Skip to main content
eScholarship
Open Access Publications from the University of California

Scholarly Works (87 results)

  • Article
Lawrence Berkeley National Laboratory (2010)

Microarray-based genomic technology has been widely used for microbial community analysis, and it is expected that microarray-based genomic technologies will revolutionize the analysis of microbial community structure, function and dynamics. A new generation of functional gene arrays (GeoChip 3.0) has been developed, with 27,812 probes covering 56,990 gene variants from 292 functional gene families involved in carbon, nitrogen, phosphorus and sulfur cycles, energy metabolism, antibiotic resistance, metal resistance, and organic contaminant degradation. Those probes were derived from 2,744, 140, and 262 species for bacteria, archaea, and fungi, respectively. GeoChip 3.0 has several other distinct features, such as a common oligo reference standard (CORS) for data normalization and comparison, a software package for data management and future updating, and the gyrB gene for phylogenetic analysis. Our computational evaluation of probe specificity indicated that all designed probes had a high specificity to their corresponding targets. Also, experimental analysis with synthesized oligonucleotides and genomic DNAs showed that only 0.0036percent-0.025percent false positive rates were observed, suggesting that the designed probes are highly specific under the experimental conditions examined. In addition, GeoChip 3.0 was applied to analyze soil microbial communities in a multifactor grassland ecosystem in Minnesota, USA, which demonstrated that the structure, composition, and potential activity of soil microbial communities significantly changed with the plant species diversity. All results indicate that GeoChip 3.0 is a high throughput powerful tool for studying microbial community functional structure, and linking microbial communities to ecosystem processes and functioning. To our knowledge, GeoChip 3.0 is the most comprehensive microarrays currently available for studying microbial communities associated with geobiochemical cycling, global climate change, bioenergy, agricuture, land use, ecosystem management, environmental cleanup and restoration, bioreactor systems, and human health.

    Cover page: GeoChip 3.0: A High Throughput Tool for Analyzing Microbial Community, Composition, Structure, and Functional Activity
    • Article
    Lawrence Berkeley National Laboratory (2008)

    Signal-to-noise-ratio (SNR) thresholds for microarray data analysis were experimentally determined with an oligonucleotide array that contained perfect match (PM) and mismatch (MM) probes based upon four genes from Shewanella oneidensis MR-1. A new SNR calculation, called signal to both standard deviations ratio (SSDR) was developed, and evaluated along with other two methods, signal to standard deviation ratio (SSR), and signal to background ratio (SBR). At a low stringency, the thresholds of SSR, SBR, and SSDR were 2.5, 1.60 and 0.80 with oligonucleotide and PCR amplicon as target templates, and 2.0, 1.60 and 0.70 with genomic DNA as target templates. Slightly higher thresholds were obtained at the high stringency condition. The thresholds of SSR and SSDR decreased with an increase in the complexity of targets (e.g., target types), and the presence of background DNA, and a decrease in the composition of targets, while SBR remained unchanged under all situations. The lowest percentage of false positives (FP) and false negatives (FN) was observed with the SSDR calculation method, suggesting that it may be a better SNR calculation for more accurate determination of SNR thresholds. Positive spots identified by SNR thresholds were verified by the Student t-test, and consistent results were observed. This study provides general guidance for users to select appropriate SNR thresholds for different samples under different hybridization conditions.

      Cover page: Empirical Evaluation of a New Method for Calculating Signal to Noise Ratio (SNR) for Microarray Data Analysis
      • Article
      LBL Publications (2010)

      The global atmospheric concentration of CO2 has increased by more than 30percent since the industrial revolution. Although the stimulating effects of elevated CO2 (eCO2) on plant growth and primary productivity have been well studied, its influences on belowground microbial communities are poorly understood and controversial. In this study, we showed a significant change in the structure and functional potential of soil microbial communities at eCO2 in a grassland ecosystem, the BioCON (Biodiversity, CO2 and Nitrogen) experimental site (http://www.biocon.umn.edu/) using a comprehensive functional gene array, GeoChip 3.0, which contains about 28,0000 probes and covers approximately 57,000 gene variants from 292 functional gene families involved in carbon, nitrogen, phosphorus and sulfur cycles as well as other functional processes. GeoChip data indicated that the functional structure of microbial communities was markedly different between ambient CO2 (aCO2) and eCO2 by detrended correspondence analysis (DCA) of all 5001 detected functional gene probes although no significant differences were detected in the overall microbial diversity. A further analysis of 1503 detected functional genes involved in C, N, P, and S cycles showed that a considerable portion (39percent) of them were only detected under either aCO2 (14percent) or eCO2 (25percent), indicating that the functional characteristics of the microbial community were significantly altered by eCO2. Also, for those shared genes (61percent) detected, some significantly (p<0.05) changed their abundance at eCO2. Especially, genes involved in labile C degradation, such as amyA, egl, and ara for starch, cellulose, and hemicelluloses, respectively, C fixation (e.g., rbcL, pcc/acc), N fixation (nifH), and phosphorus utilization (ppx) were significantly increased under eCO2, while those involved in decomposing recalcitrant C, such as glx, lip, and mnp for lignin degradation remained unchanged. This study provides insights into our understanding of belowground microbial communities and their feedbacks to terrestrial ecosystems at eCO2.

        Cover page: Alternations of Structure and Functional Activity of Below Ground Microbial Communities at Elevated Atmospheric Carbon Dioxide
        • Article
        LBL Publications (2009)
        The response of Desulfovibrio vulgaris Hildenborough to salt and H2O2 stresses were examined by physiological, global transcriptional, metabolite, and mutagenesis analyses. The growth of D. vulgaris was inhibited by 250 mM NaCl or 1 mM H2O2. Salt adaptation (long-term NaCl exposure) increased the expression of genes involved in amino acid biosynthesis and transport, electron transfer, hydrogen oxidation, and general stress responses (e.g., heat shock proteins, phage shock proteins, and oxidative stress response proteins). Genes involved in carbon metabolism, cell motility, and phage structures were decreased in expression. Comparison of transcriptomic profiles of D. vulgaris responses to salt adaptation with those of salt shock (short-term NaCl exposure) showed some similarity as well as a significant difference. Metabolite assays showed that glutamate and alanine accumulated under salt adaptation, suggesting that they may be used as osmoprotectants in D. vulgaris. Addition of amino acids (glutamate, alanine, tryptophan) or yeast extract to the growth medium relieved salt-related growth inhibition. A conceptual model is proposed to link the observed results to currently available knowledge for further understanding the mechanisms of D. vulgaris adaptation to elevated NaCl. Under H2O2 conditions, PerR regulon genes were significantly up-regulated, indicating the importance role of PerR in oxidative stress response. In addition, some Fur regulon genes were also strongly induced. Increased gene expression of thiol-peroxidase genes ahpC as well as thioredoxin reductase and thioredoxin genes indicated the involvement of thiol switch in the oxidative stress response. rbr2 was the only significantly up-regulated H2O2 scavenging enzymes. The oxidative stress response of mutants Delta perR and Delta fur demonstrated that ahpC and rbr2 were regulated by both Fur and PerR. The links between the up-regulated genes involved in H2O2 scavenging, protein fate, DNA metabolism and lipid metabolism and the down-regulated genes involved in sulfate reduction, energy production and translation were demonstrated by the gene co-expression network. The proteomics data provided further evidence at the translational level and complemented the transcriptomics data. Taken together, diverse stress resistance mechanisms may be used in D. vulgaris for detoxification of H2O2 with the up-regulation of DNA repair systems and the down-regulation of energy metabolism and protein synthesis.
          Cover page: Desulfovibrio vulgaris Hildenborough responses to salt and H2O2 stresses
          • Article
          LBL Publications (2008)
          Many environmental sites (e.g., groundwater, seawater, contaminated sites) contain low microbial biomass, which can make it difficult to effectively analyze these samples using genomic techniques requiring large amounts of DNA. Functional gene microarrays, for example, require microgram quantities of DNA. Whole community genome amplification (WCGA) amplifies the entire community in a sample using random priming. As such, the use of WCGA would be beneficial for samples containing low concentrations of DNA or that would be difficult to resample. Two DNA polymerases are known to catalyze this reaction: phi29 and Bst. Phi29 has been previously shown to be effective for amplifying microbial community DNA with a low amplification bias. This study evaluated the use of Bst for WCGA. Initial experiments compared the amplification of Desulfovibrio vulgaris using phi29 and Bst. An aliquot of DNA (10 ng) was amplified using random heptamers containing two additional 5-nitroindole residues on the 5' end and a phosphorothioate linkage on the 3' end. Amplified DNA (1 mu g) and the same amount of unamplified DNA were labeled with Cy5-dUTP and Cy3-dUTP, respectively. The unamplified DNA was pooled and then co-hybridized with the amplified DNA to a D. vulgaris microarray at 45 oC for 10 h. All hybridizations were done in triplicate. The signal-to-noise ratio (SNR) was calculated for the unamplified sample and all genes with SNR <3 in any of the triplicates was removed and the remaining genes were used for analysis. The average amplified to unamplified signal intensity was 1.02 (+0.005) and 1.05 (+0.005) for Bst and phi29, respectively. The representational bias (DjTotal) values were similar for both the Bst and phi29 amplifications (0.181 and 0.157, respectively). The percentage of genes whose ratios were > 1.5, 2, 3, and 4 were 12.56, 2.79, 0.15, and 0.03percent, respectively, for Bst and 12.22, 3.08, 0.23, and 0.03percent, respectively, for phi29. These results suggest that Bst would be a viable substitute for phi29. Further experiments are underway to compare the amplification of other pure culture DNA and examine microbial community DNA amplification.
            Cover page: Use of Bst DNA polymerase for whole genome amplification
            • 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 (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
                LBL Publications (2003)
                  Cover page: Transcriptional Analysis of Responses of Desulfovibrio vulgaris to NaCl Stress Using Whole-Genome Oligonucleotide Microarrays
                  • Article
                  • Peer Reviewed
                  UC Berkeley Previously Published Works (2015)
                  Understanding the structure, functions, activities and dynamics of microbial communities in natural environments is one of the grand challenges of 21st century science. To address this challenge, over the past decade, numerous technologies have been developed for interrogating microbial communities, of which some are amenable to exploratory work (e.g., high-throughput sequencing and phenotypic screening) and others depend on reference genes or genomes (e.g., phylogenetic and functional gene arrays). Here, we provide a critical review and synthesis of the most commonly applied "open-format" and "closed-format" detection technologies. We discuss their characteristics, advantages, and disadvantages within the context of environmental applications and focus on analysis of complex microbial systems, such as those in soils, in which diversity is high and reference genomes are few. In addition, we discuss crucial issues and considerations associated with applying complementary high-throughput molecular technologies to address important ecological questions.
                    • Article
                    • Peer Reviewed
                    LBL Publications (2017)
                    Sediment microbial fuel cells (SMFCs) can stimulate the degradation of polycyclic aromatic hydrocarbons in sediments, but the mechanism of this process is poorly understood at the microbial functional gene level. Here, the use of SMFC resulted in 92% benzo[a]pyrene (BaP) removal over 970 days relative to 54% in the controls. Sediment functions, microbial community structure, and network interactions were dramatically altered by the SMFC employment. Functional gene analysis showed that c-type cytochrome genes for electron transfer, aromatic degradation genes, and extracellular ligninolytic enzymes involved in lignin degradation were significantly enriched in bulk sediments during SMFC operation. Correspondingly, chemical analysis of the system showed that these genetic changes resulted in increases in the levels of easily oxidizable organic carbon and humic acids which may have resulted in increased BaP bioavailability and increased degradation rates. Tracking microbial functional genes and corresponding organic matter responses should aid mechanistic understanding of BaP enhanced biodegradation by microbial electrochemistry and development of sustainable bioremediation strategies.
                      Cover page: Interconnection of Key Microbial Functional Genes for Enhanced Benzo[a]pyrene Biodegradation in Sediments by Microbial Electrochemistry
                      Top