元蛋白质组学在微生物功能研究中的应用

后基因组时代,仅依靠基因组方法来研究原位微生物群落的功能已远远不够,在这种背景下元蛋白质组学研究逐渐兴起。应用元蛋白质组学技术可大规模研究原位微生物群落的蛋白质表达,分析生态系统中微生物的功能,寻找新的功能基因和代谢通路,为微生物群体的基因和功能多样性研究提供数据。同时,还可鉴定与微生物功能相关的蛋白质,这些蛋白质未来可以作为生物标记物为环境可持续发展铺路。综述了元蛋白质组学的发展概况及其在微生物功能研究中的重大作用,强调了元蛋白质组学方法在分析新功能基因及其相关基因,揭示微生物多样性与微生物群体功能之间的关系等方面起到的作用,并对其应用前景进行了展望。     

宏蛋白质组学技术在废水生物处理工艺研究领域中的应用

随着后基因组时代的到来,宏蛋白质组学逐渐兴起并在生命科学基础领域和临床医药领域成功运用,宏蛋白质组学技术现已成为各研究领域炙手可热的方法之一.宏蛋白质组学技术在废水生物处理研究领域中的应用刚起步,但已展示其强大功能.本文主要综述近年来国内外宏蛋白质组学在废水生物处理研究领域的研究进展,回顾及总结了宏蛋白质组学的研究策略及应用,如鉴定功能性蛋白质/酶、揭示污染物的微生物降解途径、推断废水生物处理系统的关键代谢途径、及探讨不同污泥微生物群落微生态变化等.
     

Endospores of halophilic bacteria of the family Bacillaceae isolated from non-saline Japanese soil may be transported by Kosa event (Asian dust storm)

Background

Natural microbial communities are extremely complex and dynamic systems in terms of their population structure and functions. However, little is known about the in situ functions of the microbial communities.

Results

This study describes the application of proteomic approaches (metaproteomics) to observe expressed protein profiles of natural microbial communities (metaproteomes). The technique was validated using a constructed community and subsequently used to analyze Chesapeake Bay microbial community (0.2 to 3.0 μm) metaproteomes. Chesapeake Bay metaproteomes contained proteins from pI 4–8 with apparent molecular masses between 10–80 kDa. Replicated middle Bay metaproteomes shared ~92% of all detected spots, but only shared 30% and 70% of common protein spots with upper and lower Bay metaproteomes. MALDI-TOF analysis of highly expressed proteins produced no significant matches to known proteins. Three Chesapeake Bay proteins were tentatively identified by LC-MS/MS sequencing coupled with MS-BLAST searching. The proteins identified were of marine microbial origin and correlated with abundant Chesapeake Bay microbial lineages, Bacteroides and α-proteobacteria.

Conclusion

Our results represent the first metaproteomic study of aquatic microbial assemblages and demonstrate the potential of metaproteomic approaches to link metagenomic data, taxonomic diversity, functional diversity and biological processes in natural environments.     

Metaproteomics to understand how microbiota function: The crystal ball predicts a promising future

In the medical, environmental, and biotechnological fields, microbial communities have attracted much attention due to their roles and numerous possible applications. The study of these communities is challenging due to their diversity and complexity. Innovative methods are needed to identify the taxonomic components of individual microbiota, their changes over time, and to determine how microoorganisms interact and function. Metaproteomics is based on the identification and quantification of proteins, and can potentially provide this full picture. Due to the wide molecular panorama and functional insights it provides, metaproteomics is gaining momentum in microbiome and holobiont research. Its full potential should be unleashed in the coming years with progress in speed and cost of analyses. In this exploratory crystal ball exercise, I discuss the technical and conceptual advances in metaproteomics that I expect to drive innovative research over the next few years in microbiology. I also debate the concepts of ‘microbial dark matter’ and ‘Metaproteomics-Assembled Proteomes (MAPs)’ and present some long-term prospects for metaproteomics in clinical diagnostics and personalized medicine, environmental monitoring, agriculture, and biotechnology.     

Characterization of metaproteomics in crop rhizospheric soil

Soil rhizospheric metaproteomics is a powerful scientific tool to uncover the interactions between plants and microorganisms in the soil ecosystem. The present study established an extraction method suitable for different soils that could increase the extracted protein content. Close to 1000 separate spots with high reproducibility could be identified in the stained 2-DE gels. Among the spots, 189 spots representing 122 proteins on a 2-DE gel of rice soil samples were successfully identified by MALDI-TOF/TOF-MS. These proteins mainly originated from rice and microorganisms. They were involved in protein, energy, nucleotide, and secondary metabolisms, as well as signal transduction and resistance. Three characteristics of the crop rhizospheric metaproteomics seemed apparent: (1) approximately one-third of the protein spots could not be identified by MALDI-TOF/TOF/MS, (2) the conservative proteins from plants formed a feature distribution of crop rhizospheric metaproteome, and (3) there were very complex interactions between plants and microorganisms existing in a crop rhizospheric soil. Further functional analysis on the identified proteins unveiled various metabolic pathways and signal transductions involved in the soil biotic community. This study provides a paradigm for metaproteomic research on soil biology.     

Metaproteomic strategies and applications for gut microbial research

The human intestine hosts various complex microbial communities that are closely associated with multiple health and disease processes. Determining the composition and function of these microbial communities is critical to unveil disease mechanisms and promote human health. Recently, meta-omic strategies have been developed that use high-throughput techniques to provide a wealth of information, thus accelerating the study of gut microbes. Metaproteomics is a newly emerged analytical approach that aims to identify proteins on a large scale in complex environmental microbial communities (e.g., the gut microbiota). This review introduces the recent analytical strategies and applications of metaproteomics, with a focus on advances in gut microbiota research, including a discussion of the limitations and challenges of these approaches.

     

基于宏组学方法认识微生物群落及其功能

进入后基因组学时代,测序技术飞速发展,测序成本明显下降,形成了涵盖宏基因组学、宏转录组学和宏蛋白质组学的宏组学技术,推动了对微生物群落的多样性、结构及潜在基因功能方面的深入研究。最近随着整合的宏组学技术的提出及应用,全面系统分析微生物群落动态变化及其代谢功能已成为可能,这将成为微生物生态学研究的新趋势。本文综述了宏组学在研究海洋湖泊、深海热泉、人体肠道、牛瘤胃生境、森林土壤与堆肥生境等环境中微生物群落的结构和功能方面的最新进展与成功应用案例。     

Metaproteomic data analysis at a glance: advances in computational microbial community proteomics

Introduction: Within the last decade, the study of microbial communities has gained increasing research interest also driven by the recognition of the important role of these consortia in human health and disease. Metaproteomics, the analysis of the entire set of proteins from all microorganisms present in one ecosystem, has become a prominent technique for studying the relation between taxonomic diversity and functional profile of microbial communities.

Areas covered: The aim of this review is to address opportunities and challenges of metaproteomics from a computational perspective. Appealing to an audience of microbial ecologists and proteomic researchers alike, we provide an overview on state-of-the-art software and databases by which metaproteome data can be readily analyzed.

Expert commentary: While tailored protein databases, combined search algorithms and iterative workflows are means to improve the identification yield, software tools for taxonomic and functional analysis are challenged by the vast amount of unannotated sequences in metaproteomics.     

Systems biology approach to bioremediation

Bioremediation has historically been approached as a 'black box' in terms of our fundamental understanding. Thus it succeeds and fails, seldom without a complete understanding of why. Systems biology is an integrated research approach to study complex biological systems, by investigating interactions and networks at the molecular, cellular, community, and ecosystem level. The knowledge of these interactions within individual components is fundamental to understanding the dynamics of the ecosystem under investigation. Understanding and modeling functional microbial community structure and stress responses in environments at all levels have tremendous implications for our fundamental understanding of hydrobiogeochemical processes and the potential for making bioremediation breakthroughs and illuminating the 'black box'.     

Metaproteomics Analysis Reveals the Adaptation Process for the Chicken Gut Microbiota

The animal gastrointestinal tract houses a large microbial community, the gut microbiota, that confers many benefits to its host, such as protection from pathogens and provision of essential metabolites. Metagenomic approaches have defined the chicken fecal microbiota in other studies, but here, we wished to assess the correlation between the metagenome and the bacterial proteome in order to better understand the healthy chicken gut microbiota. Here, we performed high-throughput sequencing of 16S rRNA gene amplicons and metaproteomics analysis of fecal samples to determine microbial gut composition and protein expression. 16 rRNA gene sequencing analysis identified Clostridiales, Bacteroidaceae, and Lactobacillaceae species as the most abundant species in the gut. For metaproteomics analysis, peptides were generated by using the Fasp method and subsequently fractionated by strong anion exchanges. Metaproteomics analysis identified 3,673 proteins. Among the most frequently identified proteins, 380 proteins belonged to Lactobacillus spp., 155 belonged to Clostridium spp., and 66 belonged to Streptococcus spp. The most frequently identified proteins were heat shock chaperones, including 349 GroEL proteins, from many bacterial species, whereas the most abundant enzymes were pyruvate kinases, as judged by the number of peptides identified per protein (spectral counting). Gene ontology and KEGG pathway analyses revealed the functions and locations of the identified proteins. The findings of both metaproteomics and 16S rRNA sequencing analyses are discussed.     

宏组学方法在污水处理系统中的应用进展

污水生物处理由微生物生理过程驱动,宏组学方法能够获得不同水平的分子信息,为认识污水处理系统中的微生物提供了新途径。本文对宏基因组学、宏转录组学、宏蛋白质组学与代谢组学等宏组学方法的发展进行综述,着重介绍各组学及整合宏组学在污水处理系统中的研究现状,并指出其应用前景。     

Metaproteomic Survey of Six Aquatic Habitats: Discovering the Identities of Microbial Populations Active in Biogeochemical Cycling

Our goal is to strengthen the foundations of metaproteomics as a microbial community analysis tool that links the functional identity of actively expressed gene products with host phylogeny. We used shotgun metaproteomics to survey waters in six disparate aquatic habitats (Cayuga Lake, NY; Oneida Lake, NY; Gulf of Maine; Chesapeake Bay, MD; Gulf of Mexico; and the South Pacific). Peptide pools prepared from filter-gathered microbial biomass, analyzed by nano-liquid chromatography–mass spectrometry (MS/MS) generating 9,693?±?1,073 mass spectra identified 326?±?107 bacterial proteins per sample. Distribution of proteobacterial (Alpha and Beta) and cyanobacterial (Prochlorococcus and Synechococcus spp.) protein hosts across all six samples was consistent with the previously published biogeography for these microorganisms. Marine samples were enriched in transport proteins (TRAP-type for dicarboxylates and ATP binding cassette (ABC)-type for amino acids and carbohydrates) compared with the freshwater samples. We were able to match in situ expression of many key proteins catalyzing C-, N-, and S-cycle processes with their bacterial hosts across all six habitats. Pelagibacter was identified as the host of ABC-type sugar-, organic polyanion-, and glycine betaine-transport proteins; this extends previously published studies of Pelagibacter's in situ biogeochemical role in marine C- and N-metabolism. Proteins matched to Ruegeria confirmed these organism's role in marine waters oxidizing both carbon monoxide and sulfide. By documenting both processes expressed in situ and the identity of host cells, metaproteomics tested several existing hypotheses about ecophysiological processes and provided fodder for new ones.     

Environmental microbiome engineering for the mitigation of climate change

Environmental microbiome engineering is emerging as a potential avenue for climate change mitigation. In this process, microbial inocula are introduced to natural microbial communities to tune activities that regulate the long-term stabilization of carbon in ecosystems. In this review, we outline the process of environmental engineering and synthesize key considerations about ecosystem functions to target, means of sourcing microorganisms, strategies for designing microbial inocula, methods to deliver inocula, and the factors that enable inocula to establish within a resident community and modify an ecosystem function target. Recent work, enabled by high-throughput technologies and modeling approaches, indicate that microbial inocula designed from the top-down, particularly through directed evolution, may generally have a higher chance of establishing within existing microbial communities than other historical approaches to microbiome engineering. We address outstanding questions about the determinants of inocula establishment and provide suggestions for further research about the possibilities and challenges of environmental microbiome engineering as a tool to combat climate change.     

Insights from quantitative metaproteomics and protein-stable isotope probing into microbial ecology

The recent development of metaproteomics has enabled the direct identification and quantification of expressed proteins from microbial communities in situ, without the need for microbial enrichment. This became possible by (1) significant increases in quality and quantity of metagenome data and by improvements of (2) accuracy and (3) sensitivity of modern mass spectrometers (MS). The identification of physiologically relevant enzymes can help to understand the role of specific species within a community or an ecological niche. Beside identification, relative and absolute quantitation is also crucial. We will review label-free and label-based methods of quantitation in MS-based proteome analysis and the contribution of quantitative proteome data to microbial ecology. Additionally, approaches of protein-based stable isotope probing (protein-SIP) for deciphering community structures are reviewed. Information on the species-specific metabolic activity can be obtained when substrates or nutrients are labeled with stable isotopes in a protein-SIP approach. The stable isotopes (¹³C, ¹⁵N, ³⁶S) are incorporated into proteins and the rate of incorporation can be used for assessing the metabolic activity of the corresponding species. We will focus on the relevance of the metabolic and phylogenetic information retrieved with protein-SIP studies and for detecting and quantifying the carbon flux within microbial consortia. Furthermore, the combination of protein-SIP with established tools in microbial ecology such as other stable isotope probing techniques are discussed.     

Metaproteomics as a tool for studying the protein landscape of human-gut bacterial species

Host-microbiome interactions and the microbial community have broad impact in human health and diseases. Most microbiome based studies are performed at the genome level based on next-generation sequencing techniques, but metaproteomics is emerging as a powerful technique to study microbiome functional activity by characterizing the complex and dynamic composition of microbial proteins. We conducted a large-scale survey of human gut microbiome metaproteomic data to identify generalist species that are ubiquitously expressed across all samples and specialists that are highly expressed in a small subset of samples associated with a certain phenotype. We were able to utilize the metaproteomic mass spectrometry data to reveal the protein landscapes of these species, which enables the characterization of the expression levels of proteins of different functions and underlying regulatory mechanisms, such as operons. Finally, we were able to recover a large number of open reading frames (ORFs) with spectral support, which were missed by de novo protein-coding gene predictors. We showed that a majority of the rescued ORFs overlapped with de novo predicted protein-coding genes, but on opposite strands or in different frames. Together, these demonstrate applications of metaproteomics for the characterization of important gut bacterial species.     

A metaproteomic approach gives functional insights into anaerobic digestion

Aims: The objective of this work was to provide functional evidence of key metabolic pathways important for anaerobic digestion processes through the identification of highly expressed proteins in a mixed anaerobic microbial consortium. Methods and Results: The microbial communities from an anaerobic industrial‐like wastewater treatment bioreactor were characterized using phylogenetic analyses and metaproteomics. Clone libraries indicated that the bacterial community in the bioreactor was diverse while the archaeal population was mainly composed of Methanocorpusculum‐like (76%) micro‐organisms. Three hundred and eighty‐eight reproducible protein spots were obtained on 2‐D gels, of which 70 were excised and 33 were identified. The putative functions of the proteins detected in the anaerobic bioreactor were related to cellular processes, including methanogenesis from CO₂ and acetate, glycolysis and the pentose phosphate pathway. Metaproteomics also indicated, by protein assignment, the presence of specific micro‐organisms in the bioreactor. However, only a limited overlap was observed between the phylogenetic and metaproteomic analyses. Conclusions: This study provides some direct evidence of the microbial activities taking place during anaerobic digestion. Significance and Impact of Study: This study demonstrates metaproteomics as a useful tool to uncover key biochemical pathways underpinning specific anaerobic bioprocesses.