microbial ecology - HG2

Microenvironments

large changes in chemistry over very small distances

nutrient access limited by diffusion (like oxygen for ex)

natural vs lab growth rates

microbes have intermittent exposure to nutrients —> feat or famine lifestyle

e.coli divide in lab way faster than in the human gut naturally

this is bc resources and conditions, non-uniform distribution, microbes have to grow in mixed communities

lowk obvi!

Enrichment Culture

culture-DEPENDENT

whos out there and what r they doing

isolate individual microbes/communities w particular metabolic abilities

Steps:

• inoculum from habitat w unknown # of organisms

• enrichment conditions promote growth of specific microbes

• positive result = organism w selected property was present (DK HOW MUCH tho)

• negative = inconclusive (cant prove it DOESN’T exist)

Workflow:

• medium w select nutrients —> sample w hella organisms —> organism of interest multiplies —> BACK dilute to first tube till enough to plate —> pure culture of organism of interest

Enrichment Bias

liquid enrichment cultures —> rapidly growing microbes that perform the selected metabolism dominate

• even if they arent the most abundant microbes in the OG environment that can do that type of metabolism

fast > amount

Lab environments let the slow ones grow faster

we end up indirectly selecting for the fastest growing bacterium in the sample that can do that metabolism.

Avoiding enrichment bias

Dilute initial inocolum and grow multiple independent enrichment cultures —> rare but fast “weed species” don’t take over every culture

Cell sorting —> separate individual cells of inoculum into wells of 96 well plates —> microbes in inoculum not in competition with eachother for nutrients

• can grow at a range of time to find the fast vs slow one

Enrichment for bacteria that degrade plastic waste

PET = polyethylene terephthalate = pollutant in landfills/ocean

Bacteria can use PET as a carbon source, they make PETase and MHETase that can break it down

Single Gene Analysis

(culture independent)

looks at diversity of microbes in a sample (who’s out there)

isolate genomic DNA from environment —>

amplify selected genes from DNA sample (16s rRNA) —>

obtain sequences —>

BLAST sequence to ID known organisms w 16S sequence matches —>

create phylogenetic tree of all sequences present including ones that don’t correspond to existing species

most abundant is usually ones that haven’t been cultured in the lab → unknown

Perturbed microbial community changing over time

deepwater horizon spill in gulf of mexico —> they did single gene analysis of bacterial 16s rRNA to characterize changes in the community after

organismal diversity decreased, got dominated by 2 types that consume oil as a carbon source

community became diverse again after oil was gone

Metagenomics vs Single gene analysis

metagenomics (culture independent):

• not only ID nif genes, but also know what 16s sequence is on the same DNA fragment

• can answer question “who in this habitat can fix N2”

Single gene is.. well.. for a single gene!

Metatranscriptomics

gives a better idea of what microbes were actually doing when sample was collected

total DNA and RNA collected

RNA rev trans —> DNA —> sequenced —> genes transcribed in environment revealed —> metagenome made —> transcripts associated with contiguous 16S rRNA

lets us say than an organism w/particular 16S was or wasn’t transcribing particular genes at the time of sample collection

Q: what were they doing!

Chemical assays

reveals processes happening in an environmental sample

need a killed control to account for abiotic processes that could prouce the measured compounds

assays give rates of reactions occurring in samples from specific environments

DONT tell u WHAT organism is present or WHO IS DOING IT

Stable Isotope Probing

assosciates reactions w organisms b4 performing them

measures use of substrates containing C or N + version of substrate with stable isotope needed (C 13 or N 15)

who is eating what.

KEY Qs

What are the methods and limitations of techniques

to assess which microbes are present in various habitats?

What can a metagenome (potentially) tell you that single-

gene community analysis can’t?

What are the methods and limitations of techniques to

determine what reactions are occurring in habitats?

What methods can reveal (or at least suggest) which

organism in a habitat is performing a particular reaction?

What is enrichment bias, and what methods are used

to avoid it?

any surface exposed to nutrients will be colonized

surfaces = nutrient, protection, means of staying in a favorable habitat vs getting washed away

abiotic surfaces: glass/rocks

organic surfaces: particles of decaying plants

other organisms as surfaces: plant roots, insects, each other

microcolonies = clusters of few cells that develop from a single attached cell

surface colonization can be sparse, detectable via microscope or hella dense

biofilms on surfaces can become microbial mats (cm thick) w diff types of microbes living in diff layers (tops of stromatolites)

Biofilms

predominant mode of bacterial growth in natural environments

group of bacteria enclosed in adhesive/self produced matrix

• made up of exopolysaccharides (EPS), proteins, nucleic acids

• adheres to abiotic or living things or free floats (flocs) in water

bacteria living in biofilms are in physiologically different states than if they were free

they can have diff growth rates, transcriptional profiles

they can hav enhanced tolerance toward antibiotics and enanced interactions w other microbes

environmental biofilms are usually multispecies communities, studies have been done on single tho.

Biofilms and human health / industry

biofilms grow on catheters, IV lines, and artificial joints in patients, fouling in fuel storage tanks, in water pipes: corrosion and water borne disease spread

biofilms help corrode submerged objects like boats/piers

dental plaque is also a biofilm w 100s of bacteria and archaea

organisms in biofilm can produce acid as a metabolic byproduct —> ex: tooth decay