Comprehensive lecture notes for the Metagenomics module covered in MCB3026F. These notes cover all content taught in lectures as well as additional materials (powerpoints, textbooks) required to succeed. These notes were created by a student who achieved a distinction in this course.
Lecture 1: Introduction to metagenomics
Microorganisms functions
- microorganisms comprise the majority of life forms on Earth
- the diversity of microorganisms reflects a staggering array of functions, many of which are necessary for all other life to
exist as they drive most of the biogeochemical cycles on Earth
- microorganisms drive the following: most of the biochemical cycles on earth, process waste, promote growth and
reproduction of plants and animals and produce antibiotics, ferment food and maintain human health
- in earth science, a biogeochemical cycle or substance turnover or cycling of substances is a pathway by which a chemical
substance moves through biotic (biosphere) and abiotic (lithosphere, atmosphere and hydrosphere) compartments of Earth
The basic Carbon Cycle
- one of the biogeochemical cycles which involves all living organisms on Earth
- blue arrows indicate aerobic environments and red arrows indicate anaerobic environments
- atmospheric carbon dioxide can be fixed into organic matter by plants
- all living organisms respire by breaking down organic matter, converting it to CO2 and this is called respiration to
generate energy for the production of organic matter again
- in the anaerobic side, the atmospheric carbon dioxide is fixed into
organic matter and this is carried out primarily by microorganisms
- anaerobic respiration and fermentation are slowly carried out by
microorganisms where organic matter is converted back to CO2
- also have microorganisms able to convert CO2 to methane (occurs
in anaerobic environments deep in the sediment and at hydrothermal
vents)
- methane then is oxidized again by microorganisms and converted
back to CO2
The Nitrogen Cycle
- another biogeochemical cycle in which atmospheric nitrogen is slowly reduced by nitrogen-fixing bacteria; take
atmospheric nitrogen and convert it to ammonia
- ammonia then can be mobilized in the soil by nitrifying bacteria which convert it from this reduced form to an oxidized
form in the form of nitrates
- these nitrates are an important source of nitrogen for plants, herbivores consume the plants and then animals eat other
animals, so the nitrogen gets passed as organic nitrogen
- the waste of animals and plants puts ammonia back into the soil and
degradation of waste or organic matter by bacteria also recycles the
ammonia back into the soil
- the ammonia can also be converted back into atmospheric nitrogen by
denitrifying bacteria
- key aspects of the nitrogen cycle are carried out my microorganisms;
denitrifying bacteria, nitrifying bacteria and the nitrogen-fixing bacteria
are essential for the cycling of nitrogen throughout the biosphere of the
earth
Services provided by microorganisms
- microorganisms in the sea remove carbon dioxide from the atmosphere and produce oxygen
- the ozone later in the stratosphere, which protects the earth from harmful UV rays, was created by the reaction of oxygen
molecules produced on the earth by microbes
- the human body contains more bacteria cells than human cells and most of these live in the GIT
- certain bacteria convert dinitrogen gas to ammonia in special structures on the roots of leguminous plants
- soil bacteria are the source of most of the antibiotics and many other drugs used in medicine today
- microbes ferment many foods such as cheese, beer and wine
- microorganisms come in many shapes and sizes, get coccoid, rod-shaped, chains of rods, chains of coccoid and
filamentous bacteria
Culture vs diversity of microorganisms
- most microorganisms live in communities or assemblages of more than 1 species
,- they carry out their own functions and metabolically interact with other microorganisms
- the true magnitude of diversity within microbial communities only recently appreciated
- was late in coming because much of microbial research is based on culturing organisms in the lab, and it was discovered
only in the last few decades that most microorganisms do not grow under standard culture conditions
Diversity of Life on Earth
- described species over 1.5 million, over 30 million species predicted to exist, 1000s cultivated in the lab
- how do we characterize the genome of species we cannot cultivate?
- how can we determine whether the genes that are expressed in nature follow the same patterns as that in the lab?
- in nature, you have mixed cultures of bacteria that are competing for resources with each other, the waste products of one
group are generally nutrients of another group
- the abiotic conditions of microenvironments change constantly, it’s a very complicated environment compared to what
happens in the lab
- unsure how real lab understanding of microbes is in relation to what happens in the environment
Metagenomics
- also known as environmental genomics, ecogenomics and community genomics
- it is the study of genetic material recovered directly from environmental samples
Why it is revolutionary
- if you look at classical microbiology, take a sample from the environment, grow the bacteria on media, look for specific
colonies, then spread out and purify to get a pure culture of that a particular bacterium, then isolate the bacterium grow it
in liquid, isolate the genomic DNA and can look at it on agarose gel
- once we have the genomic DNA, we can amplify it using 16S ribosomal RNA primers and from there derive the DNA
sequence of the 16S ribosomal RNA genes from that particular bacterial species, compare that to other sequenced species in
the database
- are able to identify the bacterium isolated from the environment in terms of its taxonomy, its genus and species
- if you want to study microorganisms in food samples with previous techniques, you need to use classical microbiology and
other molecular techniques, but these methods take time, are laborious and expensive
- moreover, you have to identify only a small part of the microorganisms because you only have the culturable bacteria and
the number of identifications is limited
- unsure how many of those have not been cultivated or cultured on the lab-based medium that actually occur in the
environment
What is Metagenomics
- traditional microbial genomics: use cultures to isolate microbe of interest; sequence the genome of one organism at a time
- metagenomics: extract sequence data from microbial communities as they exist in nature, bypass the need for culture
techniques, sequence all DNA in the sample and select DNA based on universal sequences
- metagenomics is a new area of microbial genomics that aims to sequence the full or partial genomes of all members of a
microbial community (consortium)
- the term microbial community refers to the complex microbial ecosystems that exist almost everywhere in nature
0 for example, a project in soil metagenomics might extract DNA from a soil sample in a corn field and attempt to sequence
all the DNA found in the sample
- by directly sequencing the DNA, researchers bypass the need to culture organisms
- since only a very small minority of single-cell organisms have been successfully cultured in the lab, metagenomics
becomes a very powerful technique for sequencing genes from organisms that cannot be cultured
- alternatively, homologous genes from a variety of organisms in the microbial community can be selectively sequenced via
PCR using tags that exist in known organisms
- have a freshwater environment and may be interested in the organisms
that exist on the s shoreline within the soil, take a sample of environment,
extract the environmental DNA
- if interested in the eukaryotes, can amplify the DNA using 18S ribosomal
RNA gene primers or that DNA gets sequences using high-throughput
sequencing technology; able to carry out a gene community analysis using
marker genes which are specific for different genera of bacteria
- can see where we have samples, which types of microorganisms exist in
these various sampling sites
- colour-code the different genera, can see their abundancies based on
various pie charts
- by pooling and studying the genomes of all the organisms in a community, all of the functions encoded in the
community’s DNA (metagenome) can be studied
, - has revolutionized microbiology because it offers a window on an enormous and previously unknown world of
microorganisms
What is Metagenomics
- some questions metagenomics may answer:
- are certain adaptations observable across environmental gradients?
- how do different species interact?
- can lateral gene transfer be detected?
- though metagenomics is still in its infancy, it holds great promise for answering fundamental questions about the structure
and dynamics of microbial communities
- metagenomics has already yielded several interesting discoveries
- for example: a study of ocean surface water uncovered a new class of rhodopsin genes in alpha-proteobacteria
- rhodopsin’s are proteins that response to light and serve a range of purposes in a wide variety of organisms, including the
detection of light in the retinal cells of humans and other animals
- further studies of the newly discovered bacterial rhodopsin’s found that the light response of the protein was tuned to
match that of the light that was reaching the alpha-proteobacteria at different ocean depths
- metagenomics can also be used to observe the interactions of individual members of microbial communities for example
fluorescence in situ hybridization was used to visualize archaeal and bacterial species in an ocean sediment community
Photoheterotrophy, biocatalysts and enzymes
- genetic information on potentially novel biocatalysts/enzymes, genomic linkages between function and phylogeny for
uncultured organisms, and evolutionary profiles of community function and structure
- can also be complemented with metatranscriptomic or metaproteomic analyses to describe expressed activities
- powerful tool for generating novel hypotheses of microbial function
- led to discoveries of proteorhodopsin-based photoheterotrophy and ammonia-oxidizing Archaea
- Photoheterotrophs generate ATP using light in one of two ways; they use a bacteriochlorophyll-based reaction center, or
they use a bacteriorhodopsin
- some organisms have purple-rhodopsin-based proton pumps that supplement their energy supply
- the archaeal version is called bacteriorhodopsin while the eubacterial version is called proteorhodopsin
- the pump consists of a single protein bound to Vitamin A derivative, retinal
- the pump may have accessory pigments associated with the protein
- when light is absorbed by the retinal molecule, the molecule isomerizes
- this drives the protein to change shape and pump a proton across the membrane
- the hydrogen ion gradient can then be used to generate ATP, transport solutes across the membrane or drive a flagellar
motor
- enzymes and catalysis both effect the rate of a rection
- all known enzymes are catalysis but not all catalysts are enzymes
- the difference between catalysts and enzymes is that enzymes are largely organic in nature and are biocatalysts while non-
enzymatic catalysts can be inorganic compounds
- neither catalysts nor enzymes are consumed in the reactions they catalyze
Applications
- discovery of novel enzymes and catalysts with industrial uses by screening thousands of microbial species simultaneously
- looks for pharmacologically interesting genes such as antibiotics that exist in organisms that have never been identified
before because they cannot be cultured in a lab
- the practical applications of metagenomics are vast
- the screening of genes from thousands of microbial species will undoubtedly yield many novel enzymes and catalysts with
industrial applications
- such approaches are becoming increasingly important as the number of unpatented variants of pre-existing industrial
enzymes diminishes
- for example, in the case of high-performance detergent bacillus protease, there are patents for substitutions along almost
all of its 275 amino acid chain
- the possibility of finding novel enzymes in metagenomics screens is high when one considers that samples of ocean water
from the Sargasso Sea yielded over one million new open reading frames
- metagenomics will also aid industry by circumventing the need to culture microbial organisms
Timeline of metagenomics
- started when the concept of cloning DNA directly from the environment was initially suggested
- was first implemented by Schmidt who constructed a lambda phage library from a seawater sample and screened it for
16S ribosomal RNA genes
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