Xiangzhen Li

Analysis of 16S rRNA Amplicon Sequencing Options on the Roche/454 Next-Generation Titanium Sequencing Platform

Background 16S rRNA gene pyrosequencing approach has revolutionized studies in microbial ecology. While primer selection and short read length can affect the resulting microbial community profile, little is known about the influence of pyrosequencing methods on the sequencing throughput and the outcome of microbial community analyses. The aim of this study is to compare differences in output, ease, and cost among three different amplicon pyrosequencing methods for the Roche/454 Titanium platform Methodology/Principal Findings The following three pyrosequencing methods for 16S rRNA genes were selected in this study: Method-1 (standard method) is the recommended method for bi-directional sequencing using the LIB-A kit; Method-2 is a new option designed in this study for unidirectional sequencing with the LIB-A kit; and Method-3 uses the LIB-L kit for unidirectional sequencing. In our comparison among these three methods using 10 different environmental samples, Method-2 and Method-3 produced 1.5–1.6 times more useable reads than the standard method (Method-1), after quality-based trimming, and did not compromise the outcome of microbial community analyses. Specifically, Method-3 is the most cost-effective unidirectional amplicon sequencing method as it provided the most reads and required the least effort in consumables management. Conclusions Our findings clearly demonstrated that alternative pyrosequencing methods for 16S rRNA genes could drastically affect sequencing output (e.g. number of reads before and after trimming) but have little effect on the outcomes of microbial community analysis. This finding is important for both researchers and sequencing facilities utilizing 16S rRNA gene pyrosequencing for microbial ecological studies.

Responses of the functional structure of soil microbial community to livestock grazing in the Tibetan alpine grassland

Microbes play key roles in various biogeochemical processes, including carbon (C) and nitrogen (N) cycling. However, changes of microbial community at the functional gene level by livestock grazing, which is a global land-use activity, remain unclear. Here we use a functional gene array, GeoChip 4.0, to examine the effects of free livestock grazing on the microbial community at an experimental site of Tibet, a region known to be very sensitive to anthropogenic perturbation and global warming. Our results showed that grazing changed microbial community functional structure, in addition to aboveground vegetation and soil geochemical properties. Further statistical tests showed that microbial community functional structures were closely correlated with environmental variables, and variations in microbial community functional structures were mainly controlled by aboveground vegetation, soil C/N ratio, and NH4 (+) -N. In-depth examination of N cycling genes showed that abundances of N mineralization and nitrification genes were increased at grazed sites, but denitrification and N-reduction genes were decreased, suggesting that functional potentials of relevant bioprocesses were changed. Meanwhile, abundances of genes involved in methane cycling, C fixation, and degradation were decreased, which might be caused by vegetation removal and hence decrease in litter accumulation at grazed sites. In contrast, abundances of virulence, stress, and antibiotics resistance genes were increased because of the presence of livestock. In conclusion, these results indicated that soil microbial community functional structure was very sensitive to the impact of livestock grazing and revealed microbial functional potentials in regulating soil N and C cycling, supporting the necessity to include microbial components in evaluating the consequence of land-use and/or climate changes.

The microbial gene diversity along an elevation gradient of the Tibetan grassland.

Tibet is one of the most threatened regions by climate warming, thus understanding how its microbial communities function may be of high importance for predicting microbial responses to climate changes. Here, we report a study to profile soil microbial structural genes, which infers functional roles of microbial communities, along four sites/elevations of a Tibetan mountainous grassland, aiming to explore the potential microbial responses to climate changes via a strategy of space-for-time substitution. Using a microarray-based metagenomics tool named GeoChip 4.0, we showed that microbial communities were distinct for most but not all of the sites. Substantial variations were apparent in stress, N and C-cycling genes, but they were in line with the functional roles of these genes. Cold shock genes were more abundant at higher elevations. Also, gdh converting ammonium into urea was more abundant at higher elevations, whereas ureC converting urea into ammonium was less abundant, which was consistent with soil ammonium contents. Significant correlations were observed between N-cycling genes (ureC, gdh and amoA) and nitrous oxide flux, suggesting that they contributed to community metabolism. Lastly, we found by Canonical correspondence analysis, Mantel tests and the similarity tests that soil pH, temperature, NH4+–N and vegetation diversity accounted for the majority (81.4%) of microbial community variations, suggesting that these four attributes were major factors affecting soil microbial communities. On the basis of these observations, we predict that climate changes in the Tibetan grasslands are very likely to change soil microbial community functional structure, with particular impacts on microbial N-cycling genes and consequently microbe-mediated soil N dynamics.

Enzyme activities along a climatic transect in the Judean Desert

Abstract Soil enzymes have an important influence on nutrient cycling. We examined spatial and temporal patterns in dehydrogenase, arylsulfatase, alkaline and acid phosphatase activities, and their relationships with organic carbon and microbial biomass nitrogen at three sites in Israel representing different climatic regions: Mediterranean (humid), mildly arid and arid. The sites were selected along a climatic transect from the Judean Mountains in the west to the Dead Sea in the east of Israel. With increasing aridity, soil organic carbon, soil microbial biomass nitrogen, dehydrogenase, phosphatase and different pools of arylsulfatase activities decreased significantly. A sharp change in enzyme activities existed between 260- and 120-mm mean annual rainfall. The arylsulfatase activity of the microbial biomass in the 0–2- and 5–10-cm soil layers usually accounted for more than 50% of the total activity, and the fraction of total activity in the 0–2-cm soil layer of the arid sites was significantly greater than that of the humid site. Dehydrogenase and total and microbial biomass arylsulfatase activities were sensitive indicators of the climatic change along the transect. At the humid and mildly arid sites, the activities of dehydrogenase were less in the winter than in the summer and spring, whereas total and microbial biomass arylsulfatase activities were less in both summer and winter. At the arid site, lower values were observed in the summer at 0–2-cm soil depth. At all sites, lower alkaline phosphatase activities at 0–2 cm were observed in the summer, but there were no significant seasonal differences in acid phosphatase activities. These different seasonal patterns of enzyme activities are attributed to the enzyme source, and specific seasonal soil moisture and temperature conditions at the studied sites. The low dehydrogenase and microbial biomass arylsulfatase activities in the winter at the humid and mildly arid sites are explained by the cold and wet soil conditions, and the low enzyme activity in the summer at the arid site is attributed to the dry and hot soil conditions.

Genome-wide dynamic transcriptional profiling in clostridium beijerinckii NCIMB 8052 using single-nucleotide resolution RNA-Seq

BackgroundClostridium beijerinckii is a prominent solvent-producing microbe that has great potential for biofuel and chemical industries. Although transcriptional analysis is essential to understand gene functions and regulation and thus elucidate proper strategies for further strain improvement, limited information is available on the genome-wide transcriptional analysis for C. beijerinckii.ResultsThe genome-wide transcriptional dynamics of C. beijerinckii NCIMB 8052 over a batch fermentation process was investigated using high-throughput RNA-Seq technology. The gene expression profiles indicated that the glycolysis genes were highly expressed throughout the fermentation, with comparatively more active expression during acidogenesis phase. The expression of acid formation genes was down-regulated at the onset of solvent formation, in accordance with the metabolic pathway shift from acidogenesis to solventogenesis. The acetone formation gene (adc), as a part of the sol operon, exhibited highly-coordinated expression with the other sol genes. Out of the > 20 genes encoding alcohol dehydrogenase in C. beijerinckii, Cbei_1722 and Cbei_2181 were highly up-regulated at the onset of solventogenesis, corresponding to their key roles in primary alcohol production. Most sporulation genes in C. beijerinckii 8052 demonstrated similar temporal expression patterns to those observed in B. subtilis and C. acetobutylicum, while sporulation sigma factor genes sigE and sigG exhibited accelerated and stronger expression in C. beijerinckii 8052, which is consistent with the more rapid forespore and endspore development in this strain. Global expression patterns for specific gene functional classes were examined using self-organizing map analysis. The genes associated with specific functional classes demonstrated global expression profiles corresponding to the cell physiological variation and metabolic pathway switch.ConclusionsThe results from this work provided insights for further C. beijerinckii strain improvement employing system biology-based strategies and metabolic engineering approaches.

High-efficiency motor neuron differentiation from human pluripotent stem cells and the function of Islet-1

Efficient derivation of large-scale motor neurons (MNs) from human pluripotent stem cells is central to the understanding of MN development, modelling of MN disorders in vitro and development of cell-replacement therapies. Here we develop a method for rapid (20 days) and highly efficient (~70%) differentiation of mature and functional MNs from human pluripotent stem cells by tightly modulating neural patterning temporally at a previously undefined primitive neural progenitor stage. This method also allows high-yield (>250%) MN production in chemically defined adherent cultures. Furthermore, we show that Islet-1 is essential for formation of mature and functional human MNs, but, unlike its mouse counterpart, does not regulate cell survival or suppress the V2a interneuron fate. Together, our discoveries improve the strategy for MN derivation, advance our understanding of human neural specification and MN development, and provide invaluable tools for human developmental studies, drug discovery and regenerative medicine.

Single-nucleotide resolution analysis of the transcriptome structure of Clostridium beijerinckii NCIMB 8052 using RNA-Seq

BackgroundClostridium beijerinckii is an important solvent producing microorganism. The genome of C. beijerinckii NCIMB 8052 has recently been sequenced. Although transcriptome structure is important in order to reveal the functional and regulatory architecture of the genome, the physical structure of transcriptome for this strain, such as the operon linkages and transcript boundaries are not well understood.ResultsIn this study, we conducted a single-nucleotide resolution analysis of the C. beijerinckii NCIMB 8052 transcriptome using high-throughput RNA-Seq technology. We identified the transcription start sites and operon structure throughout the genome. We confirmed the structure of important gene operons involved in metabolic pathways for acid and solvent production in C. beijerinckii 8052, including pta-ack, ptb-buk, hbd-etfA-etfB-crt (bcs) and ald-ctfA-ctfB-adc (sol) operons; we also defined important operons related to chemotaxis/motility, transcriptional regulation, stress response and fatty acids biosynthesis along with others. We discovered 20 previously non-annotated regions with significant transcriptional activities and 15 genes whose translation start codons were likely mis-annotated. As a consequence, the accuracy of existing genome annotation was significantly enhanced. Furthermore, we identified 78 putative silent genes and 177 putative housekeeping genes based on normalized transcription measurement with the sequence data. We also observed that more than 30% of pseudogenes had significant transcriptional activities during the fermentation process. Strong correlations exist between the expression values derived from RNA-Seq analysis and microarray data or qRT-PCR results.ConclusionsTranscriptome structural profiling in this research provided important supplemental information on the accuracy of genome annotation, and revealed additional gene functions and regulation in C. beijerinckii.

A Molybdopterin Oxidoreductase Is Involved in H2 Oxidation in Desulfovibrio desulfuricans G20

Three mutants deficient in hydrogen/formate uptake were obtained through screening of a transposon mutant library containing 5,760 mutants of Desulfovibrio desulfuricans G20. Mutations were in the genes encoding the type I tetraheme cytochrome c(3) (cycA), Fe hydrogenase (hydB), and molybdopterin oxidoreductase (mopB). Mutations did not decrease the ability of cells to produce H(2) or formate during growth. Complementation of the cycA and mopB mutants with a plasmid carrying the intact cycA and/or mopB gene and the putative promoter from the parental strain allowed the recovery of H(2) uptake ability, showing that these specific genes are involved in H(2) oxidation. The mop operon encodes a periplasm-facing transmembrane protein complex which may shuttle electrons from periplasmic cytochrome c(3) to the menaquinone pool. Electrons can then be used for sulfate reduction in the cytoplasm.

Prokaryotic Communities in Pit Mud from Different-Aged Cellars Used for the Production of Chinese Strong-Flavored Liquor

ABSTRACT Chinese strong-flavored liquor (CSFL) accounts for more than 70% of all Chinese liquor production. Microbes in pit mud play key roles in the fermentation cellar for the CSFL production. However, microbial diversity, community structure, and cellar-age-related changes in pit mud are poorly understood. Here, we investigated the prokaryotic community structure and diversity in pit-mud samples with different cellar ages (1, 10, 25, and 50 years) using the pyrosequencing technique. Results indicated that prokaryotic diversity increased with cellar age until the age reached 25 years and that prokaryotic community structure changed significantly between three cellar ages (1, 10, and 25 years). Significant correlations between prokaryotic communities and environmental variables (pH, NH4 +, lactic acid, butyric acid, and caproic acid) were observed. Overall, our study results suggested that the long-term brewing operation shapes unique prokaryotic community structure and diversity as well as pit-mud chemistry. We have proposed a three-phase model to characterize the changes of pit-mud prokaryotic communities. (i) Phase I is an initial domestication period. Pit mud is characterized by abundant Lactobacillus and high lactic acid and low pH levels. (ii) Phase II is a transition period. While Lactobacillus abundance decreases dramatically, that of Bacteroidetes and methanogens increases. (iii) Phase III is a relative mature period. The prokaryotic community shows the highest diversity and capability to produce more caproic acid as a precursor for synthesis of ethyl caproate, the main flavor component in CSFL. This research provides scientific evidence to support the practical experience that old fermentation cellars produce high-quality liquor.

Functional Potential of Soil Microbial Communities in the Maize Rhizosphere

Microbial communities in the rhizosphere make significant contributions to crop health and nutrient cycling. However, their ability to perform important biogeochemical processes remains uncharacterized. Here, we identified important functional genes that characterize the rhizosphere microbial community to understand metabolic capabilities in the maize rhizosphere using the GeoChip-based functional gene array method. Significant differences in functional gene structure were apparent between rhizosphere and bulk soil microbial communities. Approximately half of the detected gene families were significantly (p<0.05) increased in the rhizosphere. Based on the detected gyrB genes, Gammaproteobacteria, Betaproteobacteria, Firmicutes, Bacteroidetes and Cyanobacteria were most enriched in the rhizosphere compared to those in the bulk soil. The rhizosphere niche also supported greater functional diversity in catabolic pathways. The maize rhizosphere had significantly enriched genes involved in carbon fixation and degradation (especially for hemicelluloses, aromatics and lignin), nitrogen fixation, ammonification, denitrification, polyphosphate biosynthesis and degradation, sulfur reduction and oxidation. This research demonstrates that the maize rhizosphere is a hotspot of genes, mostly originating from dominant soil microbial groups such as Proteobacteria, providing functional capacity for the transformation of labile and recalcitrant organic C, N, P and S compounds.