06: microbial metabolism ii

8/12/2019 06: Microbial Metabolism II

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Transcribed by Kevin Lin Lecture Date: July 8, 2014

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[Microbiology] [5] [Microbial Metabolism II] by [Dr. Boylan](but he is behind schedule by 1 lecture)

[1] [Microbial Metabolism I and II][Dr. Boylan] Up to this point, weve looked at a bacterial cell. Looked at bacterialcytology. Seen what these cells are made of. Whats inside the bacterial cells.

Ribosomes, etc., appendages, the cell membrane, the cell wall with capsule. Spent alot of time goin g over then structure of the peptidoglycan especially, thatsimportant to know. Everything you can, Id tell you about it because its gonna comeup over and over again in this course. And today were going to take a look at whatgoes on inside a bacterial cell.

Metabolism. Now I know youre already well versed in this topic because you hadthe course in building blocks just a little over a year ago. Youve gone through theglycolysis, the TCA cycle, electron transport, etc, things like that. I just want tomention because they do indeed happen in bacteria as well, with a couple ofmodifications, thats all. But just because Im gonna review it now, doesnt mean you

dont have to know it. Whatever I say -, Im trying point out some of the highlights o fmetabolism that youve already gone through step by step. Im not going to ask youfor every step in glycolysis or the TCA cycle, electron transport. Basically, what arethey used for, what are these systems used for when bacteria and even our cellsgrow. How they get food, how do they break down food, how do they get energy todo what they have to do? So thats metabolism. And I guess were already about anhour behind, but Ill try to finish up if I can today. If not, I have one more lecturebefor e the first exam. And Ill finish up that time next week, if I dont do it today. Souhh

[2] [Metabolism][Dr. Boylan] Metabolism. Metabolism are really all of the biochemical reactionsthat occur inside of cells. Bacteria (prokaryotes) and also eukaryotes (us, animals,and plants). There are two parts of metabolism. Anabolism, which refers to theactual synthesis of the macromolecules in our cells the proteins, the lipids, thecarbohydrates built up of building blocks like amino acids and simple sugars, etc.,nucleotides. Thats anabolism. Making these large structures inside of our cell.Anabolic steroids. You take them to build yourself up, you get muscular. So thoseare the steps involved in biosynthesis. And these steps to make things in cells andbacteria and in us require energy. And catabolism are the part of the metabolismthat produces energy. For the cell to use for anabolism. To produce things, tosynthesize things.

So energy requirements in general. Well see those in t he next slide. All cells needenergy. And as you know, in metabolism, you go back and look at glycolysis andother systems that occur in the cell. Series of oxidation-reduction reactions. So onesubstrate is oxidized, another is reduced, over and over again. A whole chain ofreactions occur as youll see in glycolysis briefly, coming up. So oxidation -reductionreactions occur in metabolism.


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[3] [Energy (ability to do work)][Dr. Boylan] Why do you need energy? Why do bacteria need energy? For anabolicreactions, for transport, that is to get things from outside the cell, foods from outsidein the cellular environment to the interior of the cell, in the cytoplasm, where theycan use the food thats been transported for growth and energy. Motility use

flagella. That takes energy. Cell division itself, where one cell grows and dividesinto two, requires energy in the form of ATP. And maintenance, just staying alive.Even when were asleep at night, were in bed. To stay alive, we still are unde rgoingmetabolism. We dont just give up. Were on a very low rate of metabolism, butbacteria, too. To maintain, to stay alive, when were not growing that well. Needenergy just for that.

How do the cells derive energy? Oxidation-reduction reactions. One substrate isoxidized, another is reduced. Oxidation of substrates (food, metabolites) is reallyhydrolysis of them. Its catabolism, oxidation, breakdown of foods to produceenergy. The energy is produced and trapped. And as you know, the storage

compound for cellular energy is ATP. So these things, like bacteria digest glucoseand other sugars. Theyre fiddled(?) energy. They have internal energy in them, butyou have to break them down. You have to hydrolyze or catabolize glucose andother sugars to produce energy. And as you break them down step by step inglycolysis and the TCA cycle, electron transport, thats how you get energy, ATP.Oxidation reactions, catabolic reactions.

[4] [Catabolism, figure][Dr. Boylan] This is just an overview of what were go through very briefly, onceagain. Just a review of the importance of different steps involved in catabolism. Wehave the proteins, sugars, and lipids can be broken down. Heres a bacterial cellgrowing in a very rich environment of large proteins and polysaccharides and otherthings it cannot take inside. Theyre just too large. So first they have to degradethem into amino acids, simple sugars, and then take them inside. Glycerol. Thenmany are converted to glucose. And this pathway here, when cells break downglucose to produce pyruvate, is of course glycolysis right here. Youre familiar withglycolysis. The breaking down of glucose. Thats the typical example in all coursesin biochemistry and building blocks. How ce lls begin to derive energy. Were gonnalook a little bit at that glycolysis. You can see the glucose, one molecule of glucose.How many carbons are in glucose? 6. How many are in pyruvate? 3. Right. Soyoure breaking down glucose to pyruvate in g lycolysis. Another name for it is theEmbden-Meyerhof pathway, remember they use that. You get two molecules ofpyruvate. two 3-carbon compounds, pyruvate. And this reaction, glycolysis, canoccur anaerobically. Youll learn more about what that means in about twentyminutes or so. Anaerobic, it does not require oxygen. It does not require molecularoxygen. Right now, we require, we need oxygen to grow. Right now, werebreathing air. And theres about 20% free oxygen, O 2. We need that, we wouldn tsurvive without oxygen. But this reaction can go on inside a cell. This pathway,glycolysis, doesnt need oxygen. Later on, well see oxygen is important, butglycolysis itself, can occur under anaerobic conditions. It can occur in aerobic as


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well, b ut well talk about both of those later on. But the key here is glycolysis canoccur under anaerobic conditions. You dont need oxygen.

And then the pyruvate, once you get to pyruvate in cells, there are many different,what they call fates, for pyruvate. Pyruvate can go off in many different directions.

Many, many things can be done by cells (and bacteria cells in particular) topyruvate. And one of the most important of course is using pyruvate to go into theTCA cycle, the Krebs cycle. Combines with acetyl-CoA to go into another Krebs. Andthis occurs only aerobically. Aerobically. So, well go back to point these things lateron as well. So TCA cycle cannot occur under anaerobic conditions. Only with air,oxygen, free molecule oxygen. And then from there, the TCA cycle, the molecules arebroken up further to get an electron transport chain to get ATP. And thats wheremost of the energy- . ATP is derived in cells by electron transport. So thats theoverview, I think youre familiar with that . Glycolysis. Glucose to pyruvate.pyruvate, well see, can go into the TCA/Krebs cycle, or it can do many other things.Ill point out some of them. And then from the TCA cycle going around, around, and

around, and then being changed, molecules being produced, sending them into theelectron transport chain, where you get energy production. So here is how you getthe ATP. And they say, it used to always be the number of ATPs that you can getunder respiration such as shown here, these pathways, What s the total number?Anybody remember? 30-something? 38, right. But now some people say 36 or 38ATP. Thats a tremendous amount of energy. 38 ATPs (are shown below rightthere) can be derived from the breakdown of glucose. Thats sugar. Glucose itself .Reserve of energy. You break it down. You break it down, step by step. Energy isreleased. Finally the energy is shuttled through the electron transport chain. ATPsare produced, 36 to 38 ATPs. These catabolic pathways are breaking down stuff toproduce energy for greater use in growing, the anabolic steps.

[computer pop-up: batter power is low (7% left)]Oh, thats not right. *closes pop -up*

[5] [Glycolysis][Dr. Boylan] Glycolysis. Were going to look at ATP yield. Glycolysis well see th eATP is produced. But if you recall, the ATP produced in glycolysis is very minimal.Not many at all. But when you produce ATP, during the glycolysis pathway, itscalled substrate level phosphorylation. And well also look at the important ofmolecul e NAD. So were not gonna go through every step, but look at the whatshappening with ATP and NAD in this reaction, glycolysis.

[6] [Glycolysis (in 2 parts)][Dr. Boylan] So here you see glycolysis, two parts here. Glycolysis, so part one, parttwo, I guess. The 6-carbon compound, glucose. A series of hydrolytic or catabolicreactions occur. One molecule is oxidized, another reduced. Oxidation-reduction,redox reaction, over and over again. Then the 6-carbon compound, fructose, as yourecall, broken down into two 3-carbon compounds, and the pathway continues to


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get pyruvic acid. So pyruvic acid is what is called the end point or the end productof glycolysis.

Lets look at the ATP produced and used up in glycolysis. To begin glycolysis,remember one of the reasons you were doing this, having this pathway is to produce

ATP. But initially, to begin glycolysis, as you recall, you need ATP. Kind of jumpstartthe pat hway. You use ATP here converted into ADP when you use it. And heresanother step here where you have to use ATP. So to begin glycolysis, you need twomolecules of ATP.

[computer runs out of batteries, screen goes black]Is Dr. Li still here? I dont know. If its not plugged in or we didnt plug it in. Letssee. Or maybe we just didnt plug it in. [computer back on]Okay, saved by Dr. Li, thank you.

So here we have glycolysis, and initially you use two ATP. Break it down. But lookat that here. Once its broken down into 3 -carbon compounds. Remember it sort ofsplits these pathways going in twice here. Once you get to fructose-6-phosphate,its broken down two 3 -carbon compounds. You get ATP produced here, and ATPproduced here. Im not ev en gonna ask you where the steps are involved in ATPproduction, but you get ATP produced. But since this is going on twice, you get twoATP on one side and two ATP on the other. You produce four ATP in glycolysis. Sooverall in glycolysis, you use up two ATP to start it. You produce four ATP. The netyield of ATP in glycolysis is only two. Not a lot of energy in glycolysis. This has totake place, this reaction, but only two ATP of the 36 or 38 were gonna see can beformed in these pathways.

One other thing to look at here is the use of this molecule in NAD. Here you can seein glycolysis, it is reduced from NAD to the reduced form. NAD is used up. Imentioned that, thats critical to know, too. Because NAD is critical for glycolysis.Its necessary. Its used up as is shown here. Its reduced, but it is, well see,recycled. It is recycled. So itll kind of act like an enzyme, because its used over andover again. So were gonna see later on where this NAD, this oxidized NAD, isreduced is regenerated, is reproduced, so it can be used over and over again inglycolysis. So two ATP net yield. NAD reduced, but has to be recycled, reused lateron, reproduced as we will see. And further metabolism.

[7] [Tricarboxylic acid (TCA) cycle][Dr. Boylan] Heres the TCA cycle. Remember, its called an amphibolic cycle. Putthat word down. This is critical. Dentists always say when they think back upontheir basic science, years in dental school. Oh boy, I remember the TCA cycle, theKrebs cycle. I still use that every day of my life. That seems to be the big whippingboy. I know, but its critically important. I mean, you dont have to know every stepof it, but this is what keeps us alive, the TCA cycle. Its involved in both catabo lismand anabolism. Its involved in further energy production in catabolic reactions, as


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well as producing these compounds that are used by cells to make other things. Soits involved in both types of central reactions that occur in metabolism. Catabo lismand anabolism. So thats basically what its used for. And here we see one fate ofpyruvate is to enter the TCA cycle. Forget about all of these, but you see twohydrogen atoms come off here, here, here, and all these hydrogen atoms are gonna

go in to the electron transport chain. And thats where most of the ATPs are gonnabe formed. So over and over again, energy is going to be produced because of thesehydrogen atoms being produced here. And also you have all of these moleculesbeing formed, too. They are sometimes withdrawn from the cycle, right? And theyare used for the building blocks for our proteins and our nucleotides and ourpolysaccharides. So that would be energy production. Also, finishing the buildingblocks of macromolecules as theyre withdrawn from the TCA cycle. So whathappens to these hydrogen atoms? As you know

[8] [untitled, figure][Dr. Boylan] Hydrogen atoms are composed of a proton, positive charge, and an

electron, negative charge. And one easy way to remember this, in case you cantremember it off of your head. A story about these two hydrogen atoms that werewalking along the street in the opposite direction in the sidewalk. And they hadntseen each other for quite a while. And they bump into each other on the street,these two hydrogen atoms. And the first one says to the second one, How are youdoing? The second one says, Well, not too well. I think Ive lost my electron. Andthe first one says, Are you sure? The second one says, Im positive. [chuckles].Thats how I remember it. Electrons, protons. Hydrogen atoms.

So here we have these hydrogen atoms, and as they-. And then the electrons andprotons are separated from each other in this pathway here. Electron transport.The electron are transported. The hydrogens, the protons, are sort of put outside ofthe bacterial cell. Just like in mitochondria, the protons are put outside that innermembrane, and theyre kind of dying to get back in, trying to force themselves backin. And the electrons are inside being transported along the electron transportchains by the cytochromes, the dehydrogenases, and other components of electrontransport. Theyre being transported while the protons are kind of hanging aroundoutside. The cell in the case of bacteria outside the cell membrane, trying to getback in, neutralize that electron charge. So electrons are being transported downthe electron transport chain. And once again, [mumbles], but you can see, ATPs arebeing formed. Up to 36, 38 A TPs when all of this goes on inside a cell thats growingaerobically. So finally you get the third ATP, in addition to the others in glycolysisand over and over again, up to 36 or 38. And finally, why do we need oxygen tosurvive? I remember reading about bacteria when I took a micro course. Iremember reading about these bacteria called anaerobes that can grow withoutoxygen, and I thought, thats impossible! How can you grow without oxygen, freemolecular oxygen? I mean, all cells need oxygen for their carbohydrates and theyrecarboxyl groups, but as far as free molecular O 2, we need it. And why do we need it?Its because of this last step of electron transport. Thats why we simple needoxygen among many other [things] to survive, really. Oxygen. And I wanna point


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this out in a better slide that shows this terminal step of electron transport. So yougo to glycolysis, you go to the TCA, the electron transport, and finally after electrontransport is over, what do you get? Water, H 2O. Thats the end product of all thosemetabolic pathways that occur in bacterial cells that are growing aerobically. Andoxygen well see play an important role. And finally saying this is enough. Were

done with this pathway now, these hosts of other sequences of different pathwaysare now over.

[9] [Respiration][Dr. Boylan] Okay, here it is, here again. And this is called respiration. Respiration,where phosphorylation occurs. Where ATP is produced, thats called respiration. Itcan be aerobic or anaerobic, respiration, ATP production. Aerobic respiration here.And heres that last step once again in electron transport. Electron reactions.Finally, those electrons are being transported and those protons that were outsidethe membrane just dying to get in, from the hydrogen atoms that began the wholeprocess recombine. Get together with oxygen, molecular oxygen to form water. So

another way to refer to this process in aerobic respiration where ATP is formed, thelast step where water is formed, as you say, the oxygen is the final electron acceptor.In aerobic respiration, where oxygen is used. Oxygen itself is the final electronacceptor, by definition, aerobic respiration. And thats why it finally picks of theelectrons. It picks up the protons, too, butyou can say its the final protonacceptor, too, but by convention, we say oxygen in aerobic respiration is the finalelectron acceptor. Form water, water is the end product. And then youre done.

In anaerobic respiration, well see these are bacteria in the absence of oxygen. Theylack the cytochromes required to use oxygen as the final electron acceptor. Soanaerobic bacteria, as the name indicates, these cannot use oxygen. Another fact, tomany anaerobes, oxygen is a poison. And we ll see where that can occur, too. Theycant deal with oxygen. So bacteria that grow anaerobically use something -. Theystill have to go through glycolysis. They dont go through TCA cycle, but they gothrough glycolysis, they go through electron transport. But at the end, instead ofusing oxygen, when theyre anaerobic bacteria growing without air, they will useother electron acceptors, such as nitrate, sulfate, or carbonate. So by definitionaerobic respiration use oxygen as the final electron acceptor and anaerobicrespiration and a lot of bacteria were gonna talk about are strict anaerobes thatcan occur severe oral infections like periodontal disease the final electron acceptorin their respiration is either nitrate, sulfate, or carbonate. NOT oxygen. So anotherway to put it, the final electron acceptor in anaerobic respiration is an inorganiccompound other than oxygen. Because oxygen is an inorganic compound, right?But these are inorganic compounds that acceptor electrons and they makerespiration. Thats why these bacteria can grow anaerobically, they dont needoxygen. Oxygen can harm them. They use other things. They have to carry outglycolysis and electron transport. A different electron transport chain in anaerobes,but they use it to derive energy in their electron transport chains as well. Inanaerobes.


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[10] [Classification of bacteria by effect of oxygen on growth][Dr. Boylan] So you can classify bacteria by the effect of oxygen on their growth.Obligate aerobes means some bacteria will only grow in the presence of oxygen, inair, O 2. Air and oxygen, we use them interchangeably now. Obligate aerobes.Obligate anaerobes, will not grow or will not grow well theres a whole gradient of

different strict aerobes to strict anaerobes of bacteria. But anaerobes mean they donot grow in the presence of oxygen. As a matter of fact, oxygen is a poison to manystrict anaerobes. Obligate or strict anaerobes. Wer e gonna talk about theimportance of these two enzymes, and which should determine whether or not abacterial cell is an aerobe or an anaerobe. These two enzymes well see in the nextslide are critically important in that role.

There are bacteria known as facultative anaerobes. They can grow in the present orabsence of oxygen, of air. Most bacteria are really facultative anaerobes. Can growwith or without air.

Microaerophilic bacteria are those that grow with a very small percentage ofoxygen. Right now, what is the percentage (I think I asked this before), thepercentage of oxygen were breathing? Its about 20%, right? Okay and in ourmouth, its maybe 10%, 12%. And then farther back in the mouth and down there inthe gingiva, the gingival sulcus, between the teeth and the gums, there may not beany free oxygen at all. Thats a good anaerobic environment. So some -. 20% isoptimal for most bacteria if theyre aerobes. These [Microaerophilic bacteria] growto about 5% oxygen best. They thrive, they grow much better under 5%. We havespecial incubators well see that enable them to grow. You cant grow them on aPetri plate and stick them in a regular incubator and have them grow well. You haveto reduce the oxygen content from about 20% to 5%. Microaerophilic.

And some bacteria, we characterize them as capnophilic. Whenever you see -philic of course that always means love they love something. And these arebacteria that love capno - they love carbon dioxide. This is another thing. Manybacteria are stimulated in their growth. They grow much better when you enrichtheir environment with carbon dioxide. And we also have incubators that do that aswell. You put the plate in the incubator, you have this tank of carbon dioxide, a tubelead from the tank of CO 2 into the incubator, and CO 2 builds up inside, interior of theincubator to about 5% as well. 5% CO 2 and they grow much better than they wouldin the absence of CO 2, these capnophilic bacteria.

[11] [Classification according to affect of oxygen on growth][Dr. Boylan] Here, an obligate aerobe would be Bacillus , that genus. Obligateanaerobe would be Clostridium . Examples of facultative anaerobes are Streptococci that cause caries, E. coli , and most bacteria. So micro aerophiles well talk aboutwhen we get to infectious diseases, as well as capnophilic bacteria. But majority ofbacteria in the oral cavity are either obligate anaerobes, not Clostridium necessarily,but those down in the gingival sulcus. And well talk about the cause of periodontal


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disease or facultative anaerobes that can grow with or without air. Streptococci ,youll learn an awful lot about that particular group of bacteria. E. coli as well.

[12] [Reduction of molecular O 2, figure][Dr. Boylan] So what is it about oxygen that kills-, as I said oxygen can act as kind

of a poison to anaerobes. Well sometimes, well always , really, in metabolicreactions, imagine a bacterial cell growing and doubling every 20 minutes. Theremust be a ferocious amount of activity going on inside that cytosol of the bacterium.Within 20 minutes, they can lose all the cell wall, and the flagella, the DNA and RNA.Every now and then, some things in metabolism that are harmful for cells unlesstheyre gotten rid of instantaneously within nanoseconds. And some of these thingsthat are dangerous that are toxic to bacteria are reductions, molecular oxygen beingreduced. Heres oxygen. Heres what we call the superoxide radical. And here, thenegative charge. Hydrogen peroxide. And even the hydroxyl anion. One, two, three.These three things are very dangerous to bacteria. If they survive even a second orlonger, theyre gonna do some damage to the components of our cells, like our DNA,

RNA, proteins, etc. So you have to immediately get rid of these dangerous reducedcompounds of oxygen. Superoxide anion, hydrogen peroxide (which is used as youknow a disinfectant and antiseptic), and hydroxyl ion. So thats bad. Why do thesethings that are dangerous and they are produced in both aerobes and anaerobes, sowhy is it that aerobes can exist when theyre produced, whereas anaerobes cannotexist and survive when these compounds here are produced.

[13] [Why are anaerobes killed by exposure to oxygen?][Dr. Boylan] Well, heres what happens. Why are anaerobes killed by exposure tooxygen? Heres a superoxide radical, and heres H 2O2, and also OH, the hydroxyl onit are oxidizing agents. Oxidizing agents were always told we have to takevitamins and things to get rid of our oxidizing agents so they dont harm ourselves.The superoxide radical, hydrogen peroxide, and also the hydroxyl ions, should havethat here, are oxidizing agents. Generated during metabolism, split nanosecond,theyre gone.

Aerobes, but not anaerobes, contain these two enzymes that help aerobes survive.Superoxide dismutase and catalase. Aerobes have these two enzymes, anaerobes donot, they lack them. Aerobes can do, when they grow, is what they call the enzymesuperoxide dismutase that mismutates, or changes, the superoxide radical, shownhere. This enzyme here, cause a disreaction when you get H 2O2 plus oxygen. Nowthis is also dangerous, so right away, aerobes breakdown H 2O2 using the enzymecatalase to form water and oxygen. D anger of oxidizing agents to cells. So thatswhat happens. Aerobes have these important enzymes that maintain their safetyand a lot of them survive, whereas anaerobes do not, and these hydroxyl ions andsuperoxide radicals build up and they destroy, danger, destroying DNA, protein,other components of the cell. And the cells will die. So anaerobes will die veryquickly in the presence of these molecules shown here because they cant handlethem, to let them build up and these molecules shown here destroy components ofthe anaerobic cell.


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[14] [Fermentation reactions][Dr. Boylan] Okay we talked about aerobic respiration and anaerobic respiration.And theres one other type of pathway carried out by bacteria. And its veryimportant to all of you, I think, and to all of us, really. In addition to aerobic and

anaerobic respiration, bacteria can also carry out fermentations. Fermentations.And Im gonna mention a couple of them. One of them, well two of them shown hereare lactic acid and alcoho lic fermentation. Im gonna show you the pathway offermentation for the production of these molecules, lactic acid and alcohol. Lacticacid is important because thats the one that causes the demineralization of ourenamel. When bacteria, such as Streptococci we will discuss and others, producelactic acid when theyre growing and the dental plaque, this biofilm on our teeth,lactic acid begin the demineralization, begin to dissolve our enamel. Eventually leadto holes and cavities and caries. So that s the result of bacterial fermentation. Lacticacid we will see. And alcoholic fermentation is also reactions where ethyl alcohol isproduced, which is very important to all of us, particularly on the weekends when

we have alcoholic beverages. But also many products that bacteria produce infermentation reactions are used to help identify them in the microbiologylaboratory. Many, many tests are available to identify bacteria based upon whatkind of products they produce when they ferment sugars. Fermentation, how is itdefined? Its defined as a process in which the final electron acceptor is an organiccompound. So fermentation reactions are reactions and pathways in which the finalelectron acceptor is some organic molecule. Not oxygen, not nitrate or sulfate orcarbonate, but organic, a carbon-containing compound acts as the final electronacceptor in fermentations. Lets take a look at a couple here.

[15] [untitled, pyruvate figure][Dr. Boylan] This is well, you can see how involved this m ust be. Heres whatbacteria can do. Pyruvate. They get pyruvate. Talked about all the end products ofpyruvate, the end products. Some bacteria even carry out this one, lactate. All thesethings can be formed. Alcohol. Others, butanediol, etc, etc. So these are just a few ofthe many types of reactions can occur in bacteria. To help differentiate them fromeach other. Not all carry out all these reactions, of course. But one or two, or few ofthem. But its by determining what they produce whe n they change pyruvate tothese different compounds. Identify formate, identify acetate, identify butyrate.Then we can help identify bacteria. Series of reactions over and over again, to seeyes or no? Do they produce this fermentation end product or not? So fermentationis often useful in helping identify bacteria by the end products that are formedduring bacterial fermentation.

[16] [A fermentation reaction][Dr. Boylan] Heres a very simple one thats important to you. Pyruvate. And herewe have this is a fermentation. This is a complete fermentation reaction.Glycolysis produce pyruvate. Here we see the pyruvate in one simple step. Lookwhat happens here. Its converted from pyruvate to lactate. Lactic acid or lactate.Lactate always the salt. This is the culprit in caries. This is what demineralizes your


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teeth. This is what is produced in the dumb plaque on the surface of your teeth. Soheres pyruvate. Heres an enzyme, lactate dehydrogenase (E for enzyme) thatimmediately converts pyruvate to form some bacteria like the Streptococci tolactate. Thats a fermentation. Thats called lactic acid fermentation. And thatpathway is over. Lactic acid fermentation, just one simple step. Look at also here,

we see that this fermentation pathway, this fermentation reaction, NAD isreproduced. Remember I talked about its important for glycolysis to have the NADregenerated. NADH was reduced, for glycolysis has to be regenerated, NAD. Nowthis can go back into glycolysis and be used over and over again. So fermentationpathway, one simple step. Lactic acid form. Many Streptococci and other bacteriacarry out this fermentation and produce tons and tons lactate on our teeth. A lot.To demineralize them. So thats a fermentati on.

What is the final electron acceptor? Remember my definition of fermentation? Inthis pathway, which compound shown here is the final electron acceptor? Gotta beone of these three, pyruvate, NADH, or lactate. Actually it is Where is the final

ele ctron in this reaction? Its in here. We wanna oxidize it. So the final electronacceptor, an organic compound, that picks up that final electron is pyruvate. Sohere, in lactic acid fermentation, pyruvic acid is the final electron acceptor. It picksup the electron and is converted to lactate.

[17] [Ethanolic Fermentation][Dr. Boylan] How about this one. Heres another fermentation where ethyl alcoholis produced. And actually this occurs more in yeast and fungi. A few bacteria cancarry out this fermentation, but as far as in the world of microbes, mainly its theyeast that ferm ent sugars, grapes, sugars, and other sugars to form alcohol. Its afermentation reaction. We need alcohol in our practices as well. We use alcohol fordisinfection. So its not just for drinking. Here pyruvate formed. The fate ofpyruvate here, CO 2 lost, acetaldehyde. Heres our friend, NADH, that was producedin glycolysis, has to be oxidized to NAD, and you form ethyl alcohol, ethanol.Pyruvate decarboxylase is the enzyme involved here. What is the final electronacceptor in alcoholic fermentation? What picks of the electron from NADH?Acetaldehyde, an organic compound. So fermentation reactions in which the finalelectron acceptor is an organic compound. Previously with pyruvate, with lactatefermentation. In alcohol fermentation, acetaldehyde picks it up. Acetaldehyde is thefinal electron acceptor. Fermentation. Now, you get NAD regenerated in thesefermentation. Thats really great for the glycolysis to go on, its essential. Butbacteria that carry out fermentation dont really get much energy at all in the formof ATP. The only ATP they get is formed in glycolysis. So remember glycolysis inbacteria that ferment, glucose, pyruvate is formed, and then they convert it toethanol, in this case for example. But no more ATP is formed. So they dont growvery well at all in lab. They dont have robust growth because they dont producemuch energy, but that net yield from glycolysis helps them survive. And glycolysiscan also occur anaerobically. It can occur under anaerobic conditions, even thoughwe see the final electron acceptor is an organic compound.


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[18] [Ethanolic fermentation - 2][Dr. Boylan] Here we have it again. Okay, I guess I was gonna ask you the samequestions. Two enzymes involved. This is a fermentation pathway. So we haveaerobic respiration, anaerobic respiration, we have fermentation. Those are the bigthree that occur in bacteria. And once again, many important compounds are

formed. We just mentioned a couple of them here. The lactate, the ethanol infermentations. And many, many other compounds are formed to help identify thebacteria.

[19] [Mixed Acid Fermentation][Dr. Boylan] Heres an example of that. Here pyruvic acid. You can see somebacteria and do an awful lot of things, form an awful lot of different end products.Here its called mixed acid fermentation. So some bacteria, pyruvate, they can dothree or four or five different things with it. Form different acids, even. And so itscalled mixed acid, many different acids may be the end products, such as acetic acid,succinic acid. And some even produce gases such as hydrogen and CO 2. So lets talk

now how we can help fermentations identify bacteria. Here we have a test tube.This tube here if you worked in lab, you know what it is. Its a glass inverted, a littletube inside the broth in this test tube. And you put that tube in and some of themedia goes in there, some of the broth goes in there. And when you take this tubeand sterilize it and bring it back, this whole tube thats upside -down will open hereat the bottom, is completely filled with the broth and has this particular color. Sothis has a little gas already. Then, here we have three different tubes. In this casehere, I would say I dont know if they look like that

Initially, they all look the same. Initially, these three different tubes were purple.Take my word for it. You inoculate some bacteria into this tube that do not carryout fermentation at all. The color of the tube stayed the same, nothing happened.No acids produced, no gas. Whatever bacteria were inoculated in that tube were notfermenters. In the middle one here, we see that indeed they did ferment whateversugar was in the test tube. The Durham fermentation test tube upside-down. Andwhen they did that, its acid, because the color of the media changed from purple toyellow as a result of acid production, the pH went way down. You can see becauseof all the acid being formed. And also some gas was produced. So H 2 and CO 2 wereproduced. Some bacteria can do this, others cannot. And the other one on the left,only acid was produced, no gas. So just to point out the different usages offermentation test to, you know, say yes gas, yes no gas, yes gas and acid, or nofermentation at all. And those are properties that help you pinpoint a microbialgenus, species, and even well see later, even more accurately stereotype that are -,as well.

[20] [Butylene glycol fermentation][Dr. Boylan] And heres one I want to mention only because its commonly done inthe lab, called butylene glycol fermentation. I dont want to mention, once again, thewhole pathway, but look at this compound here, Acetoin. Youve probably neverheard of this anywhere else, I dont know. Its a very rare com pound, but some


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bacteria when they breakdown pyruvic acid, they form butylene glycol, and in theprocess they form the compound called acetoin. Forget about the structure of it, butthe thing is, its a compound thats produced in fermentation by some bacteria thatseasily identifiable. A very simple biochemical test. You have the bacteria growing ina broth test tube overnight. The next morning, you say, I wonder if acetoin was

produced overnight or not? You add a particular reagent, and if it did produceacetoin, the color would change to red. Doesnt matter [the color], but its a colortest. And if those bacteria didnt produce acetoin, did not carry out thisfermentation pathway, the red color would not appear. So once again, anotherfermentat ion case, not the end product, but the intermediate in the pathway thatseasily identifiable by Voges-Proskauer test. And we know for example, here are twobacteria, Enterobacter and E. coli. These are very hard to distinguish between in thelab. They look alike, they grow alike, so many different tests that are identical tothese two Gram-negative bacteria. But this is one here that helps differentiate thesetwo Gram- negative bacteria. They look alike, stain alike, everything. And thatsbecause Enterobacter , that particular genus does produce acetoin. E. coli does not.

Very, very useful because E. coli, as we will see, can do a lot good, but also a lot ofdamage. It can contaminate water, and thats indicative of sewages. Often runningthe test to see whether or not E. coli is present in what we think is contaminatedwater, sewage in our drinking water. Look for E. coli, if the Voges-Proskauer test isnegative, it wasnt E. coli there, maybe the water is safe to drink. It could besomething els e, but maybe its safe to drink, if E. coli is not there because acetoinwas not found.

[21] [A few bacterial fermentation pathways][Dr. Boylan] This is just another slide. You can look over it after. Ive mentionedsome of these already, but just to show you, so many different pathways of pyruvatein bacteria.

[22] [Classification by optimal temperature of growth][Dr. Boylan] Another way to identify bacteria. Okay, now weve gone through twotypes of respiration and fermentation. Some other ways to classify bacteria inaddition to the effect of oxygen on their growth, aerobes, anaerobes, facultative,microaerophiles, etc is the temperature of growth that they prefer. Some bacteria,believe it or not, can grow very well in the cold. In the refrigerator, even. Theyrecalled psychrophiles. They grow best at about, maybe, theres a curve... maybe from2 degrees, 4 degrees, 8 degrees. They grow very well at 4 degrees. Psychrophiles,cold. And theres one or two bacteria were gonna talk a lot later on in the othercourse that grow well in refrigerator and are pathogenic. And you really have to beaware of them, because they grow better in the cold at 4 degrees centigrade, atrefrigerator temperature than they do at 37 degrees in a regular incubator.Thermophiles grow best at above 50 centigrade. We found some even in hotsprings. They can grow at incredibly temperatures. And better than say, our bodytemperature. Mesophiles are the ones that grow in between these extremes. Ourbody temperature is about 37 degrees centigrade. Most bacteria that inhabit us,such as those that live inside of us, are mesophiles. They like to live at 37 degrees


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centigrade. Most pathogens well talk about are mesophiles. Most of them, majority.They lik e body temperature because once they get inside of us, its the temperaturethey like to grow in. They like to attack our tissues, they can hydrolyze our tissues,break them apart to get food, so they can grow and the end result, they do a lot ofdamage to our body and cause infections.

[23] [Anabolism][Dr. Boylan] ATP requirements.

[24] [Minimal media][Dr. Boylan] I guess, I dont know. I started off doing research when I was ingraduate school with bacteria and trying to learn all about metabolism. But it alwaysamazed me that heres an organism, E. coli, shown here. E. coli can grow on thisvery, very simple medium we prepare in the lab that Dr. Saxena will talk aboutprobably on Friday, called minimal media. Media, plural. Its growth medium iswhatever we use to grow bacteria in the lab. Its called medium. All it takes for E.

coli to grow is glucose and 5 salts. Thats incredible! Glucose, the source of carbon.No other sugar has to be added to their growth medium for them to grow. But youhave to have salts to, you know, synthesize and produce their phosphates, sodium,chloride ions, of course, for cells to grow. So 5 salts and only one carbon source,and from this simple medium, E. coli can produce everything it needs to grow. Itsflagella, its capsule, its peptidoglycan, its cell membrane. Just from these simplesalts and one carbon source, glucose. This E. coli that grows on this simple mediumis called a prototroph. And auxotroph is a term Ill use later. So E. coli can grow onthis simple medium, minimal medium. Its a prototroph. Sometimes on these slides,we find bacteria like E. coli that need one additional supplement to grow, anadditional growth factor. And thats called an auxotroph. So an auxotroph is abacterium that needs a growth factor that its prototroph, or parent E. coli, did notneed. So lets say E. coli grows on this medium, its a prototroph. If you also have toadd the amino acid histidine to grow, that would be an auxotroph. You have to addhistidine to grow. And any other amino acid that would help, that they need togrow. Or any other product. Auxotroph, something that require that the parent E.coli did not need other than the minimal medium.

[25] [Energy uses of a growing E. coli population of cells][Dr. Boylan] Heres how the energy thats produced now in catabolism, so all thatwe just went through, especially the electron transport. How is the ATP used. Andfrom growing E. coli on that simple medium, and following every step it uses inproducing its polysaccharides and nucleic acids, every step. We know how muchenergy is involved in the growth of E. coli, from a simple salt, one carbon medium, tolarge population of cells. We know that the ATP that they produced, more than halfis used for synthesis of proteins. And this is true in our cells, too. We found this outinitially in bacteria. How is energy used, from catabolism, used for anabolicreactions in cells and in bacteria? And its the same in our cells, roughly. 56% ofATPs produced are used for protein synthesis. Polysaccharides, 8%. Lipids, 0.3%.And more energy is needed for RNA synthesis than for DNA synthesis. DNA


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synthesis does not require much energy, not require much ATP at all. Other big usesare active transport, getting things from outside to inside cells. And mRNA turnover4%. So to me, thats really incredible. And why is it? A Darwinian question, why isit that protein synthesis requires so much ATP? What is that step that thatsinvolved in protein synthesis where ATP is used? Remember? Think about protein

synthesis now. Think about that ribosome, and theres that string of mRNA on thatribosome. And all of a sudden, you have all the amino acids there, lined up in a new-forming protein. And then you have tRNA coming down, right? Transfer RNA withthe new amino acid to add on to the ones that are already there to form this protein.Every time the amino acid from the tRNA is transferred to the growing polypeptidechain, an ATP is used. So protein synthesis is an incredibly energy demandingpathway. The production of proteins.

So I didnt finish. I still have a couple more slides to show when I see you next week.After Dr. Saxena sees you on Friday.

06: microbial metabolism ii

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