Transcribed by Erica Manion 7/14/2014

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Microbiology - Lecture 08 Bacteriophages by Dr. Boylan

[Microbial Metabolism powerpoint, slide #25 Energy uses of a growing E. coli population
of cells]
[Dr. Boylan] Good morning everybody. So, I know that you had a couple lectures on Friday
by Dr. Saxeena, but before that, I gave a lecture on bacterial metabolism and I didnt quite
finish it. So Id like to finish that one today, the last six slides on that topic, they will be on
the exam on Friday. Then Ill go on to the next topic which is scheduled for today, which is
bacteriophages, and however far we get today in lecture, that will be the end of the material
on Fridays exam, no matter where it is after fifty minutes. We stop there, that will be the
last material on the exam, and tomorrow Ill pick it up and talk more about bacteriophages
and viruses.
So we ended last time talking about, um [plays around with presentation], we talked about
bacterial metabolism, everything that goes on inside the bacterial cell. We talked about
aerobic bacteria, anaerobic bacteria, the final electron acceptors in these processes, and
then ATP is formed, fermentation products formed and fermentation carried out by
bacteria, very important, fermentation reactions that you should be aware of. Particularly
lactic acid fermentation, but alcohol also, and others. And now we talked about all those
ATP, all the energy that is produced in catabolic metabolism, how is it used for biosynthesis
and building things, and other things that have to go on inside a cell for it to survive. All the
ATP that are formed in catabolic reactions, this slide shows how they are used by bacteria
to grow. Once again, over half of the ATPs, half of the energy produced in bacterial
metabolism, is used for protein synthesis. Proteins! Structural proteins, enzymatic
proteins, because once again every time you recall [fiddles with microphone] Is it on? Its
on, yeah. Can you hear me in the back, down in front? Uh, every time a peptide bond is
formed in the ribosome, you need an ATP. tRNA brings those amino acids down, hooks
them onto the previous polypeptide and an ATP is used. So ATP is used for protein
synthesis to form that peptide bond, and other uses of ATP are shown here as well. So
anyhow, looks like its erratic today [clicks pointer several times]. This wont work at all!

[Slide #26 Bacterial Transport Processes]
Some of the energy produced by catabolism is used for bacterial transport processes. In
other words, transport. How bacteria get food from outside the cell and the environment in
which they are growing, the broth or in the body or wherever, how do they get materials
from outside the cell, what they need to grow on, into the interior of the cell, into the
cytoplasm, where they are going to use this to metabolize it. So they have to transport
these things from outside [the cell] inside [the cell]. And some of these processes whereby
bacteria transport metabolites from outside the cell inside do require energy as we will see.
And we will look on the next slide at these four processes, which show how bacteria
transport materials that they need to grow and divide and to live in this process. The fifth
process Im going to show is called group translocation, and that is shown on the next slide,
but another name for it is PTS, but I forgot to add it to the next slide. The PTS system,
standing for the phophoenolpyruvate transferase system. Well show you these five things
on the next slide.


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[Slide #27 Transport Processes]
So here we have our transport processes.

How do we get things from outside the cell, out here on the left, to the interior of the cell, on
the right here. And here, what we have is part of the cell wall, cell membrane, of a gram
negative bacterium. So here is the inside of a bacterial cell, the cytoplasm, heres a cell
membrane, phospholipid bilayer, and gram negative bacterium as you know has a
periplasm between the cell membrane and the outer membrane. So heres the periplasmic
space. And here we have the outer membrane of a gram negative bacterium, and as we
mentioned previously, in the periplasm, that space, or periplasmic space, you have the
peptidoglycan. Another name for peptidoglycan is murein, and if you read a lot of the
literature from Europe, they use the term murein for peptidoglycan. They mean the same
thing. As you recall in gram negative bacteria, only 10-15% of the cell wall is
peptidoglycan, and it is found in the periplasm.

So the question is - how do we get these different metabolites here on the left inside the
cell?

So we see in the first one, simple diffusion, the mode of entry is diffusion. Water, oxygen,
and carbon dioxide can easily pass through the outer membrane, periplasm, cell
membrane, back and forth really, between the exterior and interior of the cell. No energy at
all is required for this process. Free diffusion of these important compounds for all our
cells existence.
Glycerol: this three carbon compound, on the other hand, needs a little bit of help to pass
from the outside to the inside. So we can get through the outer membrane. Remember
these porins, the porin proteins in the outer membrane passes [glycerol] through the
periplasm, gets to the cell membrane, but it cant just diffuse in like these things on the top
line can, it needs a little bit of help. No energy, but help. Transported by the glycerol
transporter protein. So it gets to the cell membrane, this protein is there, hooks on to
glycerol, takes it inside, and no energy is needed for this process.

But the next two processes, shown here, the transport of lactose and here the transport of
galactose, those two we will cover next, do require energy. In this one, lactose, how does it
get inside the cell? It passes through this outer membrane, a porin for it, passes through the
periplasm, it gets to the cell membrane where there is a permease for it. Thats an enzyme
that binds to lactose, but in this case energy is needed to pump that lactose into the cell.
The energy is produced, in this case, by protons. Remember those protons that are right
outside the cell membrane of bacteria? They are waiting to get inside and hook up with
their electrons. Well, here the proton force, the protons, not just one but many are ready to
woosh through the cell membrane and that energy when they forge their way through the
membrane is enough energy to transport the lactose from outside to inside. And so proton
gradient, many, many protons outside here, very few in here, so the way they get in, when
they finally build up to a large enough number they force their way in and that energy of
proton gradient is formed, proton energy as well, you get lactose transported by the proton
gradient energized active transport. So energy is needed. Ill get back to shock sensitive in
a minute.
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The other example of an energy requirement is shown by the transport of galactose, this
sugar. It gets inside the periplasm and it binds to a protein in the protoplasm called
galactose binding protein. And for different sugars there are other binding proteins
depending what they are. But the binding protein takes it to the cell membrane, and in
order for it to get inside the cell as galactose, pass through the membrane, here energy is
required in the form of ATP. So in this case bacteria use up energy in the form of ATP to
transport a particular sugar. Enzymes, transporter protein enzymes are there for that
process. First they bind to a protein, the protein they bind to brings it to the cell membrane,
another protein or enzyme in the membrane brings it in, but ATP is needed. ATP is needed,
broken down to ADP and inorganic phosphate. And you get the sugar transported. Now
why does this process have to go on?

Now, active transport, what do you mean by active transport? Well, the terminology I like
to use, these sugars here, lactose and galactose, are going against the concentration
gradient. Theyre going from an area outside the cell where they are in very low supply, low
concentration, of these sugars in the environment. Inside the bacterial cell, theres a fairly
high concentration of these sugars already. But bacteria say I dont care, I want even more
sugar, I want more galactose, I want more lactose. Even though its going from a low area of
concentration to a higher! In order to do that, from low to high, it needs energy. Active
transport. Its kind of like when youre on a platform waiting for a subway to arrive. Ten
other people are with you and a subway car comes in, doors open, theres nobody in the
car. Its easy for you to pass into that subway car, to diffuse your way in. No problem,
nobody is there. The next day, you go and you are waiting for that same subway, subway
comes in and the doors open, five hundred people are already on that subway. But yet you
want to get in because you are late for microbiology lecture you know? So you have to force
your way in. There is a little bit of opening between people, and you push and you shove
and finally you squeeze your way in there. You have expended energy because you are
going from an area where there are only ten people on the platform to where there are five
hundred people, from an area of low concentration to high, basically the same principle
here is what Im trying to point out. You need energy to go from an area of low
concentration to one of higher. Bacteria dont care because they dont know where their
next meal is coming from. They are greedy. Lets get as much in as we can right now, if we
are growing in the presence of lactose or galactose, lets bring as much in as we can
possible take. As far as energy, either proton transport or ATP.

But notice these sugars, when they enter the bacterial cell cytoplasm, they are the exact
same form that they were outside. Lactose is lactose, galactose is still in the form of
galactose. Thats not the case with this last example. Why are these two examples shown
here and here, one is shock insensitive, one is shock sensitive. Well when they do these
studies to find out whats going on, and how these things are transported, they get these
bacterial cells and they shock them in the test tube. They put them under pressure and do
something to them to shock them so they lose their outer membrane. So shock, means
really, treating the bacteria to lose their outer membrane. And you can see here when they
lose their outer membrane, these bacteria are shocked, transport will still occur, these
bacteria that were insensitive, because the protons were already there helping. But when
you shock the other cells that carry out lactose transport by ATP, you shock these cells,
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they lose their outer membrane, everything inside the periplasm is going to be lost as well
as that galactose binding protein. So galactose once again is shock sensitive, because when
you shock the cells to lose the outer membrane, everything outside the cell membrane is
lost, there will be no binding protein, and in this case lactose wont get inside the cell.
But I think for us, today, maybe the more important one is this one called group
transportation, and the example shown here is the transport of glucose. But other sugars
also can be transported by group translocation. Translocation, across the membrane, from
one location to another. Group, yes we show glucose here but group, meaning there are
many other sugars that can also be transported by group translocation, or as shown on the
previous slide by the PTS, the phophoenolpyruvate transferase system, same thing. So what
is the advantage of this one? Here we have glucose, outer membrane that passes through,
gets to the cell membrane, wants to get inside the cell. There is an enzyme in the cell
membrane called Enzyme II which is ready to pick up glucose. So glucose gets here,
Enzyme II, but for glucose to pass through the membrane by this system, it needs some
energy. If you want to go back and read a lot about this you can, but all I want to point out
here is Enzyme II. To help glucose, this simple sugar, get inside the cell, it needs a
phosphate group NOT from ATP, but from this other compound called HPr-P. Histidine
containing protein, is phosphorylated. So a protein inside cell has a lot of histidine in it and
attached to histidine in this protein is a lot of phosphate. Now remember ATP has two high
energy phosphate bonds, recall that? But phosphoenol pyruvate and some other
compounds in the cell also have high energy phosphate bonds. With this type of
translocation of a sugar inside the cell, not ATP but some other molecule breaks its high
energy phosphate bond to enable the sugars to transport from outside to inside. What is
the advantage of this system? Its shown right here.
When glucose enters the cell it is already phosphorylated. It is already phosphorylated, you
see glucose6- P. These other sugars are not, they are still the same as they were before.
Why is that an advantage? Recall from glycolysis. Remember glycolysis, ATP is used to
jumpstart the system and ATP is needed for the glucose to go to glucose-6-P, it needs that
ATP. If this glucose-6-P is already formed when it comes into the cell, the bacteria save an
ATP. They dont have to phosphorylate glucose because it is already phosphorylated when
its transported. So they say the advantage of the group translocation system is sugars
enter the cell already phosphorylated, saving an ATP. Thats an immense amount of energy
for the bacteria that transport sucrose and glucose in that way. Lets just imagine there are
these, in a dental plaque, all these Streptococcus mutans, the culprit in causing caries, they
love, sucrose and sugar is their favorite thing to eat. And all of a sudden you take a sip of a
soda with a lot of sugar, a lot of sucrose. Immediately the sucrose surrounds all the
bacteria in the plaque, especially the Strep mutans, and it takes in that sucrose right away
by this system. It takes in sucrose, it doesnt have much time to do so but it takes all of it in
as quickly as it can. So it takes in sucrose to the interior of the Strep mutans cells and Its
already phosphorylated and if you take in hundreds of molecules of sucrose in the bacterial
cell, all those ATPs will be saved and used for other things and not have to be wasted by
just transporting sugar. So the advantage, once again, of group translocation, and glucose
and other sugars can be used, is that when the molecule enters the cell, it is already
phosphorylated. You dont need another ATP to form glucose-6-P. It is already
phosphorylated and can go right into glycolysis. It is an amazing energy saving reaction
compared to the others that we have seen, especially this one here where you use ATP.
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[Slide #28 Sucrose and Streptococcus mutans]
An example of how this is used, PTS or group translocation, is shown here in sucrose and
Streptococcus mutans. Once again it is the gram positive coccus that forms cavities. The
primary pathogen of caries. Loves sucrose in or diet. Loves sucrose so much that when it
grows in its presence it hydrolyses sucrose by these two enzymes. What are the two
compounds that are formed when sucrose is hydrolyzed? What are the two sugars found in
sucrose? Glucose and fructose, right? Glucose and fructose. So these enzymes break apart
the sucrose and then they transport the sucrose through by PTS system, by group
translocation. The Enzyme II in the cell membrane brings it in as sucrose-6-P. So the
previous slide we saw glucose taken in as glucose-6-P, here we see sucrose taken in as
sucrose-6-P, already phosphorylated by the transport! No need for another ATP.
Hydrolyzed inside the cell to glucose-6-P, so this sugar, now is broken down, sucrose-6-P to
glucose-6-P by the enzyme sucrose-6-P hydrolase. So sucrose is brought in, already
phosphorylated, its cleaved by this enzyme, and you have the glucose-6-P ready for
glycolysis. No need to use more ATP.

The genes for Enzyme II, the one that brings in sucrose or glucose in the previous example,
and also the genes for sucrose-6-P hydrolase, this enzyme, are induced by sucrose itself.
Remember group translocation, various sugars can be brought in by this way, we saw
glucose, sucrose, other sugars These sugars when bacteria are growing in their presence
can tell if it is sucrose or glucose or maltose or arabinose or whatever, therefore right away
gene induces an Enzyme II for the transport of that particular sugar. So Enzyme II varies
for every sugar. Every different sugar has its own Enzyme II. And its induced, the gene for
its production, is induced by the sugar itself. You know all about inducible compounds,
inducible enzymes. And it gets inside in that way. And also the enzyme that hydrolyzes
sucose-6-P is also induced by the presence of sucrose and that is true of all the sugars that
are transported in that way. But a lot of bacteria carry out this transport by this system and
once again it is to their advantage to do so because of all the energy that is saved up for
other purposes and growth and division of the bacteria.


[Slide #29 2 Aminosugars and a Tetrapeptide in Peptidoglycan]
Id like to cover at least one more topic in this area of metabolism and I want to give a brief
review for the exam on Friday about the bacterial peptidoglycan that weve seen a few
times already before I point out what I want to in this regard. Once again, we have the
building block or the monomer formed in peptidoglycan. The two aminosugars, amino
because they have the N-acetyl group here, N-acetyl. This is glucosamine, this is muramic
acid. The only difference between these two sugars is the lactic acid moiety. Lactic acid
moiety or group, here. Then we have the, in this case four amino acids, L-alanine, D-
glutamic acid, L-lysine or DAP and D-ala. Thats the building block, the brick. Thats
formed to put together a wall or a building, the building blocks that are linked to each other
over and over again to form the polymer, the peptidoglycan around the cell.

[Slide #30 Structure of peptidoglycan (murein)]
As you recall, we have the blues here represent the muramic acid, which are the ones to
which the amino acids are attached. And you see amino acid three in one strand is attached
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to amino acid four. So this will be, for example, either lysine or DAP linked to D-alanine on
an adjacent peptidoglycan residue, over and over agian. Layer and layer upon layer in
gram positive bacteria, and maybe just a monolayer in gram negative. So thats the bond
thats crosslinked, the peptide bond that is prevented from being formed by penicillin. And
youll find out more, a little bit, when Dr. Tierno talks about how penicillin works next
week. Crucial bond. If that bond did not exist, there would not be bacteria as we know of
anyhow. Why? Because once again the peptidoglycan would continue to be synthesized but
if they couldnt form that essential bond, they get so big that they would lyse, theyd burst
without the intact cell wall because of the intact peptidoglycan. So thats a very important
bond, that peptide bond there.

[Slide #31 No title, image of crosslinked peptidoglycan]
Here it is again, and heres what we just saw. But as I pointed out on this slide last week
also, every now and then you see something a little bit different in this slide. And that is
youll see 1, 2, 3, 4, not just 4 amino acids, but a 5th amino acid. L-ala, D-glutamic acid, DAP,
D-ala, D-ala. And this is actually the way these sub monomers are built inside of the cell.
They are built as five amino acids, as shown here. But always this last amino acid, this last
D-ala, these D-amino acids that are unique to bacteria, these D forms of the isomers of
amino acids, always this bond is lost as shown here. You see here, this bond is formed,
when these bonds are formed, that last D-ala is always lost. And we are going to see why.
Why is that last D-ala in the pentapeptide lost through a synthesis.

[Slide #32 Peptidoglycan Synthesis]
To review this once again, peptidoglycan synthesis has three stages. Cytoplasmic,
membrane associated, and then outside the cell where the preexisting peptidoglycan is
already formed. Heres a bacterial cell, as it takes in nutrients ad grows and gets larger
before it divides, it has to keep building up and extending its cell wall and cell membrane,
so theres already some there, some peptidoglycan. But as it gets bigger you have to add
more on to it step after step. As the cell sort of stretches and gets larger. So peptidoglycan
monomers are constantly being added on to this peptidoglycan framework around the cell.
And I want to point out some sites of action of some cell wall antibiotics that inhibit PG
synthesis.

[Slide #33 Peptidoglycan Synthesis]
Heres the key one. I dont want you to memorize everything in this but the overall purpose
of showing this slide[mumbled]
Peptidoglycan synthesis, as it said on the previous slide, occurs in three stages. Heres our
cell membrane, heres the cell membrane of a bacterial cell, phospholipid bilayer, heres the
interior of the cell where all the building blocks are formed, and heres the outside where
the actual peptidoglycan, the cell wall, is being produced outside the cell. Heres the cell
membrane and this is outside. And here we have a part of the peptidoglycan thats already
there in this cell. In the first stage in the cytoplasm, all the different parts of the monomer
of peptidoglycan are formed. You dont have to know the synthesis sequence rather, but
you know, you do go through 1, 2, 3, 4, 5, 6, 7, so all these stages occur in the cytoplasm, in
the interior. Heres the building block, two sugars, glucosamine, muramic acid. Heres the
pentaglycine, heres the five amino acids, and as you recall, this must be the bacterium
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Staphlococcus aureus as I mentioned before because this is a little unusual, it has a
pentaglycine bridge between amino acids 3 and 4 on the adjacent strands of peptidoglycan.
Pentaglycine, 5 glycine.
So this is built inside the bacterial cell, but it has to get outside the cell and it cant do that
on its own because heres this phospholipid bilayer, its very hydrophobic. So it cant do it
by itself, this monomer, it needs some help to pass through the cell membrane. And
essentially that is the function of this compound, this squiggly [?] compound right here. I
call it before the bactoprenol, go back and look at the different names of it, also known
here as undecaprenol phosphate. 11-five carbon compound [muttered, was hard to
understand], undecaprenol phosphate. This is the compound, the bactprenol I call it, that
helps the monomers go through the membrane, its hydrophobic interior, of the cell
membrane, and get to the outside. Its kind of like, once again, youre putting these parts of a
building together, youre assembling them in New Jersey, you have to bring them here to
New York, it has to go across the Hudson River. You dont build a building in New Jersey
and bring the whole thing over, you cant do that. Building blocks are assembled at one
place, transported across another milieu, in this case the Hudson River, by a ferry boat say,
and then brought here and then put together, outside of New Jersey. Well, the ferry boat, or
whatever takes the materials across the Hudson River, is like what the bactoprenol does. It
assists in the transport from one site to another. Heres another interesting point that is
going to come up. Here is this building block now, its outside the cell, you have to add it on
to what is already there. The peptoglycan is already there. In the next slide, if you look at it
carefully later youll see, thats what happens here. This new monomer added on to whats
already there, to once again increase step by step by step, the size of the peptidoglycan. As
the bacterial cell grows, more and more strands of new peptidoglycan being added on,
monomers passing though the membrane added on, one after the other. But heres the
question Im asking, how can this possibly occur? And finally, once again, the strands are
from adjacent strands of peptidoglycan are bound to each other. Heres the question
though, how can this possibly occur outside the cell? A peptide bond is being formed.
Remember lysine or DAP to D-ala, but it is occurring outside the cell. There is no ATP
outside the cell. Now you know from protein synthesis, every time an amino acid is bound
to another amino acid to form a peptide bond, ATP is used. There is no ATP here, how can
they possibly make this peptide bond between the lysine and the DAP, in this case the
pentaglycine attached to the D-ala. How does it do it?
This is where the breakage of the D-ala D-ala bond occurs. So here we have you see five
amino acids, including two D-ala, here, that D-ala, that terminal D-ala cleaved. And when
that bond is broken, when that one peptide bond is broken it produces enough energy for
the peptide bond between the adjacent strands to form outside the cell. So energy that is
required for a peptide bond, which that is, to be formed, but not ATP since its not even
around, but breaking one peptide bond produces enough energy to form another one to put
the peptidoglycans together. I dont know, they figured out how to do this. And then over
and over again, they synthesize one more peptidoglycan, add that on. One more thing Ill
mention now, because when we get to antibiotics there will be another uh, theres going to
be an antibiotic youre going to learn about later on that inhibits this production, it inhibits
the action of bactoprenol. What happens when you inhibit the action of bactoprenol? No
cell wall synthesized, cells lyse, cells die. Anything that inhibits cell wall synthesis is going
to lead to the death of the cell. And they are called bactericidal antibiotics, they kill the cell.
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The cell wall is so important for the life of the cell. So there are some antibiotics that inhibit
the action of bactoprenol. Everything has to stay inside, no cell wall would be formed
outside. And one other one to learn about also, a very important antibiotic, is involved with
the attachment of the sugar backbone and as each monomer is added on to the pre-existing
backbone, a glycosidic bond has to be formed there between the monomer and all the
peptidoglycan that is already there. That glycosidic bond between the sugars is formed,
that also, youll see, is the site of attack of another antibiotic, which is a very, very useful
antibiotic, one of the most important ones that we use because, we only use this one, as
youll find out, only under the most dire circumstances when every other antibiotic seems
to fail in treating an infection, there is one we have in the back of the refrigerator. Only use
in extreme emergency, when the bacteria seem to be not responding to any other antibiotic
used. Ill tell you the name of it right now, it is vancomycin. Youll hear about these. And so
a lot of these things, thats why we talk about the cell wall in such detail, especially
peptidoglycan, it is essential to the life of the cell. If it is not being produced or the strands
are not being linked together the cell will die. Many antibiotics we use to kill bacteria, to
treat infections, attack some point in peptidoglycan biosynthesis

[Slide #34 Cell Division]
And one more use for energy is cell division. Weve got a simple slide here, cell wall,
membrane, chromosomes. Heres one cell, the mother cell, dividing into two daughter cells.
The septum is formed here in the middle, as you see here the membrane goes first,
invaginates, followed by the cell wall. Here is the chromosome being replicated and to be
sure each of the two daughter cells has haploid chromosome, the chromosomes attach to a
protein in the cell membrane as you know, and this helps assure equal distribution of the
chromosomes or genomes of bacteria. Septum formation also requires some energy as well.

Ok, so lets take a two minute break and Ill put on the slide for bacteriophage, just two
minutes. And whatever I get to today, well end there as far as the first exam on Friday is
concerned.

[Bacteriophage powerpoint, slide #1 Bacteriophages by Dr. Boylan]
Ok, two minutes! Ok here we go, bacteriophages! I have a very fast watch today. We are
going to talk about bacteriophages, and tomorrow we are going to talk about viruses. Well,
thats a little contradictory because bacteriophages, well see, are indeed viruses. Ill talk
about what viruses are for those of you who have not had micro, dont have any idea what
viruses are.

So now were done with bacteria. As far as the genera properties of bacteria in the
overview of microbiology course that we are giving. Now overview of viruses, and then in
your course of infectious disease youll learn much more detail about how these viruses
replicate inside of our cells. But right now we are just going to give an overview of what
viruses are and a little bit about how they function and how they do replicate themselves,
but only briefly how they replicate themselves.
So bacteria phages, phage means virus, and phage means also eater. So these are viruses
that eat bacteria.

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[Slide #2 What are Viruses?]
So bacteriophages are bacterial viruses, and you can just say phage. So bacteriophage =
phage = bacterial virus. They are all interchangeably used, Ill probably use phage most of
the time. Indicates a bacterial virus, completely different from the viruses that infect plants,
animals, infect us. Well see the differences of those tomorrow. So these are just viruses
that bind to bacteria.
In general what are viruses? They are tiny, infectious agents. They are small, obligate
intracellular parasites. Viruses are not alive, they are not living. They are molecules of
protein and nucleic acid, maybe an enzyme here or there, but they are protein and nucleic
acid. They can not grow on their own. You put them on the richest medium on a petri plate
in the lab, they wont grow. They can only grow inside of living cells. They are the ultimate
parasite. In other words, they dont do anything on their own essentially, but they allow the
cells they infect, in this case bacteria, to really produce all the components of the virus as it
replicates. So they are the ultimate parasite. They dont have ATP or ribosomes or anything
like that. They need a host to replicate, they lack the machinery for generating energy and
large molecules, they dont have any ATP or ribosomes or electron transport. They use
what is in the cell they infect to reproduce. They need a host cell to replicate, in this case a
bacterium. The viral genome of all viruses is either DNA or RNA. So if you are going to
work with a virus and someone gives you one to study, your first question you ask them is
it a DNA virus or is it an RNA virus? Its one or the other. Viruses dont have both types of
nucleic acids in them, they are either a DNA virus with a DNA genome or RNA viruses that
have an RNA genome, but bacteria dont have both types of nucleic acids in them. So they
are one or the other. The capsid, well see, is the protein coat made up of capsomeres. The
most important part of a virus is its nucleic acid, be it DNA or RNA, and to protect it, it
builds a protein coat around it, a protein shell that surrounds it like a tent around it. The
genome is in the interior of the tent. We call it the capsid, made up of protein. The
individual proteins that come together to make up the capsid are called capsomeres. And
the nucleocapsid is the name given to the capsid with the enclosed genome. In other words,
the intact, the whole virus can be referred to as a nucleocapsid.

[Slide #3 Bacteriophages (phages)]
Bacteriophages or phages are bacterial viruses. And there are two classifications of
bacteriophage. One is lytic phage, the other is temperate phage. So only two types of
bacteriophage that we know of. Viruses, once again, that infect bacteria.

The lytic phages, as the name indicates, an example of this lytic phage is T2. Now, there are
many lytic phages. T2 is one that is shown on the right. They infect bacterial cells, these
viruses do. They replicate in the interior of the bacterial cell to very high numbers, perhaps
hundreds, maybe within 20 minutes, 30 minutes, you might have hundreds of new viruses
inside the bacterial cell, and then they cause the cell to lyse or burst. Thats called the lytic
cycle. So lytic phages such as T2 undergo lytic cycle where they infect the cell, begin
replication of themselves right away, grow to such large numbers that they then lyse that
cell, they are all released and go on to infect other bacteria. Over and over and over again.
Temperate phages, example of one of them is lambda, as shown here, these infect bacterial
cells. Temperate phages are always DNA phages. Youll see we do have RNA phages, we do
have DNA phages. We arent going to talk much about RNA phages today. But there are
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some viruses that infect bacteria that have RNA in their genome. For a long time we
thought they could never exist, RNA phages, but now we know there are quite a few in the
world.
But anyhow - temperate phages, such as lambda, are always DNA viruses. They infect the
cell, and the DNA, instead of immediately trying to destroy the cell, integrates into the host
genome and resides there. So with temperate phages, they inject their DNA, and instead of
immediately trying to direct the production of new viruses, new lambda phages,
They say no Im gonna hang out in this bacterial cell for a while, and Im going to integrate
myself into the chromosome of the bacterial cell. Its kind of like you have your house and
one day you have Bullitzer come along, and destroys the whole house in a matter of
minutes thats lytic. Temperate phage is kind of like your brother-in-law coming to live
with you. Sooner or later, hes not working, hes a bum, sooner or later hes going to
destroy your house. So thats kind of what these temperate phages are like. They hang out
in the cell. They integrate. They sneak into, become a part of the chromosome of the
bacterial cell, and stay there generation after generation. But because these bacteria do
have this genome there, they dont cause the cell to lyse immediately, but later on. Your
brother-in-law is eventually going to wreck your house, well well see what happens here.
There is more on this coming up. Eventually they could lyse the cell. But temperate phages
such as lamda are happy to become a part of the cell and not destroy it right away after it
infects it.

[Slide #4 Lysis or Lysogeny]
Lysis, infection by phage, produced many progeny and breaks open or lyses the host
bacterium, we just mentioned that. Lysogeny is the name given to the, after infection, the
phage DNA integrates into the host genome and resides there passively. No progeny are
produced, no lysis of the host, but eventually, like a ticking time bomb, like your brother-in-
law, sooner or later it might break free, excise itself from the bacterial chromosome and
direct the production of more temperate phage and subsequently lyse. This is called
lysogeny. By the way this should all be in red here [referring to the word lysogeny].
Lysogeny. The bacterial cell is lysogenic. The bacterial cell has a prophage, has the DNA of
the phage in its chromosome, its lysogenic. It has the capacity to be lysed when the brother-
in-law decides to wreck the house or when that piece of DNA decides to excise from the
chromosome of the bacterium and direct the production of more viruses that will
eventually lyse the cell.

[Slide #5 No title]
So overall, heres the process. Heres the bacterial cell. Heres the phage. It injects its nucleic
acid, DNA (or RNA) into the cell, and with the lytic cycle, you see what happens. That as
soon as the DNA comes into the cell, new viral particles are being produced. They are going
to be formed, and within a short period of time after they are formed they lyse the bacterial
cell. This cell is going to be destroyed.
With temperate phage, the DNA is injected, circularizes, back to that, and it becomes a part
of the bacterial chromosome. Its called the lysogenic state now, lysogeny. But this
chromosome of this bacterial cell has the DNA of the phage in there. Sooner or later it
might break free and go on and lyse the cell as shown on the left, but for the time being, it
doesnt.
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[Slide #6 Bacteriophages]
Why study bacteriophages? In general, we learn how viruses replicate. It was a simple
system. Once again go back to bacteria being simple cells. Study how viruses replicate in
bacterial cells, the processes which they undergo. Bacteria are easy to study, the viruses
that infect them are easy to study, learn about the simple steps in viral replication from
bacteria, and then later on when we have to study viruses that infect us, human cells that
are much more complicated, we have something to build on. We have the basic
information of how viruses replicate from what we learned about how they replicate in
bacteria. Lets see if the same process occurs in our cells as well. So that was a very good
advantage of working with bacteriophages way back when, and still today. Another
important reason I think to study phages is that someday you may be able to use
bacteriophages to treat bacterial infections. Youre going to learn next week I think, one of
the most important problems in the world today, is the emergence of bacteria that are
resistant to antibiotics. Bacterial resistance, they call it. Over and over again, more and
more bacteria, which we could treat readily with many different antibiotics are now
becoming resistant. So they survive in the presence of these antibiotics. And they are very
dangerous bacteria. Thats a major problem. How about another way to treat infections
caused by bacteria? Well maybe some day we will be able to use bacterial viruses that are
lytic and are very specific for Staph aureus or Strep mutans or some other bacterium. Very
specific and wed make up a cocktail of these bacteriophage for Staph aureus and wed
drink that. And its going to go and look only and search out and look for and bind only
Staph aureus, Staph aureus that may be resistant to penicillin. And they are going to only
look for Staph aureus causing that infection. Infect those Staph aureus, lyse them, help with
the infection. So its called bacteriophage therapy. And it might be essential in the future I
dont know. Its an important field of study because once again its an alternate to using
antibiotics to potentially treat infections. Its nothing really new, we knew about the
possibility of using bacteriophages this way to treat infections about 100 years ago. But
because antibiotics came along, the miracle drug, that kind of research was put on the shelf.
But now its coming back, its coming back. This study, whats happening? And what are the
problems or advantages of using bacteriophage to treat bacteria causing infections? They
are always very specific. You dont have to worry about them infecting a lot of different
types of bacteria, of your own microbial biota, the specifics. So bacteriophage therapy is
another way. And there are other reasons too but I think thats an important one. We also
learned about viruses, what they are made up of. Two macromolecules. There are also RNA
phages but we did not discuss them.

[Slide #7 Protein components of a phage]
Heres an example of a bacteriophage. And there are many different morphologies really.
Heres one of the T2. T2 or T even, its a coliphage. Its a phage that effects E. coli, so it is
called a coliphage. And Im not sure what T stands for but there are many T2 or T4 or T6,
many different phage like this, but T2, a phage that affects E. coli called a coliphage is
shown here.
And here is the what you see here is all the different protein parts. Up here we have the
head or the capsid. This is the capsid of this T2 phage. All the other parts are part of what
is called the tail. So heres the head, all the rest is the tail of the phage here. Inside the head
or the capsid is the nucleic acid. Once again the nucleic acid is the most important part of
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the virus. Just like we are all different because of our nucleic acid, our DNA, so viruses also.
Its their DNA or RNA that determines what they are. And so that nucleic acid has to be
protected from the environment, harsh conditions that will try to destroy the nucleic acid,
so the capsid surrounds the nucleic acid and protects it from being destroyed when it
infects one cell after another. In this t phage there is a neck, a collar, a sheath, a baseplate.
And theres also in the baseplate, there are prongs, these three little indentations here.
Prongs they call it, theyre not labeled on this slide. And the tail fiber. The capsid itself is a
protein coat, you see the nucleic acid is inside of it. The capsomeres are the individual
proteins that come together, self assemble more or less to build the capsid. One or several
proteins and uh, the T2 capsid also has icosahedral symmetry. Icosahedral. That means it
has 20 different surfaces. These triangles here, it has 20 different triangles that surround it.
So its kind of like a geodesic tent in a way. Its all these 20 different triangles of the
capsomeres put together in such a way to form this capsid. Icosahedral symmetry, its
called. Icosahedron, this head. And it looks kind of like a sphere. Other components of the
T2 phage are shown here, I mentioned them. There is the tail fiber, the tips here, on the
virus that are the adhesins. Kind of like the pili of the bacteria. They are called adhesins.
This is what makes the virus stick to a certain bacterial cell. This coliphage here will only
bind to E. coli, because whatever proteins are found in the tail fiber will bind to something
in the E. coli bacterial cell. They recognize some receptor. So the tail fibers enable the virus
to bind to the particular host, in this case, E. coli. Tail fibers, and it binds to a receptor in the
bacterial cell.

[Slide #8 Phage Replication]
And it is shown here. [Notices shuffling of students] Whats going on? What? Oh, Im sorry,
youre right I thought it was 11:42. Well get through it tomorrow but we will stop there for
the exam.