Publications

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    ABSTRACT: Human gut microbiota plays an important role in the pathogenesis of cirrhosis complications. Although the phylogenetic diversity of intestinal microbiota in patients with liver cirrhosis has been examined in several studies, little is known about their functional composition and structure.
    BMC genomics. 09/2014; 15(1):753.
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    ABSTRACT: Deciphering microbial communities and their role in Earth’s biosphere is crucial for addressing challenges in human health, agriculture, bioremediation and other natural processes. While next-generation sequencing platforms are still under development to improve accuracy, read length and sequencing depth, microarray-based methods have become an attractive alternative for 16S rRNA gene microbial community comparisons. The hybridization method is well-established in the laboratory. Thus main areas of improvement lie with the development of improved bioinformatics and statistical procedures for microarray data, rather than with improvements to the platform itself. In this communication we applied recently-developed bioinformatics tools to re-analyze G3 PhyloChip™ DNA microarray data acquired from deep ocean samples collected during the 2010 Deepwater Horizon oil spill in the Gulf of Mexico. We show that data collected with the G3 PhyloChip™ assay can be analyzed at various stages of resolution, from individual probes to pairs of probes to quartets of probes and finally at the commonly used probe-set level where each probe-set is associated with one operational taxonomic unit (OTU). Our analysis methods comprised topological data analysis to facilitate the detection of outlier bio-specimens and the reconstruction of empirical OTUs (eOTUs) in an unsupervised manner, without the need of pre-defined reference OTUs (rOTUs). We observed that the quartet level provided sufficient resolution for identifying a subtle outlier sample with TDA while the eOTU reconstruction was useful for annotation of the taxa associated with significant population changes in the elevated hydrocarbon waters. The presented methods will improve the deduction of important biological processes from G3 PhyloChip experiments.
    edited by Zhili He, 08/2014; Caister Academic Press.
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    ABSTRACT: Acidithiobacillus thiooxidans (A. thiooxidans), a chemolithoautotrophic extremophile, is widely used in the industrial recovery of copper (bioleaching or biomining). The organism grows and survives by autotrophically utilizing energy derived from the oxidation of elemental sulfur and reduced inorganic sulfur compounds (RISCs). However, the lack of genetic manipulation systems has restricted our exploration of its physiology. With the development of high-throughput sequencing technology, the whole genome sequence analysis of A. thiooxidans has allowed preliminary models to be built for genes/enzymes involved in key energy pathways like sulfur oxidation.
    BMC Microbiology 07/2014; 14(1):179. · 3.10 Impact Factor
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    ABSTRACT: Determining the composition and function of subgingival dental plaque is crucial to understanding human periodontal health and disease, but it is challenging because of the complexity of the interactions between human microbiomes and human body. Here, we examined the phylogenetic and functional gene differences between periodontal and healthy individuals using MiSeq sequencing of 16S rRNA gene amplicons and a specific functional gene array (a combination of GeoChip 4.0 for biogeochemical processes and HuMiChip 1.0 for human microbiomes). Our analyses indicated that the phylogenetic and functional gene structure of the oral microbiomes were distinctly different between periodontal and healthy groups. Also, 16S rRNA gene sequencing analysis indicated that 39 genera were significantly different between healthy and periodontitis groups, and Fusobacterium, Porphyromonas, Treponema, Filifactor, Eubacterium, Tannerella, Hallella, Parvimonas, Peptostreptococcus and Catonella showed higher relative abundances in the periodontitis group. In addition, functional gene array data showed that a lower gene number but higher signal intensity of major genes existed in periodontitis, and a variety of genes involved in virulence factors, amino acid metabolism and glycosaminoglycan and pyrimidine degradation were enriched in periodontitis, suggesting their potential importance in periodontal pathogenesis. However, the genes involved in amino acid synthesis and pyrimidine synthesis exhibited a significantly lower relative abundance compared with healthy group. Overall, this study provides new insights into our understanding of phylogenetic and functional gene structure of subgingival microbial communities of periodontal patients and their importance in pathogenesis of periodontitis.The ISME Journal advance online publication, 27 March 2014; doi:10.1038/ismej.2014.28.
    The ISME Journal 03/2014; · 8.95 Impact Factor
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    ABSTRACT: Nitrate is an important nutrient and electron acceptor for microorganisms, having a key role in nitrogen (N) cycling and electron transfer in anoxic sediments. High-nitrate inputs into sediments could have a significant effect on N cycling and its associated microbial processes. However, few studies have been focused on the effect of nitrate addition on the functional diversity, composition, structure and dynamics of sediment microbial communities in contaminated aquatic ecosystems with persistent organic pollutants (POPs). Here we analyzed sediment microbial communities from a field-scale in situ bioremediation site, a creek in Pearl River Delta containing a variety of contaminants including polybrominated diphenyl ethers (PBDEs) and polycyclic aromatic hydrocarbons (PAHs), before and after nitrate injection using a comprehensive functional gene array (GeoChip 4.0). Our results showed that the sediment microbial community functional composition and structure were markedly altered, and that functional genes involved in N-, carbon (C)-, sulfur (S)-and phosphorus (P)- cycling processes were highly enriched after nitrate injection, especially those microorganisms with diverse metabolic capabilities, leading to potential in situ bioremediation of the contaminated sediment, such as PBDE and PAH reduction/degradation. This study provides new insights into our understanding of sediment microbial community responses to nitrate addition, suggesting that indigenous microorganisms could be successfully stimulated for in situ bioremediation of POPs in contaminated sediments with nitrate addition.The ISME Journal advance online publication, 27 March 2014; doi:10.1038/ismej.2014.42.
    The ISME Journal 03/2014; · 8.95 Impact Factor
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    ABSTRACT: Clostridium cellulolyticum can degrade lignocellulosic biomass, and ferment the soluble sugars to produce valuable chemicals such as lactate, acetate, ethanol and hydrogen. However, the cellulose utilization efficiency of C. cellulolyticum still remains very low, impeding its application in consolidated bioprocessing for biofuels production. In this study, two metabolic engineering strategies were exploited to improve cellulose utilization efficiency, including sporulation abolishment and carbon overload alleviation. The spo0A gene at locus Ccel_1894, which encodes a master sporulation regulator was inactivated. The spo0A mutant abolished the sporulation ability. In a high concentration of cellulose (50 g/l), the performance of the spo0A mutant increased dramatically in terms of maximum growth, final concentrations of three major metabolic products, and cellulose catabolism. The microarray and gas chromatography-mass spectrometry (GC-MS) analyses showed that the valine, leucine and isoleucine biosynthesis pathways were up-regulated in the spo0A mutant. Based on this information, a partial isobutanol producing pathway modified from valine biosynthesis was introduced into C. cellulolyticum strains to further increase cellulose consumption by alleviating excessive carbon load. The introduction of this synthetic pathway to the wild-type strain improved cellulose consumption from 17.6 g/l to 28.7 g/l with a production of 0.42 g/l isobutanol in the 50 g/l cellulose medium. However, the spo0A mutant strain did not appreciably benefit from introduction of this synthetic pathway and the cellulose utilization efficiency did not further increase. A technical highlight in this study was that an in vivo promoter strength evaluation protocol was developed using anaerobic fluorescent protein and flow cytometry for C. cellulolyticum. In this study, we inactivated the spo0A gene and introduced a heterologous synthetic pathway to manipulate the stress response to heavy carbon load and accumulation of metabolic products. These findings provide new perspectives to enhance the ability of cellulolytic bacteria to produce biofuels and biocommodities with high efficiency and at low cost directly from lignocellulosic biomass.
    Biotechnology for Biofuels 02/2014; 7(1):25. · 5.55 Impact Factor
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    ABSTRACT: Unraveling the drivers of community structure and succession in response to environmental change is a central goal in ecology. Although the mechanisms shaping community structure have been intensively examined, those controlling ecological succession remain elusive. To understand the relative importance of stochastic and deterministic processes in mediating microbial community succession, a unique framework composed of four different cases was developed for fluidic and nonfluidic ecosystems. The framework was then tested for one fluidic ecosystem: a groundwater system perturbed by adding emulsified vegetable oil (EVO) for uranium immobilization. Our results revealed that groundwater microbial community diverged substantially away from the initial community after EVO amendment and eventually converged to a new community state, which was closely clustered with its initial state. However, their composition and structure were significantly different from each other. Null model analysis indicated that both deterministic and stochastic processes played important roles in controlling the assembly and succession of the groundwater microbial community, but their relative importance was time dependent. Additionally, consistent with the proposed conceptual framework but contradictory to conventional wisdom, the community succession responding to EVO amendment was primarily controlled by stochastic rather than deterministic processes. During the middle phase of the succession, the roles of stochastic processes in controlling community composition increased substantially, ranging from 81.3% to 92.0%. Finally, there are limited successional studies available to support different cases in the conceptual framework, but further well-replicated explicit time-series experiments are needed to understand the relative importance of deterministic and stochastic processes in controlling community succession.
    Proceedings of the National Academy of Sciences 02/2014; · 9.81 Impact Factor
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    ABSTRACT: Microorganisms play critical roles in many important biogeochemical processes in the Earth's biosphere. However, understanding and characterizing the functional capacity of microbial communities is still difficult due to the extremely diverse and often uncultivable nature of most microorganisms. In this study, we developed a new functional gene array, GeoChip 4, for analyzing the functional diversity, composition, structure, metabolic potential/activity, and dynamics of microbial communities. GeoChip 4 contained approximately 82,000 probes covering 141,995 coding sequences from 410 functional gene families related to microbial carbon (C), nitrogen (N), sulfur (S), and phosphorus (P) cycling, energy metabolism, antibiotic resistance, metal resistance/reduction, organic remediation, stress responses, bacteriophage, and virulence. A total of 173 archaeal, 4,138 bacterial, 404 eukaryotic and 252 viral strains were targeted, providing the ability to analyze targeted functional gene families of microorganisms included in all four domains. Experimental assessment using different amounts of DNA suggested that as little as 500 ng environmental DNA was required for good hybridization, and the signal intensities detected were well correlated with the DNA amount used. GeoChip 4 was then applied to study the effect of long-term warming on soil microbial communities at a Central Oklahoma site, with results indicating that microbial communities respond to long-term warming by enriching carbon-degradation, nutrient-cycling (nitrogen and phosphorous), and stress response gene families. To the best of our knowledge, GeoChip 4 is the most comprehensive functional gene array for microbial community analysis. This article is protected by copyright. All rights reserved.
    Molecular Ecology Resources 02/2014; · 7.43 Impact Factor
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    Qichao Tu, Zhili He, Jizhong Zhou
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    ABSTRACT: Shotgun metagenome sequencing has become a fast, cheap and high-throughput technology for characterizing microbial communities in complex environments and human body sites. However, accurate identification of microorganisms at the strain/species level remains extremely challenging. We present a novel k-mer-based approach, termed GSMer, that identifies genome-specific markers (GSMs) from currently sequenced microbial genomes, which were then used for strain/species-level identification in metagenomes. Using 5390 sequenced microbial genomes, 8 770 321 50-mer strain-specific and 11 736 360 species-specific GSMs were identified for 4088 strains and 2005 species (4933 strains), respectively. The GSMs were first evaluated against mock community metagenomes, recently sequenced genomes and real metagenomes from different body sites, suggesting that the identified GSMs were specific to their targeting genomes. Sensitivity evaluation against synthetic metagenomes with different coverage suggested that 50 GSMs per strain were sufficient to identify most microbial strains with ≥0.25× coverage, and 10% of selected GSMs in a database should be detected for confident positive callings. Application of GSMs identified 45 and 74 microbial strains/species significantly associated with type 2 diabetes patients and obese/lean individuals from corresponding gastrointestinal tract metagenomes, respectively. Our result agreed with previous studies but provided strain-level information. The approach can be directly applied to identify microbial strains/species from raw metagenomes, without the effort of complex data pre-processing.
    Nucleic Acids Research 02/2014; · 8.81 Impact Factor
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    ABSTRACT: Loss of microbial diversity is considered a major threat because of its importance for ecosystem functions, but there is a lack of conclusive evidence that diversity itself is reduced under anthropogenic stress, and about the consequences of diversity loss. Heavy metals are one of the largest, widespread pollutant types globally, and these represent a significant environmental stressor for terrestrial microbial communities. Using combined metagenomics and functional assays, we show that the compositional and functional response of microbial communities to long-term heavy metal stress results in a significant loss of diversity. Our results indicate that even at a moderate loss of diversity, some key specialized functions (carried out by specific groups) may be compromised. Together with previous work, our data suggest disproportionate impact of contamination on microbes that carry out specialized, but essential, ecosystem functions. Based on these findings, we propose a conceptual framework to explicitly consider diversity of functions and microbial functional groups to test the relationship between biodiversity and soil functions.
    Environmental Microbiology 01/2014; · 6.24 Impact Factor
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    ABSTRACT: Soil transplant into warmer regions has been shown to alter soil microbiology. In contrast, little is known about the effects of soil transplant into colder regions, albeit that climate cooling has solicited attention in recent years. To address this question, we transplanted bare fallow soil over large transects from southern China (subtropical climate zone) to central (warm temperate climate zone) and northern China (cold temperate climate zone). After an adaptation period of 4 years, soil nitrogen components, microbial biomass and community structures were altered. However, the effects of soil transplant on microbial communities were dampened by maize cropping, unveiling a negative interaction between cropping and transplant. Further statistical analyses with Canonical correspondence analysis and Mantel tests unveiled annual average temperature, relative humidity, aboveground biomass, soil pH and NH4 (+) -N content as environmental attributes closely correlated with microbial functional structures. In addition, average abundances of amoA-AOA (ammonia-oxidizing archaea) and amoA-AOB (ammonia-oxidizing bacteria) genes were significantly (P < 0.05) correlated with soil nitrification capacity, hence both AOA and AOB contributed to the soil functional process of nitrification. These results suggested that the soil nitrogen cycle was intimately linked with microbial community structure, and both were subjected to disturbance by soil transplant to colder regions and plant cropping.
    Environmental Microbiology 01/2014; · 6.24 Impact Factor
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    ABSTRACT: Development of tools for targeted genome editing and regulation of gene expression has significantly expanded our ability to elucidate the mechanisms of interesting biological phenomena, and to engineer desirable biological systems. Recent rapid progress in the study of clustered, regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas) system in bacteria has facilitated the development of newly facile and programmable platforms for genome editing and transcriptional control in a sequence-specific manner. The core RNA-guided Cas9 endonuclease in the type II CRISPR system has been harnessed to realize gene mutation, DNA deletion and insertion, as well as transcriptional activation and repression, with multiplex targeting ability, just by customizing 20 nt RNA components. Here we describe the molecular basis of the type II CRISPR/Cas system and summarize applications and factors affecting its utilization in model organisms. We also discuss the advantages and disadvantages of Cas9-based tools in comparison with widely-used customizable tools, such as Zinc-finger nucleases and transcription activator-like effector nucleases.
    Applied and Environmental Microbiology 01/2014; · 3.95 Impact Factor
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    ABSTRACT: Human saliva microbiota is phylogenetically divergent among host individuals yet their roles in health and disease are poorly appreciated. We employed a microbial functional gene microarray, HuMiChip 1.0, to reconstruct the global functional profiles of human saliva microbiota from ten healthy and ten caries-active adults. Saliva microbiota in the pilot population featured a vast diversity of functional genes. No significant distinction in gene number or diversity indices was observed between healthy and caries-active microbiota. However, co-presence network analysis of functional genes revealed that caries-active microbiota was more divergent in non-core genes than healthy microbiota, despite both groups exhibited a similar degree of conservation at their respective core genes. Furthermore, functional gene structure of saliva microbiota could potentially distinguish caries-active patients from healthy hosts. Microbial functions such as Diaminopimelate epimerase, Prephenate dehydrogenase, Pyruvate-formate lyase and N-acetylmuramoyl-L-alanine amidase were significantly linked to caries. Therefore, saliva microbiota carried disease-associated functional signatures, which could be potentially exploited for caries diagnosis.
    PLoS ONE 01/2014; 9(2):e76458. · 3.53 Impact Factor
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    ABSTRACT: Understanding the diversity, composition, structure, function, and dynamics of human microbiomes in individual human hosts is crucial to reveal human-microbial interactions, especially for patients with microbially mediated disorders, but challenging due to the high diversity of the human microbiome. Here we have developed a functional gene-based microarray for profiling human microbiomes (HuMiChip) with 36,802 probes targeting 50,007 protein coding sequences for 139 key functional gene families. Computational evaluation suggested all probes included are highly specific to their target sequences. HuMiChip was used to analyze human oral and gut microbiomes, showing significantly different functional gene profiles between oral and gut microbiome. Obvious shifts of microbial functional structure and composition were observed for both patients with dental caries and periodontitis from moderate to advanced stages, suggesting a progressive change of microbial communities in response to the diseases. Consistent gene family profiles were observed by both HuMiChip and next generation sequencing technologies. Additionally, HuMiChip was able to detect gene families at as low as 0.001% relative abundance. The results indicate that the developed HuMiChip is a useful and effective tool for functional profiling of human microbiomes.
    PLoS ONE 01/2014; 9(3):e90546. · 3.53 Impact Factor
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    ABSTRACT: Subsurface sediments of the Sonora Margin (Guaymas Basin), located in proximity of active cold seep sites were explored. The taxonomic and functional diversity of bacterial and archaeal communities were investigated from 1 to 10 meters below the seafloor. Microbial community structure and abundance and distribution of dominant populations were assessed using complementary molecular approaches (Ribosomal Intergenic Spacer Analysis, 16S rRNA libraries and quantitative PCR with an extensive primers set) and correlated to comprehensive geochemical data. Moreover the metabolic potentials and functional traits of the microbial community were also identified using the GeoChip functional gene microarray and metabolic rates. The active microbial community structure in the Sonora Margin sediments was related to deep subsurface ecosystems (Marine Benthic Groups B and D, Miscellaneous Crenarchaeotal Group, Chloroflexi and Candidate divisions) and remained relatively similar throughout the sediment section, despite defined biogeochemical gradients. However, relative abundances of bacterial and archaeal dominant lineages were significantly correlated with organic carbon quantity and origin. Consistently, metabolic pathways for the degradation and assimilation of this organic carbon as well as genetic potentials for the transformation of detrital organic matters, hydrocarbons and recalcitrant substrates were detected, suggesting that chemoorganotrophic microorganisms may dominate the microbial community of the Sonora Margin subsurface sediments.
    PLoS ONE 01/2014; 9(8):e104427. · 3.53 Impact Factor
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    ABSTRACT: Microbe plays an important role in driving biogeochemical cycles, thus it is of great interest to understand microbial responses and feedbacks to global changes. We have recently analyzed functional potentials of soil microbial community via a high-throughput, microarray-based metagenomic tool named GeoChip 3.0 to illustrate microbial responses to global changes simulated by soil transplant and/or maize cropping. Here we describe detailed experimental design, data collection and pre-processing to support our published studies by Liu et al. [5] and Zhao et al. [14].
    Genomics Data. 01/2014;
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    ABSTRACT: To examine microbial responses to climate change, we used a microarray-based metagenomics tool named GeoChip 4.0 to profile soil microbial functional genes along four sites/elevations of a Tibetan mountainous grassland. We found that microbial communities differed among four elevations. Soil pH, temperature, NH4+–N and vegetation diversity were four major attributes affecting soil microbial communities. Here we describe in details the experiment design, the data normalization process, soil and vegetation analyses associated with the study published on ISME Journal in 2014 [1], whose raw data have been uploaded to Gene Expression Omnibus (accession number GSM1185243).
    Genomics Data. 01/2014;
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    ABSTRACT: Microbial electrolysis cells (MECs) use exoelectrogenic microorganisms to convert organic matter into H2, although yields can vary significantly with environmental conditions, likely due to variations in microbial communities. This study was undertaken to better understand how microbial communities affect reactor function. Using wastewater as inoculum, 15 MEC reactors were operated for >50 days and subsequently five reactors were selected for further analysis. Solution (26 mL) was collected every 3e4 days for DNA extraction. DNA was hy- bridized to GeoChip, a comprehensive functional gene array, to examine differences in the reactor microbial communities. A large variety of microbial functional genes were observed in all reactors. Performances ranged from poor (0.1 ` 0.1 mL) to high (12.2 ` 1.0 mL) H2 pro- duction, with a maximum yield of 5.01 ` 0.43 mol H2/molglucose. The best performance was associated with higher cytochrome c genes, considerably higher exoelectrogenic bacteria (such as Shewanella, Geobacter), less methanogens and less hydrogen-utilizing bacteria. The results confirmed the possibility to obtain an effective community for hydrogen production using wastewater as inoculum. Not like fermentation, hydrogen production was significantly controlled by electron transporting process in MECs. GeoChip findings suggested that biofilm formattino can be highly stochastic and that presence of dissimilatory metal-reducing bacteria and antagonistic methanogens is critical for efficient hydrogen production in MEC reactors.
    International Journal of Hydrogen Energy 01/2014; · 3.55 Impact Factor
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    ABSTRACT: Soil microbial communities are extremely complex, composed of thousands of low-abundance species (<0.1% of total). How such complex communities respond to natural or human-induced fluctuations, including major perturbations such as global climate change, remains poorly understood, severely limiting our predictive ability of soil ecosystem functioning and resilience. In this study, we compared twelve whole-community shotgun metagenomic datasets from a grassland soil in Midwest USA; half representing soil that was undergoing infrared warming by 2°C for 10 years, which simulated the effects of climate change, and the other half representing the adjacent soil that received no warming and thus, served as control. Our analyses revealed that the heated communities showed significant shifts in composition and predicted metabolism, and these shifts were community-wide as opposed to being attributable to a few taxa. Key metabolic pathways related to carbon turnover, e.g., cellulose degradation (∼13%) and CO2 production (∼10%), and nitrogen, e.g., denitrification (∼12%), were enriched under warming, which was consistent with independent physicochemical measurements. These community shifts were interlinked, in part, with higher primary productivity of the aboveground plant communities stimulated by warming, revealing that most of the additional, plant-derived soil carbon was likely respired by microbial activity. Warming also enriched for a higher abundance of sporulation genes and genomes with higher G+C% content. Collectively, our results indicate that the microbial communities of the temperate grassland soils play important roles in mediating the feedback responses to climate change and advance understanding of the molecular mechanisms of community adaptation to environmental perturbations.
    Applied and Environmental Microbiology 12/2013; · 3.95 Impact Factor
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    ABSTRACT: Cellulosomes are key for lignocellulosic biomass degradation in cellulolytic Clostridia. Better understanding of the mechanism of cellulosome regulation would allow us to improve lignocellulose hydrolysis. It is hypothesized that cellulosomal protease inhibitors would regulate cellulosome architecture and then lignocellulose hydrolysis. Here, a dockerin-containing protease inhibitor gene (dpi) in Clostridium cellulolyticum H10 was characterized by mutagenesis and physiological analyses. The dpi mutant had a decreased cell yield on glucose, cellulose and xylan, lower cellulose utilization efficiency, and a 70% and 52% decrease of the key cellulosomal components, Cel48F and Cel9E, respectively. The decreased cellulolysis is caused by the proteolysis of major cellulosomal components, such as Cel48F and Cel9E. Disruption of cel9E severely impaired cell growth on cellulose while loss of cel48F completely abolished cellulolytic activity. These observations are due to the combinational results of gene inactivation and polar effects caused by intron insertion. Purified recombinant Dpi showed inhibitory activity against cysteine protease. Taken together, Dpi protects key cellulosomal cellulases from proteolysis in H10. This study identified the physiological importance of cellulosome-localized protease inhibitors in Clostridia.
    Molecular Microbiology 12/2013; · 4.96 Impact Factor

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