mcellbi c112 last section before final (ps7 material)

Microenvironments

Rapid environmental changes over a very small distance experienced by microbes. Affected by access to oxygen, nutrients, proximity to other microbes, temperature, etc.

Do microbes experience a growth curve in a natural environment?

No, because their resources are limited. Instead, microbes function under intermitten periods of growth and starvation depending on access to nutrients.

What affects microbial growth in their natural environment?

1. Resources and growth conditions (typically suboptimal)

2. Uneven distribution of nutrients in their habitat

3. Competition with other microbes in a mixed community

What are two ways that microbial populations and communities can be assessed?

Culture-dependent analyses (i.e. enrichment cultures) or culture-independent analyses (i.e. single-gene community analysis, metagenomics, or metatranscriptomics)

Enrichment cultures

Selecting from a sample derived from a microbial community for a certain trait (i.e. nitrogen-based metabolism) and isolating these colonies on selective plates

How can you create an enrichment culture?

Dilute and re-dilute selective liquid medium with your sample of choice, selecting for the microbes which can use the nutrients provided. Back-dilute and then isolate colonies on solid medium.

Enrichment bias

The problem with enrichment cultures in which "weed" species tend to dominate in the enrichment due to their fast-growing nature in lab conditions. Often results in the exclusion of the most abundant or ecologically significant organisms in the inoculum (not accurate to the natural habitat)

How can we avoid enrichment bias?

Dilute initial inoculum by a lot and grow multiple enrichment cultures so that the probability of a weed species taking over the enrichment culture is less across all cultures, or use cell sorting to separate individual cells into wells so there is no longer competition for nutrients.

Unique enzymes which degrade PET plastic

PETase and MHETase

What gene is most often used in single-gene analysis of an environmental sample of microbes?

16S rRNA which is highly conserved across the tree of life, allowing for easy differentation between species.

How did the microbial community located in the Gulf of Mexico change during and after the Deepwater Horizon oil spill?

During the plume of oil, samples were dominated by species which could use oil as a carbon and energy source. A few months after the plume cleared, the community returned to a level of diversity similar to before the oil spill, although the types and proportions of bacteria were different before and after the spill.

Single-gene analysis

Amplifying an environmental sample's total DNA for a single gene (ex: 16S rRNA), then sequencing the DNA and generating a phylogenetic tree to give a snapshot of most of the community, along with identifying any new species.

Metagenomics

Amplifying an environmental sample's total DNA using next-gen sequencing methods, then assembling the partial genomes back together and annotating them to know what types of genes are present within each species. Can also discover new genes and link certain genes to 16S rRNA sequences.

Metatranscriptomics

Collects total DNA and RNA from a population. RNA is reverse transcribed to DNA and sequenced to reveal what genes in the environment are actively being transcribed. When paired with a metagenomics analysis, can reveal who is in an environment and what they are doing.

Stable isotope probing

Measures the use of substrates with carbon or nitrogen via isotope tagging (13C or 15N) which will become integrated into the DNA of cells which metabolizes it. Sequence tagged 16S rRNA to determine the species which were metabolizing that substrate.

What techniques can assess which microbes are PRESENT?

Enrichment cultures, single-gene analysis, metagenomics

What are the limitations of enrichment cultures?

1. Can only prove that a certain organism was present

2. Cannot draw conclusions on abundance of population

3. Cannot prove a certain metabolism DOESN'T exist (negative result is inconclusive)

4. Prone to enrichment bias

5. Time consuming

6. Cannot identify all species in a sample

What are the limitations of single-gene analysis?

Cannot tell you anything about that species directly, only that it's present. Will often identify unknown species whose genomes haven't been sequenced yet.

What can a metagenome tell you that single-gene analysis can't?

What other operons/genes are on the same DNA fragment as the 16s rRNA, which links that species with that gene. Therefore if a species' DNA is sequenced and a nitrogen fixation gene is on the same fragment as its 16s rRNA, you can conclude that that species is a nitrogen fixer.

Chemical assays of metabolic reactions

Gives rates of reactions occuring from samples from different environments to measure if various metabolisms (ex: sulfate reduction, photosynthesis) are being performed. Measures the rates of growth against the presence of the substrate being metabolized. Always include a killed control to account for abiotic processes that may have produced the measured compound.

What methods can reveal (or at least suggest) which organisms in a habitat are performing a particular reaction?

Metagenomics paired with metatranscriptomics to pinpoint the species which are actively transcribing RNA for certain reactions.

Enrichment cultures separated into per-cell plates can also be tested for various metabolisms/reactions since they are "pure" one-species cultures within each plate.

Microcolonies

Clusters of a few cells developed from a single attached cell on a surface

Biofilms

Mats of bacteria enclosed in an adhesive, self-produced matrix of exopolysaccharides (EPS), proteins, and nucleic acids growing on surfaces which can become many cm in thickness, with different microbes living in different layers (diverse community).

Flocs

Free-floating biofilms in aquatic environments

Traits of bacteria living in biofilms

Different (often slower) growth rates, different transcriptional profiles (aka different genes being transcribed), enhanced antibiotic tolerance, and enhanced microbe-microbe interactions.

EPS

Exopolysaccharides, produced by bacteria and make up the majority of the skeleton of a biofilm

What is a biofilm made of?

EPS, proteins, and nucleic acids

Why are biofilms a common concern in human health and industry?

Enhanced tolerance towards antibiotics poses a health concern in hospitals. Can cause fouling of fuel tanks or water pipes, and corrodes submerged objects like boats and piers.

How does a biofilm grow?

By cell division AND by attracting additional cells from the liquid into the biofilm matrix

Planktonic cells

Free-living and free-swimming bacteria NOT in a biofilm

Can biofilms dissolve or are they permanent?

They can dissolve on the cue of environmental signals like nutrient deprivation

How do cells attach to a surface to begin growing into a biofilm?

Flagella help sense the surface because as it rotates, the "hitting" of the flagella against the surface notifies the bacteria. Flagella also function as an anchor to the surface along with pili. OM proteins can also sense surfaces.

EPS are [hydrophilic/hydrophobic] and protect biofilm cells from...?

They are hydrophilic and prevent cells from drying out by absorbing water from the environment.

Biofilm habitat is more favorable for which microbial processes?

They create localized gradients allowing for habitat diversity, increase resource capture, allow retention of external enzymes like siderophores, and allow for cooperation and synergistic microconsortia to form.

What types of gradients exist in a biofilm?

Oxygen gradient (copiotrophic), nutrient gradient (oligotrophic), pH gradient, gradient of secondary metabolites made from other microbes

Why do biofilms enhance antibiotic tolerance?

Larger or charged antibiotics reduce in concentration as they get deeper into the biofilm, reaching a sublethal concentration. Smaller uncharged antibiotics diffuse uninhibited and do not lose concentration, but resistance genes are often transferred horizantally between bacteria in the biofilm. Bacteria also become tolerant by slow growth, since antibiotics typically inhibit active growth processes (i.e. protein synthesis, cell wall synthesis).

Crystal violet assay

Identifies mutants in biofilm formation because crystal violet bonds nonspecifically to cell surfaces and EPS. Washing a matrix of mutants with crystal violet will identify mutants who cannot form biofilms because there will be no staining.

What genes are activated in cells beginning to transition to the biofilm lifestyle?

- Synthesize EPS

- Stop motility or switch to surface motility

- Express adhesin proteins

- Secrete extracellular enzymes for nutrient digestion

c-di-GMP

Small signaling molecule which regulates motility/sessility in bacteria. Increased c-di-GMP is linked with increased sessility, while decreased c-di-GMP is linked with increased motility.

Diguanylate cyclase (DGC)

Enzymes which synthesize c-di-GMP

Phosphodiesterase (PDE)

Enzymes which hydrolyze c-di-GMP

How are the activities of DGC and PDE regulated?

By chemical and physical signals which bind to the enzymes and change their conformation.

Increased c-di-GMP generally leads to...

Increased biofilm formation, increased virulence, and decreased liquid motility

Degenerated DGC and PDE proteins function as...

Sensors which can sense the c-di-GMP molecule rather than cleaving it due to their degenerated nature.

What are some effectors which bind to c-di-GMP?

PilZ domain proteins (controls pili activity), degenerated GGDEF (DGC type enzymes) and EAL (PDE type enzymes), transcription factors affected DNA, and RNA riboswitches which make RNA more or less accessible for translation

Wsp system

Extended two-component signaling system which activates biofilm formation by sensing a surface and triggering a phosphorylation cascade to increase production of c-di-GMP

WspA

IM receptor which is activated by unknown surface receptors in the OM. Triggers phosphorylation of connected histidine kinase WspE.

WspE

Histidine kinase which is phosphorylated when WspA is activated upon sensing of an external surface. Phosphorylates the response regulator WspR

WspR

Response regulator connected to a DGC enzyme. Upon phosphorylation by WspE, stimulates the DGC domain and causes production of c-di-GMP.

How are biofilms dissolved?

Some species will release enzymes to degrade biofilm polymers or adhesins. Other times, a drop in c-di-GMP levels triggers biofilm degredation and release of planktonic cells.

Why would cells want to exit the biofilm state and return to being planktonic cells?

It can often be important for colonizing a new environment or a different part of a host organism (often seen in pathogens).

Sterilization

Killing or removing ALL living cells, spores, and viruses on an object.

Disinfection

Killing or removing microorganisms, but not necessarily bacterial or fungal spores, from an inanimate surface or object (therefore, you can use harsher chemicals than on living tissue). Used in medical and lab settings.

Sanitizing

Reducing microbial numbers on surfaces using chemicals which are less harsh than disinfectants. Often useful in the food industry.

Antisepsis

Removal of pathogens from the surface of LIVING tissue like skin. Chemicals must be safe for skin use, but may not always be safe for internal use (ex: neosporin, betadine, 70% ethanol).

Autoclave

Combines heat and pressure to sterilize liquids without them evaporating. Often used in lab settings. Achieves sterility.

Pasteurization

Precisely controlling heat to reduce microbial populations in food products such as milk. Results in a reduced spread of pathogens, reduced spoilage, and longer shelf life.

What is killed when milk is pasteurized?

All pathogens we know of that can be transmitted in infected milk, but does NOT kill all bacteria.

Flash pasteurization

Liquids are run through a heat exchanger at 71ºC for 15 seconds and then rapidly cooled. Shelf life 2 weeks refrigerated.

Ultra-high temperature pasteurization

Liquids are run through a heat exchanger at 135ºC for 1-2 seconds and then rapidly cooled. Different in that it kills endospores! Shelf life 6-9 months unrefrigerated.

Antimicrobial drugs

Chemicals used in humans which kill or inhibit the growth of microorganisms. Can be naturally produced, semisynthetic, or synthetic, but must have selective toxicity.

Selective toxicity

Ability to kill or inhibit a pathogenic microbe at some concentration where it does not adversely affect the host

Bacteriostatic

Growth is inhibited after drug is administered, but bacteria are not killed. Removal of drug will cause bacteria to resume growth

Bactericidal

Bacteria are killed by the drug but not lysed. Cells will NOT resume growth if drug is removed, but intact cells will be detectable by OD or microscopy.

Bacteriolytic

Bacteria are killed and lysed by the drug. Cells are not detectable by OD or microscopy.

MIC

Minimal inhibitory concentrtaion, or the lowest concentration of a drug agent that inhibits growth completely.

How can you find the MIC?

Make a series of cultures with the same concentration of cells and administer a range of concentrations of your antibiotic to them. Identify the point at which the cells are prevented from growing.

Calibrated test strips

Streamlines MIC measurements. Bacteria of choice are spread on hard agar which permits growth, and the strip is placed in the center. Allow bacteria to grow. The strip releases antibiotic in a gradient, and the site where growth abruptly ceases indicates the MIC.

How is antibiotic dosing calculated?

By how long it takes for the concentration of the drug in the body drops below the MIC. If it takes 8 hours for the concentration to drop below the MIC, the dosage will be about every 8 hours.

Spectrum of action

Which types of microbes an antibiotic is able to kill/inhibit growth

Mechanism of action (MOA)

The specific molecular target that is bound by an antibiotic and how it inhibits a critical process

What do penicillins target?

Cell wall biosynthesis

Penicillin MOA

Beta-lactam ring mimics the typical D-ala-D-ala residues of PG transpeptidases and binds them, inhibiting/deactivating them.

Penicillin is [bacteriostatic/bactericidial/bacteriolytic]

Bacteriolytic

Does penicillin have selective toxicity?

Yes, because human cells don't have PG or PG synthesis enzymes.

Penicillin spectrum of action

Mostly Gram positive, but can sometimes kill Gram negative species (they can't always cross the OM).

Why would chemists want penicillins to be more acid-resistant?

Because they are often administered orally, and the stomach acids could break down the compound before it is able to do its job.

What do quinolones target?

DNA integrity when bacterial DNA gyrases (a type of topoisomerase) cuts and rebinds DNA together during replication. The drug stops the bacterial DNA gyrase from repairing the cut in DNA by stabilizing the intermediate.

Quinolones are [bacteriostatic/bactericidial/bacteriolytic]

Bactericidal

Quinolone spectrum of action

Gram positive and Gram negative bacteria

Do quinolones have selective toxicity?

Yes, because bacterial DNA gyrase and eukaryotic topoisomerases are structurally different enough that quinolone doesn't affect the eukaryotic structure.

Example of a quinolone (clinical drug name)

Ciprofloxacin

What does a napthacene ring system target?

Protein synthesis by preventing tRNAs from entering the bacterial ribosome.

Example of a napthacene ring system (clinical drug name)

Tetracycline

Napthacene ring system spectrum of action

Broad (both Gram positive and Gram negative, plus some other things depending on substitutions along the ring system)

Napthacene ring systems are [bacteriostatic/bactericidial/bacteriolytic]

Bacteriostatic -> the drug only blocks protein elongation as long as it's present

How can you discover the MOA of an unknown antimicrobial compound?

Measure its MIC towards a specific bacterium, then plate that bacteria on plates with a concentration above the MIC. These plates will select for resistance genes, and whole-genome sequencing and complementation can be used to identify the mutated gene that causes resistance.

Why is it bad if you screen for resistance mutants for a new drug compound and find that your screen yields many mutants?

High frequency of random mutations that confer resistance in bacteria in the lab is a sign that pathogens in the wild will also rapidly develop resistance to the antibiotic, lowering its usefulness.

Where do antibiotics come from?

Accidental discovery of penicillin by Alexander Fleming when a plate of Staphylococcus was contaminated with a Penicillium fungus, causing nearby colonies of staph to die.

Why do bacteria and other microbes produce antimicrobial compounds?

They secrete these compounds as attack or defense molecules as a way to better compete for resources in the same environment.

How can you identify if an unknown bacterium is producing antimicrobial compounds?

Plate a thin lawn of known bacteria (ex: E. coli) on permissive medium. Always add a control, which is a paper disc soaked in a known antibiotic. Place the unknown bacteria sample onto the plate and isolated from any other samples, and let the plate grow. If the unknown bacteria has created a "zone" of inhibition around it where E. coli cannot grow, then your unknown bacterium produces an antimicrobial compound that E. coli is susceptible to.

What triggers antibiotic production in microbes?

Nutrient deprivation when competition for resources is particularly strained.

How can you test if target bacteria are susceptible to an antimicrobial compound produced by a known bacteria (ex: Streptomyces)?

Streak Streptomyces vertically on the plate, and streak target bacteria perpendicular right up to the Streptomyces line. If growth is inhibited near the line of Streptomyces, that bacteria is susceptible.

Sterile sites on the human body

Blood, brain/cerebrospinal fluid, heart, liver, kidneys

Nonsterile sites on the human body

Skin, GI tract, urinary tract, vagina/vaginal cavity, respiratory tract

Finding microbes in sterile parts of the body is...

Unusual and often a sign of disease

Microbiome

Genes present in microbes in a particular habitat

Microbiota

Microbes present in a particular habitat

Core human microbiome

Microbial genes present in/on the body found in most/all humans

Variable human microbiome

Microbial genes present in/on the body found in a smaller subset of humans. Can be affected by diet, lifestyle, physiology, immune system, nearby community members, environment, and parentage.