HMB304 - Lecture 6 - Genetic derepression of biosynthetic gene clusters in Streptomyces as a method of antibiotic discovery

what are antibiotics produced by?

-they are produced by BGCs → biosynthetic gene clusters

how many BGCs does each streptomyces species encode?

-they encode 20-50 BGCs

what does each cluster in BGCs do?

-each cluster directs the production of one metabolite (antibiotic)

what is found based on the genome sequencing of wild streptomyces?

-that there are thousands of uncharacterized BGCs waiting to be tested

-only 10% of these clusters have been expressed in lab conditions

what do traditional culturing methods lead to?

-they lead to re-discovery, where the same BGCs are typically “turned on”

what characteristic are most BGCs?

-most BGCs are transcriptionally silent

“one strain, many compounds… but you might be the same compounds as everyone else”

why are BGCs not expressed sometimes?

-they aren’t expressed sometimes because antibiotic biosynthesis is energetically expensive for the cell

what is the process of BGCs being repressed?

1. nucleoid-associated proteins bind DNA and affect the entire genomes architecture

2. global regulators directly or indirectly affect the expression of many BGCs

3. cluster-situated regulators encode within a cluster and regulate its expression

repress → repress//activate → activate

if you want to manipulate Streptomyces to produce more antibiotics, how would you to modify their regulation?

-by manipulating the localized components (nucleoid-associated proteins, global regulators, cluster-situated regulators) by placing them under the control of strong, constitutive promoters ensures that the biosynthetic machinery runs continuously at maximum capacity, bypassing normal cellular checks

what are the pros of manipulating nucleoid-associated protein (NAP)?

-Delete! Which affects many BGCs and are highly conserved

what are the cons of manipulating nucleoid-associated protein (NAP)?

-it globally upregulates many types of genes; energy intensive

what are the pros of manipulating global regulator (GR)?

-delete or overexpress; has a broad affect and less energy than NAP

what are the cons of manipulating global regulator (GR)?

-we need to know which global regulators exist (sequenced genome)

what are the pros of manipulating cluster-situated regulator (CSR)?

-overexpress; it only targets one BGCs; less energy int.; easy link between BGC and compound

what are the cons of manipulating cluster-situated regulator (CSR)?

-has limited induction, and we need to know your CSR (sequenced genome)

what is Lsr2?

-it is a nucleoid associated protein that binds AT-rich DNA

-represses horizontally-acquired or “foreign” sequences

-since Streptomyces BGCs are often foreign, or shared between species, these sequences are often AT-rich and repressed by this

-represses antibiotic production

what can Lsr2 do?

-it can polymerize along the DNA, and/or compact sections of the chromosome to exclude transcriptional machinery

what happens if we delete Lsr2 from a model streptomyces species?

-BGCs are more expressed since with it, it represses antibiotic production

what is Lsr2 knock-down?

-it is used to induce antibiotic production

-we express a version of Lsr2 with a deficient DNA-binding domain, and it acts like a sponge for the native Lsr2

-a portable system that can be used on many unstudied/wild streptomyces species

-it has a narrow spectrum of activity

which wild species had activity with the Lsr2 knock-down?

-a wild species called Bim8 did, against the lab indicator strains

what can be used to avoid the same rediscovery problem?

-we can use the antibiotic resistance platform (ARP) to combat this

what are the steps to finding the antibiotic?

1. we need to first find the BGC (use “break it to find it”") → sequenced the wild type and mutant strains to assemble a genome and predict BGCs

2. Find the differences between the wild-type and mutant strains

3. then confirm the BGC (antibiotic connection with clean genetics) → overexpress cluster-situated gene or delete core biosynthetic gene

4. then detect abundance of all molecules and find one that is new in the Lsr2 KD

5. then purify compounds (more chemistry)

what is “break it to find it?

-it is generated mutants that were no longer antibiotic-producing

what did the chemical analysis of normal Bim8 compared to Bim8 with Lsr2 knock-down show?

-it showed that the new compound was a macrolactone (large ring shape)

-the structure turned out to be an antibiotic call luminamicin

what is luminamicin?

-an antibiotic that was actually discovered in the 80s, but had very weak antibiotic activity

-wasn’t tested against N.gonorrhoeae until now

what else is apart of the family of structurally related macrolactones besides luminamicin?

-nargenicin has a similar strucuture

→ its an experimental compound with activity against antibiotic-resistant S. aureus (MRSA)

→ has limited activity against N. gonorrhoeae

^→ and unlike nargenicin, luminamicin cannot inhibit the growth of S. aureus

(similar structures, different bacteria inhibited)

what is nargenicin?

-its an experimental compound with activity against antibiotic-resistant S. aureus (MRSA)

-has limited activity against N. gonorrhoeae

-is a macrolactone

-it binds the catalytic site of the DnaE subunit

of the different gene types in a BGC, which one do you think is the most useful for predicting an antibiotic's mechanism of action?

-self-resistance gene (BGCs) are the most useful

what are examples of resistance genes that are often mobilized by mechanisms of antibiotic resistance?

-done through

• target modification

• antibiotic modification

what are examples of resistance genes that are often encoded in BGCs to protect the producing organism

1. shield/sponge/decoy

2. functional alternative (‘bind’ to antibiotic)

what is the process of finding out luminamicin BGC self-resistance mechanism?

1. check if the gene it encodes actually is a gene for self-resistance?

→yes! Expressing dnaEL in a sensitive bacteria helps it grow on luminamicin

→ expressing dnaE (native polymerase) does not help

2. test if the DnaE-like protein can also function as a polymerase, and how sensitive it is to luminamicin (does luminamicin inhibit DnaE (from N. gonorrhoeae)?)

why can’t luminamicin bind the streptomyces DnaE-like protein?

-the proteins (DnaE & DnaEL) look exactly the same, except in their tails

→ PHP-domain

what was the new mechanism of action found in luminamicin regarding DnaE and DnaEL?

-that luminamicin binds the PHP domain of DnaE from N. gonorrhoeae

-so, it has the same target protein as nargenicin, but a different mechanism/binding site!

→ DnaE (N.gonorrhoeae) + luminamicin → collect flow-through (excess antibiotic) & wash,wash,wash → elute any “bound” antibiotic

why is having a new mechanism of action important binding site for treating antibiotic-resistant infections?

-it reduces the likelihood of cross-resistance

what are BGCs regulated by?

-they are regulated hierarchically by nucleoid associated proteins, global repressors, and cluster-situated regulators

what is genetic derepression?

-it is a mechanism of antibiotic discovery

what can self-resistance genes give us a clue about?

-it can give us a clue about a compound’s target and mechanism of action

can old antibiotics be good antibiotics?

-old antibiotics can be good antibiotics! We should mine both streptomyces as natural resources and old literature/structures