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Open Access Publications from the University of California

Scholarly Works (83 results)

  • Article
LBL Publications (2009)

Functional gene arrays (FGA) are microarrays that contain probes for genes encoding proteins or enzymes involved in functions of interest and allow for the study of thousands of genes at one time. The most comprehensive FGA to date is the GeoChip, which contains ~;;24,000 probes for ~;;10,000 genes involved in the geochemical cycling of C, N, P, and S, as well as genes involved in metal resistance and reduction and contaminant degradation. This chapter details the methods necessary for GeoChip analysis. Methods covered include preparation of DNA (whole community genome amplification and labeling), array setup (prehybridization steps), hybridization (sample and hybridization buffers), and post hybridization steps (slide washing and array scanning).

    Cover page: GeoChips for Analysis of Microbial Functional Communities
    • Article
    LBL Publications (2010)

    Increasing nitrogen deposition, increasing atmospheric CO2, and decreasing biodiversity are three main environmental changes occurring on a global scale. The BioCON (Biodiversity, CO2, and Nitrogen) ecological experiment site at the University of Minnesota's Cedar Creek Ecosystem Science Reserve started in 1997, to better understand how these changes would affect soil systems. To understand how increasing nitrogen deposition affects the microbial community diversity, heterogeneity, and functional structure impact soil microbial communities, 12 samples were collected from the BioCON plots in which nitrogenous fertilizer was added to simulate the effect of increasing nitrogen deposition and 12 samples from without added fertilizer. DNA from the 24 samples was extracted using a freeze-grind protocol, amplified, labeled with a fluorescent dye, and then hybridized to GeoChip, a functional gene array containing probes for genes involved in N, S and C cycling, metal resistance and organic contaminant degradation. Detrended correspondence analysis (DCA) of all genes detected was performed to analyze microbial community patterns. The first two axes accounted for 23.5percent of the total variation. The samples fell into two major groups: fertilized and non-fertilized, suggesting that nitrogenous fertilizer had a significant impact on soil microbial community structure and diversity. The functional gene numbers detected in fertilized samples was less that detected in non-fertilizer samples. Functional genes involving in the N cycling were mainly discussed.

      Cover page: Effects of Nitrate Exposure on the Functional Structure of a Microbial Community in a Uranium-contaminated Aquifer
      • Article
      LBL Publications (2009)

      GeoChip is a comprehensive functional gene array that targets key functional genes involved in the geochemical cycling of N, C, and P, sulfate reduction, metal resistance and reduction, and contaminant degradation. Studies have shown the GeoChip to be a sensitive, specific, and high-throughput tool for microbial community analysis that has the power to link geochemical processes with microbial community structure. However, several challenges remain regarding the development and applications of microarrays for microbial community analysis.

        Cover page: Geochip: A high throughput genomic tool for linking community structure to functions
        • Article
        • Peer Reviewed
        LBL Publications (2018)
        Increased amount of nano-silver will be released into domestic and industrial waste streams due to its extensive application. However, great controversy still exists on the effects of silver nanoparticle (Ag-NP) on biological wastewater treatment processes and a toxicology model has not been built yet. Four sequencing batch reactors with activated sludge has been run for over three months with different silver species at a concentration of 1mg Ag/L in influent. Both freshly prepared Ag-NPs and aged Ag-NPs were tested with released silver ion as control. Results in this study showed that Ag-NPs, especially freshly prepared Ag-NPs, can help to maintain or even increase the diversity of microbial community in activated sludge and the biomass concentration even under long-term treatment. It indicates that the hormesis model need to be considered for the toxicology of Ag-NPs.
          Cover page: Contradictory effects of silver nanoparticles on activated sludge wastewater treatment
          • Article
          • Peer Reviewed
          LBL Publications (2017)
          Essential gene functions remain largely underexplored in bacteria. Clostridium cellulolyticum is a promising candidate for consolidated bioprocessing; however, its genetic manipulation to reduce the formation of less-valuable acetate is technically challenging due to the essentiality of acetate-producing genes. Here we developed a Cas9 nickase-assisted chromosome-based RNA repression to stably manipulate essential genes in C. cellulolyticum. Our plasmid-based expression of antisense RNA (asRNA) molecules targeting the phosphotransacetylase (pta) gene successfully reduced the enzymatic activity by 35% in cellobiose-grown cells, metabolically decreased the acetate titer by 15 and 52% in wildtype transformants on cellulose and xylan, respectively. To control both acetate and lactate simultaneously, we transformed the repression plasmid into lactate production-deficient mutant and found the plasmid delivery reduced acetate titer by more than 33%, concomitant with negligible lactate formation. The strains with pta gene repression generally diverted more carbon into ethanol. However, further testing on chromosomal integrants that were created by double-crossover recombination exhibited only very weak repression because DNA integration dramatically lessened gene dosage. With the design of a tandem repetitive promoter-driven asRNA module and the use of a new Cas9 nickase genome editing tool, a chromosomal integrant (LM3P) was generated in a single step and successfully enhanced RNA repression, with a 27% decrease in acetate titer on cellulose in antibiotic-free medium. These results indicate the effectiveness of tandem promoter-driven RNA repression modules in promoting gene repression in chromosomal integrants. Our combinatorial method using a Cas9 nickase genome editing tool to integrate the gene repression module demonstrates easy-to-use and high-efficiency advantages, paving the way for stably manipulating genes, even essential ones, for functional characterization and microbial engineering.
            • Article
            • Peer Reviewed
            LBL Publications (2016)
            The coral holobiont often resides in oligotrophic waters; both coral cells and their symbiotic dinoflagellates possess ammonium assimilation enzymes and potentially benefit from the nitrogen fixation of coral-associated diazotrophs. However, the seasonal dynamics of coral-associated diazotrophs are not well characterized. Here, the seasonal variations of diazotrophic communities associated with three corals, Galaxea astreata, Pavona decussata, and Porites lutea, were studied using nifH gene amplicon pyrosequencing techniques. Our results revealed a great diversity of coral-associated diazotrophs. nifH sequences related to Alphaproteobacteria, Deltaproteobacteria, and Gammaproteobacteria were ubiquitous and dominant in all corals in two seasons. In contrast with the coral P. decussata, both G. astreata and P. lutea showed significant seasonal changes in the diazotrophic communities and nifH gene abundance. Variable diazotroph groups accounted for a range from 11 to 49% within individual coral samples. Most of the variable diazotrophic groups from P. decussata were species-specific, however, the majority of overlapping variable groups in G. astreata and P. lutea showed the same seasonal variation characteristics. Rhodopseudomonas palustris- and Gluconacetobacter diazotrophicus-affiliated sequences were relatively abundant in the summer, whereas a nifH sequence related to Halorhodospira halophila was relatively abundant in spring G. astreata and P. lutea. The seasonal variations of all diazotrophic communities were significantly correlated with the seasonal shifts of ammonium and nitrate, suggesting that diazotrophs play an important role in the nitrogen cycle of the coral holobiont.
              • Article
              • Peer Reviewed
              LBL Publications (2017)
              Microbes play crucial roles in various biogeochemical processes in the ocean, including carbon (C), nitrogen (N), and phosphorus (P) cycling. Functional gene diversity and the structure of the microbial community determines its metabolic potential and therefore its ecological function in the marine ecosystem. However, little is known about the functional gene composition and metabolic potential of bacterioplankton in estuary areas. The East China Sea (ECS) is a dynamic marginal ecosystem in the western Pacific Ocean that is mainly affected by input from the Changjiang River and the Kuroshio Current. Here, using a high-throughput functional gene microarray (GeoChip), we analyzed the functional gene diversity, composition, structure, and metabolic potential of microbial assemblages in different ECS water masses. Four water masses determined by temperature and salinity relationship showed different patterns of functional gene diversity and composition. Generally, functional gene diversity [Shannon-Weaner's H and reciprocal of Simpson's 1/(1-D)] in the surface water masses was higher than that in the bottom water masses. The different presence and proportion of functional genes involved in C, N, and P cycling among the bacteria of the different water masses showed different metabolic preferences of the microbial populations in the ECS. Genes involved in starch metabolism (amyA and nplT) showed higher proportion in microbial communities of the surface water masses than of the bottom water masses. In contrast, a higher proportion of genes involved in chitin degradation was observed in microorganisms of the bottom water masses. Moreover, we found a higher proportion of nitrogen fixation (nifH), transformation of hydroxylamine to nitrite (hao) and ammonification (gdh) genes in the microbial communities of the bottom water masses compared with those of the surface water masses. The spatial variation of microbial functional genes was significantly correlated with salinity, temperature, and chlorophyll based on canonical correspondence analysis, suggesting a significant influence of hydrologic conditions on water microbial communities. Our data provide new insights into better understanding of the functional potential of microbial communities in the complex estuarine-coastal environmental gradient of the ECS.
                • Article
                • Peer Reviewed
                LBL Publications (2017)
                Diazotrophic communities make an essential contribution to the productivity through providing new nitrogen. However, knowledge of the roles that both mangrove tree species and geochemical parameters play in shaping mangove rhizosphere diazotrophic communities is still elusive. Here, a comprehensive examination of the diversity and structure of microbial communities in the rhizospheres of three mangrove species, Rhizophora apiculata, Avicennia marina, and Ceriops tagal, was undertaken using high-throughput sequencing of the 16S rRNA and nifH genes. Our results revealed a great diversity of both the total microbial composition and the diazotrophic composition specifically in the mangrove rhizosphere. Deltaproteobacteria and Gammaproteobacteria were both ubiquitous and dominant, comprising an average of 45.87 and 86.66% of total microbial and diazotrophic communities, respectively. Sulfate-reducing bacteria belonging to the Desulfobacteraceae and Desulfovibrionaceae were the dominant diazotrophs. Community statistical analyses suggested that both mangrove tree species and additional environmental variables played important roles in shaping total microbial and potential diazotroph communities in mangrove rhizospheres. In contrast to the total microbial community investigated by analysis of 16S rRNA gene sequences, most of the dominant diazotrophic groups identified by nifH gene sequences were significantly different among mangrove species. The dominant diazotrophs of the family Desulfobacteraceae were positively correlated with total phosphorus, but negatively correlated with the nitrogen to phosphorus ratio. The Pseudomonadaceae were positively correlated with the concentration of available potassium, suggesting that diazotrophs potentially play an important role in biogeochemical cycles, such as those of nitrogen, phosphorus, sulfur, and potassium, in the mangrove ecosystem.
                  • Article
                  • Peer Reviewed
                  LBL Publications (2015)
                  Patterns of variation in plant and animal diversity along precipitation gradients have been extensively studied, but much less is known about how and to what extent precipitation affects the biogeographic distribution of microbial diversity in arid areas across large spatial scales. Here we collected soils from 54 sites along a 3700 km transect covering a wide range of grassland ecosystems with distinct aridity gradients. We quantified the bacterial community diversity and the effects of climate, edaphic parameter and geographic distance on the bacterial community structure using high-throughput 16S rRNA gene sequencing. Of the 35 phyla detected, 6 were dominant: Actinobacteria, Acidobacteria, Alphaproteobacteria, Deltaproteobacteria, Bacteroidetes and Planctomycetes. Aridity was a major factor influencing bacterial diversity, community composition and taxon abundance. Although the pattern of bacterial species richness is markedly different from that of plant species richness, most soil bacteria were endemic to particular bioregions like macro-organisms. Community similarity significantly declined with environmental distance and geographic distance (r = -0.579 and -0.773, respectively). Geographic distance (historical contingencies) contributed more to bacterial community variation (36.02%) than combined environmental factors (24.06%). Overall, our results showed that geographic distance and climatic factors concurrently govern bacterial biogeographic patterns in arid and semi-arid grassland.
                    Cover page: Scale-dependent effects of climate and geographic distance on bacterial diversity patterns across northern China's grasslands
                    • Article
                    • Peer Reviewed
                    LBL Publications (2018)
                    Trace elements (TEs) availability, biochemical activity and functional gene diversity was studied in a Cu-contaminated soil, revegetated for six years with a mixed stand of willow, black poplar, and false indigo-bush, and amended or not with compost plus dolomitic limestone (OMDL). The OMDL amendment significantly reduced Cu and As availability and soil toxicity, and increased the biochemical activity and microbial functional diversity assessed with the GEOCHIP technique, as compared to the unamended soil (Unt). The OMDL soil showed significantly higher abundance of 25 functional genes involved in decomposition organic compounds, and 11, 3 and 11 functional genes involved in the N, P and S biogeochemical cycles. Functional gene abundance was positively correlated with nutrient contents but negatively correlated with Cu availability and soil toxicity. The abundance of microbial functional genes encoding for resistance to various TEs also increased, possibly due to the microbial proliferation and lower Cu exposure in the presence of high total soil Cu concentration. Genes encoding for antibiotic resistance due to the co-occurrence of TEs and antibiotic resistant genes on genetic mobile elements. Overall, phytomanagement confirmed its potential to restore the biological fertility and diversity of a severely Cu-contaminated soil, but the increase of TEs and antibiotic resistant gene abundances deserve attention in future studies.
                      Cover page: Functional activity and functional gene diversity of a Cu-contaminated soil remediated by aided phytostabilization using compost, dolomitic limestone and a mixed tree stand
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