25-26: Microbial Ecology Notes

Introduction

• Microbial communities form the foundation of Earth’s biosphere.
• They shape the environment of plants and animals.
• Microbes recycle organic material.
• Below the surface, they shape crustal rock.

Microbes in Ecosystems

• Microbes are ubiquitous and found in every habitable environment.
• They fill every potential niche imaginable.
• Microbial genome + environmental factors determine the ability of a microbe to fill a niche.
• Assimilation: Process by which organisms acquire an element to build into cells.
• Dissimilation: Process of breaking down organic nutrients to inorganic minerals.
• Biomass = Bodies of living organisms.
• To obtain energy and materials for biomass, all organisms participate in the food web.
• Levels of consumption are called trophic levels.
• Every food web depends on primary producers for two things:
  • Absorbing energy from outside the ecosystem.
  • Assimilating minerals into biomass.
• In addition, all ecosystems have consumers:
  • Grazers
  • Predators
• At each trophic level, bodies of dead organisms are consumed by decomposers.

Microbes within food webs

• Different habitats have different producers and consumers (marine vs. terrestrial).
  • Marine:
    • Producers: CO_2 fixation and biomass production are performed by the phototrophic bacteria.
    • Consumers: protists and viruses
  • Terrestrial:
    • Producers: plants
    • Consumers: herbivores

Microbial Symbiosis

• Symbiosis: Intimate association of microbe with another species
• Mutualism: Both partners benefit from specific association
• Parasitism: One partner grows at the expense of another
• Commensalism: One partner benefits, one is unaffected

Examples of Mutualism

• Lichens
  • Fungus + (alga or cyanobacterium; sometimes both)
• Rhizobium inside leguminous (bean) plants
• Mixotricha, bacterial endosymbionts, and termites
• Mycorrhizae

Lichens

• Symbiotic relationship between a fungal partner and a photosynthetic partner (green algae or cyanobacteria)
• Pioneer biota – primary colonizer for soil-less surfaces (e.g. rocks)
• Fungus provides shelter, water and minerals
• Cyanobacteria generate organic carbon and fix atmospheric nitrogen

Rhizobia/Legume Interactions: Nodulation

• Specific relationship between rhizobia and legumes
• Complicated signal exchange and strictly controlled
• Plants provide carbon source and shelter
• Rhizobia fix atmospheric nitrogen to ammonia and provide it to the plants
• A major source of available nitrogen in the biosphere
• Signal exchange between specific legume and rhizobia species to initiate nodulation
• Rhizobia enter root cortical cells through infection threads
• Differentiates into bacteroids
  • Irregular shapes with no cell wall
• Bacteroids remain in symbiosome
  • Bacterium supplies fixed nitrogen
  • Plant leghemoglobin sequesters excess oxygen

Myxotricha paradoxa

• A multiple symbiont: digestive symbiont of termites
• A large ciliate (not bacteria!) that grows in the guts of termites
• M. paradoxa itself has bacterial endosymbionts- digest the cellulose found in wood
• Also has 4 kinds of bacteria attached to its surface (2 spirochetes and 2 anchor bacteria)

Plant Pathogens

• When a pathogen does colonize a plant, its growth has effects ranging from minimal to devastating.
• The most common plant pathogens are fungi.
• However, viruses and bacteria can cause plant infections as well.

Parasitism / Plant Pathogens

• Agrobacterium induces plant galls
  • Ti plasmid induces tumor growth
• Fungi grow haustorium into plant cell
  • Doesn’t break plasma membrane
  • Absorbs nutrients from host
• Fungal diseases
  • Anthracnose
  • Dutch elm disease

Marine Microbiology

• In the open ocean, the water column (known as the pelagic zone) is subdivided into distinct regions:
  • Neuston (about 10 m): Air-water interface
    • Contains the highest microbe concentration
  • Euphotic zone (100–200 m)
    • Receives light, and so phototrophs grow
  • Aphotic zone: Below the reach of light
    • Only heterotrophs and lithotrophs can grow.
  • Benthos: Ocean floor plus sediment below surface
  • Thermal vent communities (benthic organisms)

Wastewater Treatment

• Wastewater treatment decreases the organic matter and the level of human pathogens before water is returned to local rivers.
• In a municipal treatment plant, sewage undergoes:
  • Preliminary treatment: Removes solid debris
  • Primary treatment: Fine screens and sedimentation tanks remove insoluble particles.
  • Secondary treatment: Microbial decomposition of organic content
  • Tertiary (advanced) treatment: Chlorination or other chemical applications to eliminate pathogens

Measuring the unculturable

• Many prokaryotes from marine communities are said to be uncultured, because we do not know their culture requirements.
• They are now characterized by metagenomics, the sequencing of “community DNA.”

Measuring Planktonic Communities

• Counting organisms
• DNA content
  • Fluorescence microscopy measures DAPI dye
• Measure biomass
  • Chemical assays of organic matter (protein)
• Carbon fixation
  • Incorporation of radiolabeled ^{14}CO_2
• Metagenomics: analysis of total microbial community DNA- revealed approx. 25,000 different microbial species/liter seawater

THE METAGENOMICS PROCESS

DETERMINE WHAT THE GENES ARE (Sequence-based metagenomics)

• Identify genes and metabolic pathways
• Compare to other communities and more…

DETERMINE WHAT THE GENES DO (Function-based metagenomics)

• Screen to identify functions of interest, such as vitamin or antibiotic production
• Find the genes that code for functions of interest and more…

Animal Microbial Communities

• Digestive communities
  • Thousands of species in gut, stomach
  • Cellulose digestion in termites
  • Different microbes in human stomach, intestine
  • Rumen critical for digesting cellulose
  • Largest digestive chamber in cattle, sheep

Animal Microbial Communities

• Mutualistic Zooxanthellae found in coral
  • Algal symbionts (most commonly dinoflagellates)
  • Important to biosphere- coral responsible for reef formation and coastal shelf ecosystems
  • Coral bleaching: algal symbionts die or are expelled
  • Global warming a threat to coral reef ecology

A microbial world lives inside of us

• Your microbiome is trillions of microorganisms living on and inside of you.
• Humans harbor over 1000 species of bacteria alone.
• You’re about about 1:1 human:bacterial cells.
• This ratio has changed many times.
• Wherever you’re exposed to the world, there’s a microbial community present.
• We’re a tube!

What does the microbiome do?

• The microbiome is important!
• The microbiome is analogous to an organ system.
• It’s easy to take for granted…until it’s hurt.
• It has many functions such as:
  • Vitamin production.
  • Immune system development.
  • Digestion.
  • Keeping out pathogens.

Who’s there?

• Each body site has its own microbial community.
• The microbiome is mostly bacteria, but there’s lots of archaea, fungi, and eukaryotes too.
• Also, tons of viruses, especially bacteriophages.

Specifically…Who’s there?

• Bacterial phyla:
  • Bacteroidetes dominate the large intestine.
    • i.e. Prevotella spp. and Bacteroides spp.
  • Firmicutes make up most of the vaginal and esophageal bacteria.
    • i.e. Lactobaclli and Veillonella spp.
  • Actinobacteria love the skin and nose.
    • i.e. Bifidobacterium spp. and Rothia spp.
  • Cyanobacteria live in your hair!
  • Many unknown, uncultured taxa.
  • Fusobacteria mainly live in the mouth.
    • i.e. Fusobacterium nucleatum.
  • Proteobacteria live almost everywhere.
    • i.e. E. coli and and Enterobacter spp.

Microbiomes are all different between people…but metabolically similar?

Body site and microbial communities discussion!

• You know that body sites contain different microbial communities…
• Why do you think this is?
• Are there biotic/abiotic factors that affect this?
• Pick a body site (i.e. gut, nose, mouth, etc.).
• Discuss the major functions of your chosen body site.
• Discuss why you think microbes would prefer to live here.
• What do you think happens if bacteria are moved from one site to another?

Let’s look at the gut. Why is it so…different?

• The human gastrointestinal tract has a very different microbial community from other body sites.
• Many microbes produce beneficial compounds:
  • Microbes digest complex molecules and makes Short-Chain Fatty Acids.
    • Butyrate feeds the gut epithelium.
    • Acetate tempers the immune system.
  • But it’s not all rosy for us:
    • Microbes produce hydrogen sulfide. This is bad.
    • They also “activate” heterocyclic amines to become carcinogens. This is also bad.
• How and why do you think the body changes the gut community..?

Why is the gut microbiome so different?

• How does the body control microbes?
  • Low pH inhibits bacterial growth.
  • Bile salts can kill bacteria.
  • The gut produces antimicrobial compounds.
• Why would we want to limit bacterial growth?
  • Low pH keeps bacteria from getting in.
  • We want to eat first!
  • Host digestion happens before microbes’.
  • Microbial fermentation helps break down plant polysaccharides downstream in the gut.

So…then how do we get a microbiome?

• Infants are colonized by maternal vaginal and fecal microbes at birth.
  • Lots of lactobacilli for digesting milk along with Bifidobacterium.
• Babies naturally drink breast milk.
  • Milk contains compounds that we can’t digest. Why?
    • Hint: It’s for the microbes!
  • Milk (and skin from breastfeeding) contains microbes too.
• Non-breastfed babies have different microbiomes…

Milk: It does a baby good.

• Human milk contains many soluble oligosaccharides we can’t digest.
  • More of these compounds than any other mammal’s milk.
• Bifidobacterium loves these compounds.
  • Provides gut barrier functions to keep out pathogens.
  • Make the gut “slippery” to pathogens.
  • Modulate the immune system.

The maturing gut microbiome.

Think-Pair-Share: Why do families share microbiomes?

• Is it genetic? Dispersal? Magic?
• Give some examples of ways microbes might be shared between people.
• Families share their microbiomes.

A massive undertaking: The Human Microbiome Project

• The Human Microbiome Project was developed to study well…the human microbiome.
• It’s an enormous project: ~$215M and samples from multiple sites on and inside 300 people.
• They sequenced >11,000 samples in two phases.
• They also sequenced ~3,000 bacterial isolates.
• The data are public (!) and freely available here: https://portal.hmpdacc.org/

Seeing the unseen

• There are several methods to identify bacteria from a sample.
  • 16S rRNA gene sequencing.
  • Whole genome sequencing.
  • Metagenomic sequencing.

16S rRNA gene sequencing in all its glory.

• All living things possess ribosomes.
• Bacteria and archaea have 70S ribosomes made of several subunits.
  • Each subunit contains ribosomal RNAs.
• The most useful piece for bacterial microbiome work is the 16S rRNA.
• There are regions of the 16S rRNA gene that vary between bacterial species.
  • The “hypervariable regions.”
• By amplifying these regions with PCR and sequencing the fragments, you can assign taxonomy to bacteria.
• This method tells you bacterial identity, but not functional characteristics.

Metagenomic “shotgun” sequencing.

• Metagenomic sequencing sequences all the DNA in a sample.
  • This sequences microbial genes, host DNA, everything.
• Uses random primers to (try and) avoid PCR biases.
• Useful for identifying the functional potential of microbes.
• Provides insights into the genomes of uncultured microbes.
• Generally, more expensive than 16S sequencing.
• Generally, less sensitive than 16S sequencing to finding the very rare taxa.

Lifestyle choices can affect your microbiome

• The microbiome is generally stable within individuals but can be quickly altered.

• A study (David et al, 2014) looked at the gut microbiome of two people over a year.

• Two major events happened over the study:

  • One subject travelled from the USA to a developing country.
  • The other subject had a Salmonella infection.

• Ley, et al. investigated mice with a mutation in the leptin gene (ob/ob).

  • This gene produces a strong obesity phenotype.

• They fed the mice identical diets.

• The gut microbiome was different between obese and lean mice.

• Is the gut microbiome involved in obesity? How?! Is it all lifestyle choice though…?

Effects of dieting on the microbiome

• Ley, et al. followed up with a study in humans where they restricted carbohydrates or fats.
• Regardless of diet, people had fewer Firmicutes and more Bacteroidetes.
• The phyla composition in people restricting calories approached that of lean “control” subjects after a year of dieting.

Microbiota fecal transplantation

• Turnbaugh, et al. ran a study in which germ-free mice were inoculated with microbes from either wild-type or obese mice.
• Mice that received “obese” microbes had huge increases in body fat.
• Mice that received “lean” microbes did not.
• The authors also looked at how many Calories remained in lean vs obese mice’s feces.
• Obese mice had less remaining Calories in feces.

Mice eat less and gain more fat with a microbiome

• Bäckhead, et al. studied weight gain in mice with (inoculated or natural) or without (germ free) a microbiome.
• Mice with a microbiome gained more weight than germ-free mice.
• Mice ate less mouse-chow and gained more fat.

Treating a damaged microbiome with… a healthy microbiome

• We often take antibiotics to help cure bacterial infections.
• Sometimes, those same treatments can make us more likely to get a different illness.
• One such infection is Clostridioides difficile Colitis (or diarrhea).
• This bacterium can be picked up from a hospital environment after taking antibiotics.
  • A “nosocomial” infection.
• It’s often recurrent and hard to treat.

The fecal microbiota transplant

• To restore the microbiome, antibiotics are administered to the patient.
• Then, feces from a healthy donor are transferred to the sick person.
  • Either orally through capsules or colonoscopy.
• This adds a “healthy” gut community to the diseased gut.
• The new microbiome should engraft and displace the C. difficile population.
• Generally, 80-95% of these procedures cure the patient of their recurrent diarrhea.

Does FMT really change the gut microbiome?

• Patients with recurrent C. difficile infections were given donor microbiomes.
• Each person was completely cured of their infection and diarrhea.
• Do you see how the gut microbiomes of patients (A-D) change to look more like the donor microbiomes?
• Would you take this treatment?