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Microbial Ecology• Learning Objectives:

– To learn how to study microbes in their natural environments– To understand techniques used to investigate microbial ecology

• Outline:– Overview of microbial ecology– Microbial ecology techniques– Example 1: Archaea in the ocean.– Example 2: Arsenic cycling in Mono Lake.– Example 3: Microbiome of the GI tract.

What is microbial ecology?

• Study of microbes and their interactions with theenvironment.

• Some examples of microbial ecology:– Quantifying sulfur oxidizers in a deep sea hydrothermal vent.– Determining biodiversity of prokaryotes in the human GI tract.– Monitoring the distribution of ctx gene in marine estuaries.

• The subject of investigation can be application based orfundamental.– Application based science (or applied science) is usually driven by

problems effecting society in some way.– Fundamental science aims to advance the understanding of a

particular process in nature.

Microbiology and biogeochemical cycling

• Microbes mediate transformation and recycling ofelements in nature.– Carbon, sulfur, nitrogen, phosphorus are some examples.– Toxic metals also undergo biogeochemical cycling (e.g. Hg, As)

• Biological, geological, and chemical processes worktogether to alter fate and transport of elements.

• Element cycles usually involve oxidation-reductionreactions during transport of an element in theenvironment.

• Elements move through different trophic levels.• Impact of biogeochemical cycling:

– Affect bioavailability of elements to higher organisms– Control energy flow within the oceans.– Nutrient cycling can also occur within an organism (GI tract)

Example of the Marine Carbon Cycle:

Edward F. DeLong and David M. Karl, Genomic perspectives in microbialoceanography, Nature 437, 336-342 (15 September 2005)

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To culture or not to culture?Culture-dependent approach: grow organisms of a specific type• Study it as a model organism for an environmental process; or

quantify abundance of specific organisms (disease causing or not).• Pros:

– You now have a system that is useful for mechanistic studies.– You can determine the abundance of a particular population of microbes

• Cons:– You can’t grow every microorganism (culture bias).– Is your model organism the one responsible for a particular process?– You can never prove a sample is negative for a particular organism.

Culture-independent approach: use molecular techniques to observeorganisms or detect “signatures” of their activities without growth.

• Pros:– You can identify microbes without knowing their culturing conditions.– The culture bias is no longer a problem

• Cons:– Environment is complex and hard to sort out– Low abundance organisms are not well represented

Common approaches used in microbial ecology:

1. Enumerating (counting) microorganisms– The goal is to determine the abundance of microorganisms.– Some approaches use cultivation approaches others do not.

2. Microcosm study– The mud in a bottle experiment– This is useful for determining rates of reactions– Unlike in the environment you can manipulate the environment

within the bottle.

3. 16S rRNA and functional gene analysis– This can be a very rapid and useful approach to identifying

organisms and diversity within a particular environmental sample.

4. Metagenomic approaches– Don’t culture. Instead, sequence the DNA straight from the

environment.

1. Enumeration ofmicroorganisms• Total counts by microscope

– DNA dye and epifluorescence microscopy• FISH: fluorescence in situ hybridization (see

figure)– Uses 16S rRNA gene probes for bacteria or

archaea– You can target specific genera

• Viable counts: plate samples on media.– Usually underestimates the total count. (Why?)– Called culture bias (bacterial enumeration

anomaly) because you can’t cultureeverything.

• Using FISH and microscopy it wasdiscovered that crenarchaea were highlyabundant in the ocean.

– The crenarchaea were thought to be eitherextremophiles or methanogens.

1. Enumeration: archaea and bacteria in the ocean

Bacteria

Archaea

• Archaea were found toabundant in the deep ocean.

• This was unusual.• Archaea were also highly

abundant in coastal regions.• This raised questions about

the function of these archaealmicroorganisms.

• FISH revealed the globaloceans contain:

– 1.3 x 1038 archaeal cells– 3.1 x 1038 bacterial cells

• One group of archaeacomprises 1 x 1038 cells!

• What is this organisms?• A representative microbe was

isolated in 2005 Archaeal dominance in the mesopelagic zone of the PacificOcean, Nature Karner 2001 vol:409 iss:6819 pg:507 -510

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Isolation of marine Crenarchaeota SCM1

• Nitrosopumilus maritimus• Isolated from an aquarium in

Seattle.• It is a chemoautotroph• Isolated with:

– filtered aquarium water– ammonium chloride– Bicarbonate– streptomycin

• It is the first ammoniaoxidizing crenarchaeota.

• Very similar to the archaea inthe open ocean.

DAPI FISH

TEM SEMIsolation of an autotrophic ammonia-oxidizing marinearchaeonMartin Könneke, Anne E. Bernhard, José R. de la Torre,Christopher B. Walker, John B. Waterbury and David A. StahlNature 437, 543-546 (22 September 2005)

2. Microcosm studies• Collect a water or sediment

sample and incubate in amedium that simulates theenvironment.

• Measure rate of substrateutilization by:

– Direct chemical analysis. Youneed a method for measuringthe chemical of interest

– Or using a chemical isotope.You measure radioactivityinstead of the chemical.

• Example: in Mono Lakearsenic is really high. Therespiration of arsenate accountsfor ~14% of the total carbonturnover in the lake.

http://www.mikelevin.com/MonoLake.html

3. PCR for 16S rRNA and functional genes

• 16S rRNA gene analysis:– Used to asses the microbial diversity within a particular sample

without growing any organisms.

• Functional gene analysis:– Use PCR to detect genes that encode for a protein that does

something of interest like ammonia oxidation, (amoA)• Diversity of PCR products can be assessed by:

– Making a clone library (brute force but low throughput)– Using electrophoresis-based fingerprinting methods (higher

throughput): DGGE

• Sequence information is analyzed by making phylogenetictrees.

3. Detection of the functional gene for arsenatereduction, called arrA

1 2 3 4 5 6 7 8 Blanks

Increasing depth in core

Gel of PCR products carried out on DNA extracted from sediment samples at 8 different depthswithin a sediment core. You can see the DNA bands become less intense for sediments thatare deeper in the core.

The next step is to figure out how many different kinds of arrA sequences are represented in theDNA band. We do this by making a clone library and sequencing a lot of the clones.

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Making a clone library• Add DNA from PCR to a

plasmid.• Ligate the two pieces together.

– One molecule of thefunctional gene ligates to onemolecule of plasmid

• Transform into E. coli.• Each colony represents one

cloned DNA fragment.• Sequence the DNA insert.

– How many should wesequence?

• Bioinformatic analysis of theDNA sequences.

– BLAST (online databasesearch program)

– Alignments and phylogenetictrees.

+

A B C

ligate

transform

plate

plasmidsDNA inserts

Colonieswith clonedPCRproducts

Sequencing and Tree drawing

• Sequencing is usually done by dye-terminator sequencing by capillaryeletrophoresis with laser detector

• You need purified plasmid DNAor PCR products

• Phylogenetic inferences to knownsequences and organisms fromonline genetic databases:

– Genbank (functional genes)– Ribosome Database Project (16S

rRNA genes)

DGGE: denaturing gradient gel electrophoresis• DGGE analysis can give us a sense

of the diversity within a particularsample without sequencing.

• You can also analyze multiplesamples at the same time.

• We put DNA from a PCR onto aspecial gradient gel.

• The DNA will migrate through thegel and separate into individualbands based on their GC content.

• The bands represent individualDNA sequences with different GCcontent.

• More bands = more diverse• The brighter bands also indicate a

more abundant organism.• You can cut out bands and

sequence the DNA.

CyanobacteriaChromatiaceaeBeta-proteobacteria,

It is now common to combine “classic” approacheswith modern genomic methods

• Determine geochemical profiles– need to measure chemical

parameters• Do experiments with

environmental samples– Mud, sediment, water– Get rate measurements– in situ activities.

• Isolate pure cultures• Characterize physiology of the

strain, do genetic studies,biochemistry, etc.

– Genome sequence– Microarrays: gene expression

• Microbial ecology tools– Develop gene detection tools

to investigate diversity offunctional genes for a process

– Identification of the pureculture in natural populations

Oremland et al. (2005) Whither or wither geomicrobiology in theera of 'community metagenomics'.Nat Rev Microbiol. 3(7):572-8

Emphasis on geochemistry

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High throughput sequencing has spawned the“modern” approach to microbial ecology.

• Extract environmental DNA• Make large insert DNA clone

libraries– Bacterial artificial chromosome

(300 kb)– fosmid (plasmid ~50 kb)

• Sequence lots of DNA• Bioinformatic analyses of sequence

data.• What to do with this data?• Goal is to understand something

about the environment:– Must develop follow-up studies– Are the genes expressed?– Are the encoded products

functional in situ?– Are there significant cycling of

elements, nutrients or energy fluxwithin an ecosystem.

Metagenomic projects

• Metagenomics: using massivehigh throughput DNAsequencing technology tosequence genomes oforganisms in an environmentalDNA sample.

Here are few projects:• Yellowstone hot springs

(various places)• Dechlorinating bioreactor• Biogas reactor• Compost• Bovine Rumen• Acid mine drainage• Marsupial (wallaby) gut• Waste water• Termite gut• Viral communities• Lots of different human guts• Neanderthal

Human Microbiome Project

• This is called the next genomic frontier for humans.• Human microbiota: the microorganisms that live in and on us.• Microbiome: the genes of the individual microbial symbionts• Gut microbiota are important to us:

– Help harvest energy from our diet and synthesize vitamins.– Drug and toxin metabolism might predispose us to certain diseases or

cancer.– Aid in the renewal of gut epithelial cells.– Affects our innate immune and adaptive immune system. Could

influence immune disorders.– Cardiac size and human physiology (germ free mice have smaller hearts)– Behavior (germ free mice are more active).

• Disruption or alteration in one or more of these gut microbialprocesses might affect our health in positive or negative ways.

• The microbiome needs to be defined.

Human microbiota

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The human microbiome• Two parts: the core and variable

microbiomes• Core human microbiome (red):

– Set of microbial genes present in a givenhabitat in all of humans.

• Variable human microbiome (blue):– Set of microbial genes in a given habitat

in a smaller subset of humans.– These genes differ among individuals

and for different diseases.• Habitat can be defined over a range of

spatial scales:– The entire body.– The gut or part of the gut

• How stable and resilient is anindividual’s microbiota?

• We don’t know the core and variablehuman microbiome yet.

diet

What we do know about the human microbiome

• Large intestine has about 1010-1011 microorganisms in the humancolon.

• From 16S rRNA surveys 90% of the prokaryotes belong to just 2divisions (70 total)

– Firmicutes and Bacteroidetes• Among individuals it appears that there is a high degree of differences

in microbial community structure (the abundance and types of taxapresent).

– The differences appear to be stable.– How is high inter-individual diversity sustained?

• The first application of functional attributes of the human microbiomeshowed the gut genes were enriched for metabolic pathways:xenobiotics (foreign substances), glycans and amino acids; theproduction of methane; and the biosynthesis of vitamins.

Is there a link between obesity and the microbiome?(Box 26.3)

• Study done in 2006 showed that germ-free miceinoculated with microbiota from normal human got biggerwithout eating more food.– The human microbiota was more efficient in extracting energy.

• Gut microbiota from genetically obese mice were moreefficient than normal mice in releasing calories from food.– Obese mice gain more fat than wild-type mice on the same diet.

• The obese mice had more Firmicutes.• An experiment with humans that restricted fat and carbs

that also lost weight (6% of body weight) had lessFirmicutes in their gut microbiota.

http://notexactlyrocketscience.files.wordpress.com

How similar are gut microbiome to othermicrobiomes?

• In comparison to a decaying whale carcass, ocean water,and agricultural soil, gut microbiomes have similargenetic composition.

• However, gut microbiomes appear to have more genes forcarbohydrate and glycan metabolism (see fig below).