Module 4: Microbial Ecosystems (Defining and Analysis) Flashcards
Life is a…
collection of ecosystems
Abiotic vs Biotic Factors
Biotic Factors = Living organisms
Abiotic Factors = Non-living factors
What are some examples of abiotic factors in an ecosystem?
Gases, minerals, water, sunlight, etc.
Ecosystems describe the __________ and __________ of materials between __________ and _____________
Ecosystems describe the interactions and exchange of materials between organisms and surrounding environment
What is the chain reaction following environmental change in ecosystems?
1) Environmental Change
2) Organisms react to the change
3) Organisms change (adapt) in response to the change
4) The response then impacts the environment and leads back to #1
Primary Producers
Organisms that utilize energy from the sun or chemical reactions to produce organic molecules from inorganic starting materials
Photosynthetic Primary Producers
Organisms that utilize photosynthesis; harness light energy from the sun to drive carbon fixation
Chemosynthetic Primary Producers
Organisms that utilize chemosynthesis; harness energy for organic molecule synthesis from chemical reactions
(light INDEPENDENT autotrophs)
What are the two types of primary producers?
1) Photosynthetic (photoautotrophs)
2) Chemosynthetic (chemoautotrophs)
What is an example of chemosynthesis?
Chemolithoautotrophic Bacteria
–> Oxidize hydrogensulfide to produce organic molecules
Reaction equation for chemolithoautotrophic bacteria that oxidize hydrogen sulfide:
CO2 + O2 + 4H2S
Consumers
“Heterotrophs”
== Organisms that ingest the stored nutrients and energy of other organisms
–> The consumption of other organisms is their source of energy and carbon!
Decomposers
Organisms that consume/breakdown dead organic matter and release simple organic products
What is the important of decomposers?
They recycle organic molecules back into the environment!
Functional Guilds
Groups of organisms that carry out similar processes
(Play a similar role in a given ecosystem)
Members of functional guilds do not…
do not have to be genetically related!
–> Guild classification is solely based upon functions and processes BUT we will often see that genetically related organisms end up in the same guilds
What are 2 examples of microbial guilds?
1) Anoxic Phototrophic Bacteria = Shared process of harvesting light energy without producing oxygen!
2) Thermophilic methanogens = Shared process of methane production!
Niche
The specific functional role of an organism within an ecosystem which includes an organism’s direct and indirect interactions with their physical habitat and co-existing organisms
What factors help define an organism’s niche?
The 1) Type, 2) Quantity, and 3) Quality of sustaining resources (that are available)
An organism’s niche tells us about…
How a given organism interacts with its environment (both abiotic and biotic)
How does evolutionary pressure impact niche?
Selection processes and competition results in specialization of organisms for their “prime” niche
== Ensures all possible niches are “occupied”
What is a major ecological rule regarding niches?
No 2 populations of organisms can occupy the same niche and co-exist sustainably
–> One population will always outcompete the other if they have identical niches
Populations with distinct niches can…
CO-EXIST
What is an example of organisms with distinct niches co-existing?
In the human gut:
1) Methanobacter smithii (M. smithii) –> Niche = methane producer
2) Escherichia coli (E. coli) –> Niche = fermenter
–> They coexist within the human gut, one does not outcompete the other because they aren’t “fighting” for the same resources due to their different functions!
BUT this does NOT mean that they don’t interact! We often see that co-existing organisms for mutually beneficial relationships! –> here M. smithii consumes the fermentation products of E. coli which in turn helps E. coli maintain a proper gradient for fermentation
Success of microbes in a given niche depends on…
Their ability to obtain and utilize available resources
Why is adaptability of greater importance to microbial survival?
Because their environment is more rapidly changing due to the often very small areas over which their optimal conditions exist (microenvironments)
Microenvironments
Specific regions of environmental conditions that exist over a very small distance!
What is the “issue” with microenvironments?
Conditions can change DRASTICALLY over a very small distance!
Microbial Microenvironments
A specific localized area within a larger environment that provides unique conditions for microbial growth
Although individual microbes may be specialized for a prime niche…
Their ultimate survival depends on their ability to ADAPT to change and exploit available resources (better than competing organisms)
What is the main method of adaptation exhibited by microbes?
GENE REGULATION
–> Allows microbes to modify their metabolism according to external conditions!
Based upon prevailing environmental conditions, we can predict…
Why? And what are the issues with this?
The types of microbes and metabolic systems present within a given habitat!
(Because there is some correlation between microbial taxonomy and metabolic capabilities)
–> Issue: This is not always perfect as certain metabolic processes are spread out across multiple phylogenies!
Biofilms
Structured communities of microbes on a surface that interact with and support each other
(And are held together by EPS)
What organisms can form biofilms?
1) Bacteria
2) Fungi
3) Protists
4) Archaea
What are biofilms held together by?
Extracellular Polysaccharides (EPS)
Most microbes on Earth exist in…
Why?
BIOFILMS
Provides advantages!
1) Increased access to nutrients
2) Protection from environmental stresses
(including antimicrobial agents!)
Process of Biofilm Formation:
1) Primary colonizer binds/adheres to surface
2) Adhered primary colonizer divides and forms a first layer/clump of cells (microcolony)
3) Microcolony begins secreting EPS
4) Secondary colonizers join the early biofilm and also begin secreting EPS
5) As more microbes add in and continue to divide the biofilm grows
6) Water-filled channels form within the EPS matrix
==Allows for transport of nutrients and waste through the biofilm
What does EPS do?
Extracellular Polysaccharides (EPS) form a gel-like matrix around microcolonies –> Leads to the development of biofilms
What is the importance of water-filled channels forming through a biofilm?
These channels allow for the flow of water, nutrients, and waste through the biofilm!
What did the E.coli K12 experiment test?
Tested whether EPS production was needed for biofilm formation
How was the E.coli K12 experiment conducted?
Two groups of E.coli K12 were tested:
1) Wild Type (WT) = Normal E.coli K12 –> No defects in EPS
2) Mutant (mut) = Mutants had defective EPS production pathways and thus did not produce it
What EPS does E.coli K12 produce?
Colanic Acid
What were the findings of the E.coli K12 experiment?
WT = Biofilm formed! Colanic acid was produced during biofilm formation ONLY
MUT = NO biofilm formed! BUT ADHESION STILL OCCURRED!
What were the conclusions from the E.coli K12 experiment?
1) EPS is needed for biofilm formation
2) EPS is NOT needed for adhesion
3) EPS is ONLY produced WHEN NEEDED (during biofilm formation)
If EPS is only produced during biofilm formation, how do microbial cells “know” a biofilm is forming?
QUORUM SENSING
–> chemical signaling allows microbes to assess population density around them AND induce specific gene expression profiles in response (such as EPS production)
Quorum Sensing
A process that allows bacteria to communicate with each other and adjust their behavior based on cell density
–> The regulation of gene expression in response to fluctuations in cell-population density
What is an example of a bacterial biofilm in HUMANS?
Pseudomonas aeruginosa in Cystic Fibrosis
What is the bacteria that forms biofilm in Cystic Fibrosis?
Where does the biofilm form?
Pseudomonas aeruginosa
–> Adheres to epithelial cells within the lungs and forms microcolonies that secrete an EPS (alginate) leading to biofilm formation
What is the EPS produced by Pseudomonas aeruginosa
ALGINATE
How does alginate contribute to the progression of cystic fibrosis?
Alignate allows for biofilm formation of Pseudomonas aeruginosa which causes…
1) Evasion of host immune system (blocks recognition of the bacteria)
2) Provides increased antibiotic resistance
On Earth the toal # of bacteria and archaea combined =
1030 cells
The amount of carbon associated with microbes =
10-50% of the carbon found in all plants combined
Microbial biomass is…
104-530x greater than human biomass
What has been the founding of microbiology?
Pure culture studies
–> The study of microorganisms in pure culture as a pure (isolated) species
What is the problem with studying microbes in pure culture only?
1)Not representative: microbes do not live in isolation in their natural environment, studying them in isolation does not provide the full picture
(May behave differently in isolation than they do in their habitat)
2) Not all microbes can be grown in a lab (cultured): Culturable microbes represent a SMALL minority of the complex microbial world
Just because a microbe grows well/in abundance in lab…
DOES NOT mean that it is abundant within the environment!
–> Some microorganisms simply grow better in lab conditions but this may not be a reflection of the real world
What are the two main types methods for studying microbes?
1) Culture-DEPENDENT techniques
2) Culture-INDEPENDENT techniques
Microbial Ecology
The study of interactions of microbes with their surroundings
What are the 3 main goals of microbial ecology study?
1) Understand the biodiversity of microbial life in nature
2) Determine the activities of microorganisms
3) Explore the effect of such activities on communities and ecosystems
What is the proposed culturability of sea water microbes?
0.001-0.1%
What is the proposed culturability of fresh water microbes?
0.25%
What is the proposed culturability of active sludge
1-15%
What is the proposed culturability of sediments?
0.25%
What is the proposed culturability of soil?
0.3%
What is CFU?
Colony Forming Unit
== A unit calculated and used to estimate the # of viable bacteria within a given sample
CFU calculation process:
1) Small amount of sample gets plated on medium
2) Plated sample is incubated (allowed to grow)
3) # of colonies grown on medium is counted
–> The # of CFUs indicates the # of bacteria within the sample
Why does the # of CFU represent the # of bacteria?
Each visible colony (CFU) on an agar plate is assumed to have originated from a single bacterium that was able to multiply and grow into a visible cluster
–> Each bacterial cell is capable of forming a colony SO the number of colonies formed represents the # of viable bacterial cells present!
Cultivation Dependent Techniques
Methods of studying microbes that relies on culturing processes
What is enrichment culture?
A type of culture-dependent technique
–> The use of carefully designed/selected culture conditions and mediums to favor the growth of a particular microorganism!
–> An isolation and growth technique!
Enrichment cultures uses_____________ to _________________
Enrichment culture uses:
1) differences in physiological properties (between microbes)
2)…to selectively grow specific group/s of microbes
What is an example of enrichment culture process?
The growth of nitrogen-fixing bacteria in nitrogen-less medium
1) growth medium without any nitrogen is inoculated by a soil sample
2) Flask has a cotton ball covering the opening == allows for air flow (O2 and N2) to enter/exit
–> Without a readily useable nitrogen source, most microbes die BUT those that can fix nitrogen from the air, SURVIVE and GROW!
–> We can then plate this solution onto agar plate with the same nitrogen-less medium and colonies of pure nitrogen fixing bacteria will grow!
Name for nitrogen fixing bacteria
Azotobacter
What components can be added to traditional media to help promote microbial growth?
1) Quorum sensing molecules == stimulates extra growth
2) Providing siderophores == molecules that bind to Fe allowing microbes to take up Fe
3) Adding specific vitamin mixes
(–> particularly helps to growth vitamin auxotrophs)
What are cultivation-INDEPENDENT-techniques?
A set of methods used to characterize microorganisms that we CANNOT (have not yet) grow in culture
Many cultivation-INDEPENDENT-techniques are…
molecular tools!
–> They build on sequencing and analysis of nucleic acids extracted from environmental samples
What genes are of specific interest in cultivation-INDEPENDENT-techniques?
WHY?
SSU rRNA genes!
Because they have a good mix of regions that are conserved across all species and regions that are extremely variable
How does the secondary structure of SSU rRNA lend itself to genetic study?
1) Highly VARIABLE regions = allows for analysis of dissimilarity between organisms for phylogenetic study
2) Highly CONSERVED regions = allows for the production of primers that have differing levels of selectivity
Universal primers
Primers that can be used to amplify SSU rRNA genes of ALL (or many at least) organisms!
–> In terms of SSU rRNA: Designed from highly conserved regions of SSU rRNA!
SSU rRNA gene sequences acts as _____________ to determine_________
BARCODES to **determine types of microorganisms present within a given sample
What are the methods of cultivation-INDEPENDENT-techniques? (ALL)
1) Direct Sequencing
2) Metagenomics (gene libraries)
3) Denaturing Gradient Gel Electrophoresis (DGGE)
4) Fluorescent in situ hybridization
5) Terminal Restriction Fragment Length Polymorphism (TRFLP)
5) Stable Isotope Probing (SIP)
6) Flow Cytometry
What are the methods of cultivation-INDEPENDENT-techniques we are NOT covering?
3) Denaturing Gradient Gel Electrophoresis (DGGE)
4) Fluorescent in situ hybridization
5) Terminal Restriction Fragment Length Polymorphism (TRFLP)
5) Stable Isotope Probing (SIP)
6) Flow Cytometry
Direct Sequencing Technique Process:
1) Collection of environmental sample
2) Transport of sample (refrigerated to prevent changes in the sample during transport)
3) DNA extraction
4) PCR (using extracted DNA as template)
5) Gene sequencing
6) Comparison of gene sequences to known sequences in gene databases
What info does direct sequencing technique provide about a collected sample?
1) Detailed info about the diversity and composition of microorganisms present in the sampled community (what microbes are present)
3) ESTIMATES of relative numbers of microbial populations in the community
Example of how direct sequencing technique provides estimate of relative #s of microbial composition in a community:
If of the sequences tested, 500/1000 SSU rRNA gene sequences tested belong to species A, we could predict that the community is composed of 50% of species A
Problems direct sequencing technique
1) Imperfect primers == primers used to target microbial groups are usually made based upon those we CAN culture!
2) # of copies of SSU rRNA genes is DIFFERENT per microbe
–> How do we differentiate between 2 copies coming from the same individual microbe and 2 copies coming from two separate microbes (of the same type)?
Metagenomics
The study of genetic material DIRECTLY from environmental samples (no PCR)
–> Usually involves the construction of genomic libraries!
What are metagenomic libraries?
Collections of DNA fragments cloned from the genetic material of a microbial community from an environmental sample
How are metagenomic libraries made?
1) Environmental sample is collected
2) DNA is extracted from the sample
3) Restriction enzymes are introduced that cut the DNA into fragments
4) Vectors are added to the mix and get cut open by the restriction enzymes as well
5) Vector and sample DNA fragments are ligated together to form full plasmids with different DNA fragments in each
6) These plasmids are transformed into host cells
–> The collection of these host cells represent a genomic library!
Once a metagenomic library is made, how is it used?
1) Gene sequence analysis
2) Functional analysis ==> Allows host cells to produce proteins (phenotype analysis)
Other than creating libraries, what other method does metagenomics include?
Direct sequencing
–> Environmental sample DNA is extracted and then IMMEDIATELY sequenced (NO PCR STEP)
