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Chapter 11. Community analysis-based methods

Description: Microbial communities are each a composite of populations whose presence and relative abundance in water or other environmental samples are a direct manifestation of environmental conditions, including the introduction of microbe-rich fecal material and factors promoting persistence of the microbes therein. As shown by culture-independent methods, different animal-host fecal microbial communities appear distinctive, suggesting that their community profiles can be used to differentiate fecal samples and to potentially reveal the presence of host fecal material in environmental waters. Cross-comparisons of microbial communities from different hosts also reveal relative abundances of genetic groups that can be used to distinguish sources. In increasing order of their information richness, several community analysis methods hold promise for MST applications: phospholipid fatty acid (PLFA) analysis, denaturing gradient gel electrophoresis (DGGE), terminal restriction fragment length polymorphism (TRFLP), cloning/sequencing, and PhyloChip. Specific case studies involving TRFLP and PhyloChip approaches demonstrate the ability of community-based analyses of contaminated waters to confirm a diagnosis of water quality based on host-specific marker(s). The success of community-based MST for comprehensively confirming fecal sources relies extensively upon using appropriate multivariate statistical approaches. While community-based MST is still under evaluation and development as a primary diagnostic tool, results presented herein demonstrate its promise. Coupled with its inherently comprehensive ability to capture an unprecedented amount of microbiological data that is relevant to water quality, the tools for microbial community analysis are increasingly accessible, and community-based approaches have unparalleled potential for translation into rapid, perhaps real-time, monitoring platforms.
Date: May 1, 2010
Creator: Cao, Y.; Wu, C.H.; Andersen, G.L. & Holden, P.A.
Partner: UNT Libraries Government Documents Department

The use of microarrays in microbial ecology

Description: Microarrays have proven to be a useful and high-throughput method to provide targeted DNA sequence information for up to many thousands of specific genetic regions in a single test. A microarray consists of multiple DNA oligonucleotide probes that, under high stringency conditions, hybridize only to specific complementary nucleic acid sequences (targets). A fluorescent signal indicates the presence and, in many cases, the abundance of genetic regions of interest. In this chapter we will look at how microarrays are used in microbial ecology, especially with the recent increase in microbial community DNA sequence data. Of particular interest to microbial ecologists, phylogenetic microarrays are used for the analysis of phylotypes in a community and functional gene arrays are used for the analysis of functional genes, and, by inference, phylotypes in environmental samples. A phylogenetic microarray that has been developed by the Andersen laboratory, the PhyloChip, will be discussed as an example of a microarray that targets the known diversity within the 16S rRNA gene to determine microbial community composition. Using multiple, confirmatory probes to increase the confidence of detection and a mismatch probe for every perfect match probe to minimize the effect of cross-hybridization by non-target regions, the PhyloChip is able to simultaneously identify any of thousands of taxa present in an environmental sample. The PhyloChip is shown to reveal greater diversity within a community than rRNA gene sequencing due to the placement of the entire gene product on the microarray compared with the analysis of up to thousands of individual molecules by traditional sequencing methods. A functional gene array that has been developed by the Zhou laboratory, the GeoChip, will be discussed as an example of a microarray that dynamically identifies functional activities of multiple members within a community. The recent version of GeoChip contains more than 24,000 50mer oligonucleotide probes ...
Date: September 15, 2009
Creator: Andersen, G.L.; He, Z.; DeSantis, T.Z.; Brodie, E.L. & Zhou, J.
Partner: UNT Libraries Government Documents Department

Developing microbe-plant interactions for applications in plant-growth promotion and disease control, production of useful compounds, remediation, and carbon sequestration

Description: Interactions between plants and microbes are an integral part of our terrestrial ecosystem. Microbe-plant interactions are being applied in many areas. In this review, we present recent reports of applications in the areas of plant-growth promotion, biocontrol, bioactive compound and biomaterial production, remediation and carbon sequestration. Challenges, limitations and future outlook for each field are discussed.
Date: March 1, 2009
Creator: Wu, C. H.; Bernard, S.; Andersen, G.L. & Chen, W.
Partner: UNT Libraries Government Documents Department

Selective progressive response of soil microbial community to wild oat roots

Description: Roots moving through soil enact physical and chemical changes that differentiate rhizosphere from bulk soil, and the effects of these changes on soil microorganisms have long been a topic of interest. Use of a high-density 16S rRNA microarray (PhyloChip) for bacterial and archaeal community analysis has allowed definition of the populations that respond to the root within the complex grassland soil community; this research accompanies previously reported compositional changes, including increases in chitinase and protease specific activity, cell numbers and quorum sensing signal. PhyloChip results showed a significant change in 7% of the total rhizosphere microbial community (147 of 1917 taxa); the 7% response value was confirmed by16S rRNA T-RFLP analysis. This PhyloChip-defined dynamic subset was comprised of taxa in 17 of the 44 phyla detected in all soil samples. Expected rhizosphere-competent phyla, such as Proteobacteria and Firmicutes, were well represented, as were less-well-documented rhizosphere colonizers including Actinobacteria, Verrucomicrobia and Nitrospira. Richness of Bacteroidetes and Actinobacteria decreased in soil near the root tip compared to bulk soil, but then increased in older root zones. Quantitative PCR revealed {beta}-Proteobacteria and Actinobacteria present at about 10{sup 8} copies of 16S rRNA genes g{sup -1} soil, with Nitrospira having about 10{sup 5} copies g{sup -1} soil. This report demonstrates that changes in a relatively small subset of the soil microbial community are sufficient to produce substantial changes in function in progressively more mature rhizosphere zones.
Date: October 1, 2008
Creator: DeAngelis, K.M.; Brodie, E.L.; DeSantis, T.Z.; Andersen, G.L.; Lindow, S.E. & Firestone, M.K.
Partner: UNT Libraries Government Documents Department

Bacterial diversity analysis of Huanglongbing pathogen-infected citrus, using PhyloChip and 16S rRNA gene clone library sequencing

Description: The bacterial diversity associated with citrus leaf midribs was characterized 1 from citrus groves that contained the Huanglongbing (HLB) pathogen, which has yet to be cultivated in vitro. We employed a combination of high-density phylogenetic 16S rDNA microarray and 16S rDNA clone library sequencing to determine the microbial community composition of symptomatic and asymptomatic citrus midribs. Our results revealed that citrus leaf midribs can support a diversity of microbes. PhyloChip analysis indicated that 47 orders of bacteria from 15 phyla were present in the citrus leaf midribs while 20 orders from phyla were observed with the cloning and sequencing method. PhyloChip arrays indicated that nine taxa were significantly more abundant in symptomatic midribs compared to asymptomatic midribs. Candidatus Liberibacter asiaticus (Las) was detected at a very low level in asymptomatic plants, but was over 200 times more abundant in symptomatic plants. The PhyloChip analysis was further verified by sequencing 16S rDNA clone libraries, which indicated the dominance of Las in symptomatic leaves. These data implicate Las as the pathogen responsible for HLB disease. Citrus is the most important commercial fruit crop in Florida. In recent years, citrus Huanglongbing (HLB), also called citrus greening, has severely affected Florida's citrus production and hence has drawn an enormous amount of attention. HLB is one of the most devastating diseases of citrus (6,13), characterized by blotchy mottling with green islands on leaves, as well as stunting, fruit decline, and small, lopsided fruits with poor coloration. The disease tends to be associated with a phloem-limited fastidious {alpha}-proteobacterium given a provisional Candidatus status (Candidatus Liberobacter spp. later changed to Candidatus Liberibacter spp.) in nomenclature (18,25,34). Previous studies indicate that HLB infection causes disorder in the phloem and severely impairs the translocation of assimilates in host plants (5,27,40). Tatineni and colleagues discovered that the HLB bacteria were ...
Date: March 1, 2009
Creator: Shankar Sagaram, U.; DeAngelis, K.M.; Trivedi, P.; Andersen, G.L.; Lu, S.-E. & Wang, N.
Partner: UNT Libraries Government Documents Department

Greengenes: Chimera-checked 16S rRNA gene database and workbenchcompatible in ARB

Description: A 16S rRNA gene database (http://greengenes.lbl.gov) addresses limitations of public repositories by providing chimera-screening, standard alignments and taxonomic classification using multiple published taxonomies. It was revealed that incongruent taxonomic nomenclature exists among curators even at the phylum-level. Putative chimeras were identified in 3% of environmental sequences and 0.2% of records derived from isolates. Environmental sequences were classified into 100 phylum-level lineages within the Archaea and Bacteria.
Date: February 1, 2006
Creator: DeSantis, T.Z.; Hugenholtz, P.; Larsen, N.; Rojas, M.; Brodie,E.L; Keller, K. et al.
Partner: UNT Libraries Government Documents Department

Long term biosustainability in a high energy, low diversity crustal biome

Description: Geochemical, microbiological, and molecular analyses of alkaline saline groundwater at 2.8 kilometers depth in Archaean metabasalt revealed a microbial biome dominated by a single phylotype affiliated with thermophilic sulfate reducers belonging to Firmicutes. These sulfate reducers were sustained by geologically produced sulfate and hydrogen at concentrations sufficient to maintain activities for millions of years with no apparent reliance on photosynthetically derived substrates.
Date: October 1, 2006
Creator: Lin, L-H.; Wang, P-L.; Rumble, D.; Lippmann-Pipke, J.; SherwoodLollar, B.; Boice, E. et al.
Partner: UNT Libraries Government Documents Department

Characterization of coastal urban watershed bacterial communities leads to alternative community-based indicators

Description: Microbial communities in aquatic environments are spatially and temporally dynamic due to environmental fluctuations and varied external input sources. A large percentage of the urban watersheds in the United States are affected by fecal pollution, including human pathogens, thus warranting comprehensive monitoring. Using a high-density microarray (PhyloChip), we examined water column bacterial community DNA extracted from two connecting urban watersheds, elucidating variable and stable bacterial subpopulations over a 3-day period and community composition profiles that were distinct to fecal and non-fecal sources. Two approaches were used for indication of fecal influence. The first approach utilized similarity of 503 operational taxonomic units (OTUs) common to all fecal samples analyzed in this study with the watershed samples as an index of fecal pollution. A majority of the 503 OTUs were found in the phyla Firmicutes, Proteobacteria, Bacteroidetes, and Actinobacteria. The second approach incorporated relative richness of 4 bacterial classes (Bacilli, Bacteroidetes, Clostridia and a-proteobacteria) found to have the highest variance in fecal and non-fecal samples. The ratio of these 4 classes (BBC:A) from the watershed samples demonstrated a trend where bacterial communities from gut and sewage sources had higher ratios than from sources not impacted by fecal material. This trend was also observed in the 124 bacterial communities from previously published and unpublished sequencing or PhyloChip- analyzed studies. This study provided a detailed characterization of bacterial community variability during dry weather across a 3-day period in two urban watersheds. The comparative analysis of watershed community composition resulted in alternative community-based indicators that could be useful for assessing ecosystem health.
Date: March 1, 2010
Creator: Wu, C.H.; Sercu, B.; Van De Werhorst, L.C.; Wong, J.; DeSantis, T.Z.; Brodie, E.L. et al.
Partner: UNT Libraries Government Documents Department

PhyloChip microarray analysis reveals altered gastrointestinal microbial communities in a rat model of colonic hypersensitivity

Description: Irritable bowel syndrome (IBS) is a chronic, episodic gastrointestinal disorder that is prevalent in a significant fraction of western human populations; and changes in the microbiota of the large bowel have been implicated in the pathology of the disease. Using a novel comprehensive, high-density DNA microarray (PhyloChip) we performed a phylogenetic analysis of the microbial community of the large bowel in a rat model in which intracolonic acetic acid in neonates was used to induce long lasting colonic hypersensitivity and decreased stool water content and frequency, representing the equivalent of human constipation-predominant IBS. Our results revealed a significantly increased compositional difference in the microbial communities in rats with neonatal irritation as compared with controls. Even more striking was the dramatic change in the ratio of Firmicutes relative to Bacteroidetes, where neonatally irritated rats were enriched more with Bacteroidetes and also contained a different composition of species within this phylum. Our study also revealed differences at the level of bacterial families and species. The PhyloChip is a useful and convenient method to study enteric microflora. Further, this rat model system may be a useful experimental platform to study the causes and consequences of changes in microbial community composition associated with IBS.
Date: December 1, 2010
Creator: Nelson, T.A.; Holmes, S.; Alekseyenko, A.V.; Shenoy, M.; DeSantis, T.; Wu, C.H. et al.
Partner: UNT Libraries Government Documents Department

Simrank: Rapid and sensitive general-purpose k-mer search tool

Description: Terabyte-scale collections of string-encoded data are expected from consortia efforts such as the Human Microbiome Project (http://nihroadmap.nih.gov/hmp). Intra- and inter-project data similarity searches are enabled by rapid k-mer matching strategies. Software applications for sequence database partitioning, guide tree estimation, molecular classification and alignment acceleration have benefited from embedded k-mer searches as sub-routines. However, a rapid, general-purpose, open-source, flexible, stand-alone k-mer tool has not been available. Here we present a stand-alone utility, Simrank, which allows users to rapidly identify database strings the most similar to query strings. Performance testing of Simrank and related tools against DNA, RNA, protein and human-languages found Simrank 10X to 928X faster depending on the dataset. Simrank provides molecular ecologists with a high-throughput, open source choice for comparing large sequence sets to find similarity.
Date: April 1, 2011
Creator: DeSantis, T.Z.; Keller, K.; Karaoz, U.; Alekseyenko, A.V; Singh, N.N.S.; Brodie, E.L et al.
Partner: UNT Libraries Government Documents Department

Sustainable syntrophic growth of Dehalococcoides ethenogenes strain 195 with Desulfovibrio vulgaris Hildenborough and Methanobacterium congolense: Global transcriptomic and proteomic analyses

Description: Dehalococcoides ethenogenes strain 195 (DE195) was grown in a sustainable syntrophic association with Desulfovibrio vulgaris Hildenborough (DVH) as a co-culture, as well as with DVH and the hydrogenotrophic methanogen Methanobacterium congolense (MC) as a tri-culture using lactate as the sole energy and carbon source. In the co- and tri-cultures, maximum dechlorination rates of DE195 were enhanced by approximately three times (11.0±0.01 lmol per day for the co-culture and 10.1±0.3 lmol per day for the tri-culture) compared with DE195 grown alone (3.8±0.1 lmol per day). Cell yield of DE195 was enhanced in the co-culture (9.0±0.5 x 107 cells per lmol Cl{sup -} released, compared with 6.8±0.9x 107 cells per lmol Cl{sup -} released for the pure culture), whereas no further enhancement was observed in the tri-culture (7.3±1.8x 107 cells per lmol Cl{sup -} released). The transcriptome of DE195 grown in the co-culture was analyzed using a whole-genome microarray targeting DE195, which detected 102 significantly up- or down-regulated genes compared with DE195 grown in isolation, whereas no significant transcriptomic difference was observed between co- and tri-cultures. Proteomic analysis showed that 120 proteins were differentially expressed in the co-culture compared with DE195 grown in isolation. Physiological, transcriptomic and proteomic results indicate that the robust growth of DE195 in co- and tri-cultures is because of the advantages associated with the capabilities of DVH to ferment lactate to provide H2 and acetate for growth, along with potential benefits from proton translocation, cobalamin-salvaging and amino acid biosynthesis, whereas MC in the tri-culture provided no significant additional benefits beyond those of DVH.
Date: March 1, 2011
Creator: Men, Y.; Feil, H.; VerBerkmoes, N.C.; Shah, M.B.; Johnson, D.R.; Lee, P.K.H et al.
Partner: UNT Libraries Government Documents Department

Assessing the cleanliness of surfaces: Innovative molecular approaches vs. standard spore assays

Description: A bacterial spore assay and a molecular DNA microarray method were compared for their ability to assess relative cleanliness in the context of bacterial abundance and diversity on spacecraft surfaces. Colony counts derived from the NASA standard spore assay were extremely low for spacecraft surfaces. However, the PhyloChip generation 3 (G3) DNA microarray resolved the genetic signatures of a highly diverse suite of microorganisms in the very same sample set. Samples completely devoid of cultivable spores were shown to harbor the DNA of more than 100 distinct microbial phylotypes. Furthermore, samples with higher numbers of cultivable spores did not necessarily give rise to a greater microbial diversity upon analysis with the DNA microarray. The findings of this study clearly demonstrated that there is not a statistically significant correlation between the cultivable spore counts obtained from a sample and the degree of bacterial diversity present. Based on these results, it can be stated that validated state-of-the-art molecular techniques, such as DNA microarrays, can be utilized in parallel with classical culture-based methods to further describe the cleanliness of spacecraft surfaces.
Date: April 1, 2011
Creator: Cooper, M.; Duc, M.T. La; Probst, A.; Vaishampayan, P.; Stam, C.; Benardini, J.N. et al.
Partner: UNT Libraries Government Documents Department

Comparison of multiple ecogenomics methods for determining ecosystem function in uranium-contaminated environments

Description: Background: Bioremediation may offer the only feasiblestrategy for the nearly intractable problem of metal and radionuclidecontamination of soil and groundwater. To understand bioremediation incontaminated environments, it is critical to determine the organismspresent in these environments, analyze their responses to stressconditions, and elucidate functional position in the environment.Methods: We used multiple molecular techniques on both sediment andgroundwater to develop a better understanding of the functionalcapability and stress level within the microbial community inrelationship to over one hundred geochemical parameters. Due to the lowpH (3.5-4.5) and high contaminant levels (e.g., uranium) microbialdensities and activities were low. We used a phage polymeraseamplification system to construct large and small insert DNA libraries,performed metagenome sequencing, constructed clonal libraries of selectfunctional genes (SSU rRNA gene, nirK, nirS, amoA, pmoA, and dsrAB), useda SSU rDNA Phylochip microarray (9,000 taxa), and a functional gene array(23K genes). A complete comparison for community differences andsimilarities between the different techniques was assessed using severalbioinformatics techniques. Results: SSU rDNA analysis revealed thepresence of distinct bacterial phyla, including proteobacteria,acidobacteria, and planctomycetes along the contaminant gradient.Metagenome analysis identified many of the same organisms, and diversitywas lower in water than sediment. Analysis with functional gene arrays,phylochip, and specific probes for genes and organisms involved inbiogeochemical cycling of C, N, and S, metal resistance, stress response,and contaminant degradation suggested that the dominant species could bebiostimulated during in situ uranium reduction. Several other findings ofdifference and similarities between methods are presented. Conclusion:These systems biology field studies could be enabling for strategies toattenuate nletal and radionuclide contamination.
Date: January 10, 2007
Creator: Hazen, T.C.; Dehal, P.; Arkin, A.P.; Fields, M.W.; Keller, M.; Zhou, J. et al.
Partner: UNT Libraries Government Documents Department

Deep-sea oil plume enriches psychrophilic oil-degrading bacteria

Description: The biological effects and expected fate of the vast amount of oil in the Gulf of Mexico from the Deepwater Horizon blowout are unknown owing to the depth and magnitude of this event. Here, we report that the dispersed hydrocarbon plume stimulated deep-sea indigenous {gamma}-Proteobacteria that are closely related to known petroleum degraders. Hydrocarbon-degrading genes coincided with the concentration of various oil contaminants. Changes in hydrocarbon composition with distance from the source and incubation experiments with environmental isolates demonstrated faster-than-expected hydrocarbon biodegradation rates at 5 C. Based on these results, the potential exists for intrinsic bioremediation of the oil plume in the deep-water column without substantial oxygen drawdown.
Date: September 1, 2010
Creator: Hazen, T.C.; Dubinsky, E.A.; DeSantis, T.Z.; Andersen, G.L.; Piceno, Y.M.; Singh, N. et al.
Partner: UNT Libraries Government Documents Department