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1.  Deciphering the role of Paenibacillus strain Q8 in the organic matter recycling in the acid mine drainage of Carnoulès 
Background
The recycling of the organic matter is a crucial function in any environment, especially in oligotrophic environments such as Acid Mine Drainages (AMDs). Polymer-degrading bacteria might play an important role in such ecosystem, at least by releasing by-products useful for the rest of the community. In this study, physiological, molecular and biochemical experiments were performed to decipher the role of a Paenibacillus strain isolated from the sediment of Carnoulès AMD.
Results
Even though Paenibacillus sp. strain Q8 was isolated from an oligotrophic AMD showing an acidic pH, it developed under both acidic and alkaline conditions and showed a heterotrophic metabolism based on the utilization of a broad range of organic compounds. It resisted to numerous metallic stresses, particularly high arsenite (As(III)) concentrations (> 1,800 mg/L). Q8 was also able to efficiently degrade polymers such as cellulose, xylan and starch. Function-based screening of a Q8 DNA-library allowed the detection of 15 clones with starch-degrading activity and 3 clones with xylan-degrading activity. One clone positive for starch degradation carried a single gene encoding a "protein of unknown function". Amylolytic and xylanolytic activities were measured both in growing cells and with acellular extracts of Q8. The results showed the ability of Q8 to degrade both polymers under a broad pH range and high As(III) and As(V) concentrations. Activity measurements allowed to point out the constitutive expression of the amylase genes and the mainly inducible expression of the xylanase genes. PACE demonstrated the endo-acting activity of the amylases and the exo-acting activity of the xylanases.
Conclusions
AMDs have been studied for years especially with regard to interactions between bacteria and the inorganic compartment hosting them. To date, no study reported the role of microorganisms in the recycling of the organic matter. The present work suggests that the strain Q8 might play an important role in the community by recycling the scarce organic matter (cellulose, hemicellulose, starch...), especially when the conditions change. Furthermore, function-based screening of a Q8 DNA library allowed to assign an amylolytic function to a gene previously unknown. AMDs could be considered as a reservoir of genes with potential biotechnological properties.
doi:10.1186/1475-2859-11-16
PMCID: PMC3287962  PMID: 22305268
Paenibacillus; Functional redundancy; Acid Mine Drainage (AMD); Amylase; Xylanase; Polymer degradation; Organic matter; Function-based screening; Community function
2.  Molecular analysis of the diversity of vaginal microbiota associated with bacterial vaginosis 
BMC Genomics  2010;11:488.
Background
Bacterial vaginosis (BV) is an ecological disorder of the vaginal microbiota that affects millions of women annually, and is associated with numerous adverse health outcomes including pre-term birth and the acquisition of sexually transmitted infections. However, little is known about the overall structure and composition of vaginal microbial communities; most of the earlier studies focused on predominant vaginal bacteria in the process of BV. In the present study, the diversity and richness of vaginal microbiota in 50 BV positive and 50 healthy women from China were investigated using culture-independent PCR-denaturing gradient gel electrophoresis (DGGE) and barcoded 454 pyrosequencing methods, and validated by quantitative PCR.
Results
Our data demonstrated that there was a profound shift in the absolute and relative abundances of bacterial species present in the vagina when comparing populations associated with healthy and diseased conditions. In spite of significant interpersonal variations, the diversity of vaginal microbiota in the two groups could be clearly divided into two clusters. A total of 246,359 high quality pyrosequencing reads was obtained for evaluating bacterial diversity and 24,298 unique sequences represented all phylotypes. The most predominant phyla of bacteria identified in the vagina belonged to Firmicutes, Bacteroidetes, Actinobacteria and Fusobacteria. The higher number of phylotypes in BV positive women over healthy is consistent with the results of previous studies and a large number of low-abundance taxa which were missed in previous studies were revealed. Although no single bacterium could be identified as a specific marker for healthy over diseased conditions, three phyla - Bacteroidetes, Actinobacteria and Fusobacteria, and eight genera including Gardnerella, Atopobium, Megasphaera, Eggerthella, Aerococcus, Leptotrichia/Sneathia, Prevotella and Papillibacter were strongly associated with BV (p < 0.05). These genera are potentially excellent markers and could be used as targets for clinical BV diagnosis by molecular approaches.
Conclusions
The data presented here have clearly profiled the overall structure of vaginal communities and clearly demonstrated that BV is associated with a dramatic increase in the taxonomic richness and diversity of vaginal microbiota. The study also provides the most comprehensive picture of the vaginal community structure and the bacterial ecosystem, and significantly contributes to the current understanding of the etiology of BV.
doi:10.1186/1471-2164-11-488
PMCID: PMC2996984  PMID: 20819230
3.  Nested PCR and New Primers for Analysis of Sulfate-Reducing Bacteria in Low-Cell-Biomass Environments▿ †  
New primers were designed for the amplification of dsrAB genes by nested PCR to investigate the diversity of sulfate-reducing prokaryotes (SRP) in environments with low bacterial cell density. The success of the nested PCR for the determination of SRP diversity was estimated by terminal-restriction fragment length polymorphism analysis in the Reigous, a small creek at an inactive mine (Carnoulès, France), which constitutes an extreme acidic arsenic-rich environment. Nested PCR limits were evaluated in dsrAB-rich sediments, and this technique was compared to direct PCR using either known primers (DSR1F/DSR4R) or new primers (dsr619AF/dsr1905BR). The comparison of clone libraries revealed that, even if the levels of diversity observed were not identical, nested PCR did not reduce the diversity compared to that of direct DSR1F/DSR4R PCR. Clone sequences were affiliated mainly with the Desulfobacteraceae and Desulfohalobiaceae families. Many sequences (∼30%) were related to a deeply branching lineage unaffiliated with any cultured SRP. Although this dsrAB cluster was found in all libraries, the new primers better amplified this lineage, providing more information on this unknown bacterial group. Thanks to these new primers in nested PCR, the SRP community from Carnoulès could be characterized. Specific SRP populations were obtained according to environmental characteristics. Desulfomicrobiaceae-related sequences were recovered in samples with low pH, low levels of dissolved oxygen, and high As content, while sequences belonging to the deeply branching group were found in a less extreme sample. Furthermore, for the first time, dsrAB sequences related to the latter group were recovered from freshwater.
doi:10.1128/AEM.02023-09
PMCID: PMC2863462  PMID: 20228118
4.  Cellulolytic potential under environmental changes in microbial communities from grassland litter 
In many ecosystems, global changes are likely to profoundly affect microorganisms. In Southern California, changes in precipitation and nitrogen deposition may influence the composition and functional potential of microbial communities and their resulting ability to degrade plant material. To test whether such environmental changes impact the distribution of functional groups involved in leaf litter degradation, we determined how the genomic diversity of microbial communities in a semi-arid grassland ecosystem changed under reduced precipitation or increased N deposition. We monitored communities seasonally over a period of 2 years to place environmental change responses into the context of natural variation. Fungal and bacterial communities displayed strong seasonal patterns, Fungi being mostly detected during the dry season whereas Bacteria were common during wet periods. Most putative cellulose degraders were associated with 33 bacterial genera and predicted to constitute 18% of the microbial community. Precipitation reduction reduced bacterial abundance and cellulolytic potential whereas nitrogen addition did not affect the cellulolytic potential of the microbial community. Finally, we detected a strong correlation between the frequencies of genera of putative cellulose degraders and cellulase genes. Thus, microbial taxonomic composition was predictive of cellulolytic potential. This work provides a framework for how environmental changes affect microorganisms responsible for plant litter deconstruction.
doi:10.3389/fmicb.2014.00639
PMCID: PMC4243572  PMID: 25505459
cellulase; metagenomics; leaf litter; global change; microbial community composition
5.  Comparative genomics in acid mine drainage biofilm communities reveals metabolic and structural differentiation of co-occurring archaea 
BMC Genomics  2013;14:485.
Background
Metal sulfide mineral dissolution during bioleaching and acid mine drainage (AMD) formation creates an environment that is inhospitable to most life. Despite dominance by a small number of bacteria, AMD microbial biofilm communities contain a notable variety of coexisting and closely related Euryarchaea, most of which have defied cultivation efforts. For this reason, we used metagenomics to analyze variation in gene content that may contribute to niche differentiation among co-occurring AMD archaea. Our analyses targeted members of the Thermoplasmatales and related archaea. These results greatly expand genomic information available for this archaeal order.
Results
We reconstructed near-complete genomes for uncultivated, relatively low abundance organisms A-, E-, and Gplasma, members of Thermoplasmatales order, and for a novel organism, Iplasma. Genomic analyses of these organisms, as well as Ferroplasma type I and II, reveal that all are facultative aerobic heterotrophs with the ability to use many of the same carbon substrates, including methanol. Most of the genomes share genes for toxic metal resistance and surface-layer production. Only Aplasma and Eplasma have a full suite of flagellar genes whereas all but the Ferroplasma spp. have genes for pili production. Cryogenic-electron microscopy (cryo-EM) and tomography (cryo-ET) strengthen these metagenomics-based ultrastructural predictions. Notably, only Aplasma, Gplasma and the Ferroplasma spp. have predicted iron oxidation genes and Eplasma and Iplasma lack most genes for cobalamin, valine, (iso)leucine and histidine synthesis.
Conclusion
The Thermoplasmatales AMD archaea share a large number of metabolic capabilities. All of the uncultivated organisms studied here (A-, E-, G-, and Iplasma) are metabolically very similar to characterized Ferroplasma spp., differentiating themselves mainly in their genetic capabilities for biosynthesis, motility, and possibly iron oxidation. These results indicate that subtle, but important genomic differences, coupled with unknown differences in gene expression, distinguish these organisms enough to allow for co-existence. Overall this study reveals shared features of organisms from the Thermoplasmatales lineage and provides new insights into the functioning of AMD communities.
doi:10.1186/1471-2164-14-485
PMCID: PMC3750248  PMID: 23865623
Metagenomics; Acid mine drainage; Thermoplasmatales; Ferroplasma; Iron oxidation; Comparative genomics
6.  Polymicrobial Nature of Chronic Diabetic Foot Ulcer Biofilm Infections Determined Using Bacterial Tag Encoded FLX Amplicon Pyrosequencing (bTEFAP) 
PLoS ONE  2008;3(10):e3326.
Background
Diabetic extremity ulcers are associated with chronic infections. Such ulcer infections are too often followed by amputation because there is little or no understanding of the ecology of such infections or how to control or eliminate this type of chronic infection. A primary impediment to the healing of chronic wounds is biofilm phenotype infections. Diabetic foot ulcers are the most common, disabling, and costly complications of diabetes. Here we seek to derive a better understanding of the polymicrobial nature of chronic diabetic extremity ulcer infections.
Methods and Findings
Using a new bacterial tag encoded FLX amplicon pyrosequencing (bTEFAP) approach we have evaluated the bacterial diversity of 40 chronic diabetic foot ulcers from different patients. The most prevalent bacterial genus associated with diabetic chronic wounds was Corynebacterium spp. Findings also show that obligate anaerobes including Bacteroides, Peptoniphilus, Fingoldia, Anaerococcus, and Peptostreptococcus spp. are ubiquitous in diabetic ulcers, comprising a significant portion of the wound biofilm communities. Other major components of the bacterial communities included commonly cultured genera such as Streptococcus, Serratia, Staphylococcus and Enterococcus spp.
Conclusions
In this article, we highlight the patterns of population diversity observed in the samples and introduce preliminary evidence to support the concept of functional equivalent pathogroups (FEP). Here we introduce FEP as consortia of genotypically distinct bacteria that symbiotically produce a pathogenic community. According to this hypothesis, individual members of these communities when they occur alone may not cause disease but when they coaggregate or consort together into a FEP the synergistic effect provides the functional equivalence of well-known pathogens, such as Staphylococcus aureus, giving the biofilm community the factors necessary to maintain chronic biofilm infections. Further work is definitely warranted and needed in order to prove whether the FEPs concept is a viable hypothesis. The findings here also suggest that traditional culturing methods may be extremely biased as a diagnostic tool as they select for easily cultured organisms such as Staphylococcus aureus and against difficult to culture bacteria such as anaerobes. While PCR methods also have bias, further work is now needed in comparing traditional culture results to high-resolution molecular diagnostic methods such as bTEFAP.
doi:10.1371/journal.pone.0003326
PMCID: PMC2556099  PMID: 18833331
7.  Species Interactions Alter Evolutionary Responses to a Novel Environment 
PLoS Biology  2012;10(5):e1001330.
Adaptation to a novel environment is altered by the presence of co-occurring species. Species in diverse communities evolved complementary resource use, which altered the functioning of the experimental ecosystems.
Studies of evolutionary responses to novel environments typically consider single species or perhaps pairs of interacting species. However, all organisms co-occur with many other species, resulting in evolutionary dynamics that might not match those predicted using single species approaches. Recent theories predict that species interactions in diverse systems can influence how component species evolve in response to environmental change. In turn, evolution might have consequences for ecosystem functioning. We used experimental communities of five bacterial species to show that species interactions have a major impact on adaptation to a novel environment in the laboratory. Species in communities diverged in their use of resources compared with the same species in monocultures and evolved to use waste products generated by other species. This generally led to a trade-off between adaptation to the abiotic and biotic components of the environment, such that species evolving in communities had lower growth rates when assayed in the absence of other species. Based on growth assays and on nuclear magnetic resonance (NMR) spectroscopy of resource use, all species evolved more in communities than they did in monocultures. The evolutionary changes had significant repercussions for the functioning of these experimental ecosystems: communities reassembled from isolates that had evolved in polyculture were more productive than those reassembled from isolates that had evolved in monoculture. Our results show that the way in which species adapt to new environments depends critically on the biotic environment of co-occurring species. Moreover, predicting how functioning of complex ecosystems will respond to an environmental change requires knowing how species interactions will evolve.
Author Summary
Understanding how species adapt to new environments is important both for evolutionary theory and for predicting and managing ecosystem responses to changing environments. However, most research into adaptation to new environments has considered species in isolation. Whether results from these systems apply to more realistically diverse groups of species remains unclear. We exposed five species of bacteria, collected from pools around the roots of beech trees, to a novel laboratory environment either in isolation or in species mixtures for approximately 70 generations. We found that each species evolved more in diverse species mixtures than it did when cultured in isolation. Moreover, species diverged in their use of resources and how they used the waste products of other species. These changes meant that the community of bacteria that evolved together used more of the available resources and were thereby more productive than the same group of species that evolved in isolation. Our findings show that species interactions can have a major effect on evolutionary dynamics, which can in turn influence ecosystem functioning.
doi:10.1371/journal.pbio.1001330
PMCID: PMC3352820  PMID: 22615541
8.  Macrophages Inhibit Neovascularization in a Murine Model of Age-Related Macular Degeneration 
PLoS Medicine  2006;3(8):e310.
Background
Age-related macular degeneration (AMD) is the leading cause of blindness in people over 50 y of age in at least three continents. Choroidal neovascularization (CNV) is the process by which abnormal blood vessels develop underneath the retina. CNV develops in 10% of patients with AMD but accounts for up to 90% of the blindness from AMD.
Although the precise etiology of CNV in AMD remains unknown, the macrophage component of the inflammatory response, which has been shown to promote tumor growth and support atherosclerotic plaque formation, is thought to stimulate aberrant angiogenesis in blinding eye diseases. The current theory is that macrophage infiltration promotes the development of neovascularization in CNV.
Methods and Findings
We examined the role of macrophages in a mouse model of CNV. IL-10−/− mice, which have increased inflammation in response to diverse stimuli, have significantly reduced CNV with increased macrophage infiltrates compared to wild type. Prevention of macrophage entry into the eye promoted neovascularization while direct injection of macrophages significantly inhibited CNV. Inhibition by macrophages was mediated by the TNF family death molecule Fas ligand (CD95-ligand).
Conclusions
Immune vascular interactions can be highly complex. Normal macrophage function is critical in controlling pathologic neovascularization in the eye. IL-10 regulates macrophage activity in the eye and is an attractive therapeutic target in order to suppress or inhibit CNV in AMD that can otherwise lead to blindness.
Apte and colleagues examined the role of macrophages in a mouse model of choroidal neovascularization, and showed that normal macrophage function is critical in controlling pathologic neovascularization in the eye.
Editors' Summary
Background.
The most common cause of poor eyesight in later life in the developed world is known as age-related macular degeneration (AMD). The macula is the central part of the retina (the film-like membrane at the back of the eye), which is the most sensitive and important for sharp central vision. There are two types of advanced AMD: so-called wet, or neovascular, AMD (neovascular means “new vessel”) and dry, or geographic atrophy, AMD (atrophy means “to waste away”). Wet AMD occurs when abnormal, fragile new blood vessels grow under the macula behind the retina. These blood vessels often leak blood and fluid, which lift the macula. Dry AMD occurs as the light-sensitive cells in the macula (the rods and cones) break down. To study this disease further, researchers use animal models. One such animal model is made by using a laser to damage the back of the eye in a mouse, which causes the formation of new vessels. Various treatments can then be tested to see if they have any effect on the damage.
Why Was This Study Done?
One theory about AMD is that the immune system may be involved in determining how severe the damage at the back of the eye is, and how much new vessel formation occurs. The researchers wanted to look at the effect of the immune system on AMD, in particular, the effect of one type of cell called a macrophage, and a substance, IL-10, that is secreted from bone marrow cells and that affects how these macrophages work.
What Did the Researchers Do and Find?
The researchers used a mouse strain in which IL-10 was absent, induced damage in the eyes that mimicked AMD, and then looked at what role macrophages had in the eye abnormalities. They found that in the eyes of mice that lacked IL-10, there was reduced new vessel formation and increased numbers of macrophages compared to mice that had normal amounts of IL-10. Also, preventing macrophages from getting into the eyes of such mice by injecting IL-10 into the eyes made the new vessel formation worse, while direct injection of macrophages made it better.
What Do These Findings Mean?
Although animal models cannot completely replicate disease in humans, they can give us an idea of how diseases might come about and suggest possible treatment strategies. It is possible that inhibiting the effect of IL-10, or other strategies that make macrophages more efficient in the eye, may be a useful treatment for AMD. In a related Perspective (DOI: 10.1371/journal.pmed.0030364), Susan Lightman and Virginia Calder discuss the findings further, including suggesting new experiments that will need to be done as a next step.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0030310.
MedlinePlus encyclopedia entry on macular degeneration
National Institutes of Health Senior Health page of information on AMD
National Eye Institute AMD fact page
doi:10.1371/journal.pmed.0030310
PMCID: PMC1539093  PMID: 16903779
9.  Microbial Diversity in Anaerobic Sediments at Río Tinto, a Naturally Acidic Environment with a High Heavy Metal Content▿† 
Applied and Environmental Microbiology  2011;77(17):6085-6093.
The Tinto River is an extreme environment located at the core of the Iberian Pyritic Belt (IPB). It is an unusual ecosystem due to its size (100 km long), constant acidic pH (mean pH, 2.3), and high concentration of heavy metals, iron, and sulfate in its waters, characteristics that make the Tinto River Basin comparable to acidic mine drainage (AMD) systems. In this paper we present an extensive survey of the Tinto River sediment microbiota using two culture-independent approaches: denaturing gradient gel electrophoresis and cloning of 16S rRNA genes. The taxonomic affiliation of the Bacteria showed a high degree of biodiversity, falling into 5 different phyla: Proteobacteria, Firmicutes, Bacteroidetes, Acidobacteria, and Actinobacteria; meanwhile, all the Archaea were affiliated with the order Thermoplasmatales. Microorganisms involved in the iron (Acidithiobacillus ferrooxidans, Sulfobacillus spp., Ferroplasma spp., etc.), sulfur (Desulfurella spp., Desulfosporosinus spp., Thermodesulfobium spp., etc.), and carbon (Acidiphilium spp., Bacillus spp., Clostridium spp., Acidobacterium spp., etc.) cycles were identified, and their distribution was correlated with physicochemical parameters of the sediments. Ferric iron was the main electron acceptor for the oxidation of organic matter in the most acid and oxidizing layers, so acidophilic facultative Fe(III)-reducing bacteria appeared widely in the clone libraries. With increasing pH, the solubility of iron decreases and sulfate-reducing bacteria become dominant, with the ecological role of methanogens being insignificant. Considering the identified microorganisms—which, according to the rarefaction curves and Good's coverage values, cover almost all of the diversity—and their corresponding metabolism, we suggest a model of the iron, sulfur, and organic matter cycles in AMD-related sediments.
doi:10.1128/AEM.00654-11
PMCID: PMC3165421  PMID: 21724883
10.  GeoChip-Based Analysis of the Functional Gene Diversity and Metabolic Potential of Microbial Communities in Acid Mine Drainage▿ †  
Acid mine drainage (AMD) is an extreme environment, usually with low pH and high concentrations of metals. Although the phylogenetic diversity of AMD microbial communities has been examined extensively, little is known about their functional gene diversity and metabolic potential. In this study, a comprehensive functional gene array (GeoChip 2.0) was used to analyze the functional diversity, composition, structure, and metabolic potential of AMD microbial communities from three copper mines in China. GeoChip data indicated that these microbial communities were functionally diverse as measured by the number of genes detected, gene overlapping, unique genes, and various diversity indices. Almost all key functional gene categories targeted by GeoChip 2.0 were detected in the AMD microbial communities, including carbon fixation, carbon degradation, methane generation, nitrogen fixation, nitrification, denitrification, ammonification, nitrogen reduction, sulfur metabolism, metal resistance, and organic contaminant degradation, which suggested that the functional gene diversity was higher than was previously thought. Mantel test results indicated that AMD microbial communities are shaped largely by surrounding environmental factors (e.g., S, Mg, and Cu). Functional genes (e.g., narG and norB) and several key functional processes (e.g., methane generation, ammonification, denitrification, sulfite reduction, and organic contaminant degradation) were significantly (P < 0.10) correlated with environmental variables. This study presents an overview of functional gene diversity and the structure of AMD microbial communities and also provides insights into our understanding of metabolic potential in AMD ecosystems.
doi:10.1128/AEM.01798-10
PMCID: PMC3028740  PMID: 21097602
11.  Phenotypic characterization and 16S rDNA identification of culturable non-obligate halophilic bacterial communities from a hypersaline lake, La Sal del Rey, in extreme South Texas (USA) 
Aquatic Biosystems  2012;8:5.
Background
La Sal del Rey ("the King's Salt") is one of several naturally-occurring salt lakes in Hidalgo County, Texas and is part of the Lower Rio Grande Valley National Wildlife Refuge. The research objective was to isolate and characterize halophilic microorganisms from La Sal del Rey. Water samples were collected from the lake and a small creek that feeds into the lake. Soil samples were collected from land adjacent to the water sample locations. Sample salinity was determined using a refractometer. Samples were diluted and cultured on a synthetic saline medium to grow halophilic bacteria. The density of halophiles was estimated by viable plate counts. A collection of isolates was selected, gram-stained, tested for catalase, and characterized using API 20E® test strips. Isolates were putatively identified by sequencing the 16S rDNA. Carbon source utilization by the microbial community from each sample site was examined using EcoPlate™ assays and the carbon utilization total activity of the community was determined.
Results
Results showed that salinity ranged from 4 parts per thousand (ppt) at the lake water source to 420 ppt in water samples taken just along the lake shore. The density of halophilic bacteria in water samples ranged from 1.2 × 102 - 5.2 × 103 colony forming units per ml (cfu ml-1) whereas the density in soil samples ranged from 4.0 × 105 - 2.5 × 106 colony forming units per gram (cfu g-1). In general, as salinity increased the density of the bacterial community decreased. Microbial communities from water and soil samples were able to utilize 12 - 31 carbon substrates. The greatest number of substrates utilized was by water-borne communities compared to soil-based communities, especially at lower salinities. The majority of bacteria isolated were gram-negative, catalase-positive, rods. Biochemical profiles constructed from API 20E® test strips showed that bacterial isolates from low-salinity water samples (4 ppt) showed the greatest phenotypic diversity with regards to the types and number of positive tests from the strip. Isolates taken from water samples at the highest salinity (420 ppt) tended to be less diverse and have only a limited number of positive tests. Sequencing of 16S DNA displayed the presence of members of bacterial genera Bacillus, Halomonas, Pseudomonas, Exiguobacterium and others. The genus Bacillus was most commonly identified. None of the isolates were members of the Archaea probably due to dilution of salts in the samples.
Conclusions
The La Sal del Rey ecosystem supports a robust and diverse bacterial community despite the high salinity of the lake and soil. However, salinity does appear to a limiting factor with regards to the density and diversity of the bacterial communities that inhabit the lake and surrounding area.
doi:10.1186/2046-9063-8-5
PMCID: PMC3310331  PMID: 22480362
12.  Convergent Bacterial Microbiotas in the Fungal Agricultural Systems of Insects 
mBio  2014;5(6):e02077-14.
ABSTRACT
The ability to cultivate food is an innovation that has produced some of the most successful ecological strategies on the planet. Although most well recognized in humans, where agriculture represents a defining feature of civilization, species of ants, beetles, and termites have also independently evolved symbioses with fungi that they cultivate for food. Despite occurring across divergent insect and fungal lineages, the fungivorous niches of these insects are remarkably similar, indicating convergent evolution toward this successful ecological strategy. Here, we characterize the microbiota of ants, beetles, and termites engaged in nutritional symbioses with fungi to define the bacterial groups associated with these prominent herbivores and forest pests. Using culture-independent techniques and the in silico reconstruction of 37 composite genomes of dominant community members, we demonstrate that different insect-fungal symbioses that collectively shape ecosystems worldwide have highly similar bacterial microbiotas comprised primarily of the genera Enterobacter, Rahnella, and Pseudomonas. Although these symbioses span three orders of insects and two phyla of fungi, we show that they are associated with bacteria sharing high whole-genome nucleotide identity. Due to the fine-scale correspondence of the bacterial microbiotas of insects engaged in fungal symbioses, our findings indicate that this represents an example of convergence of entire host-microbe complexes.
IMPORTANCE
The cultivation of fungi for food is a behavior that has evolved independently in ants, beetles, and termites and has enabled many species of these insects to become ecologically important and widely distributed herbivores and forest pests. Although the primary fungal cultivars of these insects have been studied for decades, comparatively little is known of their bacterial microbiota. In this study, we show that diverse fungus-growing insects are associated with a common bacterial community composed of the same dominant members. Furthermore, by demonstrating that many of these bacteria have high whole-genome similarity across distantly related insect hosts that reside thousands of miles apart, we show that these bacteria are an important and underappreciated feature of diverse fungus-growing insects. Because of the similarities in the agricultural lifestyles of these insects, this is an example of convergence between both the life histories of the host insects and their symbiotic microbiota.
doi:10.1128/mBio.02077-14
PMCID: PMC4251994  PMID: 25406380
13.  In situ proteo-metabolomics reveals metabolite secretion by the acid mine drainage bio-indicator, Euglena mutabilis 
The ISME Journal  2012;6(7):1391-1402.
Euglena mutabilis is a photosynthetic protist found in acidic aquatic environments such as peat bogs, volcanic lakes and acid mine drainages (AMDs). Through its photosynthetic metabolism, this protist is supposed to have an important role in primary production in such oligotrophic ecosystems. Nevertheless, the exact contribution of E. mutabilis in organic matter synthesis remains unclear and no evidence of metabolite secretion by this protist has been established so far. Here we combined in situ proteo-metabolomic approaches to determine the nature of the metabolites accumulated by this protist or potentially secreted into an AMD. Our results revealed that the secreted metabolites are represented by a large number of amino acids, polyamine compounds, urea and some sugars but no fatty acids, suggesting a selective organic matter contribution in this ecosystem. Such a production may have a crucial impact on the bacterial community present on the study site, as it has been suggested previously that prokaryotes transport and recycle in situ most of the metabolites secreted by E. mutabilis. Consequently, this protist may have an indirect but important role in AMD ecosystems but also in other ecological niches often described as nitrogen-limited.
doi:10.1038/ismej.2011.198
PMCID: PMC3379634  PMID: 22237547
proteomics; metabolomics; Euglena mutabilis; primary production; AMD
14.  Comparative molecular analysis of chemolithoautotrophic bacterial diversity and community structure from coastal saline soils, Gujarat, India 
BMC Microbiology  2012;12:150.
Background
Soils harbour high diversity of obligate as well as facultative chemolithoautotrophic bacteria that contribute significantly to CO2 dynamics in soil. In this study, we used culture dependent and independent methods to assess the community structure and diversity of chemolithoautotrophs in agricultural and coastal barren saline soils (low and high salinity). We studied the composition and distribution of chemolithoautotrophs by means of functional marker gene cbbL encoding large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase and a phylogenetic marker 16S rRNA gene. The cbbL form IA and IC genes associated with carbon fixation were analyzed to gain insight into metabolic potential of chemolithoautotrophs in three soil types of coastal ecosystems which had a very different salt load and sulphur content.
Results
In cbbL libraries, the cbbL form IA was retrieved only from high saline soil whereas form IC was found in all three soil types. The form IC cbbL was also amplified from bacterial isolates obtained from all soil types. A number of novel monophyletic lineages affiliated with form IA and IC phylogenetic trees were found. These were distantly related to the known cbbL sequences from agroecosystem, volcanic ashes and marine environments. In 16S rRNA clone libraries, the agricultural soil was dominated by chemolithoautotrophs (Betaproteobacteria) whereas photoautotrophic Chloroflexi and sulphide oxidizers dominated saline ecosystems. Environmental specificity was apparently visible at both higher taxonomic levels (phylum) and lower taxonomic levels (genus and species). The differentiation in community structure and diversity in three soil ecosystems was supported by LIBSHUFF (P = 0.001) and UniFrac.
Conclusion
This study may provide fundamentally new insights into the role of chemolithoautotrophic and photoautotrophic bacterial diversity in biochemical carbon cycling in barren saline soils. The bacterial communities varied greatly among the three sites, probably because of differences in salinity, carbon and sulphur contents. The cbbL form IA-containing sulphide-oxidizing chemolithotrophs were found only in high saline soil clone library, thus giving the indication of sulphide availability in this soil ecosystem. This is the first comparative study of the community structure and diversity of chemolithoautotrophic bacteria in coastal agricultural and saline barren soils using functional (cbbL) and phylogenetic (16S rDNA) marker genes.
doi:10.1186/1471-2180-12-150
PMCID: PMC3438102  PMID: 22834535
cbbL; Clone libraries; 16S rRNA gene; RuBisCO; Barren soil
15.  Study of inter- and intra-individual variations in the salivary microbiota 
BMC Genomics  2010;11:523.
Background
Oral bacterial communities contain species that promote health and others that have been implicated in oral and/or systemic diseases. Culture-independent approaches provide the best means to assess the diversity of oral bacteria because most of them remain uncultivable.
Results
The salivary microbiota from five adults was analyzed at three time-points by means of the 454 pyrosequencing technology. The V1-V3 region of the bacterial 16S rRNA genes was amplified by PCR using saliva lysates and broad-range primers. The bar-coded PCR products were pooled and sequenced unidirectionally to cover the V3 hypervariable region. Of 50,708 obtained sequences, 31,860 passed the quality control. Non-bacterial sequences (2.2%) were removed leaving 31,170 reads. Samples were dominated by seven major phyla: members of Firmicutes, Proteobacteria, Actinobacteria, Bacteroidetes and candidate division TM7 were identified in all samples; Fusobacteria and Spirochaetes were identified in all individuals, but not at all time-points. The dataset was represented by 3,011 distinct sequences (100%-ID phylotypes) of ~215 nucleotides and 583 phylotypes defined at ≥97% identity (97%-ID phylotypes). We compared saliva samples from different individuals in terms of the phylogeny of their microbial communities. Based on the presence and absence of phylotypes defined at 100% or 97% identity thresholds, samples from each subject formed separate clusters. Among individual taxa, phylum Bacteroidetes and order Clostridiales (Firmicutes) were the best indicators of intraindividual similarity of the salivary flora over time. Fifteen out of 81 genera constituted 73 to 94% of the total sequences present in different samples. Of these, 8 were shared by all time points of all individuals, while 15-25 genera were present in all three time-points of different individuals. Representatives of the class Sphingobacteria, order Sphingobacteriales and family Clostridiaceae were found only in one subject.
Conclusions
The salivary microbial community appeared to be stable over at least 5 days, allowing for subject-specific grouping using UniFrac. Inclusion of all available samples from more distant time points (up to 29 days) confirmed this observation. Samples taken at closer time intervals were not necessarily more similar than samples obtained across longer sampling times. These results point to the persistence of subject-specific taxa whose frequency fluctuates between the time points. Genus Gemella, identified in all time-points of all individuals, was not defined as a core-microbiome genus in previous studies of salivary bacterial communities. Human oral microbiome studies are still in their infancy and larger-scale projects are required to better define individual and universal oral microbiome core.
doi:10.1186/1471-2164-11-523
PMCID: PMC2997015  PMID: 20920195
16.  Prevalence of Age-Related Macular Degeneration in Nakuru, Kenya: A Cross-Sectional Population-Based Study 
PLoS Medicine  2013;10(2):e1001393.
Using digital retinal photography and slit lamp examination in a population-based sample in the Nakuru District of Kenya, Andrew Bastawrous and colleagues determined the prevalence of age-related macular degeneration in adults 50 years and older.
Background
Diseases of the posterior segment of the eye, including age-related macular degeneration (AMD), have recently been recognised as the leading or second leading cause of blindness in several African countries. However, prevalence of AMD alone has not been assessed. We hypothesized that AMD is an important cause of visual impairment among elderly people in Nakuru, Kenya, and therefore sought to assess the prevalence and predictors of AMD in a diverse adult Kenyan population.
Methods and Findings
In a population-based cross-sectional survey in the Nakuru District of Kenya, 100 clusters of 50 people 50 y of age or older were selected by probability-proportional-to-size sampling between 26 January 2007 and 11 November 2008. Households within clusters were selected through compact segment sampling.
All participants underwent a standardised interview and comprehensive eye examination, including dilated slit lamp examination by an ophthalmologist and digital retinal photography. Images were graded for the presence and severity of AMD lesions following a modified version of the International Classification and Grading System for Age-Related Maculopathy. Comparison was made between slit lamp biomicroscopy (SLB) and photographic grading.
Of 4,381 participants, fundus photographs were gradable for 3,304 persons (75.4%), and SLB was completed for 4,312 (98%). Early and late AMD prevalence were 11.2% and 1.2%, respectively, among participants graded on images. Prevalence of AMD by SLB was 6.7% and 0.7% for early and late AMD, respectively. SLB underdiagnosed AMD relative to photographic grading by a factor of 1.7.
After controlling for age, women had a higher prevalence of early AMD than men (odds ratio 1.5; 95% CI, 1.2–1.9). Overall prevalence rose significantly with each decade of age. We estimate that, in Kenya, 283,900 to 362,800 people 50 y and older have early AMD and 25,200 to 50,500 have late AMD, based on population estimates in 2007.
Conclusions
AMD is an important cause of visual impairment and blindness in Kenya. Greater availability of low vision services and ophthalmologist training in diagnosis and treatment of AMD would be appropriate next steps.
Please see later in the article for the Editors' Summary
Editors' Summary
Background
Worldwide, 39 million people are blind, and 246 million people (mainly living in developing countries) have moderate or severe visual impairment. The third leading global cause of blindness (after cataracts and glaucoma) is age-related macular degeneration (AMD). This group of conditions is characterized by lesions in the macular (central) region of the retina, the tissue at the back of the eye that converts light into electrical messages and sends them to the brain. AMD, which affects older people, destroys the sharp central vision that is needed for reading or driving, leaving only dim, blurred images or a black hole at the center of vision. AMD can be diagnosed by examining digital photographs of the retina or by examining the retina directly using a special magnifying lens (slit lamp biomicroscopy). There is no cure for AMD, although injections into the eye of certain drugs, such as bevacizumab, that block the activity of vascular endothelial growth factor can slow the rate of vision loss caused by some forms of AMD.
Why Was This Study Done?
Most investigations of the prevalence (the proportion of a population with a disease) of AMD and of risk factors for AMD have studied people with European or Asian ancestry. Very little is known about AMD in African populations, and the data that are available mainly come from African populations living outside Africa. It is important to know whether AMD is an important cause of visual impairment and blindness in Africa, so that informed decisions can be made about the need for AMD programs in African countries. In this cross-sectional population-based study, the researchers investigate the prevalence of AMD among people aged 50 years or older living in Nakuru District (an ethnically diverse region of Kenya) and look for predictors of AMD in this population. In a cross-sectional population-based study, researchers observe a representative subset of a population at a single time point.
What Did the Researchers Do and Find?
The researchers randomly selected 100 clusters of 50 people aged 50 years or older for their study. Between January 2007 and November 2008, study participants had a comprehensive eye examination and completed a standardized interview that included questions about their age, gender, other demographic details, medical history, and exposure to possible risk factors for AMD. Based on digital retinal images, the prevalences of early and late AMD among the study population were 11.2% and 1.2%, respectively. The prevalences of early and late AMD judged by slit lamp biomicroscopy were 6.7% and 0.7%, respectively. After controlling for age, women had a higher prevalence of both early and late AMD than men. The overall prevalence of AMD rose with age: compared to the youngest age group, the oldest age group had a three-fold higher risk of developing late AMD. Of the people with any grade of AMD, 25.6% had some visual impairment and 2.5% were blind. Overall, 9.9% of the blindness seen in the study was attributable to AMD.
What Do These Findings Mean?
These findings identify AMD as an important cause of visual impairment and blindness in Nakuru District, Kenya. Extrapolation of these findings to the whole of Kenya suggests that 283,900 to 362,800 Kenyans had early AMD and 25,200 to 50,500 had late AMD in 2007. The accuracy of these findings is limited by the inability to obtain digital retinal images from all the participants (often because of electricity failures) and by other aspects of the study design. Moreover, because the methodology used in this study differed from some other studies of AMD, the prevalence of AMD reported here cannot be compared directly to those found in other studies. Nevertheless, these findings have several important implications. In particular, although recent evidence suggests that bevacizumab is likely to be both effective and affordable in Africa, the infrastructure required to deliver an adequate AMD service is currently prohibitively expensive in most African countries. Thus, these findings suggest that it is essential that research is undertaken to support the development of AMD treatment programs that are affordable and deliverable in Africa, and that low vision resources are provided for individuals with vision impairment.
Additional Information
Please access these websites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1001393.
The US National Eye Institute provides detailed information about age-related macular degeneration
The UK National Health Service Choices website also provides information about age-related macular degeneration, including personal stories about the condition
The UK Royal National Institute of Blind People has information on age-related macular degeneration, including a video of a person describing their experiences of the condition
AMD Alliance International provides written and audio information in several languages about age-related macular degeneration, including a large selection of personal stories; the Macular Degeneration Partnership also provides information about age-related macular degeneration, including a simulation of the condition
MedlinePlus has links to additional resources about age-related macular degeneration (in English and Spanish)
doi:10.1371/journal.pmed.1001393
PMCID: PMC3576379  PMID: 23431274
17.  Characterization of Epiphytic Bacterial Communities from Grapes, Leaves, Bark and Soil of Grapevine Plants Grown, and Their Relations 
PLoS ONE  2013;8(8):e73013.
Despite its importance in plant health and crop quality, the diversity of epiphytic bacteria on grape berries and other plant parts, like leaves and bark, remains poorly described, as does the role of telluric bacteria in plant colonization. In this study, we compare the bacterial community size and structure in vineyard soils, as well as on grapevine bark, leaves and berries. Analyses of culturable bacteria revealed differences in the size and structure of the populations in each ecosystem. The highest bacteria population counts and the greatest diversity of genera were found in soil samples, followed by bark, grapes and leaves. The identification of isolates revealed that some genera – Pseudomonas, Curtobacterium, and Bacillus – were present in all ecosystems, but in different amounts, while others were ecosystem-specific. About 50% of the genera were common to soil and bark, but absent from leaves and grapes. The opposite was also observed: grape and leaf samples presented 50% of genera in common that were absent from trunk and soil. The bacterial community structure analyzed by T-RFLP indicated similarities between the profiles of leaves and grapes, on the one hand, and bark and soil, on the other, reflecting the number of shared T-RFs. The results suggest an interaction between telluric bacterial communities and the epiphytic bacteria present on the different grapevine parts.
doi:10.1371/journal.pone.0073013
PMCID: PMC3758280  PMID: 24023666
18.  Identification of Cellulose-Responsive Bacterial and Fungal Communities in Geographically and Edaphically Different Soils by Using Stable Isotope Probing 
Many bacteria and fungi are known to degrade cellulose in culture, but their combined response to cellulose in different soils is unknown. Replicate soil microcosms amended with [13C]cellulose were used to identify bacterial and fungal communities responsive to cellulose in five geographically and edaphically different soils. The diversity and composition of the cellulose-responsive communities were assessed by DNA-stable isotope probing combined with Sanger sequencing of small-subunit and large-subunit rRNA genes for the bacterial and fungal communities, respectively. In each soil, the 13C-enriched, cellulose-responsive communities were of distinct composition compared to the original soil community or 12C-nonenriched communities. The composition of cellulose-responsive taxa, as identified by sequence operational taxonomic unit (OTU) similarity, differed in each soil. When OTUs were grouped at the bacterial order level, we found that members of the Burkholderiales, Caulobacteriales, Rhizobiales, Sphingobacteriales, Xanthomonadales, and the subdivision 1 Acidobacteria were prevalent in the 13C-enriched DNA in at least three of the soils. The cellulose-responsive fungi were identified as members of the Trichocladium, Chaetomium, Dactylaria, and Arthrobotrys genera, along with two novel Ascomycota clusters, unique to one soil. Although similarities were identified in higher-level taxa among some soils, the composition of cellulose-responsive bacteria and fungi was generally unique to a certain soil type, suggesting a strong potential influence of multiple edaphic factors in shaping the community.
doi:10.1128/AEM.07313-11
PMCID: PMC3302591  PMID: 22287013
19.  Insights into the Diversity of Eukaryotes in Acid Mine Drainage Biofilm Communities▿  
Microscopic eukaryotes are known to have important ecosystem functions, but their diversity in most environments remains vastly unexplored. Here we analyzed an 18S rRNA gene library from a subsurface iron- and sulfur-oxidizing microbial community growing in highly acidic (pH < 0.9) runoff within the Richmond Mine at Iron Mountain (northern California). Phylogenetic analysis revealed that the majority (68%) of the sequences belonged to fungi. Protists falling into the deeply branching lineage named the acidophilic protist clade (APC) and the class Heterolobosea were also present. The APC group represents kingdom-level novelty, with <76% sequence similarity to 18S rRNA gene sequences of organisms from other environments. Fluorescently labeled oligonucleotide rRNA probes were designed to target each of these groups in biofilm samples, enabling abundance and morphological characterization. Results revealed that the populations vary significantly with the habitat and no group is ubiquitous. Surprisingly, many of the eukaryotic lineages (with the exception of the APC) are closely related to neutrophiles, suggesting that they recently adapted to this extreme environment. Molecular analyses presented here confirm that the number of eukaryotic species associated with the acid mine drainage (AMD) communities is low. This finding is consistent with previous results showing a limited diversity of archaea, bacteria, and viruses in AMD environments and suggests that the environmental pressures and interplay between the members of these communities limit species diversity at all trophic levels.
doi:10.1128/AEM.02500-08
PMCID: PMC2663193  PMID: 19201960
20.  Metataxonomic profiling and prediction of functional behaviour of wheat straw degrading microbial consortia 
Background
Mixed microbial cultures, in which bacteria and fungi interact, have been proposed as an efficient way to deconstruct plant waste. The characterization of specific microbial consortia could be the starting point for novel biotechnological applications related to the efficient conversion of lignocellulose to cello-oligosaccharides, plastics and/or biofuels. Here, the diversity, composition and predicted functional profiles of novel bacterial-fungal consortia are reported, on the basis of replicated aerobic wheat straw enrichment cultures.
Results
In order to set up biodegradative microcosms, microbial communities were retrieved from a forest soil and introduced into a mineral salt medium containing 1% of (un)treated wheat straw. Following each incubation step, sequential transfers were carried out using 1 to 1,000 dilutions. The microbial source next to three sequential batch cultures (transfers 1, 3 and 10) were analyzed by bacterial 16S rRNA gene and fungal ITS1 pyrosequencing. Faith’s phylogenetic diversity values became progressively smaller from the inoculum to the sequential batch cultures. Moreover, increases in the relative abundances of Enterobacteriales, Pseudomonadales, Flavobacteriales and Sphingobacteriales were noted along the enrichment process. Operational taxonomic units affiliated with Acinetobacter johnsonii, Pseudomonas putida and Sphingobacterium faecium were abundant and the underlying strains were successfully isolated. Interestingly, Klebsiella variicola (OTU1062) was found to dominate in both consortia, whereas K. variicola-affiliated strains retrieved from untreated wheat straw consortia showed endoglucanase/xylanase activities. Among the fungal players with high biotechnological relevance, we recovered members of the genera Penicillium, Acremonium, Coniochaeta and Trichosporon. Remarkably, the presence of peroxidases, alpha-L-fucosidases, beta-xylosidases, beta-mannases and beta-glucosidases, involved in lignocellulose degradation, was indicated by predictive bacterial metagenome reconstruction. Reassuringly, tests for specific (hemi)cellulolytic enzymatic activities, performed on the consortial secretomes, confirmed the presence of such gene functions.
Conclusion
In an in-depth characterization of two wheat straw degrading microbial consortia, we revealed the enrichment and selection of specific bacterial and fungal taxa that were presumably involved in (hemi) cellulose degradation. Interestingly, the microbial community composition was strongly influenced by the wheat straw pretreatment. Finally, the functional bacterial-metagenome prediction and the evaluation of enzymatic activities (at the consortial secretomes) revealed the presence and enrichment of proteins involved in the deconstruction of plant biomass.
doi:10.1186/1754-6834-7-92
PMCID: PMC4064818  PMID: 24955113
Soil; Lignocellulose; Wheat straw; Microbial consortia; PICRUSt; Klebsiella; Trichosporon; alpha-L-fucosidases; beta-xylosidases
21.  Soil Bacterial Community Shifts after Chitin Enrichment: An Integrative Metagenomic Approach 
PLoS ONE  2013;8(11):e79699.
Chitin is the second most produced biopolymer on Earth after cellulose. Chitin degrading enzymes are promising but untapped sources for developing novel industrial biocatalysts. Hidden amongst uncultivated micro-organisms, new bacterial enzymes can be discovered and exploited by metagenomic approaches through extensive cloning and screening. Enrichment is also a well-known strategy, as it allows selection of organisms adapted to feed on a specific compound. In this study, we investigated how the soil bacterial community responded to chitin enrichment in a microcosm experiment. An integrative metagenomic approach coupling phylochips and high throughput shotgun pyrosequencing was established in order to assess the taxonomical and functional changes in the soil bacterial community. Results indicate that chitin enrichment leads to an increase of Actinobacteria, γ-proteobacteria and β-proteobacteria suggesting specific selection of chitin degrading bacteria belonging to these classes. Part of enriched bacterial genera were not yet reported to be involved in chitin degradation, like the members from the Micrococcineae sub-order (Actinobacteria). An increase of the observed bacterial diversity was noticed, with detection of specific genera only in chitin treated conditions. The relative proportion of metagenomic sequences related to chitin degradation was significantly increased, even if it represents only a tiny fraction of the sequence diversity found in a soil metagenome.
doi:10.1371/journal.pone.0079699
PMCID: PMC3835784  PMID: 24278158
22.  Selecting Microbial Strains from Pine Tree Resin: Biotechnological Applications from a Terpene World 
PLoS ONE  2014;9(6):e100740.
Resin is a chemical and physical defensive barrier secreted by many plants, especially coniferous trees, with insecticidal and antimicrobial properties. The degradation of terpenes, the main components accounting for the toxicity of resin, is highly relevant for a vast range of biotechnological processes, including bioremediation. In the present work, we used a resin-based selective medium in order to study the resin-tolerant microbial communities associated with the galls formed by the moth Retinia resinella; as well as resin from Pinus sylvestris forests, one of the largest ecosystems on Earth and a yet-unexplored source of terpene-degrading microorganisms. The taxonomic and functional diversity of the cultivated, resin-tolerant fraction of the whole microbiota were unveiled by high-throughput sequencing, which resulted in the detection of more than 40 bacterial genera among the terpene-degrading microorganisms, and a range of genes involved in the degradation of different terpene families. We further characterized through culture-based approaches and transcriptome sequencing selected microbial strains, including Pseudomonas sp., the most abundant species in both environmental resin and R. resinella resin-rich galls, and three fungal species, and experimentally confirmed their ability to degrade resin and also other terpene-based compounds and, thus, their potential use in biotechnological applications involving terpene catabolism.
doi:10.1371/journal.pone.0100740
PMCID: PMC4074100  PMID: 24971580
23.  Huanglongbing alters the structure and functional diversity of microbial communities associated with citrus rhizosphere 
The ISME Journal  2011;6(2):363-383.
The diversity and stability of bacterial communities present in the rhizosphere heavily influence soil and plant quality and ecosystem sustainability. The goal of this study is to understand how ‘Candidatus Liberibacter asiaticus' (known to cause Huanglongbing, HLB) influences the structure and functional potential of microbial communities associated with the citrus rhizosphere. Clone library sequencing and taxon/group-specific quantitative real-time PCR results showed that ‘Ca. L. asiaticus' infection restructured the native microbial community associated with citrus rhizosphere. Within the bacterial community, phylum Proteobacteria with various genera typically known as successful rhizosphere colonizers were significantly greater in clone libraries from healthy samples, whereas phylum Acidobacteria, Actinobacteria and Firmicutes, typically more dominant in the bulk soil were higher in ‘Ca. L. asiaticus'-infected samples. A comprehensive functional microarray GeoChip 3.0 was used to determine the effects of ‘Ca. L. asiaticus' infection on the functional diversity of rhizosphere microbial communities. GeoChip analysis showed that HLB disease has significant effects on various functional guilds of bacteria. Many genes involved in key ecological processes such as nitrogen cycling, carbon fixation, phosphorus utilization, metal homeostasis and resistance were significantly greater in healthy than in the ‘Ca. L. asiaticus'-infected citrus rhizosphere. Our results showed that the microbial community of the ‘Ca. L. asiaticus'-infected citrus rhizosphere has shifted away from using more easily degraded sources of carbon to the more recalcitrant forms. Overall, our study provides evidence that the change in plant physiology mediated by ‘Ca. L. asiaticus' infection could elicit shifts in the composition and functional potential of rhizosphere microbial communities. In the long term, these fluctuations might have important implications for the productivity and sustainability of citrus-producing agro-ecosystems.
doi:10.1038/ismej.2011.100
PMCID: PMC3260493  PMID: 21796220
Candidatus Liberibacter asiaticus; ecosystem functioning; GeoChip 3.0; huanglongbing; microbial diversity; nutrient cycling
24.  Core Gene Set As the Basis of Multilocus Sequence Analysis of the Subclass Actinobacteridae 
PLoS ONE  2011;6(3):e14792.
Comparative genomic sequencing is shedding new light on bacterial identification, taxonomy and phylogeny. An in silico assessment of a core gene set necessary for cellular functioning was made to determine a consensus set of genes that would be useful for the identification, taxonomy and phylogeny of the species belonging to the subclass Actinobacteridae which contained two orders Actinomycetales and Bifidobacteriales. The subclass Actinobacteridae comprised about 85% of the actinobacteria families. The following recommended criteria were used to establish a comprehensive gene set; the gene should (i) be long enough to contain phylogenetically useful information, (ii) not be subject to horizontal gene transfer, (iii) be a single copy (iv) have at least two regions sufficiently conserved that allow the design of amplification and sequencing primers and (v) predict whole-genome relationships. We applied these constraints to 50 different Actinobacteridae genomes and made 1,224 pairwise comparisons of the genome conserved regions and gene fragments obtained by using Sequence VARiability Analysis Program (SVARAP), which allow designing the primers. Following a comparative statistical modeling phase, 3 gene fragments were selected, ychF, rpoB, and secY with R2>0.85. Selected sets of broad range primers were tested from the 3 gene fragments and were demonstrated to be useful for amplification and sequencing of 25 species belonging to 9 genera of Actinobacteridae. The intraspecies similarities were 96.3–100% for ychF, 97.8–100% for rpoB and 96.9–100% for secY among 73 strains belonging to 15 species of the subclass Actinobacteridae compare to 99.4–100% for 16S rRNA. The phylogenetic topology obtained from the combined datasets ychF+rpoB+secY was globally similar to that inferred from the 16S rRNA but with higher confidence. It was concluded that multi-locus sequence analysis using core gene set might represent the first consensus and valid approach for investigating the bacterial identification, phylogeny and taxonomy.
doi:10.1371/journal.pone.0014792
PMCID: PMC3069002  PMID: 21483493
25.  Culture-independent analysis of the soil bacterial assemblage at the Great Salt Plains of Oklahoma 
Journal of basic microbiology  2011;52(1):16-26.
The Great Salt Plains (GSP) of Oklahoma is a natural inland terrestrial hypersaline environment that forms evaporite crusts of mainly NaCl. Previous work described GSP bacterial assemblages through the phylogenetic and phenetic characterization of 105 isolates from 46 phylotypes. The current report describes the same bacterial assemblages through culture-independent 16S rRNA gene clone libraries. Although from similar hypersaline mud flats, the bacterial libraries from two sites, WP3 and WP6, were quite different. The WP3 library was dominated by cyanobacteria, mainly Cyanothece and Euhalothece. The WP6 library was rich in anaerobic sulfur-cycle organisms, including abundant Desulfuromonas. This pattern likely reflects differences in abiotic factors, such as frequency of flooding and hydrologic push. While more than 100 OTUs were identified, the assemblages were not as diverse, based on Shannon indexes, as bacterial communities from oligohaline soils. Since natural inland hypersaline soils are relatively unstudied, it was not clear what kind of bacteria would be present. The bacterial assemblage is predominantly genera typically found in hypersaline systems, although some were relatives of microbes common in oligohaline and marine environments. The bacterial clones did not reflect wide functional diversity, beyond phototrophs, sulfur metabolizers, and numerous heterotrophs.
doi:10.1002/jobm.201100175
PMCID: PMC3597346  PMID: 21953014
Hypersaline; 16S rRNA; Community; Bacteria; Soil

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