Using flow cytometry to quantify microbial heterogeneity

Flow cytometry is a powerful technique for the study of single cells, and thus it is of particular utility in the study of heterogeneity in microbial populations. This review seeks to highlight the role of flow cytometric analyses in studies of microbial heterogeneity, drawing wherever possible on recently published research articles. Whilst microbial heterogeneity is well documented in both natural and laboratory environments, the underlying causes are less well understood. Possible sources for the heterogeneity that is observed in microbial systems are discussed, together with the flow cytometric tools that aid its study. The role of flow cytometry in molecular biology is discussed with reference to gene reporter systems, which enable heterogeneity of gene expression to be monitored. With the recent sequencing of a variety of microbial genomes, it is anticipated that flow cytometry will have an increasing role to play in studying the effects of gene expression and mutation on heterogeneity, and in resolving the interactions of genetics and physiology.     

Use of flow cytometric methods for single-cell analysis in environmental microbiology

Flow cytometry (FCM) is emerging as an important tool in environmental microbiology. Although flow cytometry applications have to date largely been restricted to certain specialized fields of microbiology, such as the bacterial cell cycle and marine phytoplankton communities, technical advances in instrumentation and methodology are leading to its increased popularity and extending its range of applications. Here we will focus on a number of recent flow cytometry developments important for addressing questions in environmental microbiology. These include (i) the study of microbial physiology under environmentally relevant conditions, (ii) new methods to identify active microbial populations and to isolate previously uncultured microorganisms, and (iii) the development of high-throughput autofluorescence bioreporter assays.     

Microbial biosensors.

A microbial biosensor consists of a transducer in conjunction with immobilised viable or non-viable microbial cells. Non-viable cells obtained after permeabilisation or whole cells containing periplasmic enzymes have mostly been used as an economical substitute for enzymes. Viable cells make use of the respiratory and metabolic functions of the cell, the analyte to be monitored being either a substrate or an inhibitor of these processes. Bioluminescence-based microbial biosensors have also been developed using genetically engineered microorganisms constructed by fusing the lux gene with an inducible gene promoter for toxicity and bioavailability testing. In this review, some of the recent trends in microbial biosensors with reference to the advantages and limitations are been discussed. Some of the recent applications of microbial biosensors in environmental monitoring and for use in food, fermentation and allied fields have been reviewed. Prospective future microbial biosensor designs have also been identified.     

Flow cytometry and bacterial pathogenesis

Our understanding of microbial adaptations to diverse and threatening environments is limited by the assumption that the behavior of individual bacteria can be accurately determined by measuring the behavior of populations. Recent advances in gene expression reporter systems, fluorescence microscopy and flow cytometry allow microbiologists to explore the complex interactions between bacteria and their environment with single cell resolution. The application of these technologies has been particularly useful in systems, such as host-pathogen interactions, where genetic analysis is often cumbersome. Recently, flow cytometry is increasingly being applied to study host-pathogen interactions.     

Quantification of the presence and activity of specific microorganisms in nature

Traditional techniques for assessment of microbial numbers and activity generally lack the specificity required for risk assessment following environmental release of genetically engineered microbial inocula. Immunological and molecular-based techniques, such as DNA probing and genetic tagging, were initially used to determine the presence or absence of microorganisms in environmental samples. Increasingly they are being developed for quantification of populations of specific organisms, either indigenous or introduced, in the environment. In addition, they are being used to quantify the activity of particular organisms or groups of organisms, greatly extending the range of techniques available to the microbial ecologist. This article reviews the use of traditional techniques for the quantification of microbial population size and activity and the application of molecular techniques, including DNA probing, genetic marking, use of fluorescent probes, and quantitative PCR, in combination with advanced cell detection techniques such as confocal laser scanning microscopy and flow cytometry.     

Flow cytometry in radiation research: past, present and future

Flow cytometry is an invaluable technique in research and clinical laboratories. The technique has been applied extensively to many areas of radiation research at both the experimental and clinical level. In the past few years, there has been a significant increase in the capabilities of modern flow cytometers to undertake multicolor analysis in a user-friendly manner. The developments in cytometric technology are being matched by the rapid development of new reagents, new fluorochromes and new platforms such as bead arrays. These developments are facilitating many new applications in both basic and clinical research that have relevance for many fields of biology, including radiation research. This review provides a historical overview of the application of flow cytometry to radiobiology and an update on how technology and reagents have changed and cites examples of new applications relevant to radiation researchers. In addition, some entirely new flow instrumentation is currently under development that has significant potential for applications in radiation research.     

Phospholipid fatty acid profiling of microbial communities–a review of interpretations and recent applications

Profiling of microbial communities in environmental samples often utilizes phospholipid fatty acid (PLFA) analysis. This method has been used for more than 35 years and is still popular as a means to characterize microbial communities in a diverse range of environmental matrices. This review examines the various recent applications of PLFA analysis in environmental studies with specific reference to the interpretation of the PLFA results. It is evident that interpretations of PLFA results do not always correlate between different investigations. These discrepancies in interpretation and their subsequent applications to environmental studies are discussed. However, in spite of limitations to the manner in which PLFA data are applied, the approach remains one with great potential for improving our understanding of the relationship between microbial populations and the environment. This review highlights the caveats and provides suggestions towards the practicable application of PLFA data interpretation.     

Evolution of beetle bioluminescence: the origin of beetle luciferin

Bioluminescence, the conversion of chemical energy into light in living organisms, is dependent on two principal components, an enzyme luciferase and the substrate luciferin. In beetles, the enzyme luciferase has been extensively studied, with signi?cant enzymological, sequence and structural data now available. Furthermore, the enzyme has been employed in a remarkable number of important applications, from microbial detection and medical imaging to GM gene expression studies. However, there is little information regarding the biosynthesis of beetle luciferin, and here we review the literature and speculate as to its evolutionary origins. Luciferin consists of a benzothiazole moiety attached to a thiazole carboxylic acid moiety, the former being rarely observed in nature but the latter being observed in a broad range of biologically derived molecules. Benzothiazoles are, however, observed in melanogenesis and we speculate as to whether this may be relevant to the understanding of luciferin biosynthesis in beetles. This review examines recent novel insights into beetle luciferin recycling and we assess a range of possible biosynthetic mechanisms. Copyright © 2004 John Wiley & Sons, Ltd.     

Microbial flow cytometry: An ideal tool for prospective antimicrobial drug development

Flow cytometry has tremendous applications in qualitative and quantitative analysis of characteristics of single microbial cells. Its ability to efficiently discriminate and quantify multiple parameters of microbial cells has made it a powerful tool to catalog the mechanism of action of antimicrobial peptides (AMPs) on target cells. Here, we provide a comprehensive overview and strategic design on how multi-parametric analysis of flow cytometry is unsurpassed in studying the antimicrobial process of AMPs in an accurate and rapid way. This strategy provides a conceptual framework for understanding distinct classes of AMPs and getting insights into antimicrobial mechanisms of novel AMPs.     

Analysis of free intracellular calcium by flow cytometry: multiparameter and pharmacologic applications

Flow cytometry offers numerous advantages over traditional techniques for measuring intracellular Ca(2+) in lymphoid and nonlymphoid cells. In particular, the heterogeneity of cell responses can be defined by flow cytometry, and multiparameter analyses permit the determination of intracellular Ca(2+) in surface-marker-defined target cells as well as correlation of changes in Ca(2+) with other biochemical markers, including ligand binding. This article presents several established methods for measuring intracellular Ca(2+) by flow cytometry in lymphoid and nonlymphoid cells. Examples are provided for determination of Ca(2+) in human peripheral blood leukocytes and two human epithelial cell lines grown in monolayer. In addition, applications are reviewed or presented for correlating changes in intracellular Ca(2+) with other cell parameters, including cell cycle analysis, changes in cell membrane integrity, and the induction of apoptosis markers. Finally, a number of novel sample handling capabilities useful for performing kinetic analyses of Ca(2+) changes by flow cytometry are now available and one application is presented which is finding utility in pharmacologic studies.     

Quantitative real-time PCR approaches for microbial community studies in wastewater treatment systems: Applications and considerations

Quantitative real-time PCR (qPCR) has been widely used in recent environmental microbial ecology studies as a tool for detecting and quantifying microorganisms of interest, which aids in better understandings of the complexity of wastewater microbial communities. Although qPCR can be used to provide more specific and accurate quantification than other molecular techniques, it does have limitations that must be considered when applying it in practice. This article reviews the principle of qPCR quantification and its applications to microbial ecology studies in various wastewater treatment environments. Here we also address several limitations of qPCR-based approaches that can affect the validity of quantification data: template nucleic acid quality, nucleic acid extraction efficiency, specificity of group-specific primers and probes, amplification of nonviable DNA, gene copy number variation, and limited number of sequences in the database. Even with such limitations, qPCR is reportedly among the best methods for quantitatively investigating environmental microbial communities. The application of qPCR is and will continue to be increasingly common in studies of wastewater treatment systems. To obtain reliable analyses, however, the limitations that have often been overlooked must be carefully considered when interpreting the results.     

含Dsred类似生色团的红色荧光蛋白的发展及应用

自从绿色荧光蛋白(GFP)被发现以来,荧光蛋白在生物医学领域已经成为一种重要的荧光成像工具．随着红色荧光蛋白DsRed的出现,各种优化的DsRed突变体和远红荧光蛋白也不断涌现．其中荧光蛋白生色团的形成机制对改建更优的荧光蛋白变种影响很大,对于红色荧光蛋白而言,大多数的红色荧光蛋白的生色团类型为DsRed类似生色团,在此基础上又出现了Far-red DsRed类似生色团．目前,含DsRed类似生色团的荧光蛋白主要有单体红色荧光蛋白、光转换荧光蛋白、斯托克斯红移蛋白、荧光计时器等．这些优化的荧光蛋白作为分子探针可以实现对活细胞、细胞器或胞内分子的时空标记和追踪,已经在生物工程学、细胞生物学、基础医学领域得到广泛应用．本文综述了含DsRed类似生色团的荧光蛋白的研究进展及其应用,以及由此发展起来的远红荧光蛋白在活体显微成像技术中的应用,并展望了荧光探针技术研究的新方向．     

Application of flow cytometry to algal physiology and phytoplankton ecology

Abstract Flow cytometry, originally developed for biomedical purposes, is now being put to extensive use in the study of microalgae. Successful applications to date include studies of cytochemistry, cell cycle dynamics and phytoplankton ecology. This review summarises recent work in a rapidly expanding field of research, and highlights those areas in which significant progress can be expected when the necessary equipment becomes more readily available.     

元基因组文库分析技术研究进展

随着新的分析技术的不断出现和成熟,促进了微生物分子生态学及相关学科的诞生和迅速发展。其中,元基因组文库分析技术即是近年来微生物分子生态学研究领域兴起的一种新的分析技术。就元基因组分析技术诞生的背景及该技术的原理进行了讨论,着重阐述了元基因组文库分析技术在寻找新基因、开发新的生物活性物质、研究群落中微生物多样性、人类元基因组测序等方面的应用。另外,归纳总结了目前国际上常用的诸如PCR为基础的筛选、荧光原位杂交（fluorescent in situ hybridization,FISH）、底物诱导的基因表达筛选（substrate induced gene expression screening,SIGEX）、基因芯片等元基因组文库筛选方法,并就不同方法的优缺点进行了分析和讨论,指出了目前元基因组文库分析技术存在的主要问题并对今后该技术的发展进行了展望。     

Advances and prospects of rhodopsin-based optogenetics in plant research

Microbial rhodopsins have advanced optogenetics since the discovery of channelrhodopsins almost two decades ago. During this time an abundance of microbial rhodopsins has been discovered, engineered, and improved for studies in neuroscience and other animal research fields. Optogenetic applications in plant research, however, lagged largely behind. Starting with light-regulated gene expression, optogenetics has slowly expanded into plant research. The recently established all-trans retinal production in plants now enables the use of many microbial opsins, bringing extra opportunities to plant research. In this review, we summarize the recent advances of rhodopsin-based plant optogenetics and provide a perspective for future use, combined with fluorescent sensors to monitor physiological parameters.

One-sentence summary: The current status in rhodopsin-based optogenetics in plants is reviewed and the potential applications of optogenetics combined with fluorescence sensors are discussed.     

Bacterial alkaline proteases: molecular approaches and industrial applications

Proteolytic enzymes are ubiquitous in occurrence, being found in all living organisms, and are essential for cell growth and differentiation. The extracellular proteases are of commercial value and find multiple applications in various industrial sectors. Although there are many microbial sources available for producing proteases, only a few are recognized as commercial producers. A good number of bacterial alkaline proteases are commercially available, such as subtilisin Carlsberg, subtilisin BPN' and Savinase, with their major application as detergent enzymes. However, mutations have led to newer protease preparations with improved catalytic efficiency and better stability towards temperature, oxidizing agents and changing wash conditions. Many newer preparations, such as Durazym, Maxapem and Purafect, have been produced, using techniques of site-directed mutagenesis and/or random mutagenesis. Directed evolution has also paved the way to a great variety of subtilisin variants with better specificities and stability. Molecular imprinting through conditional lyophilization is coming up to match molecular approaches in protein engineering. There are many possibilities for modifying biocatalysts through molecular approaches. However, the search for microbial sources of novel alkaline proteases in natural diversity through the "metagenome" approach is targeting a hitherto undiscovered wealth of molecular diversity. This fascinating development will allow the biotechnological exploitation of uncultured microorganisms, which by far outnumber the species accessible by cultivation, regardless of the habitat. In this review, we discuss the types and sources of proteases, protease yield-improvement methods, the use of new methods for developing novel proteases and applications of alkaline proteases in industrial sectors, with an overview on the use of alkaline proteases in the detergent industry.     

Flow cytometry and cell sorting of heterogeneous microbial populations: the importance of single-cell analyses.

The most fundamental questions such as whether a cell is alive, in the sense of being able to divide or to form a colony, may sometimes be very hard to answer, since even axenic microbial cultures are extremely heterogeneous. Analyses that seek to correlate such things as viability, which is a property of an individual cell, with macroscopic measurements of culture variables such as ATP content, respiratory activity, and so on, must inevitably fail. It is therefore necessary to make physiological measurements on individual cells. Flow cytometry is such a technique, which allows one to analyze cells rapidly and individually and permits the quantitative analysis of microbial heterogeneity. It therefore offers many advantages over conventional measurements for both routine and more exploratory analyses of microbial properties. While the technique has been widely applied to the study of mammalian cells, is use in microbiology has until recently been much more limited, largely because of the smaller size of microbes and the consequently smaller optical signals obtainable from them. Since these technical barriers no longer hold, flow cytometry with appropriate stains has been used for the rapid discrimination and identification of microbial cells, for the rapid assessment of viability and of the heterogeneous distributions of a wealth of other more detailed physiological properties, for the analysis of antimicrobial drug-cell interactions, and for the isolation of high-yielding strains of biotechnological interest. Flow cytometric analyses provide an abundance of multivariate data, and special methods have been devised to exploit these. Ongoing advances mean that modern flow cytometers may now be used by nonspecialists to effect a renaissance in our understanding of microbial heterogeneity.