Microbial Decomposer Communities Are Mainly Structured by Trophic Status in Circumneutral and Alkaline Streams

In streams, the release of nitrogen and phosphorus is reported to affect microbial communities and the ecological processes they govern. Moreover, the type of inorganic nitrogen (NO₃, NO₂, or NH₄) may differently impact microbial communities. We aimed to identify the environmental factors that structure aquatic microbial communities and drive leaf litter decomposition along a gradient of eutrophication. We selected five circumneutral (Portuguese) and five alkaline (French) streams differing in nutrient concentrations to monitor mass loss of alder leaves, bacterial and fungal diversity by PCR-denaturing gradient gel electrophoresis, fungal biomass and reproduction, and bacterial biomass during 11 weeks of leaf immersion. The concentrations of inorganic nutrients in the stream water ranged from 5 to 300 μg liter⁻¹ soluble reactive phosphorus, 0.30 to 5.50 mg liter⁻¹ NO₃-N, 2 to 103 μg liter⁻¹ NO₂-N, and <4 to 7,100 μg liter⁻¹ NH₄-N. Species richness was maximum in moderately anthropized (eutrophic) streams but decreased in the most anthropized (hypertrophic) streams. Different species assemblages were found in subsets of streams with different trophic statuses. In both geographic areas, the limiting nutrient, either nitrate or phosphate, stimulated the microbial activity in streams of intermediate trophic status. In the hypertrophic streams, fungal biomass and reproduction were significantly lower, and bacterial biomass dramatically decreased at the site with the highest ammonium concentration. The limiting nutrients that defined the trophic status were the main factor structuring fungal and bacterial communities, whatever the geographic area. A very high ammonium concentration in stream water most probably has negative impacts on microbial decomposer communities.There is evidence that increases in nitrate and phosphate concentrations stimulate microbial respiration and fungal and bacterial activity (biomass buildup, sporulation, and/or productivity) on plant litter, leading to faster leaf decomposition in freshwaters (16, 17, 26, 34). However, fungal demands of nitrate and phosphate are reported to be fulfilled at relatively low levels (1, 12), and further increases in these nutrients in the stream water do not necessarily result in enhanced fungal activity. Besides, the form in which inorganic nutrients are present in streams, their biological availability, and even their toxicity have different ecological consequences. In densely anthropized hypertrophic streams, high levels of nitrate and phosphate were associated with decreased fungal biomass and leaf breakdown, most probably because of the high concentrations of ammonium and ammonia (2). On the other hand, the positive effects of nutrients on biomass and productivity of leaf-associated fungi can be offset by other factors, such as low oxygen concentration and sedimentation, leading to retarded decomposition (26, 33, 34).Changes in inorganic nutrient concentrations in the stream water were reported to alter the structure of fungal communities on plant litter (16, 36). Nutrient additions to moderate levels increased the diversity of fungal communities in circumneutral soft-water Appalachian mountain streams (18) but not in a Mediterranean alkaline stream (1). Moreover, fungal diversity was lower in circumneutral eutrophic streams than in reference streams (10, 35). Fungal diversity has been assessed mostly through the morphological analysis of produced conidia, not taking into account nonsporulating fungi. This raises the question of whether the differential impacts of eutrophication on fungal diversity could be due partly to difficulties in measuring actual diversity. Besides, the study of bacterial diversity on decomposing leaves has been strongly restricted to a few cultivable bacteria (<1%). Molecular typing, such as denaturing gradient gel electrophoresis (DGGE) of a specific rRNA gene region, has proved useful for assessing diversity in both leaf-associated fungi and bacteria (7, 8, 9, 11, 30).We aimed to identify the environmental factors that drive the ecological processes in freshwaters impacted by eutrophication through examination of leaf litter decomposition and associated microbial communities along a gradient of nutrient enrichment. Specifically, we addressed the following two questions: (i) which are the environmental factors that mainly structure the fungal and bacterial communities and (ii) what are the relationships between concentrations of inorganic nutrients in the stream water, leaf litter decomposition, and the activity of associated microbes? We selected 10 stream sites spanning wide concentration ranges of dissolved inorganic nitrogen (NO₃-N, NO₂-N, NH₄-N, and NH₃-N) and soluble reactive phosphorus (SRP), including 5 in northwestern Portugal with circumneutral pH and 5 in southwestern France with an alkaline pH. With these two groups of stream sites, we assessed the structure of and diversity in both sporulating and nonsporulating fungal communities, using asexual spore morphology and DGGE fingerprints of the ITS2 region, and in bacterial communities, using DGGE fingerprints of the 16S rRNA gene region. Additionally, we examined leaf mass loss and microbial activity on decomposing leaves by determining bacterial and fungal biomass and fungal reproduction.     

Soil Microbial Community Responses to Multiple Experimental Climate Change Drivers

Researchers agree that climate change factors such as rising atmospheric [CO₂] and warming will likely interact to modify ecosystem properties and processes. However, the response of the microbial communities that regulate ecosystem processes is less predictable. We measured the direct and interactive effects of climatic change on soil fungal and bacterial communities (abundance and composition) in a multifactor climate change experiment that exposed a constructed old-field ecosystem to different atmospheric CO₂ concentration (ambient, +300 ppm), temperature (ambient, +3°C), and precipitation (wet and dry) might interact to alter soil bacterial and fungal abundance and community structure in an old-field ecosystem. We found that (i) fungal abundance increased in warmed treatments; (ii) bacterial abundance increased in warmed plots with elevated atmospheric [CO₂] but decreased in warmed plots under ambient atmospheric [CO₂]; (iii) the phylogenetic distribution of bacterial and fungal clones and their relative abundance varied among treatments, as indicated by changes in 16S rRNA and 28S rRNA genes; (iv) changes in precipitation altered the relative abundance of Proteobacteria and Acidobacteria, where Acidobacteria decreased with a concomitant increase in the Proteobacteria in wet relative to dry treatments; and (v) changes in precipitation altered fungal community composition, primarily through lineage specific changes within a recently discovered group known as soil clone group I. Taken together, our results indicate that climate change drivers and their interactions may cause changes in bacterial and fungal overall abundance; however, changes in precipitation tended to have a much greater effect on the community composition. These results illustrate the potential for complex community changes in terrestrial ecosystems under climate change scenarios that alter multiple factors simultaneously.Soil microbial communities are responsible for the cycling of carbon (C) and nutrients in ecosystems, and their activities are regulated by biotic and abiotic factors such as the quantity and quality of litter inputs, temperature, and moisture. Atmospheric and climatic changes will impact both abiotic and biotic drivers in ecosystems and the response of ecosystems to these changes. Feedbacks from ecosystem to the atmosphere may also be regulated by soil microbial communities (3). Although microbial communities regulate important ecosystem processes, it is often unclear how the abundance and composition of microbial communities correlate with climatic perturbations and interact to effect ecosystem processes. As such, much of the ecosystem climate change research conducted to date has focused on macroscale responses to climatic change such as changes in plant growth (43, 44), plant community composition (2, 37), and coarse scale soil processes (14, 18, 21, 26), many of which may also indirectly interact to effect microbial processes. Studies that have addressed the role of microbial communities and processes have most often targeted gross parameters, such as microbial biomass, enzymatic activity, or basic microbial community profiles in response to single climate change factors (22, 28, 29, 33, 61, 63).Climate change factors such as atmospheric CO₂ concentrations, warming, and altered precipitation regimes can potentially have both direct and indirect impacts on soil microbial communities. However, the direction and magnitude of these responses is uncertain. For example, the response of soil microbial communities to changes in atmospheric CO₂ concentrations can be positive or negative, and consistent overall trends between sites and studies have not been observed (1, 28, 34-36). Further, depending on what limits ecosystem productivity, precipitation and soil moisture changes may increase or decrease the ratio of bacteria and fungi, as well as shift their community composition (8, 50, 58). Increasing temperatures can increase in microbial activity, processing, and turnover, causing the microbial community to shift in favor of representatives adapted to higher temperatures and faster growth rates (7, 46, 60, 64, 65). Atmospheric and climatic changes are happening in concert with one another so that ecosystems are experiencing higher levels of atmospheric CO₂, warming, and changes in precipitation regimes simultaneously. Although the many single factor climate change studies described above have enabled a better understanding of how microbial communities may respond to any one factor, understanding how multiple climate change factors interact with each other to influence microbial community responses is poorly understood. For example, elevated atmospheric [CO₂] and precipitation changes might increase soil moisture in an ecosystem, but this increase may be counteracted by warming (10). Similarly, warming may increase microbial activity in an ecosystem, but this increase may be eliminated if changes in precipitation lead to a drier soil condition or reduced litter quantity, quality, and turnover. Such interactive effects of climate factors in a multifactorial context have been less commonly studied even in plant communities (45), and detailed studies are rarer still in soil microbial communities (25). Clearly, understanding how microbial communities will respond to these atmospheric and climate change drivers is important to make accurate predications of how ecosystems may respond to future climate scenarios.To address how multiple climate change drivers will interact to shape soil microbial communities, we took advantage of a multifactor climatic change experiment that manipulated atmospheric CO₂ (+300 ppm, ambient), warming (+3°C, ambient) and precipitation (wet and dry) in a constructed old-field ecosystem that had been ongoing for 3.5 years at the time of sampling. Previous work on this project has demonstrated direct and interactive effects of the treatments on plant community composition and biomass (15, 30), soil respiration (56), microbial activity (30), nitrogen fixation (21), and soil carbon stocks (20). These results led us to investigations of how the soil bacterial and fungal communities, important regulators of some of these processes, were responding using culture-independent molecular approaches. Our research addresses two overarching questions. (i) Do climatic change factors and their interactions alter bacterial and fungal abundance and diversity? (ii) Do climatic change factors and their interactions alter bacterial or fungal community composition?     

Diversity of Formyltetrahydrofolate Synthetases in the Guts of the Wood-Feeding Cockroach Cryptocercus punctulatus and the Omnivorous Cockroach Periplaneta americana

We examined the diversity of a marker gene for homoacetogens in two cockroach gut microbial communities. Formyltetrahydrofolate synthetase (FTHFS or fhs) libraries prepared from a wood-feeding cockroach, Cryptocercus punctulatus, were dominated by sequences that affiliated with termite gut treponemes. No spirochete-like sequences were recovered from the omnivorous roach Periplaneta americana, which was dominated by Firmicutes-like sequences.The guts of wood-feeding termites and Cryptocercus punctulatus cockroaches share an unusual pattern of electron flow, as high rates of CO₂-reductive acetogenesis typically supplant methanogenesis as the terminal electron sink (2, 3). Past studies have shown that from 10 to 30% of gut acetate produced in environments of termites and wood-feeding cockroaches is microbially generated from CO₂ (3, 28), ultimately powering 18 to 26% of the host insect''s own respiratory energy metabolism (25). Nevertheless, most termites emit methane (2), and termite emissions constitute approximately 4% of the global methane budget (27). Cockroaches have been proposed to represent an additional source of note (9). Interestingly, methanogenic termites and cockroaches exhibit increased acetogenesis following addition of exogenous H₂ (3, 29). This suggests that these insects are host to a robust population of bacteria that are capable of homoacetogenesis but may be primarily using alternative electron donors (and other substrates and pathways) in vivo.Acetogenic bacteria belonging to two bacterial phyla, Firmicutes and Spirochaetes, have been isolated from the guts of termites (1, 4, 11, 12, 14). Several surveys have targeted and used the gene for formyltetrahydrofolate synthetase (FTHFS), a key gene in the Wood-Ljungdahl pathway of acetogenesis (16), as a potential marker for the pathway (15, 18). For the wood-feeding termites that have been examined, the studies have revealed an abundance of FTHFS sequences that form a coherent phylogenetic cluster, together with genes from homoacetogenic termite gut spirochetes belonging to the genus Treponema (24, 26, 30). This suggests that treponemes may be among the more abundant of the homoacetogens active in these environments.Little is known about the population structure and biology of CO₂-reducing, acetogenic bacteria in the guts of either omnivorous or wood-feeding cockroaches. The wood-feeding cockroach Cryptocercus hosts an abundance of flagellate protozoa closely related to those believed to dominate polysaccharide fermentation in the guts of termites (5, 6, 22), suggesting that at least one key environmental niche is filled by similar microbes in both termites and Cryptocercidae. Additionally, Cryptocercidae cockroaches, like termites, house diverse spirochetes and are the site of intense CO₂ reduction into acetate (3, 7). Possibly, spirochetes capable of CO₂ reduction into acetate are present in the hindguts of cockroaches. However, no evidence has yet been presented for the existence of homoacetogenic treponemes in environments other than the guts of termites, and FTHFS surveys of human (21) or horse (15) fecal matter and bovine rumen samples (20) revealed only Firmicutes-like and other FTHFS alleles that are distinct from those comprising the termite treponeme cluster.Here, by examining FTHFS gene diversity in Cryptocercus punctulatus and Periplaneta americana guts, we endeavored to learn more about the distribution and origins of homoacetogenic treponemes (and their genes) that are found in wood-feeding termites. In particular, we wished to ascertain whether FTHFS genes present in either of the two cockroaches are termite treponeme-like and, if so, whether analysis reveals any obvious signal indicating recent or ancient lateral community transfer events between insect lineages.     

In Vitro Kinetics of Prebiotic Inulin-Type Fructan Fermentation by Butyrate-Producing Colon Bacteria: Implementation of Online Gas Chromatography for Quantitative Analysis of Carbon Dioxide and Hydrogen Gas Production

Kinetic analyses of bacterial growth, carbohydrate consumption, and metabolite production of five butyrate-producing clostridial cluster XIVa colon bacteria grown on acetate plus fructose, oligofructose, inulin, or lactate were performed. A gas chromatography method was set up to assess H₂ and CO₂ production online and to ensure complete coverage of all metabolites produced. Method accuracy was confirmed through the calculation of electron and carbon recoveries. Fermentations with Anaerostipes caccae DSM 14662^T, Roseburia faecis DSM 16840^T, Roseburia hominis DSM 16839^T, and Roseburia intestinalis DSM 14610^T revealed similar patterns of metabolite production with butyrate, CO₂, and H₂ as the main metabolites. R. faecis DSM 16840^T and R. intestinalis DSM 14610^T were able to degrade oligofructose, displaying a nonpreferential breakdown mechanism. Lactate consumption was only observed with A. caccae DSM 14662^T. Roseburia inulinivorans DSM 16841^T was the only strain included in the present study that was able to grow on fructose, oligofructose, and inulin. The metabolites produced were lactate, butyrate, and CO₂, without H₂ production, indicating an energy metabolism distinct from that of other Roseburia species. Oligofructose degradation was nonpreferential. In a coculture of R. inulinivorans DSM 16841^T with the highly competitive strain Bifidobacterium longum subsp. longum LMG 11047 on inulin, hardly any production of butyrate and CO₂ was detected, indicating a lack of competitiveness of the butyrate producer. Complete recovery of metabolites during fermentations of clostridial cluster XIVa butyrate-producing colon bacteria allowed stoichiometric balancing of the metabolic pathway for butyrate production, including H₂ formation.The implementation of 16S rRNA gene-based analytical techniques in the ongoing exploration of the microbial diversity of the human colon ecosystem has both broadened and sharpened the prevailing image of its population (17, 24, 32). While a rather conservative perception of the composition of the colon microbiota has dominated gut research for several decades (36), recent studies have revealed the importance of previously largely neglected bacterial groups and have reduced historically numerically overestimated subpopulations to their actual (marginal) size (8, 22, 52). The human colon has been shown to be a remarkably selective environment, which is reflected by a rather shallow microbial diversity (32). Species belonging to the bacterial divisions Firmicutes, Bacteroidetes, Proteobacteria, and Actinobacteria make up more than 98% of the bacterial population of the human colon (2, 17, 24). However, this superficial uniformity only covers an overwhelming diversity at the lower taxonomic levels; the human colon has been estimated to harbor between 500 and 1,000 species, representing over 7,000 strains, with up to 80% of them considered uncultivable using presently available methodologies (14, 28, 53).Assessing identity and abundance of the major microbial groups composing the colon microbiota is a first and indispensable step toward a better understanding of the ecosystem of the large intestine (48). However, defining a complex ecosystem such as the human colon requires more than the construction of a catalog of its members (32). A major challenge of gastrointestinal microbiology lies in linking phylogenetic subgroups with particular ecological habitats and niches (7, 8, 23). The latter requires further development of highly discriminating 16S rRNA gene-targeted probes to monitor spatial bacterial distribution, combined with renewed efforts toward species isolation through the application of innovative cultivation methods and media, and extensive metabolic characterization of representative strains (19, 35, 48).Recently, a global ecological approach, combining efforts in probe development (1, 27), species isolation (3), and metabolic characterization (4, 11, 15, 20), has led to the identification of a functional group of microorganisms, composed of species belonging to the clostridial clusters IV and XIVa, that are responsible for colon butyrate production. As butyrate is regarded as a key metabolite for the maintenance of colon health, this functional subunit of the colon microbiota could have a major influence on human well-being and might be considered as a target for prebiotic dietary interventions (25, 35, 45). Some recently described lactate- and/or acetate-converting colon butyrate producers have been reported to be able to degrade prebiotic inulin-type fructans, although the kinetics of their respective breakdown mechanisms have hardly been investigated (10, 20). The enhancement of colon butyrate production observed after consumption of oligofructose or inulin (6, 31, 40)—the so-called butyrogenic effect—as well as the limited stimulatory effect of these prebiotics on the clostridial cluster IV and XIVa colon populations (16, 30) have been attributed to cross-feeding with bifidobacteria, which are still considered the primary fructan degraders (5, 38). Anaerostipes caccae as well as Roseburia spp. have been shown to be able to (co)metabolize end products of bifidobacterial fructan fermentation (lactate and/or acetate) or to grow on short oligosaccharides and monosaccharides released by Bifidobacterium spp. during fructan degradation (4, 20).Recently, many clostridial cluster IV and XIVa butyrate producers characterized in detail have been shown to produce gases, mainly CO₂ and H₂ (12, 15, 20, 46). Consequently, they might be responsible for an enhancement of gas production as a result of fructan fermentation, through either cross-feeding or direct degradation of inulin-type fructans (15, 16). Indeed, inulin-type fructan consumption has been reported to cause some gastrointestinal discomfort related to gas production—essentially, flatulence and bloating (43)—while bifidobacteria, the main beneficiaries of dietary fructan intake, do not produce gases (19, 49). Although CO₂ and H₂ production by colon butyrate producers could have implications for human intestinal well-being, (in vitro) production has not been satisfactorily monitored up to now, probably due to limited availability of a performant apparatus for (online) gas analysis (15, 20). Moreover, the currently proposed pathway for colon butyrate production does not provide a conclusive quantitative link between bacterial (co)substrate metabolism and H₂ formation (11).This study investigated the kinetics of inulin-type fructan degradation by representatives of the genera Anaerostipes and Roseburia. A method based on online gas chromatography (GC) was developed to assess gas production qualitatively and quantitatively in a continuously sparged fermentation vessel for complete coverage of metabolite production. The competitiveness of inulin-degrading butyrate producers was investigated through coculture fermentations with Bifidobacterium longum subsp. longum LMG 11047, a strain representing a highly competitive cluster of bifidobacteria that share both high fructose consumption and oligofructose degradation rates and are able to perform partial breakdown of inulin (18, 20). A stoichiometrically balanced pathway for butyrate production, including H₂ production, is proposed.     

The Carnivorous Pale Pitcher Plant Harbors Diverse,Distinct, and Time-Dependent Bacterial Communities

The ability of American carnivorous pitcher plants (Sarracenia) to digest insect prey is facilitated by microbial associations. Knowledge of the details surrounding this interaction has been limited by our capability to characterize bacterial diversity in this system. To describe microbial diversity within and between pitchers of one species, Sarracenia alata, and to explore how these communities change over time as pitchers accumulate and digest insect prey, we collected and analyzed environmental sequence tag (454 pyrosequencing) and genomic fingerprint (automated ribosomal intergenic spacer analysis and terminal restriction fragment length polymorphism) data. Microbial richness associated with pitcher plant fluid is high; more than 1,000 unique phylogroups were identified across at least seven phyla and 50 families. We documented an increase in bacterial diversity and abundance with time and observed repeated changes in bacterial community composition. Pitchers from different plants harbored significantly more similar bacterial communities at a given time point than communities coming from the same genetic host over time. The microbial communities in pitcher plant fluid also differ significantly from those present in the surrounding soil. These findings indicate that the bacteria associated with pitcher plant leaves are far from random assemblages and represent an important step toward understanding this unique plant-microbe interaction.Characterization of the phyllosphere is fundamental to our understanding of the ecology and evolution of plant populations and plant diversity and their interactions with other organisms (46, 64, 66). The carnivorous pitcher plant genus Sarracenia is an obvious system to address basic questions in plant-microbe interaction because each pitcher (a modified leaf) of the plant contains a microcosm composed of larval insects, fungi, algae, rotifers, nematodes, and bacteria that, together, ultimately break down nutrients from insect prey for the plant (1, 10, 20, 28, 37). Each pitcher represents a naturally defined and discrete community with a finite volume and a discrete life span (each leaf lasts only one season). Several investigations have explored species interactions within Sarracenia pitchers (13, 20, 34, 54), and competition, predation and dispersal frequency appear to be important drivers of community composition in the system (1, 20, 43, 44). Studies involving community patterns on a larger scale within pitchers, however, are few, and the processes that produce these patterns remain unknown (33).     

Characterization of Airborne Microbial Communities at a High-Elevation Site and Their Potential To Act as Atmospheric Ice Nuclei

Bacteria and fungi are ubiquitous in the atmosphere. The diversity and abundance of airborne microbes may be strongly influenced by atmospheric conditions or even influence atmospheric conditions themselves by acting as ice nucleators. However, few comprehensive studies have described the diversity and dynamics of airborne bacteria and fungi based on culture-independent techniques. We document atmospheric microbial abundance, community composition, and ice nucleation at a high-elevation site in northwestern Colorado. We used a standard small-subunit rRNA gene Sanger sequencing approach for total microbial community analysis and a bacteria-specific 16S rRNA bar-coded pyrosequencing approach (4,864 sequences total). During the 2-week collection period, total microbial abundances were relatively constant, ranging from 9.6 × 10⁵ to 6.6 × 10⁶ cells m⁻³ of air, and the diversity and composition of the airborne microbial communities were also relatively static. Bacteria and fungi were nearly equivalent, and members of the proteobacterial groups Burkholderiales and Moraxellaceae (particularly the genus Psychrobacter) were dominant. These taxa were not always the most abundant in freshly fallen snow samples collected at this site. Although there was minimal variability in microbial abundances and composition within the atmosphere, the number of biological ice nuclei increased significantly during periods of high relative humidity. However, these changes in ice nuclei numbers were not associated with changes in the relative abundances of the most commonly studied ice-nucleating bacteria.Microbes are abundant in the atmosphere, with both cultivation-dependent and molecular approaches showing that the atmosphere harbors a diverse assemblage of bacteria and fungi, including taxa also commonly found on leaf surfaces (5, 49) and in soil habitats (30). The abundance and composition of airborne microbial communities are variable across time and space (14, 24, 27, 33, 47, 48, 69). However, the atmospheric conditions responsible for driving the observed changes in microbial abundances are unknown. The diversity of airborne microorganisms, and the factors influencing diversity levels, also remains poorly characterized. One reason for these limitations in knowledge is that until recently, culture-based microbiological methods have been the standard, and it is well-recognized that such methods capture only a small portion of the total microbial diversity (59). As demonstrated in a number of recent studies (6, 13, 22, 23, 33, 52, 59, 63, 73), advances in culture-independent techniques allow far more of the microbial diversity present in the atmosphere to be surveyed and the spatiotemporal variability in microbial communities to be examined.Microbes are often considered passive inhabitants of the atmosphere, dispersing via airborne dust particles. However, recent studies suggest that many atmospheric microbes may be metabolically active (3, 4, 64), even up to altitudes of 20,000 m (34). Some airborne microbes may alter atmospheric conditions directly by acting as cloud condensation nuclei (7, 25, 56) and/or ice nuclei (IN) (19, 41, 56, 57, 61); this hypothesis is supported by the observation that most ice nuclei in snow samples are inactivated by a 95°C heat treatment (16, 17). However, the overall contribution of airborne microbes to atmospheric processes such as ice nucleation remains unclear.The best-studied ice-nucleating microbes are gram-negative bacteria that have also been isolated from leaf surfaces, including Pseudomonas syringae, Pseudomonas fluorescens, Erwinia herbicola, Xanthomonas campestri, and Sphingomonas spp. (45). These bacteria have been cultured extensively, and their ice-nucleating activity has been traced to a membrane-bound glycoprotein (40, 42, 70). However, their specific influence on atmospheric processes remains, at this point, largely anecdotal. Less is known about the ice-nucleating activities of fungi, but a few studies have shown that fungi can be effective ice nucleators, capable of initiating ice nucleation at temperatures as high as −2°C (41, 61). At this point, all known ice-nucleating microorganisms are amenable to culture-based studies, but given that the vast majority of microorganisms have yet to be cultured, it is likely that other ice-nucleating microbes remain undiscovered.The work presented here addresses three overarching questions. (i) Are microbial abundances altered by changes in atmospheric conditions? (ii) How is the diversity and composition of airborne microbial communities influenced by changes in atmospheric conditions? (iii) Can we identify known and novel ice-nucleating microbes in the atmosphere by testing for correlations between taxa abundances and the concentrations of biological ice nuclei? To address these questions, we combined epifluorescence microscopy, tagged pyrosequencing, Sanger sequencing, and an ice nucleation assay with atmospheric measurements to characterize the microbial communities at a high-elevation research site.     

Detection of a Reproducible,Single-Member Shift in Soil Bacterial Communities Exposed to Low Levels of Hydrogen

Soil is exposed to hydrogen when symbiotic rhizobia in legume root nodules cannot recycle the hydrogen that is generated during nitrogen fixation. The hydrogen emitted is most likely taken up by free-living soil bacteria that use hydrogen as an energy source, though the bacteria that do this in situ remain unclear. In this study, we investigated the effect of hydrogen exposure on the bacteria of two different soils in a microcosm setup designed to simulate hydrogen-emitting root nodules. Although the size and overall composition of the soil bacterial community did not significantly alter after hydrogen exposure, one ribotype increased in relative abundance within each soil. This single-ribotype shift was identified by generating multiple terminal restriction fragment length polymorphism (T-RFLP) profiles of 16S rRNA genes from each soil sample, with gene sequence confirmation to identify terminal restriction fragments. The increased abundance of a single ribotype after hydrogen exposure, within an otherwise similar community, was found in replicate samples taken from each microcosm and was reproducible across replicate experiments. Similarly, only one member of the soil bacterial community increased in abundance in response to hydrogen exposure in soil surrounding the root nodules of field-grown soybean (Glycine max). The ribotypes that increased after hydrogen exposure in each soil system tested were all from known hydrogen-oxidizing lineages within the order Actinomycetales. We suggest that soil actinomycetes are important utilizers of hydrogen at relevant concentrations in soil and could be key contributors to soil''s function as a sink in the global hydrogen cycle.Soil is the major sink in the global hydrogen cycle and accounts for approximately 75 to 80% of uptake from the atmosphere (7, 11). Soil is such a strong sink that the atmospheric mixing ratio of molecular hydrogen, H₂, is hemispherically asymmetric because of the greater landmass in the Northern Hemisphere (11). Many nitrogen-fixing bacteria that form symbiotic relationships with legume plants cannot recycle the H₂ that is generated during N₂ fixation (2, 13). Most of the H₂ emitted from legume root nodules is taken up by the surrounding soil, within a few centimeters of the nodule surface, and is not released to the atmosphere (20). Although the H₂ emitted by the rhizobial symbionts costs the legume approximately 5% of its daily photosynthate and “represents a significant investment by the plant” (9), there is growing evidence to suggest that soil exposed to H₂ is beneficial to plant growth, separate from the benefits derived from N₂ fixation (8, 10, 28). Previously, La Favre and Focht have hypothesized that “the hydrogen which is evolved during N₂ fixation represents an additional energy input into the plant-soil ecosystem… since metabolism of H₂ by chemolithotrophic bacteria results in an input of fixed carbon to the system” (20). A number of studies have found that when H₂ is taken up by soil, net CO₂ fixation occurs at the rate of 7 to 8 nmol CO₂ per g of soil per h (22, 34). For a legume fixing 200 kg of atmospheric N₂ per hectare, over 200,000 liters of H₂ could be released into the legume''s rhizosphere over the duration of the growing season and CO₂ fixation could result in an extra 25 kg of soil carbon fixed per hectare (9, 10, 28).Many bacteria isolated from soil are able to utilize H₂ as an energy source (2, 5-7, 21), and free-living bacteria are most likely responsible for the H₂ uptake observed by soil surrounding legume roots (22). Adding a bacterial energy source, such as H₂, could affect the microbial population size, as has been observed previously (34), but more specific shifts within the bacterial community may occur if just the microorganisms able to utilize the energy source multiply. Their activity could also have downstream consequences specifically on other members of the community. Most H₂-oxidizing cultures have required enrichment with concentrations of H₂ that are not environmentally relevant and therefore cannot be assumed to be carrying out H₂ oxidation at much lower, naturally occurring concentrations (5-7). Recent surveys of microbes present in soil samples, via their nucleic acids, have revealed many novel bacterial inhabitants that have been little studied and thus may also be contributing to the repertoire of bacterial soil processes, such as H₂ uptake (16). A recent study into the effect of H₂ on soil bacteria focused on a few groups of H₂-oxidizing, autotrophic bacteria and thus ignored many other H₂ utilizers potentially present in soil (34).There are now many ways of characterizing the entire microbial community in environmental samples, either via their entire genomic content, though metagenomic analysis of soil is difficult at present, or via analysis of the lineages present according to 16S rRNA gene sequences, or ribotypes (36). A recent study comparing high-throughput pyrosequencing of 16S rRNA genes and an easily accessible profiling method, known as terminal restriction fragment length polymorphism (T-RFLP), found the simpler profiles were appropriate for comparing the dominant ribotypes in multiple samples (24). Although T-RFLP profiles only provide a simplified snapshot of the dominant members in microbial communities, compared to the deeper analyses provided by microarrays or high-throughput sequencing technologies, T-RFLP profiling is a cost-effective, reproducible, and robust method of “fingerprinting” many soil samples rapidly and efficiently (14, 24, 25, 32).In this study, we chose to assess the dominant members of the soil bacterial community via T-RFLP profiles of ribotypes present in H₂-treated and control soils to avoid a narrow focus on well-studied H₂ oxidizers. We investigated the bacterial community structure in two different soils, utilizing a microcosm setup with concentrations of H₂ calculated to occur in the rhizosphere of N₂-fixing legumes, to determine whether common responses to H₂ exposure could be predicted from soils that differ by climate, edaphic characteristics, and starting communities. Soil in microcosms has previously been shown to have similar H₂ uptake properties to soil close to H₂-emitting legume nodules (9), but we also complemented our plant-free microcosm work with an examination of soil collected from the root systems of field-grown soybean (Glycine max (L.) Merr.).     

Cultivation of Fastidious Bacteria by Viability Staining and Micromanipulation in a Soil Substrate Membrane System

Soil substrate membrane systems allow for microcultivation of fastidious soil bacteria as mixed microbial communities. We isolated established microcolonies from these membranes by using fluorescence viability staining and micromanipulation. This approach facilitated the recovery of diverse, novel isolates, including the recalcitrant bacterium Leifsonia xyli, a plant pathogen that has never been isolated outside the host.The majority of bacterial species have never been recovered in the laboratory (1, 14, 19, 24). In the last decade, novel cultivation approaches have successfully been used to recover “unculturables” from a diverse range of divisions (23, 25, 29). Most strategies have targeted marine environments (4, 23, 25, 32), but soil offers the potential for the investigation of vast numbers of undescribed species (20, 29). Rapid advances have been made toward culturing soil bacteria by reformulating and diluting traditional media, extending incubation times, and using alternative gelling agents (8, 21, 29).The soil substrate membrane system (SSMS) is a diffusion chamber approach that uses extracts from the soil of interest as the growth substrate, thereby mimicking the environment under investigation (12). The SSMS enriches for slow-growing oligophiles, a proportion of which are subsequently capable of growing on complex media (23, 25, 27, 30, 32). However, the SSMS results in mixed microbial communities, with the consequent difficulty in isolation of individual microcolonies for further characterization (10).Micromanipulation has been widely used for the isolation of specific cell morphotypes for downstream applications in molecular diagnostics or proteomics (5, 15). This simple technology offers the opportunity to select established microcolonies of a specific morphotype from the SSMS when combined with fluorescence visualization (3, 11). Here, we have combined the SSMS, fluorescence viability staining, and advanced micromanipulation for targeted isolation of viable, microcolony-forming soil bacteria.     

CO2 Uptake and Fixation by a Thermoacidophilic Microbial Community Attached to Precipitated Sulfur in a Geothermal Spring

Carbon fixation at temperatures above 73°C, the upper limit for photosynthesis, is carried out by chemosynthetic thermophiles. Yellowstone National Park (YNP), Wyoming possesses many thermal features that, while too hot for photosynthesis, presumably support chemosynthetic-based carbon fixation. To our knowledge, in situ rates of chemosynthetic reactions at these high temperatures in YNP or other high-temperature terrestrial geothermal springs have not yet been reported. A microbial community attached to precipitated elemental sulfur (S^o floc) at the source of Dragon Spring (73°C, pH 3.1) in Norris Geyser Basin, YNP, exhibited a maximum rate of CO₂ uptake of 21.3 ± 11.9 μg of C 10⁷ cells⁻¹ h⁻¹. When extrapolated over the estimated total quantity of S^o floc at the spring''s source, the S^o floc-associated microbial community accounted for the uptake of 121 mg of C h⁻¹ at this site. On a per-cell basis, the rate was higher than that calculated for a photosynthetic mat microbial community dominated by Synechococcus spp. in alkaline springs at comparable temperatures. A portion of the carbon taken up as CO₂ by the S^o floc-associated biomass was recovered in the cellular nucleic acid pool, demonstrating that uptake was coupled to fixation. The most abundant sequences in a 16S rRNA clone library of the S^o floc-associated community were related to chemolithoautotrophic Hydrogenobaculum strains previously isolated from springs in the Norris Geyser Basin. These microorganisms likely contributed to the uptake and fixation of CO₂ in this geothermal habitat.The upper temperature limit for primary production via photosynthesis is ∼73°C (7, 8, 11). At this temperature, photosynthesis is restricted to cyanobacteria of the genus Synechococcus, which generally inhabit alkaline environments (11). In acidic environments (pH < 4.0), the upper temperature limit for photosynthetic-based primary production is ∼56°C. Under these conditions, phototrophic activity is restricted to the unicellular eukaryotic red algae Cyanidium, Galdieria, and Cyanidioschyzon, collectively referred to as “cyanidia” (6, 12, 31, 48). Primary production above this temperature in acidic environments occurs through chemoautotrophy, a metabolism restricted to prokaryotes.Yellowstone National Park (YNP), WY, possesses numerous high-temperature (73 to 93°C) geothermal environments that are thought to support communities of microorganisms through chemoautotrophic-based primary production. Evidence for chemosynthesis in these environments is based on the recovery of 16S rRNA gene sequences that are affiliated with cultivated representatives of the phyla Aquificae and Crenarchaeota, many of which are capable of CO₂ fixation via the oxidation of hydrogen (H₂) and/or sulfide (HS⁻) (15, 17, 21, 24, 26, 28, 41, 46). Surprisingly, CO₂ fixation has yet to be demonstrated in situ in YNP hot spring environments (acidic or alkaline) where temperatures exceed the limits of photosynthesis and where primary production is thought to be driven by chemoautotrophic metabolism (14, 15, 28, 29).Dragon Spring, an acid-sulfate-chloride (ASC) spring located in the Norris Geyser Basin of YNP, is a likely habitat for chemoautotrophic primary production. The pH of the water is ∼3.1, and the temperature of the water at the source fluctuates from 65 to 78°C, which is well above the upper temperature limit for photosynthesis under acidic conditions. Potential electron donors for chemolithoautotrophic growth in the source water include hydrogen (H₂) and sulfide (S²⁻) at concentrations of 13 nM and 65 μM, respectively (15). In addition, submerged substrata at the spring''s source are blanketed by precipitates of elemental sulfur (S°), hereafter referred to as S^o floc (23). Inventories of bacterial and archaeal 16S rRNA genes recovered from S^o floc collected from the source of Dragon Spring indicate the presence of Crenarchaeota and Aquificae (4, 15). The latter are related to chemolithoautotrophic Hydrogenobaculum spp., representatives of which have recently been isolated from the spring (15). In the present study, we demonstrate uptake and fixation of CO₂ at a temperature of 73°C by a Hydrogenobaculum-dominated microbial community associated with S^o floc collected from the source of Dragon Spring. This is the first direct evidence of CO₂ uptake in situ by a thermoacidophilic microbial community at a temperature that precludes photosynthesis in terrestrial geothermal springs.     

Microbial Community Structure and Denitrification in a Wetland Mitigation Bank

Wetland mitigation is implemented to replace ecosystem functions provided by wetlands; however, restoration efforts frequently fail to establish equivalent levels of ecosystem services. Delivery of microbially mediated ecosystem functions, such as denitrification, is influenced by both the structure and activity of the microbial community. The objective of this study was to compare the relationship between soil and vegetation factors and microbial community structure and function in restored and reference wetlands within a mitigation bank. Microbial community composition was assessed using terminal restriction fragment length polymorphism targeting the 16S rRNA gene (total bacteria) and the nosZ gene (denitrifiers). Comparisons of microbial function were based on potential denitrification rates. Bacterial community structures differed significantly between restored and reference wetlands; denitrifier community assemblages were similar among reference sites but highly variable among restored sites throughout the mitigation bank. Potential denitrification was highest in the reference wetland sites. These data demonstrate that wetland restoration efforts in this mitigation bank have not successfully restored denitrification and that differences in potential denitrification rates may be due to distinct microbial assemblages observed in restored and reference (natural) wetlands. Further, we have identified gradients in soil moisture and soil fertility that were associated with differences in microbial community structure. Microbial function was influenced by bacterial community composition and soil fertility. Identifying soil factors that are primary ecological drivers of soil bacterial communities, especially denitrifying populations, can potentially aid the development of predictive models for restoration of biogeochemical transformations and enhance the success of wetland restoration efforts.Wetlands provide more ecosystem services (e.g., flood control, water purification, nutrient cycling, and habitat for wildlife) per hectare than any other ecosystem (16). Riparian wetlands, in particular, are sites of intense biogeochemical activity and play an important role in improving water quality, recycling nutrients, and detoxifying chemicals (41). Changing patterns of land use over the last century have resulted in the loss of over half of the wetlands in the contiguous United States (17) and about 60% of wetlands in the Midwestern United States (82). The loss of ecosystem services through conversion of wetlands to alternative (primarily agricultural) land uses exacerbates nutrient pollution and eutrophication of downstream ecosystems (57). Declines in wetland acreage have continued despite a federal policy goal of no-net-loss of wetland acreage and function adopted in 1990 (7, 55). Wetland mitigation projects provide compensation for impacted wetlands and aim to replace the critical functions provided by wetlands. Despite decades of wetland mitigation, however, restoration efforts frequently fail to reestablish desired levels of ecosystem services. Restoration outcomes remain uncertain, and more information is necessary in order to improve monitoring and assessment of wetland development (13, 18, 50, 80).One approach to wetland compensation is through mitigation banks. These sites are areas that are restored, established, enhanced, or preserved for replacement of wetlands that will be affected by future land use change. Mitigation banks are considered “third-party” compensatory mitigation, where the permittee (e.g., developer planning to destroy a wetland) is responsible for purchasing wetland credits in acreage, but the wetland bank is established and managed by another party (24). Wetland mitigation banks have unique characteristics that distinguish them from smaller individual restoration projects (7, 69, 81). Due to their size, wetland mitigation banks are especially heterogeneous and may have a great deal of within-site variability in hydrology and nutrient status, making it challenging to implement a single restoration design. Thus, wetland mitigation banks require intense management and monitoring for improved success (7, 69, 81).Restoration efforts such as mitigation banks aim to replace chemical, physical, and biological ecosystem functions of wetlands that have been lost through anthropogenic disturbance (24). Monitoring of wetland mitigation sites has largely focused on measures of macro-scale community structure (e.g., vegetation surveys) (52) along with measures of hydrology and soil type (24). Measurement of vegetation is a common proxy for wetland performance but does not provide an accurate assessment of wetland function (6, 52). Quantitative assessment is achievable, however, for ecosystem services such as water quality improvement through nitrate removal, where well-characterized microbial mechanisms underlie denitrification processes.The link between microbial community structure and function in a restoration context is a topic of current interest (33). Relating microbial community composition and dynamics to chemical, physical, and biological variables can help to reveal important ecological drivers of microbial communities and their activities (26, 35, 42). Conserved bacterial functional genes related to specific biogeochemical transformations allow evaluation of the community structure of microbial populations directly involved in these processes (49, 60, 63, 77, 79). Assessing the diversity of microorganisms that are specifically involved in denitrification is possible through amplification of the nosZ gene, which encodes the catalytic subunit of nitrous oxide reductase, the enzyme responsible for the final step of denitrification (60, 63, 66). Phylogenetically diverse microorganisms can carry out denitrification though the majority of previously described denitrifiers belong to subphyla within the Proteobacteria (53, 56, 60, 61). Denitrification is a facultative process that occurs only under anaerobic conditions (53, 75). Complete denitrification to N₂ is more prevalent in anaerobic, saturated wetland ecosystems (14, 76), and incomplete denitrification to N₂O is the less desirable, more common endpoint of denitrification under more aerobic, drier conditions (14, 62). While the environmental factors (e.g., oxygen, carbon, nitrate, and pH) that influence bulk denitrification rates have been well characterized (31, 72), the influence of these factors on the composition of denitrifier communities, particularly in a restoration context, is unclear. Understanding the relationship between the microbial populations responsible for nitrogen transformations and easily measured environmental parameters (e.g., soil chemical and physical measures) could lead to assessment metrics that are linked directly to ecosystem functions such as denitrification and bridge the current gap in functional assessment methods (36, 60, 70).The objectives of this study were (i) to compare the microbial and plant community composition in restored wetlands to the composition in adjacent reference floodplain forest wetlands; (ii) to assess the relationship between microbial community composition (based on terminal restriction fragment length polymorphism [T-RFLP]) and potential denitrification activity throughout the mitigation bank; and (iii) to examine soil factors correlated with microbial community composition using both phylogenetic and functional gene markers. As soil environmental conditions affect microbial community structure and activity, we expected that sites where wetland hydrology and soil chemistry have been successfully restored would harbor microbial assemblages that are similar in composition and denitrification function to those observed in reference wetlands within this mitigation bank.     

Vertical Distribution of Ammonia-Oxidizing Crenarchaeota and Methanogens in the Epipelagic Waters of Lake Kivu (Rwanda-Democratic Republic of the Congo)

Four stratified basins in Lake Kivu (Rwanda-Democratic Republic of the Congo) were sampled in March 2007 to investigate the abundance, distribution, and potential biogeochemical role of planktonic archaea. We used fluorescence in situ hybridization with catalyzed-reported deposition microscopic counts (CARD-FISH), denaturing gradient gel electrophoresis (DGGE) fingerprinting, and quantitative PCR (qPCR) of signature genes for ammonia-oxidizing archaea (16S rRNA for marine Crenarchaeota group 1.1a [MCG1] and ammonia monooxygenase subunit A [amoA]). Abundance of archaea ranged from 1 to 4.5% of total DAPI (4′,6-diamidino-2-phenylindole) counts with maximal concentrations at the oxic-anoxic transition zone (∼50-m depth). Phylogenetic analysis of the archaeal planktonic community revealed a higher level of richness of crenarchaeal 16S rRNA gene sequences (21 of the 28 operational taxonomic units [OTUs] identified [75%]) over euryarchaeotal ones (7 OTUs). Sequences affiliated with the kingdom Euryarchaeota were mainly recovered from the anoxic water compartment and mostly grouped into methanogenic lineages (Methanosarcinales and Methanocellales). In turn, crenarchaeal phylotypes were recovered throughout the sampled epipelagic waters (0- to 100-m depth), with clear phylogenetic segregation along the transition from oxic to anoxic water masses. Thus, whereas in the anoxic hypolimnion crenarchaeotal OTUs were mainly assigned to the miscellaneous crenarchaeotic group, the OTUs from the oxic-anoxic transition and above belonged to Crenarchaeota groups 1.1a and 1.1b, two lineages containing most of the ammonia-oxidizing representatives known so far. The concomitant vertical distribution of both nitrite and nitrate maxima and the copy numbers of both MCG1 16S rRNA and amoA genes suggest the potential implication of Crenarchaeota in nitrification processes occurring in the epilimnetic waters of the lake.Lake Kivu is a meromictic lake located in the volcanic region between Rwanda and the Democratic Republic of the Congo and is the smallest of the African Great Rift Lakes. The monimolimnion of the lake contains a large amount of dissolved CO₂ and methane (300 km³ and 60 km³, respectively) as a result of geological and biological activity (24, 73, 85). This massive accumulation converts Lake Kivu into one of the largest methane reservoirs in the world and into a unique ecosystem for geomicrobiologists interested in the methane cycle and in risk assessment and management (34, 71, 72, 85). Comprehensive studies on the diversity and activity of planktonic populations of both large and small eukaryotes and their trophic interplay operating in the epilimnetic waters of the lake are available (33, 39, 49). Recent surveys have also provided a deeper insight into the seasonal variations of photosynthetic and heterotrophic picoplankton (67, 68), although very few data exist on the composition, diversity, and spatial distribution of bacterial and archaeal communities. In this regard, the studies conducted so far of the bacterial/archaeal ecology in Lake Kivu have been mostly focused on the implications on the methane cycle (34, 73), but none have addressed the presence and distribution of additional archaeal populations in the lake.During the last few years, microbial ecology studies carried out in a wide variety of habitats have provided compelling evidence of the ubiquity and abundance of mesophilic archaea (4, 10, 13, 19). Moreover, the discovery of genes encoding enzymes related to nitrification and denitrification in archaeal metagenomes from soil and marine waters (29, 86, 88) and the isolation of the first autotrophic archaeal nitrifier (40) demonstrated that some archaeal groups actively participate in the carbon and nitrogen cycles (56, 64, 69). In relation to aquatic environments, genetic markers of ammonia-oxidizing archaea (AOA) of the marine Crenarchaeota group 1.1a (MCG1) have consistently been found in water masses of several oceanic regions (6, 14, 17, 26, 28, 30, 37, 42, 51, 52, 89), estuaries (5, 9, 26, 53), coastal aquifers (26, 66), and stratified marine basins (15, 41, 44). Although less information is available for freshwater habitats, recent studies carried out in oligotrophic high-mountain and arctic lakes showed an important contribution of AOA in both the planktonic and the neustonic microbial assemblages (4, 61, 89).The oligotrophic nature of Lake Kivu and the presence of a well-defined redoxcline may provide an optimal niche for the development of autotrophic AOA populations. Unfortunately, no studies of the involvement of microbial planktonic populations in cycling nitrogen in the lake exist, and only data on the distribution of dissolved inorganic nitrogen species in relation to phytoplankton ecology (67, 68) and nutrient loading are available (54, 58). Our goals here were to ascertain whether or not archaeal populations other than methane-related lineages were relevant components of the planktonic microbial community and to determine whether the redox gradient imposed by the oxic-anoxic interphase acts as a threshold for their vertical distribution in epipelagic waters (0- to 100-m depth). To further explore the presence and potential activity of nitrifying archaeal populations in Lake Kivu, samples were analyzed for the abundance and vertical distribution of signature genes for these microorganisms, i.e., the 16S rRNA of MCG1 and the ammonia monooxygenase subunit A (amoA) gene by quantitative PCR.     

Residues in a Highly Conserved Claudin-1 Motif Are Required for Hepatitis C Virus Entry and Mediate the Formation of Cell-Cell Contacts

Claudin-1, a component of tight junctions between liver hepatocytes, is a hepatitis C virus (HCV) late-stage entry cofactor. To investigate the structural and functional roles of various claudin-1 domains in HCV entry, we applied a mutagenesis strategy. Putative functional intracellular claudin-1 domains were not important. However, we identified seven novel residues in the first extracellular loop that are critical for entry of HCV isolates drawn from six different subtypes. Most of the critical residues belong to the highly conserved claudin motif W₃₀-GLW₅₁-C₅₄-C₆₄. Alanine substitutions of these residues did not impair claudin-1 cell surface expression or lateral protein interactions within the plasma membrane, including claudin-1-claudin-1 and claudin-1-CD81 interactions. However, these mutants no longer localized to cell-cell contacts. Based on our observations, we propose that cell-cell contacts formed by claudin-1 may generate specialized membrane domains that are amenable to HCV entry.Hepatitis C virus (HCV) is a major human pathogen that affects approximately 3% of the global population, leading to cirrhosis and hepatocellular carcinoma in chronically infected individuals (5, 23, 42). Hepatocytes are the major target cells of HCV (11), and entry follows a complex cascade of interactions with several cellular factors (6, 8, 12, 17). Infectious viral particles are associated with lipoproteins and initially attach to target cells via glycosaminoglycans and the low-density lipoprotein receptor (1, 7, 31). These interactions are followed by direct binding of the E2 envelope glycoprotein to the scavenger receptor class B type I (SR-B1) and then to the CD81 tetraspanin (14, 15, 33, 36). Early studies showed that CD81 and SR-B1 were necessary but not sufficient for HCV entry, and claudin-1 was discovered to be a requisite HCV entry cofactor that appears to act at a very late stage of the process (18).Claudin-1 is a member of the claudin protein family that participates in the formation of tight junctions between adjacent cells (25, 30, 37). Tight junctions regulate the paracellular transport of solutes, water, and ions and also generate apical-basal cell polarity (25, 37). In the liver, the apical surfaces of hepatocytes form bile canaliculi, whereas the basolateral surfaces face the underside of the endothelial layer that lines liver sinusoids. Claudin-1 is highly expressed in tight junctions formed by liver hepatocytes as well as on all hepatoma cell lines that are permissive to HCV entry (18, 24, 28). Importantly, nonhepatic cell lines that are engineered to express claudin-1 become permissive to HCV entry (18). Claudin-6 and -9 are two other members of the human claudin family that enable HCV entry into nonpermissive cells (28, 43).The precise role of claudin-1 in HCV entry remains to be determined. A direct interaction between claudins and HCV particles or soluble E2 envelope glycoprotein has not been demonstrated (18; T. Dragic, unpublished data). It is possible that claudin-1 interacts with HCV entry receptors SR-B1 or CD81, thereby modulating their ability to bind to E2. Alternatively, claudin-1 may ferry the receptor-virus complex to fusion-permissive intracellular compartments. Recent studies show that claudin-1 colocalizes with the CD81 tetraspanin at the cell surface of permissive cell lines (22, 34, 41). With respect to nonpermissive cells, one group observed that claudin-1 was predominantly intracellular (41), whereas another reported associations of claudin-1 and CD81 at the cell surface, similar to what is observed in permissive cells (22).Claudins comprise four transmembrane domains along with two extracellular loops and two cytoplasmic domains (19, 20, 25, 30, 37). The first extracellular loop (ECL1) participates in pore formation and influences paracellular charge selectivity (25, 37). It has been shown that the ECL1 of claudin-1 is required for HCV entry (18). All human claudins comprise a highly conserved motif, W₃₀-GLW₅₁-C₅₄-C₆₄, in the crown of ECL1 (25, 37). The exact function of this domain is unknown, and we hypothesized that it is important for HCV entry. The second extracellular loop is required for the holding function and oligomerization of the protein (25). Claudin-1 also comprises various signaling domains and a PDZ binding motif in the intracellular C terminus that binds ZO-1, another major component of tight junctions (30, 32, 37). We further hypothesized that some of these domains may play a role in HCV entry.To understand the role of claudin-1 in HCV infection, we developed a mutagenesis strategy targeting the putative sites for internalization, glycosylation, palmitoylation, and phosphorylation. The functionality of these domains has been described by others (4, 16, 25, 35, 37, 40). We also mutagenized charged and bulky residues in ECL1, including all six residues within the highly conserved motif W₃₀-GLW₅₁-C₅₄-C₆₄. None of the intracellular domains were found to affect HCV entry. However, we identified seven residues in ECL1 that are critical for entry mediated by envelope glycoproteins derived from several HCV subtypes, including all six residues of the conserved motif. These mutants were still expressed at the cell surface and able to form lateral homophilic interactions within the plasma membrane as well as to engage in lateral interactions with CD81. In contrast, they no longer engaged in homophilic trans interactions at cell-cell contacts. We conclude that the highly conserved motif W₃₀-GLW₅₁-C₅₄-C₆₄ of claudin-1 is important for HCV entry into target cells and participates in the formation of cell-cell contacts.     

Functional diversity and electron donor dependence of microbial populations capable of U(VI) reduction in radionuclide-contaminated subsurface sediments

In order to elucidate the potential mechanisms of U(VI) reduction for the optimization of bioremediation strategies, the structure-function relationships of microbial communities were investigated in microcosms of subsurface materials cocontaminated with radionuclides and nitrate. A polyphasic approach was used to assess the functional diversity of microbial populations likely to catalyze electron flow under conditions proposed for in situ uranium bioremediation. The addition of ethanol and glucose as supplemental electron donors stimulated microbial nitrate and Fe(III) reduction as the predominant terminal electron-accepting processes (TEAPs). U(VI), Fe(III), and sulfate reduction overlapped in the glucose treatment, whereas U(VI) reduction was concurrent with sulfate reduction but preceded Fe(III) reduction in the ethanol treatments. Phyllosilicate clays were shown to be the major source of Fe(III) for microbial respiration by using variable-temperature Mössbauer spectroscopy. Nitrate- and Fe(III)-reducing bacteria (FeRB) were abundant throughout the shifts in TEAPs observed in biostimulated microcosms and were affiliated with the genera Geobacter, Tolumonas, Clostridium, Arthrobacter, Dechloromonas, and Pseudomonas. Up to two orders of magnitude higher counts of FeRB and enhanced U(VI) removal were observed in ethanol-amended treatments compared to the results in glucose-amended treatments. Quantification of citrate synthase (gltA) levels demonstrated a stimulation of Geobacteraceae activity during metal reduction in carbon-amended microcosms, with the highest expression observed in the glucose treatment. Phylogenetic analysis indicated that the active FeRB share high sequence identity with Geobacteraceae members cultivated from contaminated subsurface environments. Our results show that the functional diversity of populations capable of U(VI) reduction is dependent upon the choice of electron donor.Uranium contamination in subsurface environments is a widespread problem at mining and milling sites across North America, South America, and Eastern Europe (1). Uranium in the oxidized state, U(VI), is highly soluble and toxic and thus is a potential contaminant to local drinking-water supplies (46). Nitrate is often a cocontaminant with U(VI) as a result of the use of nitric acid in the processing of uranium and uranium-bearing waste (6, 45). Oxidized uranium can be immobilized in contaminated groundwater through the reduction of U(VI) to insoluble U(IV) by indirect (abiotic) and direct (enzymatic) processes catalyzed by microorganisms. Current remediation practices favor the stimulation of reductive uranium immobilization catalyzed by indigenous microbial communities along with natural attenuation and monitoring (5, 24, 40, 44, 65, 68, 69). Microbial uranium reduction activity in contaminated subsurface environments is often limited by carbon or electron donor availability (13, 24, 44, 69). Previous studies have indicated that U(VI) reduction does not proceed until nitrate is depleted (13, 16, 24, 44, 68, 69), as high nitrate concentrations inhibit the reduction of U(VI) by serving as a competing and more energetically favorable terminal electron acceptor for microorganisms (11, 16). The fate and transport of uranium in groundwater are also strongly linked through sorption and precipitation processes to the bioreduction of Fe minerals, including oxides, layer-silicate clay minerals, and sulfides (7, 23, 53).In order to appropriately design U(VI) bioremediation strategies, the potential function and phylogenetic structure of indigenous subsurface microbial communities must be further understood (24, 34, 46). Conflicting evidence has been presented on which microbial groups, Fe(III)- or sulfate-reducing bacteria (FeRB or SRB), effectively catalyze the reductive immobilization of U(VI) in the presence of amended electron donors (5, 44, 69). The addition of acetate to the subsurface at a uranium-contaminated site in Rifle, Colorado, initially stimulated FeRB within the family Geobacteraceae to reduce U(VI) (5, 65). However, with long-term acetate addition, SRB within the family Desulfobacteraceae, which are not capable of U(VI) reduction, increased in abundance and a concomitant reoxidation of U(IV) was observed (5, 65). At a uranium-contaminated site in Oak Ridge, Tennessee, in situ and laboratory-based experiments successfully employed ethanol amendments to stimulate denitrification followed by the reduction of U(VI) by indigenous microbial communities (13, 24, 44, 48, 50, 57, 68). In these studies, ethanol amendments stimulated both SRB and FeRB, with SRB likely catalyzing the reduction of U(VI). This suggests that the potential for bioremediation will be affected by the choice of electron donor amendment through effects on the functional diversity of U(VI)-reducing microbial populations. As uranium reduction is dependent on the depletion of nitrate, the microbial populations mediating nitrate reduction are also critical to the design of bioremediation strategies. Although nitrate-reducing bacteria (NRB) have been studied extensively in subsurface environments (2, 15, 19, 24, 56, 58, 70), the mechanisms controlling the in situ metabolism of NRB remain poorly understood.The dynamics of microbial populations capable of U(VI) reduction in subsurface sediments are poorly understood, and the differences in the microbial community dynamics during bioremediation have not been explored. Based on the results of previous studies (13, 44, 49, 57, 68, 69), we hypothesized that the activity of nitrate- and Fe(III)-reducing microbial populations, catalyzing the reductive immobilization of U(VI) in subsurface radionuclide-contaminated sediments, would be dependent on the choice of electron donor. The objectives of the present study were (i) to characterize structure-function relationships for microbial groups likely to catalyze or limit U(VI) reduction in radionuclide-contaminated sediments and (ii) to further develop a proxy for the metabolic activity of FeRB. Microbial activity was assessed by monitoring terminal electron-accepting processes (TEAPs), electron donor utilization, and Fe(III) mineral transformations in microcosms conducted with subsurface materials cocontaminated with high levels of U(VI) and nitrate. In parallel, microbial functional groups (i.e., NRB and FeRB) were enumerated and characterized using a combination of cultivation-dependent and -independent methods.     

Role of Plant Residues in Determining Temporal Patterns of the Activity,Size, and Structure of Nitrate Reducer Communities in Soil

The incorporation of plant residues into soil not only represents an opportunity to limit soil organic matter depletion resulting from cultivation but also provides a valuable source of nutrients such as nitrogen. However, the consequences of plant residue addition on soil microbial communities involved in biochemical cycles other than the carbon cycle are poorly understood. In this study, we investigated the responses of one N-cycling microbial community, the nitrate reducers, to wheat, rape, and alfalfa residues for 11 months after incorporation into soil in a field experiment. A 20- to 27-fold increase in potential nitrate reduction activity was observed for residue-amended plots compared to the nonamended plots during the first week. This stimulating effect of residues on the activity of the nitrate-reducing community rapidly decreased but remained significant over 11 months. During this period, our results suggest that the potential nitrate reduction activity was regulated by both carbon availability and temperature. The presence of residues also had a significant effect on the abundance of nitrate reducers estimated by quantitative PCR of the narG and napA genes, encoding the membrane-bound and periplasmic nitrate reductases, respectively. In contrast, the incorporation of the plant residues into soil had little impact on the structure of the narG and napA nitrate-reducing community determined by PCR-restriction fragment length polymorphism (RFLP) fingerprinting. Overall, our results revealed that the addition of plant residues can lead to important long-term changes in the activity and size of a microbial community involved in N cycling but with limited effects of the type of plant residue itself.Modern agricultural practices include a return of plant residues to soil, as this is considered sustainable to the environment. It is now recognized that the conversion of native land into cultivated systems leads to carbon losses, which can be up to 20 to 40% (17). Postharvest plant residues therefore represent an important source of carbon, helping to replenish soil organic matter that decomposes as a result of cultivation. Decomposing plant residues are also a source of nutrients, such as nitrogen, with reduced nitrate leaching compared to mineral fertilizers, which is beneficial for water quality (3). In addition, leaving the plant residue on the soil surface limits water losses by evaporation and prevents soil erosion by wind or water (15).The biochemical composition of plant residues is one of the most important factors influencing their decomposition in soil (14, 28, 29, 51). Indeed, Manzoni et al. (28), using a data set of 2,800 observations, showed previously that the patterns of decomposition were regulated by the initial residue stoichiometry. Several other factors such as climatic conditions, soil type, or localization of the residue in the soil (incorporated or on the soil surface) were also reported previously to influence decomposition (2, 24, 29, 44). Microorganisms are the major decomposers of organic matter in soil, and therefore, the diversity and activity of the microbial community during plant residue decomposition has received much attention (6, 23, 26, 27, 35). It was shown previously that the biochemical composition of plant residues influences microbial respiration (8) and microbial community structure (7, 37). The recent development of carbon-labeling approaches has furthered our knowledge of the microorganisms that actively assimilate the carbon derived from various plant residues (10, 31). However, most of those studies focused on microorganisms involved in C mineralization, and in contrast, very little is known about the effect of plant residue decomposition on the microbial communities involved in biochemical cycles other than the carbon cycle. Thus, despite the influence of plant residues on nitrogen cycling (1, 4, 5, 16, 20), studies assessing the effect of the presence and composition of plant residues on the ecology of microbial communities involved in nitrogen cycling are rare (21, 32, 36).The dissimilatory reduction of nitrate into nitrite is the first step in the processes of denitrification and the dissimilatory reduction of nitrate to ammonium (33, 41). The reduction of nitrate by denitrification leads to losses of nitrogen, which is often a limiting nutrient for plant growth in agriculture. Two types of dissimilatory nitrate reductases, differing in location, have been characterized: a membrane-bound nitrate reductase (Nar) and a periplasmic nitrate reductase (Nap) (9, 53). Nitrate reducers can harbor either Nar, Nap, or both (40, 47). Nitrate reducers are probably the most taxonomically diverse functional community within the nitrogen cycle, with members in most bacterial phyla and also archaea (42). Because of this high level of diversity of heterotrophs sharing the ability to produce energy from nitrate reduction, nitrate reducers are an excellent model system to investigate the response of the N-cycling community to plant residue addition.The aim of this work was to determine how the incorporation of plant residues with contrasting biochemical compositions into soil affects the nitrate-reducing community. For this purpose, we monitored the dynamics of the potential activity, size, and structure of the nitrate-reducing community after the addition of wheat, rape, or alfalfa residues to soil in a field experiment. As the nature and availability of the substrate change during residue decomposition (38, 39, 48), the influence of the incorporation of different plant residues on the nitrate-reducing community was investigated at several sampling times for 11 months.     

Anaerobic Biodegradation of n-Hexadecane by a Nitrate-Reducing Consortium

Nitrate-reducing enrichments, amended with n-hexadecane, were established with petroleum-contaminated sediment from Onondaga Lake. Cultures were serially diluted to yield a sediment-free consortium. Clone libraries and denaturing gradient gel electrophoresis analysis of 16S rRNA gene community PCR products indicated the presence of uncultured alpha- and betaproteobacteria similar to those detected in contaminated, denitrifying environments. Cultures were incubated with H₃₄-hexadecane, fully deuterated hexadecane (d₃₄-hexadecane), or H₃₄-hexadecane and NaH¹³CO₃. Gas chromatography-mass spectrometry analysis of silylated metabolites resulted in the identification of [H₂₉]pentadecanoic acid, [H₂₅]tridecanoic acid, [1-¹³C]pentadecanoic acid, [3-¹³C]heptadecanoic acid, [3-¹³C]10-methylheptadecanoic acid, and d₂₇-pentadecanoic, d₂₅-, and d₂₄-tridecanoic acids. The identification of these metabolites suggests a carbon addition at the C-3 position of hexadecane, with subsequent β-oxidation and transformation reactions (chain elongation and C-10 methylation) that predominantly produce fatty acids with odd numbers of carbons. Mineralization of [1-¹⁴C]hexadecane was demonstrated based on the recovery of ¹⁴CO₂ in active cultures.Linear alkanes account for a large component of crude and refined petroleum products and, therefore, are of environmental significance with respect to their fate and transport (38). The aerobic activation of alkanes is well documented and involves monooxygenase and dioxygenase enzymes in which not only is oxygen required as an electron acceptor but it also serves as a reactant in hydroxylation (2, 16, 17, 32, 34). Alkanes are also degraded under anoxic conditions via novel degradation strategies (34). To date, there are two known pathways of anaerobic n-alkane degradation: (i) alkane addition to fumarate, commonly referred to as fumarate addition, and (ii) a putative pathway, proposed by So et al. (25), involving carboxylation of the alkane. Fumarate addition proceeds via terminal or subterminal addition (C-2 position) of the alkane to the double bond of fumarate, resulting in the formation of an alkylsuccinate. The alkylsuccinate is further degraded via carbon skeleton rearrangement and β-oxidation (4, 6, 8, 12, 13, 21, 37). Alkane addition to fumarate has been documented for a denitrifying isolate (21, 37), sulfate-reducing consortia (4, 8, 12, 13), and five sulfate-reducing isolates (4, 6-8, 12). In addition to being demonstrated in these studies, fumarate addition in a sulfate-reducing enrichment growing on the alicyclic alkane 2-ethylcyclopentane has also been demonstrated (23). In contrast to fumarate addition, which has been shown for both sulfate-reducers and denitrifiers, the putative carboxylation of n-alkanes has been proposed only for the sulfate-reducing isolate strain Hxd3 (25) and for a sulfate-reducing consortium (4). Experiments using NaH¹³CO₃ demonstrated that bicarbonate serves as the source of inorganic carbon for the putative carboxylation reaction (25). Subterminal carboxylation of the alkane at the C-3 position is followed by elimination of the two terminal carbons, to yield a fatty acid that is one carbon shorter than the parent alkane (4, 25). The fatty acids are subject to β-oxidation, chain elongation, and/or C-10 methylation (25).In this study, we characterized an alkane-degrading, nitrate-reducing consortium and surveyed the metabolites of the consortium incubated with either unlabeled or labeled hexadecane in order to elucidate the pathway of n-alkane degradation. We present evidence of a pathway analogous to the proposed carboxylation pathway under nitrate-reducing conditions.     

Presence of Novel,Potentially Homoacetogenic Bacteria in the Rumen as Determined by Analysis of Formyltetrahydrofolate Synthetase Sequences from Ruminants

Homoacetogens produce acetate from H₂ and CO₂ via the Wood-Ljungdahl pathway. Some homoacetogens have been isolated from the rumen, but these organisms are expected to be only part of the full diversity present. To survey the presence of rumen homoacetogens, we analyzed sequences of formyltetrahydrofolate synthetase (FTHFS), a key enzyme of the Wood-Ljungdahl pathway. A total of 275 partial sequences of genes encoding FTHFS were PCR amplified from rumen contents of a cow, two sheep, and a deer. Phylogenetic trees were constructed using these FTHFS gene sequences and the translated amino acid sequences, together with other sequences from public databases and from novel nonhomoacetogenic bacteria isolated from the rumen. Over 90% of the FTHFS sequences fell into 34 clusters defined with good bootstrap support. Few rumen-derived FTHFS sequences clustered with sequences of known homoacetogens. Conserved residues were identified in the deduced FTHFS amino acid sequences from known homoacetogens, and their presence in the other sequences was used to determine a “homoacetogen similarity” (HS) score. A homoacetogen FTHFS profile hidden Markov model (HoF-HMM) was used to assess the homology of rumen and homoacetogen FTHFS sequences. Many clusters had low HS scores and HoF-HMM matches, raising doubts about whether the sequences originated from homoacetogens. In keeping with these findings, FTHFS sequences from nonhomoacetogenic bacterial isolates grouped in these clusters with low scores. However, sequences that formed 10 clusters containing no known isolates but representing 15% of our FTHFS sequences from rumen samples had high HS scores and HoF-HMM matches and so could represent novel homoacetogens.Feed ingested by ruminant animals is fermented in the rumen by a complex community of microbes. This community produces, among other products, the volatile fatty acids acetate, propionate, and butyrate, which are absorbed across the rumen wall and satisfy a large part of the animals'' carbon and energy requirements. Hydrogen gas (H₂) is also formed and is the major precursor of the methane (CH₄) formed in ruminant animals. This ruminant-derived CH₄ is a contributor to global greenhouse gas emissions (46) and also represents an energy loss for the animals (34). Proposed ruminant greenhouse gas mitigation strategies include using feeds that produce less CH₄ and more volatile fatty acids (31). Alternative strategies include interventions that slow or halt methanogenesis by vaccination, using natural inhibitors found in plants, and supplementing feed with fats and oils or small-molecule inhibitors (31, 32). In the absence of methanogenesis, accumulation of H₂ could lead to a decrease in the rate of feed fermentation (31, 53) and hence a decrease in animal productivity. Other microbes that use H₂ without producing methane could be valuable in conjunction with intervention strategies that inhibit methanogens. This possibility has sparked interest in possible inoculation of ruminants with alternative H₂ users.Bacteria that use the Wood-Ljungdahl pathway to produce acetate from CO₂ are metabolically (6) and phylogenetically (48) diverse and are designated “homoacetogens.” Homoacetogens grow with H₂ or other suitable electron donors, such as formate or sugars, plus CO₂ as a terminal electron acceptor, heterotrophically with organic substrates such as sugars and methoxylated compounds, or mixotrophically with, e.g., H₂ and organic substrates. Homoacetogens have been reported to occur in a normally functioning rumen, but they are unlikely to compete with methanogens for H₂ (24, 25, 34). However, homoacetogens could play an important role in the disposal of H₂ if methanogens are not established in or are eliminated from the rumen (11, 17). At present, it is not clear whether resident rumen homoacetogens could fulfill the H₂ disposal role or whether homoacetogens would have to be added to the rumen to take over this role from the methanogens.Cultivation-based enumeration techniques have shown that the sizes of rumen acetogen populations range from undetectable to 1.2 × 10⁹ per g of rumen contents and that the prevalence of these acetogens depends on diet, animal age, and time of sampling (5, 7, 23, 24). Several homoacetogens, including Acetitomaculum ruminis (15), Eubacterium limosum (14, 17), Blautia schinkii, and Blautia producta (11), have been isolated from ruminants. Homoacetogens have also been isolated from the kangaroo forestomach, whose function is analogous to that of the rumen, which suggests that homoacetogenesis may play a role in hydrogen removal in the low-methane-emission forestomach (37).Because homoacetogens occur in different lineages of bacteria (48), traditional 16S rRNA gene-based surveys provide little information on their prevalence. The formyltetrahydrofolate synthetase (FTHFS) gene (fhs) has been used as a functional marker for homoacetogens, as the enzyme that it encodes catalyzes a key step in the reductive acetogenesis pathway (26). The structure of the enzyme of the homoacetogen Moorella thermoacetica has been reported, and putative functional features have been identified (27, 41, 42). FTHFS sequences from true homoacetogens differ from their homologs in sulfate-reducing bacteria and in other bacteria that degrade purines and amino acids via the glycine synthase-glycine reductase pathway (12, 21, 22, 26). At present, only a limited number of FTHFS sequences have been deposited in databases, and the vast majority of them are partial sequences retrieved from complex microbial communities. FTHFS sequences have been surveyed in sludge (39, 43, 54), termites (40, 44), salt marsh plant roots (21), horse manure (22), cow manure, freshwater sediment, rice field soil, and sewage (54), but so far only one study has investigated bovine ruminal FTHFS sequences (30). The rumen FTHFS sequences had low levels of similarity to the FTHFS sequences of known homoacetogens and could be sequences of novel homoacetogens. To our knowledge, no bacteria with these unique FTHFS sequences have been identified.The aims of this study were to assess the diversity of FTHFS gene sequences retrieved from rumen samples and to screen novel rumen isolates for the presence of FTHFS genes and test their ability to grow as homoacetogens. We used alignments of FTHFS sequences to define a homoacetogen similarity score based on the presence of diagnostic amino acids and developed a hidden Markov model to assess the likelihood that FTHFS sequences of unknown origin are sequences from true homoacetogens that are able to use H₂ or alternative electron donors for reductive acetogenesis.     

Phylogenetic and Multivariate Analyses To Determine the Effects of Different Tillage and Residue Management Practices on Soil Bacterial Communities

Bacterial communities are important not only in the cycling of organic compounds but also in maintaining ecosystems. Specific bacterial groups can be affected as a result of changes in environmental conditions caused by human activities, such as agricultural practices. The aim of this study was to analyze the effects of different forms of tillage and residue management on soil bacterial communities by using phylogenetic and multivariate analyses. Treatments involving zero tillage (ZT) and conventional tillage (CT) with their respective combinations of residue management, i.e., removed residue (−R) and kept residue (+R), and maize/wheat rotation, were selected from a long-term field trial started in 1991. Analysis of bacterial diversity showed that soils under zero tillage and crop residue retention (ZT/+R) had the highest levels of diversity and richness. Multivariate analysis showed that beneficial bacterial groups such as fluorescent Pseudomonas spp. and Burkholderiales were favored by residue retention (ZT/+R and CT/+R) and negatively affected by residue removal (ZT/−R). Zero-tillage treatments (ZT/+R and ZT/−R) had a positive effect on the Rhizobiales group, with its main representatives related to Methylosinus spp. known as methane-oxidizing bacteria. It can be concluded that practices that include reduced tillage and crop residue retention can be adopted as safer agricultural practices to preserve and improve the diversity of soil bacterial communities.Agricultural sustainability is linked to soil management and efficient use of natural and economic resources (25, 53). Sustainable handling of resources can be obtained by applying conservation agricultural practices, i.e., reduced tillage, crop residue retention, and crop rotation (26). Reduced tillage and crop residue retention have been proposed, as they facilitate water infiltration, reduce erosion, improve soil structure, increase soil organic matter and carbon content, and moderate soil temperatures (13, 16, 30, 33, 56). Compared with conventional tillage and crop residue removal, these practices can also decrease production costs by reducing the use of heavy machinery, fuels, water, and fertilizers (19, 23). The positive effect of these practices seems to be correlated with the improvement of soil structure and a higher availability of organic substrates for microorganisms (3, 30). Improved soil structure allows better soil aeration and diffusion of water and nutrients through the soil profile, while the retention of crop residues enhances microbial activity and the soil microbial biomass content (12, 28). These improvements in soil quality can also increase soil microbial diversity, thus protecting crops against pests and diseases through competition for soil nutrients (8).Until now, most research has focused on microbial communities affected by agricultural practices, i.e., tillage and residue management, by using indicators such as plate counting and microbial biomass or by analyzing denaturing gradient gel bacterial banding patterns (21, 22, 37). Salles et al. (46) reported the use of canonical correspondence analysis on denaturing gradient gel electrophoresis banding pattern data to understand the effect of crop and land history on Burkholderia communities. However, few studies have applied phylogenetic and multivariate analyses to understand the effect of soil management practices, i.e., tillage and residue management, on microbial communities.It is necessary to interpret the changes in microbial communities as a function of contextual environmental parameters to analyze the effect of anthropogenic activities on microbial communities (42). Once modifications in microbial communities are interpreted as a function of contextual environments, it becomes possible to determine the kind of organisms that dominate such environments and to establish whether specific practices could lead to changes in beneficial or nonbeneficial microorganisms for agro-ecosystems. Changes in microbial communities can then be related to food production, soil quality, and greenhouse gas emissions (19, 20, 36).Govaerts et al. (19, 20, 21, 22) had previously characterized the soils used in this study. They showed that soils under zero tillage (ZT) and crop residue retention (+R) have better soil quality, crop yields, and catabolic diversity and a higher diversity of microflora groups than do soils under conventional tillage (CT) with or without crop residue retention (−R). The aim of this study was to complement the results of Govaerts et al. (19, 20, 21, 22) by using phylogenetic approaches and the additive main effect and multiplicative interactions (AMMI) model (18, 60) to analyze the effect of the above treatments on soil bacterial communities.     

Tryptophan Residues in the Portal Protein of Herpes Simplex Virus 1 Critical to the Interaction with Scaffold Proteins and Incorporation of the Portal into Capsids

Incorporation of the herpes simplex virus 1 (HSV-1) portal vertex into the capsid requires interaction with a 12-amino-acid hydrophobic domain within capsid scaffold proteins. The goal of this work was to identify domains and residues in the U_L6-encoded portal protein pU_L6 critical to the interaction with scaffold proteins. We show that whereas the wild-type portal and scaffold proteins readily coimmunoprecipitated with one another in the absence of other viral proteins, truncation beyond the first 18 or last 36 amino acids of the portal protein precluded this coimmunoprecipitation. The coimmunoprecipitation was also precluded by mutation of conserved tryptophan (W) residues to alanine (A) at positions 27, 90, 127, 163, 241, 262, 532, and 596 of U_L6. All of these W-to-A mutations precluded the rescue of a viral deletion mutant lacking U_L6, except W163A, which supported replication poorly, and W596A, which fully rescued replication. A recombinant virus bearing the W596A mutation replicated and packaged DNA normally, and scaffold proteins readily coimmunoprecipitated with portal protein from lysates of infected cells. Thus, viral functions compensated for the W596A mutation''s detrimental effects on the portal-scaffold interaction seen during transient expression of portal and scaffold proteins. In contrast, the W27A mutation precluded portal-scaffold interactions in infected cell lysates, reduced the solubility of pU_L6, decreased incorporation of the portal into capsids, and abrogated viral-DNA cleavage and packaging.Immature herpesvirus capsids or procapsids consist of two shells: an inner shell, or scaffold, and an outer shell that is roughly spherical and largely composed of the major capsid protein VP5 (24, 38).The capsid scaffold consists of a mixture of the U_L26.5 and U_L26 gene products, with the U_L26.5 gene product (pU_L26.5, ICP35, or VP22a) being the most abundant (1, 12, 20, 21, 32, 38). The U_L26.5 open reading frame shares its coding frame and C terminus with the U_L26 gene but initiates at codon 307 of U_L26 (17). The extreme C termini of both VP22a and the UL26-encoded protein (pU_L26) interact with the N terminus of VP5 (7, 14, 26, 40, 41). Capsid assembly likely initiates when the portal binds VP5/VP22a and/or VP5/pU_L26 complexes (22, 25). The addition of more of these complexes to growing capsid shells eventually produces a closed sphere bearing a single portal. pU_L26 within the scaffold contains a protease that cleaves itself between amino acids 247 and 248, separating pU_L26 into an N-terminal protease domain called VP24 and a C-terminal domain termed VP21 (4, 5, 8, 9, 28, 42). The protease also cleaves 25 amino acids from pU_L26 and VP22a to release VP5 (5, 8, 9). VP21 and VP22a are replaced with DNA when the DNA is packaged (12, 29).When capsids undergo maturation, the outer protein shell angularizes to become icosahedral (13). One fivefold-symmetrical vertex in the angularized outer capsid shell is biochemically distinct from the other 11 and is called the portal vertex because it serves as the channel through which DNA is inserted as it is packaged (23). In herpes simplex virus (HSV), the portal vertex is composed of 12 copies of the portal protein encoded by U_L6 (2, 23, 39). We and others have shown that interactions between scaffold and portal proteins are critical for incorporation of the portal into the capsid (15, 33, 44, 45). Twelve amino acids of scaffold proteins are sufficient to interact with the portal protein, and tyrosine and proline resides within this domain are critical for the interaction with scaffold proteins and incorporation of the portal into capsids (45).One goal of the current study was to map domains and residues within the U_L6-encoded portal protein that mediate interaction with scaffold proteins. We show that the portal-scaffold interaction requires all but the first 18 and last 36 amino acids of pU_L6, as well as several tryptophan residues positioned throughout the portal protein.     

Use of Ichip for High-Throughput In Situ Cultivation of “Uncultivable” Microbial Species

One of the oldest unresolved microbiological phenomena is why only a small fraction of the diverse microbiological population grows on artificial media. The “uncultivable” microbial majority arguably represents our planet''s largest unexplored pool of biological and chemical novelty. Previously we showed that species from this pool could be grown inside diffusion chambers incubated in situ, likely because diffusion provides microorganisms with their naturally occurring growth factors. Here we utilize this approach and develop a novel high-throughput platform for parallel cultivation and isolation of previously uncultivated microbial species from a variety of environments. We have designed and tested an isolation chip (ichip) composed of several hundred miniature diffusion chambers, each inoculated with a single environmental cell. We show that microbial recovery in the ichip exceeds manyfold that afforded by standard cultivation, and the grown species are of significant phylogenetic novelty. The new method allows access to a large and diverse array of previously inaccessible microorganisms and is well suited for both fundamental and applied research.It has been known for over a century that the overwhelming majority of microbial species do not grow on synthetic media in vitro and remain unexplored (13, 32, 37, 39, 40, 43). The rRNA and metagenomics approaches demonstrated a spectacular diversity of these uncultivated species (11, 21, 25-27, 30, 36). Accessing this “missing” microbial diversity is of significant interest for both basic and applied sciences and has been recognized as one of the principal challenges for microbiology today (12, 29, 41). In recent years, technical advances in cultivation methodologies have recovered a diverse set of ecologically relevant species (1, 3, 5, 7, 15, 20, 24, 28, 33, 42). However, by and large the gap between microbial diversity in nature and that in culture collections remains unchanged, and most microbial phyla still have no cultivable representatives (25, 29). Earlier, we developed a novel method of in situ cultivation of environmental microorganisms inside diffusion chambers (15). The rationale for such an approach was that diffusion would provide cells inside the chamber with naturally occurring growth components and enable those species that grew in nature at the time of the experiment to also grow inside the diffusion chambers. Expectedly, this method yields a rate of microbial recovery many times larger than those of standard techniques. Even so, this method is laborious and does not allow an efficient, high-throughput isolation of microbial species en masse. This limits the method''s applicability, for example, in the drug discovery effort. Here we transform this methodology into a high-throughput technology platform for massively parallel cultivation of “uncultivable” species. Capitalizing on earlier microfluidics methods developed for microbial storage and screening (4, 16), we have designed and tested an isolation chip, or ichip for short, which consists of hundreds of miniature diffusion chambers. If each diffusion minichamber is loaded with a single cell, the resulting culture is monospecific. The ichip thus allows microbial growth and isolation into pure culture in one step. Here we demonstrate that cultivation of environmental microorganisms inside the ichip incubated in situ leads to a significantly increased colony count over that observed on synthetic media. Perhaps even more significantly, species grown in ichips are different from those registered in standard petri dishes and are highly novel.