Lecture 13: Secreted Antagonism

some bacteria antagonize the _____ and _____ of others that might otherwise occupy the same _____

growth; survival; niche

the _____ principle says that no two organisms can occupy the EXACT same niche, over time one will eventually _____

competitive exclusion; outcompete the other

bacterial antagonism can occur at a distance through _____ (for example _____)

secreted products; antibiotics

some forms of antagonism require cell-cell _____

contact

the producer of an antagonist typically possesses an _____ to avoid _____

antidote; self-intoxication

how do we measure antagonism?

competitive index

measure antagonism begins by mixing two strains of bacteria in a _____ at a defined _____

co-culture; ratio

after growing two bacteria in co-culture, you harvest the bacteria and count the number of _____ of each type (usually this is aided by _____)

colonies; selective markers

why do you need a selective marker when counting colonies for a competitive index?

to tell them apart

what are two common types of selective markers used to differentiate the co-culture?

antibiotic resistance, fluorescence

competitive index is the ratio _____ co-culture/ratio _____ co-culture

after; before

strain with a competitive index of _____ has outcompeted the other strain

>1

in the competitive index, >1 = _____

advantage

in the competitive index, <1 = _____

disadvantage

in the competitive index of 1 = _____

no advantage or disadvantage

the competitive index is more informative when comparing a _____ organism with a _____

WT; non-toxic mutant

if a WT organism outcompetes another, but a knockout mutant has a competitive index of 1, what does this mean?

that gene is necessary to outcompete the other organism

what are two ways to measure the activity of secreted antagonistic products?

Kirby-bauer and MIC

Kirby-Bauer disk diffusion assay involves the overlay of a sterile disk infused with _____ on an agar plate spread with _____

antibiotic; bacteria

the bacterial lawn on a KB assay will grown on the plate, and if the antibiotic inhibits grow, a _____ will form around the disk

zone of clearance

the size of the zone of clearance correlates to the _____ of the isolate (KB assay)

susceptibility

a zone of clearance can be used to detect the presence of antimicrobials in _____

complex cell extracts

what does MIC stand for?

minimum inhibitory concentration

the MIC is the _____ concentration of antibiotic that completely _____ bacterial growth

lowest; inhibits

how do you figure out the MIC from a strip assay?

where the growth intersects the strip

who discovered the first antibiotic? which antibiotic was it?

Alexander fleming, penicillin

which scientists were the first to PURIFY penicillin and use it to treat a bacterial infection?

ernst cain and Howard florey

in the 1940s and 50s, _____ screened thousands of soil isolates for antimicrobial natural products. what is this called?

Selman Waksman; bioprospecting

antibiotics inhibit processes _____ for bacterial viability

essential

antibiotics are useful for treating infections because they generally do not target these processes in _____

human cells

what are some examples of bacterial processes targeted by antibiotics?

peptidoglycan synthesis, protein synthesis, transcription, metabolism

_____ produce >70% of clinically used antibiotics and antifungals

actinomycetes (ex. streptomyces)

actinomycetes are _____, _____-forming bacteria that produce many clinically relevant antimicrobials

filamentous, spore

where are actinomycetes found?

ubiquitous (soil, ocean, plants, animals, etc.)

why did the discovery of new antimicrobials by streptomycetes grown in the rich lab media quickly stop?

streptomycin doesn’t produce much antibiotic in rich conditions

what are the 2 main reasons why bacteria make antibiotics?

inhibit growth of competition, protect symbiotic hosts from pathogens

Passalid beetles eat _____ and form a structure called a _____. The beetle droppings act as a food source and the bacteria found in the beetles produce antimicrobials to help _____

decaying wood; battle gallery (hot and humid!); preserve the food

leaf cutter ants harvest leaves that act as _____ for _____ found in their nests

nutrients; fungi

leafcutter ants cultivate and consume _____ that grows on decaying harvested leaves

fungi

_____ is a pathogenic fungi that can invade and take over the Leafcutter ant’s fungi

Esovopsis

the Leafcutter ants are covered in a _____ bacteria called _____

actinomycete; Pseudonocardia

Pseudonocardia produces _____ that specifically kill _____ and prevent it from overtaking the Leafcutter ant’s food source

antifungals; Escovopsis

Beewolfs are predatory to _____

honey bees

once a Beewolf paralyzes the honey bee deposit them into a _____ and _____

hole; lay and egg on them

the paralyzed honey bee serves as a _____ for the beewolf _____

food source; larvae

How do beewolf use actinomycetes?

Act as an exit marker for the larvae to leave the cave

actinomycetes is _____ for the survival of the Beewolf larvae

essential

antibiotics are only targeted toward bacteria that are _____ to the organism producing it (this is called _____)

not related; kin descrimination

there is a gradual appearance of a _____ as the organisms plated together become more _____ related

zone of inhibition; distantly

antibiotics exhibit immense _____

chemical diversity

how are antibiotics so diverse?

30 molecules have almost infinite rearrangements

why is it useful to understand how natural antibiotics are synthesized?

so we can make new ones

most antibiotics can be made with _____ different enzyme classes

two

what are the two classes of enzymes that produce antibiotics?

non ribosomal peptide synthases (NRPs) and Polyketide synthases (PKS)

NRPs are enormous _____ that make _____ antibiotics

modular enzymes; polypeptide

NRPs function like and _____ and each PCP domain gets charges with a specific _____

assembly lines; amino acid

the amino acids on an NRPs are transferred onto successive _____ domains in a _____ order, elongating the chain

PCP; fixed

antibiotics can be further _____ after synthesis

modified

NRPs are specifically responsible for making _____

cyclic polypeptides

Polyketide synthases are responsible for making _____ antibiotics

polyketide

NRPs are responsible for making _____ antibiotics

polypeptide

PKS also function like an _____, and each ACP domain gets charged with an _____ and _____ group

acetyl-CoA; R

the acetyl groups in PKS are transferred onto successive _____ domains in a _____ order, elongating the chain

ACP; fixed

modern bioprospecting can identify _____ from metagenomic sequencing that encode for _____ or _____ genes

biosynthesis gene clusters (BCGs); NRPS; PKS

once you identify a genes coding for a antibiotic synthase, you can _____ them in other organisms to isolate a _____

express; bioactive compound

after sequencing the NRPS A domain, we are now able to _____ what amino acid will be incorporated, so we can use the BGCs as _____ and synthesize the molecule ourselves using _____

predict; instructions

typically antibiotic producers encode a mechanism of _____ which is likely the source of _____ in other organism

resistance; resistance

antibiotic resistance works via _____ mechanisms but underscores the need to develop a _____ antibiotic arsenal

diverse; diverse

what are three main strategies of antibiotic resistance?

prevent entry, prevent binding, reverse binding

what is the primary function of siderophores?

capture iron

iron is a critical element for _____ but in the environment it is most present in the _____ form

cellular function; insoluble Fe3+

siderophores _____ Fe3+ and make it _____, and thus available for microbial uptake

chelate; water-soluble

siderophores are diverse _____ and many are _____ peptides

small molecules; non-ribosomal

some diserophores have higher _____ for Fe3+ than others and can _____ from them

affinity; steal

siderophore piracy are microbes that import _____ produce by _____

siderophores; OTHER organisms

siderophores play a critical role in the _____, allowing plants to grow in iron-deficit _____

rhizosphere; soil

what is iron needed for in microbes?

respiration!

bacteriocins are secreted proteins that are _____ to bacteria

toxic

are bacteriocins larger or smaller than antibiotics?

much LARGER

bacteriocins are made by standard _____ translation and most are _____ post-translationally

ribosomal; modified

what does RIPPs stand for?

ribosomal produced and post-translationally modified peptides

Bacteriocins typically only inhibit _____-related species

closely

most bacteriocins work by forming _____ in the bacterial _____ and are typically named about the species they _____

pores; bacterial membrane; target

talons are the _____ form of secreted antagonists

LARGEST

the structure of tailocins are homologous to _____

phage tails

Tailocins are ONLY produced by _____

cell lysis (trade-off!)

Tailocins are typically _____ spectrum

narrow (like phages!)

tailocins infect cells through standard _____ receptors, but instead of injecting a _____, it simply creates a _____ to extracellular space which causes a collapse of _____

phage; phage genome; conduit; PMF

how are bacteria ultimately killed by Tailocins?

collapse of PMF = no respiration

rank the secreted antagonists from smallest to largest

antibiotics, siderophores, bacteriocins, tailocins

rank the secreted antagonists by INCREASING spectrum

tailocins, bacteriocins, siderophores, antibiotics