Can a plasmid contain antibiotic synthesis and resistance genes?

[Nathan McCorkle]

Such that it would first start expressing resistance, which would
trigger antibiotic production. The cell would be protected from the
antibiotic, and as long as the antibiotic was pumped out (maybe the
resistance is an efflux pump) it would only kill neighboring
non-transformants.

This would eliminate the need for buying antibiotics.

--
-Nathan

[Sebastian Cocioba]

IIRC the antibiotic is first taken up and then gets degraded so the resistance is internal. Seems like its analogous to plugging a battery into itself. It would only waste energy? I may be wrong though. Let me find some refs.

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[Nathan McCorkle]

On Tue, Jan 29, 2013 at 1:37 PM, Sebastian Cocioba <scoc...@gmail.com> wrote:
> IIRC the antibiotic is first taken up and then gets degraded so the
> resistance is internal.

Right, resistance is internal, provided by the gene on the plasmid.
The antibiotic wouldn't have to be 'taken up' in this case, only
pumped out.

Obviously the organisms we've discovered antibiotics in, are resistant
to those they produce.

> Seems like its analogous to plugging a battery into
> itself. It would only waste energy? I may be wrong though. Let me find some
> refs.

If you call transformant selection a 'waste' of energy, otherwise
you're 'wasting' money on expensive reagent companies.


--
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[Dakota Hamill]

I would think for large scale production of certain antibiotics, they would do scale up fermentation of an organism containing the necessary genes.  In some cases, the organism which lead to the discovery of the particular compound might not be the choice organism to do the scaled up fermentation in.  So in that case, I do wonder if companies simply provide that resistance gene + production gene in the form of a plasmid, or if they actually put it into the genome of the organism and create a mutant strain.  I'm not 100% positive but I think it'd be towards creating a mutant, since I imagine plasmids could be diluted out of the population, so to speak, over the course of the fermentation.

I think they've done it with yeast for brewing etc, though that could just be years of selection pressure, not direct genome manipulation.

[Sebastian S. Cocioba]

http://www.ncbi.nlm.nih.gov/m/pubmed/17238916/

Here is a recent article on the matter of exporting the drug before it gets too crowded in the cell. Interesting and relevant. 

Sebastian S Cocioba

CEO & Founder

New York Botanics, LLC

Sent via Mobile E-Mail 

On Jan 29, 2013, at 4:56 PM, Dakota Hamill <dko...@gmail.com> wrote:

I would think for large scale production of certain antibiotics, they would do scale up fermentation of an organism containing the necessary genes.  In some cases, the organism which lead to the discovery of the particular compound might not be the choice organism to do the scaled up fermentation in.  So in that case, I do wonder if companies simply provide that resistance gene + production gene in the form of a plasmid, or if they actually put it into the genome of the organism and create a mutant strain.  I'm not 100% positive but I think it'd be towards creating a mutant, since I imagine plasmids could be diluted out of the population, so to speak, over the course of the fermentation.

I think they've done it with yeast for brewing etc, though that could just be years of selection pressure, not direct genome manipulation.

[Cathal Garvey]

In short, yes. But your resistance gene, if available, has to be one
that confers immunity without destroying the antibiotic;
beta-lactamases, for example, are exported from the cell and are highly
efficient, so you'd be wasting tons of energy with little likely gain.

The area to look at here is "lantibiotics" and other bacteriocins, as
these are often produced by relatively short operons ("short" here means
~2kb), and usually include an immunity gene that confers resistance
passively. For example, many bacteriocins interfere with cell membranes
by binding to certain surface ligands (can't recall which/where). Their
immunity genes produce something that alters or "caps" these ligands, so
the bacteriocin can't bind to the cell surface anymore.

I considered using bacteriocins to generate a "homocidal plasmid", but
the costs at the time of including a bacteriocin operon in the plasmid
were too big. Mind you, I was limiting myself to cis-genic options for
B.subtilis, whereas there were other compatible systems that affect
B.subtilis which would have been smaller and more efficient.. if the
immunity genes would have worked well in a different host.

[Mega]

If there were such plasmids, they would be bridely used I think. 

that would e.g. be the best case for chloroplast integration. 

They say that chlorplast transformation is very hard-core because if you leave one non-transformed chloroplast per plant cell it may divide quicker than those transformed. Thus without long exposure to the antibiotic, at the end you get the plant you started working with. 

Lantibiotics usually have a small spectrum IIRC, so you have to find one that is lethal to the target species.  

[Mega]

http://www.pnas.org/content/103/25/9661.abstract

(Amplification of the entire kanamycin biosynthetic gene cluster during empirical strain improvement of Streptomyces kanamyceticus)

http://www.academia.edu/200477/Heterologous_expression_of_the_kanamycin_biosynthetic_gene_cluster_pSKC2_in_Streptomyces_venezuelae_YJ003

http://www.ncbi.nlm.nih.gov/pubmed/20936279

[Mega]

Here it is written a bit clearer: 

http://link.springer.com/article/10.1007%2Fs00253-007-1096-4?LI=true

They isolated a cosmid from a kanamycin producing bacterium, and put the genes in another bacterium that does not produce kanamycin by default.

Result: Kanamycin (A) was produced.

[Mega]

May I cite

Analysis of kanamycin biosynthetic genes in pSKC2 pSKC2 has a total of 32 kb and revealed 28 open-readingframes including kanamycin biosynthetic genes (

kanA

,

kanB

,

kanC 

,

kanD

,

kanE 

,

kanF 

,

kanI 

,

kanK 

, and

kacL

),resistance genes (

kanM 

and

kanH 

), and regulatory genes(

kanL

and

kanG 

; Fig.1a).

(http://www.academia.edu/200477/Heterologous_expression_of_the_kanamycin_biosynthetic_gene_cluster_pSKC2_in_Streptomyces_venezuelae_YJ003#outer_page_3)

Of course, one would leave out the regulatory stuff. That makes 9 Genes in total (I guesstimate some 9,5 kbp or more) for the production. Quite big for a would-be standard-plasmid.... 

You probably would take the same resistance proteins because as far as I understood they don't degrade kanamycin, they rather methylate ribosomal RNA to be restistant

 

[Mega]

And one last post for those who want it synthesized now :D 

One protein has around 1000 bp as usual.

http://www.ncbi.nlm.nih.gov/nuccore/51981291

The overwiev

KanA

http://www.ncbi.nlm.nih.gov/protein/51981314

KanB

http://www.ncbi.nlm.nih.gov/protein/51981313

KanC

http://www.ncbi.nlm.nih.gov/protein/51981315

KanD

http://www.ncbi.nlm.nih.gov/protein/51981318

KanE

http://www.ncbi.nlm.nih.gov/protein/51981316

KanF

http://www.ncbi.nlm.nih.gov/protein/51981322

KanI

http://www.ncbi.nlm.nih.gov/protein/51981312

KanK

http://www.ncbi.nlm.nih.gov/protein/51981309

KanL

http://www.ncbi.nlm.nih.gov/protein/51981308

Maybe one could make a fusion protein to comine the active sites in one protein, or something alike, to make the construct smaller?

[Koeng]

anyone got this strain to PCR it?

[Andreas Sturm]

Well, streptomyces is a kind of actionmycetes. They are ubiquitous (spread over the whole globe). They make that smell that smells like "rain on a warm street".

There may be a chance to do the PCR from soil?

[Andreas Sturm]

Or, of course you could ask the original authors for a sample. Of course that depends if there is aa email adress given in the paper (or: in older papers a postal adress). And, how old the paper is (are the scientists still alive, do they still have access to the  same lab where the strain is deposited?)
However: Worst thing they can say is no.

[jlund256]

Many antibiotics are made by bacteria, as part of their war on competing bacteria.  So they make antibiotics that they themselves are resistant to.  I don't know the field well enough to know whether any antibiotic synthesis genes are carried on plasmids, though there is nothing preventing it.

 

[Patrik D'haeseleer]

Have a look at toxin-antitoxin systems, which are often found on plasmids. If the toxin degrades slower than the antitoxin, this prevents the plasmid from getting lost from the bacteria - a nice "selfish gene" scenario.

[Andreas Sturm]

Thank you!!! 

That was the key word I'm missing!! 

MazEF system: 

MazF

is a toxin and MazE is an antitoxin that antagonizes MazF; (2)

MazF is long-lived, whereas MazE is a labile protein degraded

in vivo by the ATP-dependent ClpPA serine protease

Is anyone able to make an educated guess, whether chloroplasts have an  ATP - dependent ClpPA serine protease  like E.Coli that would allow the use of this system? 

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[Andreas Sturm]

Is anyone able to make an educated guess, whether chloroplasts have an  ATP - dependent ClpPA serine protease  like E.Coli that would allow the use of this system? 

Came to think of it: 

If not so, I can just add a Serine-dependent protease by PCR :D  

[Andreas Sturm]

Very good... 

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC92497/

Te relBE operon does exactly this. relE, the toxin is 288 nucleotides long, while relB is 240 ncts long. relB is the antidote. 

In this study they tested if it could also be used in yeast. 

[Cathal Garvey]

Slight distinction between toxin/antitoxin systems and antibiotics
though; the latter will kill neighbouring cells that don't harbour the
plasmid, whereas the former will only kill a carrying cell if it loses
the plasmid. There will be a certain number of survivors anyway, who
survive the toxin by chance, good timing, or mutation.

On 31/01/13 07:21, Patrik D'haeseleer wrote:
> Have a look at toxin-antitoxin systems

> <https://en.wikipedia.org/wiki/Toxin-antitoxin_system>, which are often

[Mega]

But if the toxon is secreted, then it's self-selecting, I assume.

[Cathal Garvey]

Not necessarily; does the toxin enter other cells? Does it get degraded
quickly by extracellular proteases?

Toxin/antitoxin systems more often appear to be selfish plasmid
strategies to force individual cells to keep the plasmid. Antibiotics,
on the other hand, are usually a cellular strategy (which might or might
not be plasmid-encoded) to kill off the competition.

Some antibiotics of course serve both purposes; bacteriocins are often
encoded with their own resistance gene, and so they will act as a
toxin/antitoxin and an antibiotic system. By contrast, most
"traditional" antibiotics don't actually have a "resistance" or
"immunity" gene, because they are made by a species that isn't affected
by them.

For example, penicillium fungi don't suffer any harm from penicillins,
so they don't have or need a gene to make them resistant; therefore, the
only natural resistance genes for penicillins that I know of are
beta-lactamases, which destroy the antibiotic rather than just making
the cell immune.

So, toxin/antitoxin will increase the stability of a plasmid
dramatically, but AFAIK they won't have any effect on surrounding cells;
if they did, they'd be reclassified as bacteriocins or antibiotics.

[Andreas Sturm]

The toxin has a half-life of 30 minutes while the antitoxin is much shorter. Was it half a minute or 5 minutes? 

So, toxin/antitoxin will increase the stability of a plasmid
dramatically, but AFAIK they won't have any effect on surrounding cells;
if they did, they'd be reclassified as bacteriocins or antibiotics.

Well, if I add  e.g. a chloroplast export signal peptide (are there any? There must be?!), it escapes the chloroplasts. The signal peptide is cleaved off, and it enters another chloroplast without resistance. And destroys it. 

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Some antibiotics of course serve both purposes; bacteriocins are often
encoded with their own resistance gene, and so they will act as a
toxin/antitoxin and an antibiotic system. By contrast, most
"traditional" antibiotics don't actually have a "resistance" or
"immunity" gene, because they are made by a species that isn't affected
by them.
For example, penicillium fungi don't suffer any harm from penicillins,
so they don't have or need a gene to make them resistant; therefore, the
only natural resistance genes for penicillins that I know of are
beta-lactamases, which destroy the antibiotic rather than just making
the cell immune.

So, toxin/antitoxin will increase the stability of a plasmid
dramatically, but AFAIK they won't have any effect on surrounding cells;
if they did, they'd be reclassified as bacteriocins or antibiotics.

On 31/01/13 16:42, Mega wrote:
> But if the toxon is secreted, then it's self-selecting, I assume.
>

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For example, penicillium fungi don't suffer any harm from penicillins,
so they don't have or need a gene to make them resistant; therefore, the
only natural resistance genes for penicillins that I know of are
beta-lactamases, which destroy the antibiotic rather than just making
the cell immune.
So, toxin/antitoxin will increase the stability of a plasmid
dramatically, but AFAIK they won't have any effect on surrounding cells;
if they did, they'd be reclassified as bacteriocins or antibiotics.

On 31/01/13 16:42, Mega wrote:
> But if the toxon is secreted, then it's self-selecting, I assume.
>

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So, toxin/antitoxin will increase the stability of a plasmid
dramatically, but AFAIK they won't have any effect on surrounding cells;
if they did, they'd be reclassified as bacteriocins or antibiotics.
On 31/01/13 16:42, Mega wrote:
> But if the toxon is secreted, then it's self-selecting, I assume.
>

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[Cathal Garvey (Phone)]

If the toxin and antitoxin are expressed in the chloroplast, and you export only the toxin, would that not kill the cell? The toxin in question can kill yeast, could probably do it to plants too?

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[Nathan McCorkle]

On Thu, Jan 31, 2013 at 12:04 PM, Cathal Garvey (Phone)
<cathal...@cathalgarvey.me> wrote:
> If the toxin and antitoxin are expressed in the chloroplast, and you export
> only the toxin, would that not kill the cell? The toxin in question can kill
> yeast, could probably do it to plants too?
>

Hmm, you could say the same thing about added antibiotic too then, I
think... if the chloroplast is resistant, but the main cell isn't.
Maybe there are some toxins/antibiotics that only affect
bacteria/chloroplast? Or maybe if a plant cell's chloroplast died, it
would simply die as a result? If plant cells live in defined media in
dark conditions, that might tell us if killing/halting just the
chloroplast might be enough.

--
-Nathan

[Nathan McCorkle]

On Thu, Jan 31, 2013 at 8:53 AM, Cathal Garvey
<cathal...@cathalgarvey.me> wrote:
> Not necessarily; does the toxin enter other cells? Does it get degraded
> quickly by extracellular proteases?
>
> Toxin/antitoxin systems more often appear to be selfish plasmid
> strategies to force individual cells to keep the plasmid. Antibiotics,
> on the other hand, are usually a cellular strategy (which might or might
> not be plasmid-encoded) to kill off the competition.

Well looking through some papers, it seems that plastid transformation
is often selected with spectinomycin resistance genes, where the
spectinomycin is applied externally. So it must be around the main
cell before getting to the plastid for degradation.

It seems theoretically possible that you could just use this 18kb
spectinomycin operon, which is biosynthesis and resistance
http://www.ncbi.nlm.nih.gov/nuccore/EU255259

Found via
The Gene Cluster for Spectinomycin Biosynthesis and the
Aminoglycoside-Resistance Function of spcM in Streptomyces spectabilis
Kyoung-Rok Kim, Tae-Jong Kim, Joo-Won Suh
http://home.kookmin.ac.kr/~bior/Curr.%20Microbiol%2057-371.pdf

--
-Nathan

[Andreas Sturm]

If the toxin and antitoxin are expressed in the chloroplast, and you export only the toxin, would that not kill the cell? The toxin in question can kill yeast, could probably do it to plants too?

Well, e.g. kanamycin is just toxic to plant cells because it destroys their chloroplasts. (It Inhibits the bacterial translational machinery of plastids) .

But IIRC, it is harmless for eukaryotic ribosomes.



The toxin however, would probably have som toxicity to the plant's nuclear machinery. But maybe it would be too weak to kill it entirely. Or the antitioxin would have to be released into the cytosol too, not having a chloroplast import protein thus not invading other chloroplasts?

[Andreas Sturm]

Thanks for that Nathan!

Now I have the sequences of biosynthesis pathway for kanamycin and streptomycin  antibiotics ;)

However, both are kind of big. KanSynth ~ 10 kbp ; strepto Synth ~ 18 kbp.


The toxin/antitoxin has just some <1kbp in total!!

[Andreas Sturm]

What may be feasible (but labour intensive): Make the cell produce the antidote with nuclear localization signal and toxin with chloroplast transit peptide and kanamycin resistance (for cell selection!).

At the same time you'd have to transform the chloroplasts with the genes of interest and the resistance.

[jarlemag]

One thing seems to have been forgotten in the discussion: Evolution.

Using externally supplied antibiotics, a selection pressure is constantly applied, selecting for cells with resistance to the antibiotic.
If the antibiotic is internally produced, cells could survive not only by expressing a resistance gene, but also by turning off/losing the resistance gene.
As both antibiotic production and resistance has an energy cost, one would expect cells which do not express the antibiotic to have an advantage over cells doing so and thus be selected for. If the antibiotic producing activity is lost, there is then no selection pressure for the resistance gene, and that might rapidly be lost too.

However, as long as there are enough non-revertant cells expressing the antibiotic, the cells in a population might keep each other in check.

This seems like an interesting topic for modelling/simulation. What could the evolutionary dynamics look like in this situation?
Many cells are expressing an antibiotic, thus maintaining selection pressure for the antibiotic resistance genes. However, any one cell might retain the antibiotic resistance genes while dropping the antibiotic production gene, possibly gaining an advantage. If enough cells do this, the advantage of having the antibiotic resistance genes disappear. In the initial condition, any single cell would thus lose out if it dropped both its antibiotic production and resistance genes. However, the population as a whole would benefit (increase growth) by doing so. Could the population overcome the disadvantage, at the single cell level, of mutations that would confer benefit at the population level?

Thoughts?

Best regards,
JP

[Andreas Sturm]

This seems like an interesting topic for modelling/simulation. What could the evolutionary dynamics look like in this situation?
Many cells are expressing an antibiotic, thus maintaining selection pressure for the antibiotic resistance genes.

This shouldn't be simulated but done ;)

Actually, printing out the two genes needed (relBE) would cost "just" ~250$ (cheaper at the chinese, maybe 160$ or so)



 

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[Nathan McCorkle]

On Fri, Feb 1, 2013 at 1:54 PM, jarlemag <jarle...@gmail.com> wrote:
> One thing seems to have been forgotten in the discussion: Evolution.
>
> Using externally supplied antibiotics, a selection pressure is constantly
> applied, selecting for cells with resistance to the antibiotic.
> If the antibiotic is internally produced, cells could survive not only by
> expressing a resistance gene, but also by turning off/losing the resistance
> gene.

I think you meant by turning off production of the antibiotic. Turning
off resistance would kill the cell. You can try to get around this by
placing your selection genes next to other genes that would cause a
lethal auxotrophy. The idea being that if recombination happens,
there's a greater chance it will mess with the nearby stuff, which
would again cause the cell to die.

> As both antibiotic production and resistance has an energy cost, one would
> expect cells which do not express the antibiotic to have an advantage over
> cells doing so and thus be selected for. If the antibiotic producing
> activity is lost, there is then no selection pressure for the resistance
> gene, and that might rapidly be lost too.
>
> However, as long as there are enough non-revertant cells expressing the
> antibiotic, the cells in a population might keep each other in check.
>

well in the case of spectinomycin, one of the proteins involved in
synthesis of the antibiotic (it transferred a methyl) also conferred
resistance (in addition to the resistance pump gene)

so if synthesis was lost, some resistance would be lost too, so I
guess to make this more efficient you need to find a way to increase
the systems interconnectedness.

Maybe you could make another synthesis protein ALSO function as a
transcription factor for resistance... etc

--
-Nathan

[jarlemag]

Yes, that's what I meant - sorry for the mistype.
Good idea regarding gene placement, I'd never thought of that before.

I feel like an important point was missing from my previous post, though: Maybe it's too obvious to warrant mention, but in nature one would expect the selection
pressure for maintaing antibiotic production + resistance would be coming from competition with those organisms which the antibiotic inhibits/kills.

- J