CLASS: Fundamentals I 11:00-12:00 Scribe: Lauren Morris

DATE: 08-19-2010 Proof: Spencer Terry

PROFESSOR: Pritchard LECTURE TITLE Page 1 of 7

I. Microbial Polysaccharides [S1]

a. READ SLIDE

II. Classification of Bactria by Gram-Staining [S2]

a. This Danish physician, Christian Gram, about 120 years ago, was experimenting with some of these new coal-tar dyes that became available. These German organic chemists about them were just churning out all these neat so-called coal-tar dyes.

i. They were made by diazetizing analine with all these other compounds.

ii. And one of these was crystal violet. It’s a nice beautiful symmetrical molecule. If you’ve ever worked with this, a tiny little speck of it gets on you, then it gets all over you

b. What he found is that he could group bacteria into two major groups on the basis of staining properties. Many of you who had microbiology will have done this gram staining.

i. The idea is you first of all, put the bacteria on a slide, fix them there either by warming it briefly in a flame or putting a little alcohol on it and letting it dry (thus attach bacteria to the slide)

ii. You then stain the bacteria with crystal-violet which makes everything purple

iii. Then you use a mordant, Iodide, to help fix the stain there.

iv. You put a little alcohol on the slide and let it run over the stained bacteria, and you’ll see it washes out the stain of some bacteria

v. Since it’s hard to see colorless bacteria, usually people do a counter stain, and that counter stain usually is safranin which turns the bacteria pink although some people use a stain that turns the bacteria green.

vi. The net effect is some bacteria end up being one color and other bacteria another color.

c. The blue bacteria are gram-positive, and the ones that are pink are gram-negative

d. This image is a mixture of streptococci. They are gram-positive; they are blue. The E.coli are gram-negative so it’s real easy to tell them apart

e. For the first hundred years ago, people didn’t really know how this worked. They knew it had to do something with the cell walls of the bacteria

III. Gram-Positive Bacteria Figure [S3]

a. Gram-positive bacteria differ from gram-negative in that it has only one membrane. Gram-positive will have this typical lipid bilayer, symmetrical bilayer by the way,

i. Then on the surface they’ll be this peptidoglycan layer

ii. Now this image is a schematic from your textbook and a lot of people are not sure they believe this anymore, and in some cases they know it’s not true

iii. The basic idea is there is a thick layer of peptidoglycan on the outside of the membrane

IV. Gram-negative Bacteria Figure [S4]

a. In the Gram-negative bacteria there are two membranes

i. the symmetrical bilayer is the inner membrane

ii. then there is a layer of peptidoglycan

iii. then there’s this outer membrane, and the outer membrane is not symmetrical.

b. The bottom is the typical phopholipid, but the outer leaflet (they talk about leaflets sometimes) is something called lipopolysaccharide

i. The lipid part is embedded in the membrane and then there are these long polysaccharide chains [points to the tall grey lines on the outer membrane] and that’s lipopolysacchride – that’s very important in disease.

V. Lipopolysaccharide Figure [S5]

a. Ok, that’s showing you the same thing so I’ll skip along

VI. Peptidoglycan Structure Figure [S6]

a. Peptidoglycan, we’ll talk about it’s structure

i. Let me just remind you what peptidoglycan is: bacteria have an internal pressure of about 5 atm, and if they weren’t in a strong little bag or sac, they would blow up and pop.

ii. The sac their in is composed of something called peptidoglycan that adds strength and shape to the bacteria cell wall. It consists of these fairly long chains of polysaccharide

b. Here we have an N-acetylglucosamine [points to the pink and blue structures at top of slide]

i. Except it has this little ether group attached to it – and what this is called is N-acetylmuramic It is ether linked to this 3-Carbon unit here

ii. These polysaccharide chains that go on are cross-linked with these peptide bridges

iii. The composition of these peptide bridges is a little different in different bacteria, but most cases, the 1st amino acid (AA) that this lactic acid group here is attached to is Alanine

iv. Then it’s attached to something called Isoglutamate – that’s a glutamic acid, but normally in proteins a glutamic acid would be linked through this alpha-carboxyl group. Here it is linked through the gamma-carboxyl – that’s why they call it isoglutamate

c. By the way this is a D-AA

i. Normally in proteins, you never ever find a D-AA, but bacteria occasionally use D-AA

ii. For all other proteins there is just L-AA

d. Then it is linked to Lys, or a compound very similar to Lys in some bacteria, and finally a D-Ala

i. These things can then be cross-linked – this is called a Stem Peptide

ii. Stem peptides can be cross-linked by the so called bridge peptides, which differ in composition depending on what the bacteria is

iii. Often in gram-neg there is no cross link peptide connected directly; but in things like Staph aureus, you may recognize this as a Gly or you may not, but there’s 5 Gly in a row – that’s the bridge peptide in Staph aureus

iv. For some reason textbooks always show this [indicating the part of slide above the arrow pointing towards “in Staphylococcus aureus only”]. It’s different in different bacteria, but textbooks always use Staph aureus as an example, probably because it was the first one to be figured out.

VII. N-Acetylmuramic Acid Figure [S7]

a. A close up of N-acetylmuramic acid. Again, it’s the glucosamine structure with this lactic acid group linked here

VIII. Gram-Positive/Negative Cell Wall Figures [S8]

a. Repeating stuff

b. The idea is these stem peptides can be cross-linked

c. Again, this is Staph aureus showing you the 5 Gly in a row

d. QUESTION: So Staph aureus is the only one with peptidoglycan stems cross-linked? ANSWER: No, almost all gram-positives are cross-linked, but they’re not crosslinked with 5 Gly. Ex Group B Strep is cross-linked with 2 Ala. Stem peptides are almost identical with gram positives. They’re a little different in gram neg.

e. QUESTION: What is the difference between linking of gram pos and gram neg? ANSWER: It depends on the bacterias. In general, gram neg tend to be directly cross-linked. I’m gonna show you in a moment, whereas gram pos often are cross-linked through a bridge peptide.

IX. Transpeptidase Reaction [S9]

a. To answer your question; there’s a reaction called a Transpeptidase Reaction

i. It is a really important reaction and has been enormously studied

ii. The example shown here is for Staph aureus. See those 5 Gly?

iii. The idea is that this enzyme then causes the formation of a bond between this amino group and the carboxylic acid group of this 2nd Ala and at the same time cleaves off this Ala [references the dotted line]

iv. Release an Ala to make a linkage there

v. So all these polysaccharide chains that have the stem peptides coming off, many will be cross-linked

vi. So basically the peptidoglycan of a bacteria is like one big cross-link bag or basket

X. Biosynthesis of Peptidoglycan [S10]

a. This is illustrating the biosynthesis of peptidoglycan

b. What happens to begin with, is when you make a UDP N-acetalglucosamine, and an enzyme catalyzes the addition of a phospholino pyruvate to it, essentially what you do is you make UDP-N-acetalmuramic acid.

i. And then you add sequentially, you know building up the stem peptide now, an Ala, a Glutamine, a Lys

ii. And the interesting thing is you don’t add one Ala after another, you add them two at once,

iii. Notice they’re D-Ala, not L-Ala. It’s real critical that they’re D

iv. Then this is linked to a Dolichol-phosphate. We talked dolichol when we talked about glycoproteins – bacteria make dolichols too, usually either shorter than the ones that animals make

v. And then you add another GlcNAc to make this basic subunit of peptidoglycan

vi. Then you buildup the cross-link. Remember this only happens in Staph – different AA are used in gram pos

XI. Biosynthesis of Peptidoglycan (con’t) [S11]

a. You have this basic subunit it can then be, as I said, cross-linked to another subunit, and again, so on and so on

b. you build up this big cross-linked network or mesh of peptidoglycans

XII. Lysoszyme [S12]

a. There’s the peptidoglycan there

b. This man, Alexander Fleming, is doubly famous for a couple discoveries he made

i. There’s a “myth”, maybe not a myth – one day, back in 1922, he had a bad cold and his nose was running and he was working on his cultures and his nose dripped in his culture plate petri dish

ii. Instead of throwing it out he put it in the incubator to see what would happen. The next day he came back and found that the bacteria had all lysed where his nose had dripped into the plate – there’s something in his nasal secretion that killed all the bacteria.

iii. He thought this was a way to cure disease, so he started testing different bacteria – some bacteria lysed readily with this component in the nasal secretion, and other bacteria didn’t

iv. He was disappointed because not all human pathogens were harmed by whatever this substance was – “lysozyme” – it’s a small protein, studied extensively

v. Then he thought, “maybe that’s why the bacteria aren’t pathogens” - We have lysozyme in our saliva, in our tears, nasal secretions, and all secretions, in our blood and everywhere

vi. So if a bacteria was dissolved by lysozyme, it wouldn’t be a human pathogen. Bacteria that are pathogens for us all modify their peptidoglycans so they are not attacked by lysozyme.

c. They have neat tricks for changing this peptidoglycan, like taking off this acetyl group, and then lysozyme doesn’t cut it anymore

i. Where lysozyme cuts is right after this NAM here [references the lysozyme cleavage point] – if you cleave the peptidoglycan, then this sac that’s holding the bacteria with this 5 atm pressure inside isn’t intact anymore, and the bacteria blows up and lyses

d. Most bacteria out there doesn’t make people sick, you could probably eat a handful of dirt with thousands of different bacteria and you are not gonna get sick. Only a relatively small number of bacteria are human pathogens. The ones that are have got to defeat our defenses, things like lysozyme.

XIII. Penicillin [S13]

a. Another discovery the man made and another “myth”:

i. a mold in the air fell into one of his dishes, and instead of throwing it out, let it grow through the weekend, came back and found something like this [references petri dish]

ii. that’s staph aureus there, there’s the mold [white circle]. Notice that clear zone – the mold is obviously secreting something that’s dissolving/killing the bacteria

iii. that substance turned out to be Penicillin

iv. originally he couldn’t isolate this, so not much was done for several years because they couldn’t get enough of it

b. this substance did kill human pathogens, unlike lysozyme

c. later they worked out the structure of Penicillin – 5 membered ring with S, C, N, 2 methyl groups and a C, and then this really unusual 4 member ring – there’s a carbonyl group attached to a N (that’s an amide) that’s cyclic (special name = beta-lactam)

XIV. Penicillin (con’t) [S14]

a. These guys, before WWII, an awful lot of soldiers were wounded died from infections that they got after they were wounded. It was enormously important to come up with antibiotics that would kill things like Staph and strep that cause so many deaths, so there was a big push to try to purify the penicillin

b. Fleming originally used bedpans from a hospital trying to grow the mold – didn’t work well. England was being bombed (1940s) so they moved the research to the States. Here they discovered that they could grow this mold in enormous fermentors on an enormous scale in something called corn-steep liquor.

i. Corn can be dry milled or wet milled and the wet milling has a by-product called corn-steep liquor which is either thrown away or dried up and fed to cattle

ii. It turns out it was a wonderful substrate for growing penicillin – so they made vast quantities of penicillin and that turned out to be a wonder drug, killing all strep and all staph and worked great for several years until bacteria figured out a way to defeat it.

XV. PENICILLIN FUNCTIONS BY… [S15]

a. Let me just tell you how penicillin works

i. This is a transpeptidase reaction I’m showing you now for the 3rd or 4th time.

ii. The amino group binding to the carboxyl group of a nearby stem peptide