Genetic variability in a bacteria species

Questions on the method used for the study. The first bacterial cell did not have any resistance. This was marked in a color to identify its origin right?
You mentioned that several plates were used in the experiment. Were the bacterial cells from the first generation of clones that were marked in the first plate used for all other plates? Are the clones from the first cell easily identified to determine what generation produced the resistance on each plate. or are the other plates used for each generation produced from the first plate?If only a few of the colonies survived, do you mean the entire plate or is it individuals in the colonies on each plate that survived?Can the study pinpoint exactly when it occurred for resistance in generation time or the original bacteria cell?

Hi crash, Where does the statement found Here say what you said? I don't think that 1. means 1 bacteria.I understand the statement to mean more than one as they are to be spread out on a plate.It nowhere says all these bacteria was from a single bacteria. That is an assumption.Since the so called single individual that is your founding individual was not exposed to penecilin you have no way of knowing whether that individual had an immunity to penecilin. Yes that is obvious as all did not survive when exposed to penecilin. Well no there has been no proof that the trait was acquired during the "log phase".It is proof that something has occured.Either the original single individuall bacteria as you put forth had to have no immunity and some of its offspring gained immunity or the original single individual bacteria posessed immunity and then some of the offspring lost that immunity and their offspring did not have the immunity.We are in agreement that the bacteria that survived had the immunity prior to exposure to penecilin.We just don't agree on how that immunity began to exist.My question then is if the immunity was not in the DNA how did any bacteria survive until today?It makes no difference what antibiotic science can come up with there are bacteria that are already immune to that antibiotic.That supports that the DNA has those immunities already in it. One assertion does not prove another assertion.There are two possibilities.One possibility is that the immunity trait was acquired.The other possibility is that the original individual had immunity and some of the offspring and their offspring did not receive that immunity due to a bad mutation.God Bless,

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"John 5:39 (KJS) Search the scriptures; for in them ye think ye have eternal life: and they are they which testify of me."

You are correct. The Lederbergs' experiment does not show the source of the mutation. It only shows that the observed mutations were not induced by the antibiotic or after the antibiotic was introduced. The limitations of the experiment are no surprise; they are appropriate for the hypothesis being tested.That seems to be of little consequence in light of other more detailed experiments. Other experiments have been done showing mutations of bacteria genome over a large number of generations.One such experiment is described here:The reference linked to above describes several experiments where changes to the genomes of organisms over 100s to thousand of generations where detected. I don't see how any of the results are compatible with your theory that changes were already present in the original organisms.

From the original Lederberg paper:"In a typical experiment, a dense broth culture was grown from a single colony on plain agar."
http://profiles.nlm.nih.gov/BB/A/B/F/J/_/bbabfj.pdfSo you start out with an isolated, single bacterium that then produces a colony big enough to see. This colony is then transferred to a liquid medium to increase the population size. It is the only practical conclusion. If the original bacterium used to grow the original colony was resistant then all of it's descendants (barring the in 1 a few million mutations that would knockout the resistance gene) would also carry that resistance. Instead, only 1 in 10 billion of the descendants of that single bacterium are resistant to spectinomycin, as they discussed in the paper above. Therefore, the resistance had to originate in one of the descendants of that single bacterium. The descendants of that single bacterium were tested. If the resistance gene were present in the parent then it should also be present in the offspring. Yes, there was. Log phase represents the period during which bacteria are dividing exponentially (hence the use of "log" as in "logarithmic"). Since there was more than one bacteria from each spot on the master plate that was resistant it represents a mutation during the period of division, not during the stationary phase where the bacteria are not dividing. If the mutation occurred during stationary phase then we should not see the same spot producing colonies on multiple plates at the same position. This is why the bacteriophage resistance is a nice corrolary in the Lederberg paper. Bacteriophage resistance often results from an indel in the tonB gene. This results in a lack of the full tonB gene product which happens to be the binding site for bacteriophage, so these mutations confer bacteriophage resistance. It is interesting to note that these mutants occurred in 1 in every 10 million divisions.So we would expect the same for the antibiotic resistance gene, if it was present in the founding bacterium. One in every 10 million descendants would have a mutation that knocks this gene out and makes it non-functional. Therefore, one in every 10 million descendants of a resistant bacterium should be antibiotic sensitive. Given such long odds for this occuring in the first few generations it is rejected in favor of the resistance coming about in the descendants and not in the founding bacterium. The difference being is that we have the numbers on our side, while you don't. Why would bacteria have mutations conferring resistance to antibiotics that they have never seen? Sounds like mutation is random with respect to fitness afterall.Edited by Taq, : No reason given.

You understand that this is just a summary of the Lederberg's experiment, right? That this doesn't even begin to describe their actual techniques or results? That the images are cartoons, not actual science data?This is a summary that would be appropriate for 8th graders, roughly, so naturally it's not a complete description of their actual experiment. If you want to know what they actually did you need to go to the actual paper the Lederberg's wrote, which is this: Just a moment...It's no mark against you that you didn't go out looking for other sources - the one you found is a good place to start - but a lot of these questions you have can be addressed by going to the primary source, which is going to be the scientific literature that the Lederbergs had published. (It's an interesting and very accessible paper - I hadn't realized the Lederbergs invented replica plating.)To answer your question - I know that's what they did because that's the standard technique for setting up culture - you quadrant streak an agar plate to produce isolated colonies:Do you see the isolated colonies up at the top? By the pattern we know that those colonies each formed from an individual cell. If you take a sample of one of those colonies and transfer to liquid media, you have a soup of bacteria that are clones of each other. That soup is what the Lederbergs used, highly diluted, to inoculate their original plates. Each colony is the result of a single bacterium. That's how they grow. If they produced a plate with 30 colonies on it, they added 30 cells.That's so reliable that it's an established way to find out how many bacteria are in a liquid culture - take a sample, dilute it about a hundred-thousand-fold, spread it on a plate, and count how many colonies you get after a day of incubation. Each visible colony indicates the presence of a single cell; multiply it by the dilution factor and that's how many cells are in your original sample. It's a summary of the experiment. It's not going to say everything. You need to read the original paper. Certainly the single individual was exposed to the streptomycin (what they actually used), as were all its descendants. If the individual was resistant to the antibiotic then all of its clonal descendents would have been.Remember bacteria don't breed like people; there's nothing that "skips a generation." They don't obey the Mendelian genetics you may have learned in school because they have only a single chromosome, and therefore only a single allele per trait. Thus proving that antibiotic resistance was the acquired trait. The experiment is the proof, ICANT. If resistance was the widespread trait and loss of resistance the new thing, then each colony would have been primarily comprised of individuals who had retained the resistance, and therefore every single colony would have replicated on antibiotic media.That's not what happened. That proves that lack of resistance was the widespread trait and that resistance to the antibiotic was the new, acquired trait - acquired by random mutation.The proof of this is that some colonies replicated on antibiotic media and some did not, and that the same colonies died when exposed to antibiotic on the original plate. They survived by not living somewhere where they were exposed to streptomycin. Quite simple, actually. Esterichia coli lives primarily in the gut of animals - see, that's what "coli" in the name means, "colon" - which is a place where Streptomyces griseus, the bacterium in which streptomycin originally evolved, does not generally reside. Any particular mutation is very rare, so if the population had been originally resistance and susceptibility was the acquired trait, only a very small number of individuals would have lost the resistance to the antibiotic and resistant individuals would have dominated every single colony, which means that we would observe that every colony successfully replicated to antibiotic media.But they didn't. Some did and others did not. That proves that antibiotic resistance was the acquired trait, not the reverse. Like I've told you three times, now.

Well it doesn't but then that site isn't the original paper, it is a simplified precis. The actual original replication experiment paper can be found here (PDF). There will be more than one bacterium in the culture plated out, but it is unlikely that there will be more than 1 resistant bacterium so close together as to appear as only 1 colony unless the resistance strain already has a high frequency. Not there, but this is standard practice. To make sure that there is one originating bacterium the sample to be plated out will usually be grown from a colony from a streaked plate, a technique specifically developed to isolate colonies originating from a single bacterium.In the original paper they say 'In a typical experiment, a dense broth culture was grown from a single colony on plain agar'.When discussing a streptomycin resistance based experiment (Page 7 of the PDF) they mention having to grow up massive amounts of bacteria because the mutation was so rare. 20 or 30 plates with 3 X 10⁹ bacteria plated out on them and only 3 resistants. Can you explain to us how this could be the case if the mutant was pre-existing (even as just 1 bacterium) in the initial starting colony as you posit?TTFN,WK

Hi WK, Where does this paper mention the experiment that I referenced from Berkeley?This experiment was concerning accumulating resistance.The one I referenced when exposed to penecilin some colonies died and others did not. But when exposed to the penecilin none gained resistance they were either immune or they died.When you click explore at the bottom of the page I referenced it takes you HereWhich says: Is this lesson being taught at Berkeley correct?Back to the bacteria.How many different mutations are possible in bacteria?Because if there is one mutation in 10 billion that survive why would that one mutation be a trait of immunity?Couldn't it just as easily be a netural mutation?Couldn't it just as easily be a delentious mutation?Couldn't it just as easily be the ability to digest nylon?If mutations are truly random as proposed isn't it possible that there are hundreds of mutations that could arise rather than a mutation of immunity to penecilin?But my further question is why isn't it possible for the DNA to contain the immunity?There are people who have immunities that are not active in their offspring but is active in later descendants. Why could that not be possible in bacteria?God Bless,

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"John 5:39 (KJS) Search the scriptures; for in them ye think ye have eternal life: and they are they which testify of me."

OK, now I'm confused. How does the Lederberg's 1952 paper documenting their experiments not relate to the the Berkley site offering a precis of the basic methodology of the Lederberg's experiments published in 1952?Did you actually read and understand either of them? No, it wasn't, it showed the evolution of resistance. Except you didn't reference anything other than the Berkley page which refers to the Lederberg's 1952 work, the only applicable paper is the one I linke to, the fact that you are incapable of recognising that the two are the same is your own problem. The antibiotic in the Lederberg's paper is Streptomycin rather than penicillin, but otherwise there is no difference. I don't know how it is taught at Berkley I only know what that page shows. Like I already said, it is a very simplified explanation of the experiment. The only bit they leave out that is relevant to our discussion is that before the initial plating out the bacterial cuture came from a clonal isolate and was susceptible to the phage/antibiotic. A vastly huge number, essentially incalculable. If you have a more specific question I might be able to be a bit more specific. Well why would you assume that was the survival rate of a mutation? And why would you assume only one mutation occurred in the experiment? With a relatively small bacterial genome and a sufficient period of incubation, not neccessarily a massively long time, there is a very good probability that every single possible point mutation that the genome could undergo has occurred at least once. There is little to no reason to suppose that there is only one possible mutation which gives rise to resistanceIn a selection experiment the reason it survives is because in the presence of antibiotics resistance is a very beneficial trait. Unless it is highly detrimental otherwise I don't know why you think it should necessarily be lost from the population once it has arisen. They do, why you think they don't is beyond me. The Lederberg's paper shows that resistance to bacteriophages and streptomycin can also arise, and the chances are that in some cases you could simply use the replicate plate method to screen for as many selective conditions as you like, that was one of the entire points of the Lederberg's methodology. Because there is no way that makes sense. If the DNA was there why wasn't the original bacteria resistant? If it is turned on in response to the antibiotic why aren't the majority of the colonies on the replicate plates resistant. Well this is vague enough only to be very unhelpfull, care to provide an actual specific example? One point I would make is that people are diploid and bacteria aren't. Bacteria don't have recessive traits like sexual organisms, there is essentially no skipping of generations for bacterial traits.I think you need to take more time to understand these experiments rather than just trying to shotgun nonsensical questions in hopes that one of your wild shots will find some target. If you understood these experiments at all you would understand why many of your questions make no sense.TTFN,WK

The paper is the experiment you referenced from Berkeley. Are you asking why a paper written in 1952 doesn't reference a website you're looking at in 2010? I trust even you can solve that conundrum. There's no way to count, really; the E. coli genome is comprised of 4.6 million base pairs and 4300 genes. Any one of those base pairs could be deleted, duplicated, or substituted, or any number of base pairs could be added at any position on the chromosome. Any one of those genes could be mutated. It's a bit like asking "how many different ways could you insert a typo into Moby Dick." Nobody said that "only one mutation in 10 billion survive". But only one mutation in 10 billion might provide resistance to antibiotic, which is why that bacterium survived and others did not - natural selection. Yes, absolutely; those mutations probably did arise, but the environment selected for antibiotic resistance not nylon digestion, so the nylon digestive bacteria died when antibiotic was introduced to the plate. People are diploid and bacteria are not. Human beings have two copies each of 23 different chromosomes. Bacteria have only one copy of a single chromosome.If something's in their DNA, it gets expressed. There's no "skipping a generation" in bacteria, it's not physically possible for that to happen. As I've repeatedly told you bacteria don't follow Mendelian genetics.

Will try not to overlap with previous posts . . . Because that mutation allowed the bacteria to survive in the presence of antibiotic. I would think that this should be obvious. If it was a neutral mutation in an environment containing antibiotic then it would have been killed by the antibiotic. If you used the same master plate to stamp out colonies on plates containing bacteriophage you would find that there are bacteriophage resistant mutants on the same master plate, but they are not the same bacteria that are antibiotic resistant. All of the bacteria are mutating, but not all are gaining the same mutations. That's the whole point. It is in the DNA. It is in the DNA of the descendants of the founding bacterium, but it was not in the DNA of the founding bacterium. Therefore, the DNA necessary for antibiotic resistance had to come about through mutation of ancestral DNA. Not only that, but these mutations occurred in the ABSENCE of antibiotics demonstrating that mutations are random with respect to fitness. Because people are diploid organisms. Bacteria are haploid. Bacteria use clonal reproduction. People use sexual reproduction.

These other mutations do, indeed, occur. But the population sizes of bacteria used in these experiments are huge, millions upon millions of bacteria are involved. The reason that the penicillin mutation is the one we detect is that its the one we're looking for. Bacteria, unlike people, only have a single copy of their DNA. They are haploid rather than diploid, in the biological parlance.In a diploid organism there are two copies of each gene. Often one version of the gene will be dominant, which means it will exert its effect even if there is only one copy of it, and one version of the gene will be recessive which means that it will only exert its effect if both copies are this variant. So, what happens when you have a hidden immunity in humans (or other animals) is that both parents carry the recessive gene, but also have a dominant gene so the recessive gene has no effect, and then both pass this recessive copy to their offspring. The offspring, now having two recessive copies, has the immunity.(An example of this is the CCR5 Δ32 gene which gives near total immunity to HIV-1 infection)Now, because bacteria aren't diploid, but haploid they have only one copy of each gene and thus express all of the genes they carry* and cannot have hidden immunity in the same way.* - strictly, it's more complicated than that because gene expression is under environmental control but that's not relevant in this case.

they don't skip generations is confusing to me. My understanding is bacteria replicate in generation time. When is it determined on a agar dish that the bacteria begin their second generation?If you take samples from the first dish and transfer them to another dish, is this still the first generation replicating?The study that found 2 or 3 immune ? out of billions that were grown does not specify if they all come from one generation of replication.
Isn't each row replicated considered another generation of bacteria?

It shouldn't be. Bacteria are haploid organisms - one copy of one chromosome. You're a diploid organism, you have two copies of 23 chromosomes, half from each parent. That's why you're not a clone of either of your parents, you merely share traits with both of them.But bacteria reproduce clonally, by cell division. There's not really a notion of "generations" in bacteria because they're constantly dividing and doubling, and when a single bacterium undergoes fission, what you have is two completely identical individuals where one used to be. Which one was the "first" and which one is of the "next generation"? It's impossible to say. Both of those bacteria are going to continue to double themselves at the same rate.So we don't really talk about "generations" of bacteria, we talk about how long it takes for a population to double in size. In something like E. coli that can be about 20-40 minutes. No. There's not really anything that can be considered a "generation" of bacteria because bacteria can go on doubling and doubling until they run out of a nutrient.

I understand that during the lag phase that the cell is actively metabolizing for cell division but it also does this again in the stationary phase. What stage must be completed before samples are taken to determine antibiotic resistance?

No, that's exactly wrong. The bacteria metabolize for growth during the log phase, when their population expands at an exponential rate (and can therefore be logarithmically modeled.) During lag phase the bacteria are upregulating and downregulating appropriate metabolic pathways in response to the conditions of their new environments; it lasts about 20 minutes because that's how long protein translation takes, start to finish.In stationary phase the bacteria have exhausted some nutrient and are no longer growing exponentially; they're engaging in enough cell division to replace individuals who die, and they're also expressing secondary metabolic activity (that's not really relevant to what we're talking about, but it's in stationary phase that bacteria produce their own antibiotics and other secondary metabolites.) To determine resistance, bacteria are incubated on antibiotic-positive media. If colonies are present, that indicates resistance. If colonies are absent then log phase never even occurred - the bacteria didn't live long enough.The presence of colonies means that the bacteria underwent a log phase. Otherwise there wouldn't be enough individuals to have colonies.