by the rodents and the cricket; most were single-seeded (79%) and seeds were moved up to 19 m. Over 40% of seeds were re-cached from primary cache locations, while about 12% germinated in the primary caches. Seed removal rates varied widely amongst tree species, from 3% in Beilschmiedia assamica to 97% in Actinodaphne obovata. Seed predation was observed in nine species. Chisocheton cumingianus (57%) and Prunus ceylanica (25%) had moderate levels of seed predation while the remaining species had less than 10% seed predation. We hypothesized that seed traits that provide information on resource quantity would influence rodent choice of a seed, while traits that determine resource accessibility would influence whether seeds are removed or eaten. Removal rates significantly decreased (p < 0.001) while predation rates increased (p = 0.06) with seed size. Removal rates were significantly lower for soft seeds (p = 0.002), whereas predation rates were significantly higher on soft seeds (p = 0.01). Our results show that murid rodents play a very important role in affecting the seed fates of tropical trees in the Eastern Himalayas. We also found that the different rodent groups differed in their seed handling behavior and responses to changes in seed characteristics.     

Evidence for Cyclic Photophosphorylation during CO(2) Fixation in Intact Chloroplasts: Studies with Antimycin A, Nitrite, and Oxaloacetate

This study examines the effect of antimycin A and nitrite on ¹⁴CO₂ fixation in intact chloroplasts isolated from spinach (Spinacia oleracea L.) leaves. Antimycin A (2 micromolar) strongly inhibited CO₂ fixation but did not appear to inhibit or uncouple linear electron transport in intact chloroplasts. The addition of small quantities (40-100 micromolar) of nitrite or oxaloacetate, but not NH₄Cl, in the presence of antimycin A restored photosynthesis. Antimycin A inhibition, and the subsequent restoration of photosynthetic activities by nitrite or oxaloacetate, was observed over a wide range of CO₂ concentration, light intensity, and temperature. High O₂ concentration (up to 240 micromolar) did not appear to influence the extent of the inhibition by antimycin A, nor the subsequent restoration of photosynthetic activity by nitrite or oxaloacetate. Studies of O₂ exchanges during photosynthesis in cells and chloroplasts indicated that 2 micromolar antimycin A stimulated O₂ uptake by about 25% while net O₂ evolution was inhibited by 76%. O₂ uptake in chloroplasts in the presence of 2 micromolar antimycin A was 67% of total O₂ evolution. These results suggest that only a small proportion of the O₂ uptake measured was directly linked to ATP generation. The above evidence indicates that cyclic photophosphorylation is the predominant energy-balancing reaction during photosynthesis in intact chloroplasts. On the other hand, pseudocyclic O₂ uptake appears to play only a minimal role.