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JOANNE STUBBE: OK, so what I want to do today

is hopefully finish up or get pretty close to finishing up

module 6, where we've been focused on bacterial uptake

of iron into cells.

In the last lecture, I briefly introduced you

to gram-positive and gram-negative

big peptidoglycan, small peptidoglycan,

outer-cell membrane.

They both have the same goals.

They've got to get--

They take up iron the same way from a siderophore, which

is what we talked about last time, or by a heme.

And we'll talk a little bit about that.

And that's what you focused on in your problem set.

But they have different apparati to do

that, because of the differences between the outer--

because of the cell walls' distinctions

between gram-negative and gram-positive.

So we were talking, at the end of the class,

about, this was for the siderophores

which we talked about.

We need to take them up.

These are common to all uptake systems.

You have some kind of ATPase system and ABC ATPase.

We're not going to talk about that in detail,

but it uses ATP to bring these molecules

and also heme molecules across the plasma membrane.

And then, in all cases, you have this issue

of how do you get the iron out of whatever the carrier is,

be it a siderophore where the carriers can bind very tightly

or heme where you also have to do something

to get the iron out of the heme so that it can be used.

And so what I want to just say, very briefly--

and this you all should know now.

So now we're looking at heme uptake.

I'm not going to spend a lot of time drawing the pictures out,

but, if you look at the PowerPoint cartoon, what

you will see is there is a protein like this, which

hopefully you now have been introduced to from your problem

set.

So this could be IsdB or IsdH.

And we'll come back to that, subsequently.

And it sits on the outside of the peptidoglycan.

So this is the protein.

The key thing that is present in all these Isd proteins

is-- let me draw this differently-- is a NEAT domain.

OK?

And we'll come back to that later on.

But this domain--

So you have a big protein, and there's

one little domain that's going to suck the heme out.

And so what happens is we'll see in Staph. aureus, which

is what we're going to be focused on,

you have hemoglobin.

And somehow-- and I'm going to indicate heme

as a ball of orange, with a little planar

thing as the protoporphyrin IX.

OK, are you all with me?

And then somehow this gets sucked out

into the NEAT domain, where--

And again, all of these gram-positive and gram-negative

systems are slightly different, but in the Staph. aureus system

we'll be talking about today and you

had to think about in the problem set you basically

have a cascade of proteins which have additional NEAT

domains from which, because this is such a large peptidoglycan,

you need to transfer the heme to the plasma-membrane

transporter.

And what's interesting about these systems

and is distinct is that they end up,

they're covalently bound to the peptidoglycan.

And I'm going to indicate peptidoglycan as "PG."

And we'll talk about that reaction today--

the enzyme that catalyzes those reactions.

And all of these guys end up covalently bound

to the peptidoglycan-- which is distinct from all

of the experiments you looked at in your problem set.

Nobody can figure out how to make the peptidoglycan

with these things covalently bound.

So what you're looking at is a model for the actual process.

OK, so, also-- so that's the gram-positive.

And in the gram-negative, one has two ways of doing this.

And again, these parallel the ways with siderophore uptake.

So you have an outer membrane--

So this is the outer membrane.

And you have a beta barrel, with a little plug in it.

And so these beta barrels, they're at, like, 20 or 30

of these things in the outer membranes.

And they can take up siderophores,

as we talked about last time, but they can also

take up hemes.

OK?

So each one of these is distinct,

although the structures are all pretty much the same.

And so what you see in this case is,

there are actually two ways that you can take heme up.

So you can take up heme directly.

And we'll see that what we'll be looking at

is hemoglobin, which has four alpha 2 beta 2.

So this could be hemoglobin.

That's one of the major sources, and it is the major source

for Staph. aureus.

And so this can bind directly to the beta barrel--

gets extracted.

The heme gets extracted.

The protein doesn't get through.

And so the heme is transferred through this beta barrel.

OK.

So that's one mechanism.

And then there's a second mechanism.

And the second mechanism involves a hemophore.

And the hemophore is going to pick up the heme.

And so every organism is distinct.

There are many kinds of hemophores.

And I have a definition of all of these--

the nomenclature involved.

And so, after class today, I'll update these notes,

because that's not in the original--

the definitions aren't in the original PowerPoint.

OK?

So what you have, over here, is the hemophore

that somehow extracts the heme out

of hemoglobin or haptoglobin.

We'll see that's another thing.

So this gets extracted and then gets

transferred, in that fashion.

And so these hemophores come in all flavors and shapes.

They're different-- for example, in Pseudomonas or M.

tuberculosis.

And we're not going to talk about them further,

but the idea is they all use these beta-barrel proteins

to be able to somehow transfer the heme across.

And what happens, just as in the case-- if you go back

and you look at your notes from last time,

there's a periplasmic binding protein

that takes the heme and shuttles it, again,

to these ABC transporters.

OK?

So, in this system, again, you have

a periplasmic binding protein.

And this goes to the ABC transporter,

which uses ATP and the energy of hydrolysis of ATP,

to transfer this into the cytosol.

OK, so this is the same.

That remains the same.

And the transporters are distinct.

And then, again, once you get inside the cell,

what do you have to do?

You've got to get the iron out of the heme.

So the problems that you're facing

are very similar to the siderophores.

So, in all cases--

So the last step is, in the cytosol,

you need to extract the iron.

And you can extract--

usually, this is in a plus-3 oxidation state.

So you extract the iron.

And this can be done by a heme oxygenase, which

degrades the heme.

OK.

In some cases, people have reported

that you can reduce the iron 3 to iron 2, when the heme can

come out, but that still probably is not an easy task

because you've got four--

you've got four nitrogens, chelating to the heme,

and the exchange, the ligand exchange, rates

are probably really slow.

So I would say the major way of getting

the iron out of the heme is by degradation of the heme.

And we're not going to talk about that in detail at all,

either.

OK.

So that's the introductory part.

And here's the nomenclature, which

I've already gone through.

I've got all these terms defined.

And if you don't remember that, or you don't remember it

from the reading, you have a page with all the names--

which are confusing.

And so the final thing I wanted to say,

before we go on and actually start looking at peptidoglycans

and gram-positive bacteria and heme uptake

in Staph. aureus, which is what I was going to focus on

in this little module, is to just show you,

bacteria desperately need iron.

So what do they do?

This is what they do.

OK, so, here you can see-- and some bacteria

make three or four kinds of siderophores.

Others only make one or two kinds of siderophores,

but what they've done is they've figured out

how to scavenge the genes that are required

for these beta barrels.

So they can take up a siderophore

that some other bacteria makes.

OK?

And that's also true of yeast.

Yeast don't make siderophores, but most yeast have,

in their outer membranes, ways of picking up

siderophores and bringing it into the cell, since--

and remember we talked about the fact

there were 500 different kinds of siderophores.

But you can see that the strategy is exactly the same.

You have a beta barrel.

You have-- these are all periplasmic binding proteins.

This picture is screwed up, in that they forgot the TonB.

Remember, there's a three-component machine,

TonB, ExbB and D, which is connected to a proton motive

force across a plasma membrane, which

is key for getting either the heme or the iron

into the periplasm.

And you use a periplasmic binding protein,

which then goes through these ATPase transp--

ABC-ATPase transporters.

So what I showed you was heme uptake, iron uptake, but in all

of these cases, like Staph. aureus we'll be talking about,

we can also get iron out of transferrin.

We've talked about that.

That's the major carrier in humans.

The siderophores can actually extract the iron

from the transferrin.

And remember the KD was 10 to the minus 3,

so somehow, again, you've got to get iron transferred

under those conditions.

And that's how these guys survive.

So they're pretty desperate to get iron.

And inside, once they get inside the cell,

you have all variations of the theme to get the iron out.

But they're all sort of similar.

Somehow, you've got to get rid of whatever is tightly binding

it.

And if you're creative, you can reuse

whatever is tightly binding it, to go pick up some more metal.

OK.

So that just summarizes what I just said.

And so, in two seconds, I'm going to show you,

now-- we've spent one whole lecture,

a little more than a lecture, talking

about iron uptake in humans via DMT1,

the iron-2 transporter, and the transferrin transfer receptor.

So, in the plus-two and plus-three states,

we just started looking at the strategies by bacteria