Description
ABSTRACT
System approaches to elucidate ecosystem functioning constitute an emerging area of research within microbial ecology. Such approaches aim at investigating all levels of biological information (DNA, RNA, proteins and metabolites) to capture the functional interactions occurring in a given ecosystem and track down characteristics that could not be accessed by the study of isolated components. In this context, the study of the proteins collectively expressed by all the microorganisms present within an ecosystem (metaproteomics) is not only crucial but can also provide insights into microbial functionality. Overall, the success of metaproteomics is closely linked to metagenomics, and with the exponential increase in the availability of metagenome sequences, this field of research is starting to experience generation of an overwhelming amount of data, which requires systematic analysis. Metaproteomics has been employed in very diverse environments, and this review discusses the recent advances achieved in the context of human biology, soil, marine and freshwater environments as well as natural and bioengineered systems.
Keyword: environmental proteomics, ecosystem function, marine and freshwater environment, soil, aquatic environment.
TABLE OF CONTENTS
TITLE PAGE
APPROVAL PAGE
DEDICATION
ACKNOWLEDGEMENT
ABSTRACT
TABLE OF CONTENT
CHAPTER ONE
- INTRODUCTION
- BACKGROUND OF THE STUDY
- PROBLEM STATEMENT
- AIM OF THE STUDY
- SIGNIFICANCE OF THE STUDY
- SCOPE OF THE STUDY
CHAPTER TWO
LITERATURE REVIEW
2.0 LITERATURE REVIEW
2.1 REVIEW OF THE STUDY
2.2 AN OVERVIEW OF METAPROTEOMICS
2.3 PROTEOMIC OF MICROBIAL COMMUNITY
2.4 METAPROTEOMICS AND THE HUMAN INTESTINAL MICROBIOME
2.5 METAPROTEOMICS IN ENVIRONMENTAL MICROBIOME STUDIES
CHAPTER THREE
3.0 METHODS
3.1 PROTEIN EXPRESSION IN THE HUMAN MICROBIOME
3.2 PROTEIN EXPRESSION IN SOIL
3.3 PROTEIN EXPRESSION IN MARINE AND FRESHWATER
CHAPTER FOUR
4.0 RESULT
CHAPTER FIVE
- CONCLUSIONS
- RECOMMENDATION
5.3 REFERENCES
CHAPTER ONE
1.0 INTRODUCTION
1.1 BACKGROUND OF THE STUDY
Microorganisms occupy virtually every habitat on our planet, and their activities largely determine the environmental conditions of today’s world. Indeed, microorganisms are heavily involved in biogeochemistry, ensuring the recycling of elements such as carbon and nitrogen (Madsen, 2011). In addition, microorganisms are extensively used to degrade anthropogenic waste prior to release into the environment (Hussain et al., 2010). In their natural habitat, microorganisms coexist in mixed communities, the complexity of which is specific to each environment, for example from six estimated individual taxa for an acid mine drainage biofilm (Ram et al., 2005), up to 106 estimated taxa per gram of soil. As most of the microorganisms present in the environment have not been cultured, their investigation requires the use of molecular techniques that bypass the traditional isolation and cultivation of individual species (Amann et al., 1995). Moreover, even when isolation is possible, a single species removed from its natural environment might not necessarily display the same characteristics under laboratory conditions as it does within its ecological niche. Therefore, the study of mixed microbial communities within their natural environment is key to the investigation of the diverse roles played by microorganisms, and to the identification of the microbial potential for biotechnological application, including but not limited to: pharmaceutical, diagnostics, waste treatment, bioremediation and renewable energy generation. An emerging field of research in microbial ecology encompasses system approaches, whereby all levels of biological information are investigated (DNA, RNA, proteins and metabolites) to capture the functional interactions occurring in a given ecosystem and identify characteristics that could not be accessed by the study of isolated components (Röling et al., 2010). Recent technological advances, including the development of high-throughput ‘omics’ methods, make such system approaches possible, where mixed microbial communities are viewed as one meta-organism. Metaproteomics are employed to determine respectively the DNA sequences of the meta-organism under study, the collectively transcribed RNA, the translated proteins and the metabolites resulting from cellular processes. All of the generated data can then be used to identify the metabolic pathways and cellular processes at work within an ecosystem.
1.2 Problem statement
Aquatic ecosystems support a substantial source of the earth’s biological diversity. They are an essential reservoir and share an enormous proportion of earth’s biological productivity. Both aquatic resources and its biodiversity are interrelated to each other and they perform a myriad of functions and are valuable and essential for the sustainability of biotic communities. Aquatic biodiversity in both freshwater and marine environments are under continuous decline because of overexploitation of species, introduced exotic plant or animal, pollution sources from cities, industries and agricultural zones, loss and changes in ecological niche. Their conservation and management in the form of bio reserve points and bioregional management and worldwide monitoring are needed for the protection of the aquatic biodiversity. This study is presenting information on biodiversity in aquatic habitats and their resources, in marine and fresh water ecosystems, their importance conservation and restoration mechanisms using metaproteomics methods.
1.3 Aim of the study
The main aim of the study is to develop and apply metaproteomic platforms to better understand environmental systems and their robustness to change.
1.4 Scope of the study
The scope of this study covers studying the quantity of the molecular cellular components (e.g. DNA, mRNA, proteins and metabolites) in environmental samples can reveal significant information on ecosystem function.
1.5 Significance of the study
This study will provide a means we can gain a much more comprehensive understanding of environmental responses to processes such as climate change or pollution etc. The field known as Environmental Omics is mostly dominated by DNA sequencing. However, proteins are the functional entities in cells and therefore identifying and quantifying proteins gives a much more accurate insight into how ecosystems respond to environmental perturbations. Gaining a snapshot of ecosystem function through measuring the proteins in an environmental sample is referred to as metaproteomics.
The project would suit ideally a biosciences/chemistry graduate with a strong interest in novel and multidisciplinary approaches to environmental engineering, analytics, or mapping ecological responses using new, cutting edge technologies.
This study will serve as a training in quantitative analytical techniques e.g. high performance liquid, mass spectrometry. This includes experimental design and analysis of large amounts of data with bioinformatics pipelines. They will become experts in handling proteins and interpreting complex data. Metaproteomics is a tool that can be transferred to many different fields so flexibility within the project is high.
Metaproteomics is just one of many omics tools which are gaining momentum in their application in the laboratory and the field. The candidate will be developing skills that can be applied for research and development in many different field including the use of cutting edge analytical equipment.
CHAPTER FIVE
5.1 Conclusions
Overall, the field of metaproteomics is gaining momentum at an exponential rate within very diverse environments. An overview of selected studies from the ecosystems has been discussed. Advances in metaproteomics finally allow for the consideration of the integration of such data in system approaches. This was partly achieved in the aquatic environment where Lauro et al. (2011) combined metaproteomic and physicochemical data to describe the interaction between the microbial populations defining the biogeochemical cycles throughout a water column. Such an approach could feasibly be transferred to other environmental ecosystems. To date, the application of complex system approaches is still scarce and requires a coordinated experimental design that brings together expertise from each of the many technologies involved.
The technical limitations encountered throughout the metaproteomic workflow have, for the most part, been addressed in the ecosystems discussed in this work. However, it should be kept in mind that an exhaustive investigation of the entire metaproteome is unlikely due to the unfeasibility of developing a universal protein analysis protocol. Furthermore, it must be considered that a metaproteome may include intracellular, extracellular and membrane-bound proteins, and ideally, the three protein fractions should be analysed for each sample. When possible, opting for gel-free protein fractionation seems to lead to a higher level of protein identification when compared with gel-based methods. For example, when analysing the metaproteome of activated sludge, the use of 2-DGE resulted in the identification of 38 proteins, while the 2D-nano-LC method led to the identification of 5029 proteins (Williams et al. (2010)). In addition, it is now apparent that metaproteomic approaches benefit from the availability of relevant metagenomic data, either matched or unmatched. As a result of this combined protocol, a new difficulty is encountered regarding the analysis and interpretation of the vast quantity of data generated. A major hurdle in the utilization of metagenomic data, impacting directly on metaproteomics, has been recognized as the assembly and the annotation of the collected genomic fragments.
5.2 RECOMMENDATION
Future metaproteomic studies should aim to progress from proof of concept approaches to experimental designs leading to practical applications. For example, metaproteomic comparisons between healthy and diseased states within the human microbiome have yet to be carried out. Additionally, comparative in situ bioremediation investigations will need to be conducted to access the functional response of the natural mixed microbial communities to common pollutants. Furthermore, when investigating bioremediation processes, pollutants should be systematically monitored throughout the trials, to clarify the contaminant degradation status as a function of time. This in turn should allow for the proper assessment of the degrading abilities of the microbial communities investigated.
To conclude, the feasibility of metaproteomic studies has been successfully demonstrated in very diverse natural and engineered environments. However, only few studies to date employed this strategy to answer specific biological questions such as how complex communities define the biology of a given ecosystem (Mueller et al., 2010; Lauro et al., 2011), and it is now critical to move metaproteomics forward in order for this technology to achieve its full potential. To this end, future studies must be designed with an aim towards gaining some understanding of ecological concepts, and the data generated must be adequately analysed. In addition, attempts should be made to integrate such data in the context of system approaches to allow for the prediction of functional responses to environmental stimuli.
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