纳米二次离子质谱技术(NanoSIMS)在微生物生态学研究中的应用

超高分辨率显微镜成像技术与同位素示踪技术相结合的纳米二次离子质谱技术(NanoSIMS)具有较高的灵敏度和离子传输效率、极高的质量分辨率和空间分辨率(＜ 50 nm),代表着当今离子探针成像技术的最高水平.利用稳定性或者放射性同位素在原位或者微宇宙条件下示踪目标微生物,然后将样品进行固定、脱水、树脂包埋或者导电镀膜处理,制备成可供二次离子质谱分析的薄片,进一步通过NanoSIMS成像分析,不仅能够在单细胞水平上提供微生物的生理生态特征信息,而且能够准确识别复杂环境样品中的代谢活跃的微生物细胞及其系统分类信息,对于认识微生物介导的元素生物地球化学循环机制具有重要意义.介绍了纳米二次离子质谱技术的工作原理和技术路线,及其与同位素示踪技术、透射电子显微镜(TEM)、扫描电子显微镜(SEM)、荧光原位杂交技术(FISH)、催化报告沉积荧光原位杂交技术(CARD-FISH)、卤素原位杂交技术(Halogen In Situ Hybridization,HISH)等联合使用在微生物生态学研究方面的应用.     

Linking environmental processes to the in situ functioning of microorganisms by high‐resolution secondary ion mass spectrometry (NanoSIMS) and scanning transmission X‐ray microscopy (STXM)

Environmental microbiology research increasingly focuses on the single microbial cell as the defining entity that drives environmental processes. The interactions of individual microbial cells with each other, the environment and with higher organisms shape microbial communities and control the functioning of whole ecosystems. A single‐cell view of microorganisms in their natural environment requires analytical tools that measure both cell function and chemical speciation at the submicrometre scale. Here we review the technical capabilities and limitations of high‐resolution secondary ion mass spectrometry (NanoSIMS) and scanning transmission (soft) X‐ray microscopy (STXM) and give examples of their applications. Whereas NanoSIMS can be combined with isotope‐labelling, thereby localizing the distribution of cellular activities (e.g. carbon/nitrogen fixation/turnover), STXM provides information on the location and chemical speciation of metabolites and products of redox reactions. We propose the combined use of both techniques and discuss the technical challenges of their joint application. Both techniques have the potential to enhance our understanding of cellular mechanisms and activities that contribute to microbially mediated processes, such as the biogeochemical cycling of elements, the transformation of contaminants and the precipitation of mineral phases.     

单细胞尺度下的微生物学研究:意义与方法

最近研究表明,即便是处于同一种群中的微生物细胞,在基因转录和翻译、蛋白活性、以及代谢物丰度等多个水平都可能存在显著差异,说明微生物细胞间存在着多个层次上的异质性;同时,传统微生物学研究方法需要将所研究的微生物对象在实验室实现再次培养,然后对纯培养的微生物种群进行研究,这样往往造成实验室的研究结果无法真实地反映微生物细胞在自然界中的原始状态,急需发展新的原位研究手段;此外,自然界中的微生物目前只有极少部分可以在实验室中进行培养,仍有大量微生物无法通过传统方法进行发掘和研究。单细胞尺度微生物学为解决这些微生物学研究中的重要挑战提供了一种新的策略和技术思路,有望帮助我们更为直观、深入地了解每个细胞内部的状态,以及其在自然界的生理生态功能。本文对单细胞尺度微生物学研究的意义以及当前单细胞尺度微生物学的研究方法,特别是新兴的微生物单细胞组学方法进行了介绍。     

Analyzing Microbial Population Heterogeneity—Expanding the Toolbox of Microfluidic Single-Cell Cultivations

Recent research on population heterogeneity revealed fascinating insights into microbial behavior. In particular emerging single-cell technologies, image-based microfluidics lab-on-chip systems generate insights with spatio-temporal resolution, which are inaccessible with conventional tools. This review reports recent developments and applications of microfluidic single-cell cultivation technology, highlighting fields of broad interest such as growth, gene expression and antibiotic resistance and susceptibility. Combining advanced microfluidic single-cell cultivation technology for environmental control with automated time-lapse imaging as well as smart computational image analysis offers tremendous potential for novel investigation at the single-cell level. We propose on-chip control of parameters like temperature, gas supply, pressure or a change in cultivation mode providing a versatile technology platform to mimic more complex and natural habitats. Digital analysis of the acquired images is a requirement for the extraction of biological knowledge and statistically reliable results demand for robust and automated solutions. Focusing on microbial cultivations, we compare prominent software systems that emerged during the last decade, discussing their applicability, opportunities and limitations. Next-generation microfluidic devices with a high degree of environmental control combined with time-lapse imaging and automated image analysis will be highly inspiring and beneficial for fruitful interdisciplinary cooperation between microbiologists and microfluidic engineers and image analysts in the field of microbial single-cell analysis.     

Simultaneous analysis of microbial identity and function using NanoSIMS

Identifying the function of uncultured microbes in their environments today remains one of the main challenges for microbial ecologists. In this article, we describe a new method allowing simultaneous analysis of microbial identity and function. This method is based on the visualization of oligonucleotide probe-conferred hybridization signal in single microbial cells and isotopic measurement using high-resolution ion microprobe (NanoSIMS). In order to characterize the potential of the method, an oligonucleotide containing iodized cytidine was hybridized on fixed cells of Escherichia coli cultured on media containing different levels of ¹³C or ¹⁵N. Iodine signals could clearly be localized on targeted cells and the isotopic enrichment could be monitored at the single-cell level. The applicability of this new technique to the study of in situ ecophysiology of uncultured microorganisms within complex microbial communities is illustrated.     

Linking microbial phylogeny to metabolic activity at the single-cell level by using enhanced element labeling-catalyzed reporter deposition fluorescence in situ hybridization (EL-FISH) and NanoSIMS

To examine phylogenetic identity and metabolic activity of individual cells in complex microbial communities, we developed a method which combines rRNA-based in situ hybridization with stable isotope imaging based on nanometer-scale secondary-ion mass spectrometry (NanoSIMS). Fluorine or bromine atoms were introduced into cells via 16S rRNA-targeted probes, which enabled phylogenetic identification of individual cells by NanoSIMS imaging. To overcome the natural fluorine and bromine backgrounds, we modified the current catalyzed reporter deposition fluorescence in situ hybridization (FISH) technique by using halogen-containing fluorescently labeled tyramides as substrates for the enzymatic tyramide deposition. Thereby, we obtained an enhanced element labeling of microbial cells by FISH (EL-FISH). The relative cellular abundance of fluorine or bromine after EL-FISH exceeded natural background concentrations by up to 180-fold and allowed us to distinguish target from non-target cells in NanoSIMS fluorine or bromine images. The method was optimized on single cells of axenic Escherichia coli and Vibrio cholerae cultures. EL-FISH/NanoSIMS was then applied to study interrelationships in a dual-species consortium consisting of a filamentous cyanobacterium and a heterotrophic alphaproteobacterium. We also evaluated the method on complex microbial aggregates obtained from human oral biofilms. In both samples, we found evidence for metabolic interactions by visualizing the fate of substrates labeled with (13)C-carbon and (15)N-nitrogen, while individual cells were identified simultaneously by halogen labeling via EL-FISH. Our novel approach will facilitate further studies of the ecophysiology of known and uncultured microorganisms in complex environments and communities.     

Metabolomics methods for the synthetic biology of secondary metabolism

Many microbial secondary metabolites are of high biotechnological value for medicine, agriculture, and the food industry. Bacterial genome mining has revealed numerous novel secondary metabolite biosynthetic gene clusters, which encode the potential to synthesize a large diversity of compounds that have never been observed before. The stimulation or "awakening" of this cryptic microbial secondary metabolism has naturally attracted the attention of synthetic microbiologists, who exploit recent advances in DNA sequencing and synthesis to achieve unprecedented control over metabolic pathways. One of the indispensable tools in the synthetic biology toolbox is metabolomics, the global quantification of small biomolecules. This review illustrates the pivotal role of metabolomics for the synthetic microbiology of secondary metabolism, including its crucial role in novel compound discovery in microbes, the examination of side products of engineered metabolic pathways, as well as the identification of major bottlenecks for the overproduction of compounds of interest, especially in combination with metabolic modeling. We conclude by highlighting remaining challenges and recent technological advances that will drive metabolomics towards fulfilling its potential as a cornerstone technology of synthetic microbiology.     

Respiration response imaging for real-time detection of microbial function at the single-cell level

The ability to detect specific functions of uncultured microbial cells in complex natural communities remains one of the most difficult tasks of environmental microbiology. Here we present respiration response imaging (RRI) as a novel fluorescence microscopy-based approach for the identification of microbial function, such as the ability to use C(1) substrates, at a single-cell level. We demonstrate that RRI could be used for the investigation of heterogeneity of a single microbial population or for functional profiling of microbial cells from complex environmental communities, such as freshwater lake sediment.     

Gold-ISH: A nano-size gold particle-based phylogenetic identification compatible with NanoSIMS

The linkage of microbial phylogenetic and metabolic analyses by combining ion imaging analysis with nano-scale secondary ion mass spectrometry (NanoSIMS) has become a powerful means of exploring the metabolic functions of environmental microorganisms. Phylogenetic identification using NanoSIMS typically involves probing by horseradish peroxidase-mediated deposition of halogenated fluorescent tyramides, which permits highly sensitive detection of specific microbial cells. However, the methods require permeabilization of target microbial cells and inactivation of endogenous peroxidase activity, and the use of halogens as the target atom is limited because of heavy background signals due to the presence of halogenated minerals in soil and sediment samples. Here, we present “Gold-ISH,” a non-halogen phylogenetic probing method in which oligonucleotide probes are directly labeled with Undecagold, an ultra-small gold nanoparticle. Undecagold-labeled probes were generated using a thiol-maleimide chemical coupling reaction and they were purified by polyacrylamide gel electrophoresis. The method was optimized with a mixture of axenic ¹³C-labeled Escherichia coli and Methanococcus maripaludis cells and applied to investigate sulfate-reducing bacteria in an anaerobic sludge sample. Clear gold-derived target signals were detected in microbial cells using NanoSIMS ion imaging. It was concluded that Gold-ISH can be a useful approach for metabolic studies of naturally occurring microbial ecosystems using NanoSIMS.     

Search and discovery strategies for biotechnology: the paradigm shift.

Profound changes are occurring in the strategies that biotechnology-based industries are deploying in the search for exploitable biology and to discover new products and develop new or improved processes. The advances that have been made in the past decade in areas such as combinatorial chemistry, combinatorial biosynthesis, metabolic pathway engineering, gene shuffling, and directed evolution of proteins have caused some companies to consider withdrawing from natural product screening. In this review we examine the paradigm shift from traditional biology to bioinformatics that is revolutionizing exploitable biology. We conclude that the reinvigorated means of detecting novel organisms, novel chemical structures, and novel biocatalytic activities will ensure that natural products will continue to be a primary resource for biotechnology. The paradigm shift has been driven by a convergence of complementary technologies, exemplified by DNA sequencing and amplification, genome sequencing and annotation, proteome analysis, and phenotypic inventorying, resulting in the establishment of huge databases that can be mined in order to generate useful knowledge such as the identity and characterization of organisms and the identity of biotechnology targets. Concurrently there have been major advances in understanding the extent of microbial diversity, how uncultured organisms might be grown, and how expression of the metabolic potential of microorganisms can be maximized. The integration of information from complementary databases presents a significant challenge. Such integration should facilitate answers to complex questions involving sequence, biochemical, physiological, taxonomic, and ecological information of the sort posed in exploitable biology. The paradigm shift which we discuss is not absolute in the sense that it will replace established microbiology; rather, it reinforces our view that innovative microbiology is essential for releasing the potential of microbial diversity for biotechnology penetration throughout industry. Various of these issues are considered with reference to deep-sea microbiology and biotechnology.     

未培养微生物研究:方法、机遇与挑战

自然界中绝大部分的微生物仍是未培养的,称之为未培养微生物或微生物"暗物质"。对其进行研究不仅有助于认识微生物多样性及其代谢特征,加深对环境中微生物参与的生态学过程的理解,还有利于重构生命之树,揭示微生物的进化历程,具有重要的科学意义。同时未培养微生物是发现新基因资源和新活性物质的巨大宝库。随着现代分子生物学研究方法和培养技术的成熟和完善,从环境中直接破译未培养微生物的遗传信息,并实现培养逐渐成为可能。本文主要介绍了基于宏基因组技术和单细胞基因组技术或两者结合运用,研究环境中未培养微生物的主要方法和挑战,总结分析了目前已经解析的未培养微生物的主要类群,并对未来研究的机遇进行了展望。     

Relationship between fitness and heterogeneity in exponentially growing microbial populations

Despite major environmental and genetic differences, microbial metabolic networks are known to generate consistent physiological outcomes across vastly different organisms. This remarkable robustness suggests that, at least in bacteria, metabolic activity may be guided by universal principles. The constrained optimization of evolutionarily motivated objective functions, such as the growth rate, has emerged as the key theoretical assumption for the study of bacterial metabolism. While conceptually and practically useful in many situations, the idea that certain functions are optimized is hard to validate in data. Moreover, it is not always clear how optimality can be reconciled with the high degree of single-cell variability observed in experiments within microbial populations. To shed light on these issues, we develop an inverse modeling framework that connects the fitness of a population of cells (represented by the mean single-cell growth rate) to the underlying metabolic variability through the maximum entropy inference of the distribution of metabolic phenotypes from data. While no clear objective function emerges, we find that, as the medium gets richer, the fitness and inferred variability for Escherichia coli populations follow and slowly approach the theoretically optimal bound defined by minimal reduction of variability at given fitness. These results suggest that bacterial metabolism may be crucially shaped by a population-level trade-off between growth and heterogeneity.     

Engineering stilbene metabolic pathways in microbial cells

Numerous in vitro and in vivo studies on biological activities of phytostilbenes have brought to the fore the remarkable properties of these compounds and their derivatives, making them a top storyline in natural product research fields. However, getting stilbenes in sufficient amounts for routine biological activity studies and make them available for pharmaceutical and/or nutraceutical industry applications, is hampered by the difficulty to source them through synthetic chemistry-based pathways or extraction from the native plants. Hence, microbial cell cultures have rapidly became potent workhorse factories for stilbene production. In this review, we present the combined efforts made during the past 15?years to engineer stilbene metabolic pathways in microbial cells, mainly the Saccharomyces cerevisiae baker yeast, the Escherichia coli and the Corynebacterium glutamicum bacteria. Rationalized approaches to the heterologous expression of the partial or the entire stilbene biosynthetic routes are presented to allow the identification and/or bypassing of the major bottlenecks in the endogenous microbial cell metabolism as well as potential regulations of the genes involved in these metabolic pathways. The contributions of bioinformatics to synthetic biology are developed to highlight their tremendous help in predicting which target genes are likely to be up-regulated or deleted for controlling the dynamics of precursor flows in the tailored microbial cells. Further insight is given to the metabolic engineering of microbial cells with “decorating” enzymes, such as methyl and glycosyltransferases or hydroxylases, which can act sequentially on the stilbene core structure. Altogether, the cellular optimization of stilbene biosynthetic pathways integrating more and more complex constructs up to twelve genetic modifications has led to stilbene titers ranging from hundreds of milligrams to the gram-scale yields from various carbon sources. Through this review, the microbial production of stilbenes is analyzed, stressing both the engineering dynamic regulation of biosynthetic pathways and the endogenous control of stilbene precursors.     

基于比较基因组学重构细菌的代谢途径和调控网络

微生物基因组学的迅速发展为从功能基因与蛋白、网络及其调控等不同的角度,全面理解与认识微生物的代谢过程、构建细胞工厂,提供了丰富的背景信息。基于基因组序列进行代谢网络重构,有助于发现新的代谢功能基因、调控元件、甚至新的代谢途径,从而优化设计细胞内从原料到产品的生物合成路线。然而,目前公共数据库平台中代谢途径基因的功能注释,许多是错误或不完整的。以下着重介绍了近年来出现的一些用于代谢途径和调控网络重构的新型比较基因组学技术,并以近期的丙酮丁醇梭菌中木糖代谢途径的重构工作为例来说明其应用。     

Fluorinated colloidal gold immunolabels for imaging select proteins in parallel with lipids using high-resolution secondary ion mass spectrometry

The local abundance of specific lipid species near a membrane protein is hypothesized to influence the protein's activity. The ability to simultaneously image the distributions of specific protein and lipid species in the cell membrane would facilitate testing these hypotheses. Recent advances in imaging the distribution of cell membrane lipids with mass spectrometry have created the desire for membrane protein probes that can be simultaneously imaged with isotope labeled lipids. Such probes would enable conclusive tests to determine whether specific proteins colocalize with particular lipid species. Here, we describe the development of fluorine-functionalized colloidal gold immunolabels that facilitate the detection and imaging of specific proteins in parallel with lipids in the plasma membrane using high-resolution SIMS performed with a NanoSIMS. First, we developed a method to functionalize colloidal gold nanoparticles with a partially fluorinated mixed monolayer that permitted NanoSIMS detection and rendered the functionalized nanoparticles dispersible in aqueous buffer. Then, to allow for selective protein labeling, we attached the fluorinated colloidal gold nanoparticles to the nonbinding portion of antibodies. By combining these functionalized immunolabels with metabolic incorporation of stable isotopes, we demonstrate that influenza hemagglutinin and cellular lipids can be imaged in parallel using NanoSIMS. These labels enable a general approach to simultaneously imaging specific proteins and lipids with high sensitivity and lateral resolution, which may be used to evaluate predictions of protein colocalization with specific lipid species.     

Genome-scale metabolic model in guiding metabolic engineering of microbial improvement

In the past few decades, despite all the significant achievements in industrial microbial improvement, the approaches of traditional random mutation and selection as well as the rational metabolic engineering based on the local knowledge cannot meet today’s needs. With rapid reconstructions and accurate in silico simulations, genome-scale metabolic model (GSMM) has become an indispensable tool to study the microbial metabolism and design strain improvements. In this review, we highlight the application of GSMM in guiding microbial improvements focusing on a systematic strategy and its achievements in different industrial fields. This strategy includes a repetitive process with four steps: essential data acquisition, GSMM reconstruction, constraints-based optimizing simulation, and experimental validation, in which the second and third steps are the centerpiece. The achievements presented here belong to different industrial application fields, including food and nutrients, biopharmaceuticals, biopolymers, microbial biofuel, and bioremediation. This strategy and its achievements demonstrate a momentous guidance of GSMM for metabolic engineering breeding of industrial microbes. More efforts are required to extend this kind of study in the meantime.