Cycle 6: Endosymbiosis and Antibiotics

• Endosymbiosis


  A theorized process in which early eukaryotic cells were formed from simpler prokaryotes.

  
  

  Aerobic bacteria gets engulfed, ATP yield goes through the roof, because of oxidative phosphorylation. Subsequent to this, the mitochondria, and some that had mitochondria engulfed cyanobacteria created plants and algae.

  
• Ancestral bacterium


  Anaerobic, pretty sluggish, doesn't make a lot of ATP.
• Development of endomembrane system

  Infolding of the plasma membrane, led to the formation of all internal membrane systems that aren't a part of the mitochondria and chloroplast.
• Nuclear envelope and ER


  Created by infolding, the lipid composition of the PM, are very similar to the lipids that define the ER and nuclear envelope.
• Cyanobacterial genome


  :3.9Mb, 3725 genes.
• Chlamydomonas chloroplast genome (this genome is derived from the cyanobacteria genome


  )99 genes in 203kb.
• Mitochondria and chloroplasts have a complete _ and _ apparatus.

  transcriptional, translational
• Mitochondria and chloroplast were once free living bacteria. Endosymbiosis, these free living bacteria were engulfed and became modern day mitochondria and chloroplasts.


  An obvious immediate consequence is that those genome became a lot smaller.Why is the mitochondrial genome smaller than the bacterial genome it descended from?1. Genes are redundant. For example, these cells need iron. It's a cofactor for lots of enzymes, etc. The bacterial cell by itself in the past would require an iron transporter to get iron, but the entire cell that the mitochondria is in can bring in the iron itself. We don't need iron transporters anymore specifically for the mitochondria anymore. There are lots of genes important for cell function that already exist in the genome. I don't need as many genes encoded for the mitochondria because those genes already exist within the larger cell that engulfed it.2. The genes have moved.This is a process called horizontal gene transfer.
• horizontal gene transfer


  Over millions of years, the movement of genes from the mitochondria or the chloroplast, to the nucleus. What we've done is just changed the location of the gene, but not the function of the gene.

  
  

  - ONLY the location of this gene has been changed, the function however remains the same.

  - The protein encoded by the gene also remains the same, but due to the change in location of the gene, the protein needs to be imported into the organelle from the cytoplasm to do its job.
• How to detect horizontal gene transfer

  DNA hybridization can be used to detect horizontal gene transfer
• DNA Hybridization

  We're using a labelled DNA probe. Three species, A, B and C, we've isolated this DNA from these three species. We've isolated either mitochondrial DNA or nuclear DNA. Complementary base pairing with the DNA probe, binds to regions of the single stranded DNA that we've denatured.

  
  

  We isolate mitochondria DNA, do hybridization experiment, and we see when we use mitochondrial DNA that there is a hybridization result. We're picking up the labelled probe sticking to the DNA from the mitochondria.

  
• For


  species A...There is only a result in the mitochondrial DNA but not in the nuclear DNA. This tells us that horizontal gene transfer has not occurred in species A.

  
• For


  species B....instead of getting a hybridization signal in mitochondrial DNA, we get it when we hybridize using nuclear DNA.This shows that horizontal gene transfer has occurred. In species B there is no evidence found that the labelled probe gene is anywhere in the mitochondrial genome. It suggests that the gene has now moved.

  
• In


  species C....We find a hybridization in both, when we probe the mitochondrial DNA and the nuclear DNA. Seeing copies of the gene in both.

  This is becausebefore the transfer to the nucleus a copy of that gene is made, and now you have two copies in the genome of the organelles, one of the copies is sent to the nucleus. After it works out there, the other copy in the mitochondrial genome is just degraded and now you're left with just one copy in the nucleus.It's in the middle of the act, the original mitochondrial copy just has not been degraded yet.

  
• Eukaryotes without mitochondria

  Giardia

  - Very primitive eukaryote.- Lack mitochondria.- Never had them or lost them?- Belongs to a group called the diplomonads.- It's an intestinal parasite.
• cpn60


  Helper gene, chaperone, help prevent proteins from denaturing. When proteins need to be imported into the mitochondria, cpn60 helps them fold correctly.
• Where you find cpn60?

  In both cases, you find cpn60 in the nucleus. It's a mitochondrial protein, and it's found in the nucleus.
• What is Giardia and other diplomonads doing with cpn60?


  The ancestors of diplomonads had mitochondria. cpn60 was doing its job, horizontal gene transfer occurred. They lost their mitochondria after some type of horizontal gene transfer occurred. cpn60 has no function in diplomonads. They don't have mitochondria, it's just a remnant gene that sits there. The fact that it is there is proof they previously had mitochondria.
• Diplomonads


  a protist that has modified mitochondria, two equal-sized nuclei, and multiple flagella
• Why move genes to the nucleus?

  1. Control: If you have this primitive eukaryote, with a mitochondria and nucleus, it's like autonomy. You have the power to do your own thing. But you can't have an autonomous cell inside another autonomous cell (mitochondria or chloroplast and cell). You need the mitochondria doing what the cell wants them to. You need regulation and control. So by taking those gene back into the nucleus now the nucleus exerts overall control of function over these organelles.

  2. Reactive oxygen: In the mitochondria and chloroplast there is lots of oxygen and lots of electrons. Random side reactions you can't avoid, oxygen can just pick up an electron, produce a superoxide, and maybe superoxide reacts with another electron to create hydrogen peroxide. These molecules are very toxic, and there are enzymes to help get rid of them. Superoxide and hydrogen peroxide can damage DNA. So here we have DNA that's sitting pretty close to these electron transport chains. So why keep that DNA there when you have this other wonderful repository which doesn't generate any reactive oxygen, the nucleus. The chance of mutation and damage is so much less if you get that DNA out of these organelles and into the nucleus.

  3. Genetic recombination: Re-arranging of genes, major source of variation, it can't occur in organelle genomes. If you want to increase variability, get the genes into the nucleus where they can recombine into newer maybe more interesting genes.
• Why haven't all organelle genes moved to the nucleus?

  1.Rapidly degraded product. D1 for example needs to be replaced quickly if it is damaged. Therefore it is more beneficial for the chloroplast genome to encode for and create the D1 protein instead of the nuclear genome because importing the protein will take too much time.2.Unable to import protein. Having the gene present in a different location means the protein product needs to be transported into the organelle in order to functionThere are certain properties of the protein that may make it hard to transport.3.Time. HGT has not stopped. It is possible that if we check the organelle genomes in another 100 years, some its current genes may be lost due to HGT.
• Taking a closer look at the signal peptide. Through analysis of lots of these proteins there are three regions that are required for the signal sequence:

  N-region, H-region, C-region.

  
  

  All of these regions have very similar structure.

  This overall region is about 25 amino acids long, so the mature protein might be 1000's of amino acids long. So it's a very small portion. But there is not a consensus sequence.
• N-region


  seems to have positively charged amino acids. Important for the binding to the membrane either in chloroplast or mitochondria.
• H-region


  hydrophobic stretch, help movement across the membrane.
• C-region

  has positively and negatively charged amino acids. Not sure what this does.
• The protein import machinery is not looking for _______ amino acids

  specific.

  
  

  there isn't a specific consensus sequence. It just seems to have these general motifs (consequence of that is if you just mutate a single amino acid in there it probably wont impact import because there's no specific sequence).
• contact sites


  There are contact sites, where one membrane comes in contact with another one, and this is where import takes place. The protein is passing across both membrane at the same time.

  
• How protein targeting works


  Precursor protein has the signal sequence, there's a receptor that brings it over to the protein translocator machinery. There are contact sites, where one membrane comes in contact with another one, and this is where import takes place. The protein is passing across both membrane at the same time.

  There's a cleavage site at the end of the signal peptide. An enzyme will come along and cut there, and then you have the mature protein sitting in the mitochondrial matrix (in this example).

  
• Most studies of evolution are constrained by two facts


  1. Organisms grow slowly - reproduce (generation time, time of which one generation to give rise to the next).

  2. Can't go back in time and look at ancestors.
• Experimental evolution


  Gives you a way to overcome these two points (Organisms grow slowly - reproduce (generation time, time of which one generation to give rise to the next, and can't go back in time and look at ancestors.) Organisms that have a very fast generation time. Enables you to look at fast evolution and change over time.
• Model systems for experimental evolution:

  Viruses

  Bacteria

  Chlamydomonas

  Drosophila

  Yeast
• Ancestral and Evolved Strains Experiment Procedure


  One culture of bacteria gets split into two sub-cultures of bacteria. One carries a neutral gene - red colour. The cells express a red colour. This neutral gene produces a pigment. This gene doesn't insert into an important gene, but its stable, and doesn't impact the fitness of the culture. This is to distinguish this from this sub-culture. One is ancestral, the one with the neutral pigment is evolved. Leave the ancestral cultural to do what its always done.Let this culture evolve, expose this to a stress (i.e. high temp, low temp, salt..) over many generations (i.e. 2000) and then compare the two.

  
• Can evolution produce adaptation if it depends on random mutations (most of which are harmful)?
• Lenski group MSU (Long Term Evolution Experiment (LEE))

  - Their organism was E. coli. Why E. coli? It divides fast. Divides into two daughter cells in the matter of minutes/hours.- Asexual (no recombination).- Spontaneous mutation. The mutations occur naturally in replication.- Population size is also huge. Beneficial mutations are rare. The population size is large because of this.- Started with 12 identical populations. One flask, and move some of those cells into twelve identical other flasks. You want those cultures to just keep growing for many generations.-This experiment has been going on since February 1988.
• How do you keep the E. coli populations in a long term evolution experiment growing?

  Need to introduce fresh growth media.

  
  

  Transfer these 100 micro litres into fresh growth media that doesn't have any cells in it, and this enables the cells to keep growing. Because the cells had lost their growth medium, nutrients, so everyday you do this.

  Then the cells grow for another 24 hours, then they are sub-cultured again. You can freeze aliquots, so every 500 generations, a grad student would come in and do this transfer, then take our another 100 micro litres and freeze it. The reason you freeze is so that you can revive it and investigate it later.
• Results of Lenski group MSU long term evolution experiment?

  After more than 30,000 generations one line grows better than the rest. (A-3 line. It's cloudier indicating more cells).

  
  

  How can this culture have higher growth rate? How can it have more cells? If the glucose is the same in all of them how could one grow better?
• In the A-3 line from the Lenski group MSU long term evolution experiment.. how can this culture have higher growth rate? How can it have more cells? If the glucose is the same in all of them how could one grow better?

  Growth media does contain citrate (Fe-citrate). The citrate is there because its complex with iron. These cells need to take iron from the environment. Free iron tends to fall out of solution, so to keep it in there you link it with citrate.

  So one way to explain this amount of growth is that the cells underwent a mutation that enables them to use citrate as a carbon source, if you can do that then you can divide longer over 24 hours. Ecological opportunity where the A-3 line could take advantage of the citrate.

  The problem with this is that E. coli only transport citrate under anaerobic conditions. All of these flasks are growing aerobically. So whatever has happened has enabled A-3 to bring in citrate under aerobic conditions and use it as a carbon source. This is the only real explanation for increased growth rate.
• the A-3 line


  Culture density just after about 33,000 generations, it shoots up and reaches a plateau. Much higher than it used to be. Citric positive phenotype around 33,000 generations.

  Actualization: The citrate positive phenotype becomes a apparent and it occurs. These cells can now metabolize citrate.Refinement: At a higher level than the initial actualization of the citrate positive phenotype.

  
• Operon


  Genes are often organized in an operon. Many genes controlled by a single promotor. Controls the expression of multiple genes.
• The gene at the end of the citrate operon is the citrate transporter (


  CitT). Oxygen is a very strong _____________ regulator to the operon.Negative. Oxygen blocks the citrate operon's promotor and the expression of all these genes.