Microbial Ecology • Learning Objectives: – To learn how to study microbes in their natural environments – To understand techniques used to investigate microbial ecology

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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.

Microbiology and biogeochemical cycling

What is microbial ecology? • Study of microbes and their interactions with the environment. • 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 or fundamental. – 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.

Example of the Marine Carbon Cycle:

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

• Biological, geological, and chemical processes work together to alter fate and transport of elements. • Element cycles usually involve oxidation-reduction reactions during transport of an element in the environment. • 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) Edward F. DeLong and David M. Karl, Genomic perspectives in microbial oceanography, Nature 437, 336-342 (15 September 2005)

To culture or not to culture?

Common approaches used in microbial ecology:

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:

1. Enumerating (counting) microorganisms – –

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

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2. Microcosm study – – –

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 observe organisms or detect “signatures” of their activities without growth. • Pros:

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Total counts by microscope – DNA dye and epifluorescence microscopy

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– Uses 16S rRNA gene probes for bacteria or archaea – You can target specific genera

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Using FISH and microscopy it was discovered that crenarchaea were highly abundant in the ocean. – The crenarchaea were thought to be either extremophiles or methanogens.

Don’t culture. Instead, sequence the DNA straight from the environment.

1. Enumeration: archaea and bacteria in the ocean •

– Usually underestimates the total count. (Why?) – Called culture bias (bacterial enumeration anomaly) because you can’t culture everything.

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1. Enumeration of microorganisms

FISH: fluorescence in situ hybridization (see figure)

Viable counts: plate samples on media.

This can be a very rapid and useful approach to identifying organisms and diversity within a particular environmental sample.

4. Metagenomic approaches

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

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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

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

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The goal is to determine the abundance of microorganisms. Some approaches use cultivation approaches others do not.

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Archaea were found to abundant in the deep ocean. This was unusual. Archaea were also highly abundant in coastal regions. This raised questions about the function of these archaeal microorganisms. FISH revealed the global oceans contain:

Bacteria

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

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One group of archaea comprises 1 x 1038 cells! What is this organisms? A representative microbe was isolated in 2005

Archaea

Archaeal dominance in the m esopelagic zone of the Pacific Ocean, Nature Karner 2001 vol:409 iss:6819 pg:507 -510

2. Microcosm studies

Isolation of marine Crenarchaeota SCM1 • • • •

Nitrosopumilus maritimus Isolated from an aquarium in Seattle. It is a chemoautotroph Isolated with: – – – –

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DAPI

FISH •

filtered aquarium water ammonium chloride Bicarbonate streptomycin

It is the first ammonia oxidizing crenarchaeota. Very similar to the archaea in the open ocean.

Collect a water or sediment sample and incubate in a medium that simulates the environment. Measure rate of substrate utilization by:

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

– Direct chemical analysis. You need a method for measuring the chemical of interest – Or using a chemical isotope. You measure radioactivity instead of the chemical.

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TEM

SEM

Isolation of an autotrophic ammonia-oxidizing marine archaeon Martin Könneke, Anne E. Bernhard, José R. de la Torre, Christopher B. Walker, John B. Waterbury and David A. Stahl Nature 437, 543-546 (22 September 2005)

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:

Example: in Mono Lake arsenic is really high. The respiration of arsenate accounts for ~14% of the total carbon turnover in the lake.

3. Detection of the functional gene for arsenate reduction, called arrA Gel of PCR products carried out on DNA extracted from sediment samples at 8 different depths within a sediment core. You can see the DNA bands become less intense for sediments that are deeper in the core. The next step is to figure out how many different kinds of arrA sequences are represented in the DNA band. We do this by making a clone library and sequencing a lot of the clones.

– Use PCR to detect genes that encode for a protein that does something of interest like ammonia oxidation, (amoA) Increasing depth in core

• 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 phylogenetic trees.

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plasmids

DNA inserts

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Add DNA from PCR to a plasmid. Ligate the two pieces together. – One molecule of the functional gene ligates to one molecule of plasmid

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Transform into E. coli. Each colony represents one cloned DNA fragment. Sequence the DNA insert. – How many should we sequence?

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Sequencing and Tree drawing

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Making a clone library

Bioinformatic analysis of the DNA sequences. – BLAST (online database search program) – Alignments and phylogenetic trees.

ligate A

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transform

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plate

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DGGE analysis can give us a sense of the diversity within a particular sample without sequencing. You can also analyze multiple samples at the same time. We put DNA from a PCR onto a special gradient gel. The DNA will migrate through the gel and separate into individual bands based on their GC content. The bands represent individual DNA sequences with different GC content. More bands = more diverse The brighter bands also indicate a more abundant organism. You can cut out bands and sequence the DNA.

Phylogenetic inferences to known sequences and organisms from online genetic databases: – Genbank (functional genes) – Ribosome Database Project (16S rRNA genes)

Colonies with cloned PCR products

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

DGGE: denaturing gradient gel electrophoresis •

Sequencing is usually done by dyeterminator sequencing by capillary eletrophoresis with laser detector You need purified plasmid DNA or PCR products

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Determine geochemical profiles –

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Do experiments with environmental samples – – –

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Mud, sediment, water Get rate measurements in situ activities.

Isolate pure cultures Characterize physiology of the strain, do genetic studies, biochemistry, etc. – –

Cyanobacteria Chromatiaceae Beta-proteobacteria,

Emphasis on geochemistry

need to measure chemical parameters

Genome sequence Microarrays: gene expression

Microbial ecology tools –

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Develop gene detection tools to investigate diversity of functional genes for a process Identification of the pure culture in natural populations

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

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

Metagenomic projects

Extract environmental DNA Make large insert DNA clone libraries – Bacterial artificial chromosome (300 kb) – fosmid (plasmid ~50 kb)

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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 flux within an ecosystem.

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).

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Disruption or alteration in one or more of these gut microbial processes might affect our health in positive or negative ways. The microbiome needs to be defined.

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Metagenomics: using massive high throughput DNA sequencing technology to sequence genomes of organisms in an environmental DNA 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 microbiota

The human microbiome • •

Two parts: the core and variable microbiomes Core human microbiome (red): – Set of microbial genes present in a given habitat in all of humans.

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diet

Habitat can be defined over a range of spatial scales: – The entire body. – The gut or part of the gut

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How stable and resilient is an individual’s microbiota? We don’t know the core and variable human microbiome yet.

Large intestine has about 1010-1011 microorganisms in the human colon. From 16S rRNA surveys 90% of the prokaryotes belong to just 2 divisions (70 total) – Firmicutes and Bacteroidetes

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.

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What we do know about the human microbiome

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Among individuals it appears that there is a high degree of differences in microbial community structure (the abundance and types of taxa present). – The differences appear to be stable. – How is high inter-individual diversity sustained?

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

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

How similar are gut microbiome to other microbiomes?

• Study done in 2006 showed that germ-free mice inoculated with microbiota from normal human got bigger without eating more food.

• In comparison to a decaying whale carcass, ocean water, and agricultural soil, gut microbiomes have similar genetic composition. • However, gut microbiomes appear to have more genes for carbohydrate and glycan metabolism (see fig below).

– The human microbiota was more efficient in extracting energy.

• Gut microbiota from genetically obese mice were more efficient 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 less Firmicutes in their gut microbiota.

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