Lecture 12: Cell Envelope & Morphology (part 2)

glycosyltransfer is also known as _____

glycan polymerization

transpeptidation is also known as _____

peptide crosslinking

which class of PBP/s does glycosyltransfer?

Only A

which class of PBP/s does transpeptidation

A and B

there is NO ATP in the _____

periplasm

what is the prefix for all the genes involved in PG synthesis?

mur

True or False: if a single Mur gene is deleted, the bacteria can still survive

FALSE: all Mur genes are ESSENTIAL for viability

peptidoglycan needs to be inserted and cut at the right _____ at the right _____

place; time

PG insertion along the _____ of the cell cylinder ELONGATES the cell

circumference (middle)

formation of a PG _____ at the _____ divides the cell

septum; midcell

the PG septum is a _____ of PG separating the cell into two _____

disk; compartments

what is the main way scientists were able to track the location of PG synthesis?

fluorescent labeled D-amino acids

which type of amino acids do we add fluorescent labels to in order to track PG synthesis? Why?

D-aa, they are specific to PG synthesis (L-aa are used in protein synthesis)

when tracking fluorescent D-aminoa acids, they saw that PG is inserted along the cell _____ and at the _____, but NEVER at the _____

cylinder; septum; poles

how are we able to isolate a mutant defective for PG synthesis if it is essential for viability?

temperature sensitive mutants

temperature sensitive mutations are mutations that cause a protein to be _____ at _____ temperatures

destabilized/inactive; high

“ts” mutations encode mutant proteins that fold properly at the _____ temperature, but do NOT at the _____ temp

permissive; restrictive

how do you isolate a temperature sensitive mutation?

spot each mutant at permissive and restrictive temp (in same spot); if a colony is missing at restrictive, you can use the same colony on permissive plate for research

why bother spotting the colony at the permissive temp if you are looking for a mutant?

gives you a mutant you can actually work with!

in order to see how the mutation effects cell shape, you take the _____ mutants and shift them to the _____ temperature under microscopy, then you can observe how it changes is _____

ts; restrictive; morphology

what are “fts” mutants defective for?

cell division

what dos “fts” stand for?

filamentation temperature sensitive

why do “fts” mutants form long filaments?

they can elongate, but fail to divide (no septum formation)

what are “rod” mutants defect for?

cell elongation

what do “rod” mutants look like under microscopy?

circular (cannot form rod shape)

what type of mutant is this?

fts mutant

what type of mutant is this?

rod mutant

what are the TWO PG synthesis machines?

Rod complex and divisome

each of the PG synthesis machines are _____-embedded and are made of _____ proteins

membrane; mutliple

each protein in the PG synthesis machines is encoded for by an _____ or _____ gene

fts; rod

each PG synthesis machine is associated with a _____ protein

filament-forming

the rod complex is a PG synthesis machine that is dedicated to _____

elongation

the divisome is a PG synthesis machine dedicated to _____

division

what is the role of MreB in the PG Rod complex?

filament-forming protein

what is the role of RodA in the PG Rod complex?

PG glycotransferase (polymerizes glycans)

what is the role of PBP2a in the PG rod complex?

PG transpeptidase (crosslinks glycine from RodA)

what is the role of PBP1 in the PG rod complex?

both GT and TP

what is the role of MreCD in the PG rod complex?

regulatory proteins for RodA/PBP2a

what is the Divisome equivalent to the RodA in the rod complex?

FtsW

what is the Divisome equivalent to the MreCD in the rod complex?

FtsQLB

what is the Divisome equivalent to the PBP2a in the rod complex?

PBP2b

what is the Divisome equivalent to the MreB in the rod complex?

FtsZ

MreB is the bacterial homolog to _____ found in eukaryotes

actin

FtsZ is the bacterial homologous to _____ found in eukaryotes

tubulin

where is actin found in eukaryotes?

cytoskeleton

where is tubulin found in eukaryotes?

microtubules

both MreB and FtsZ form polymeric _____ that recruit and guide _____

filaments; PG synthesis

what end of the protofilament are monomers added?

plus end

what end of the protofilament are monomers removed?

minus end

monomers bound to _____ are added to the protofilament, once it gets _____ the monomer will fall off

ATP; hydrolyzed

if purified MreB are added to a solution they _____ form filaments

WILL

what does in vitro mean?

outside the cell

MreB forms small _____-like filaments when inside the cell

arc

filament proteins can sense membrane _____, then recruit and direct PG synthesis

geometry

when purified MreB proteins are added an official membrane they form _____ that align to the _____ of the membrane. They also _____ the membrane with is supportive evidence for their role in making the cell’s _____

filaments; curved face; deform; shape

in a healthy cell; MreB will moves back and forth _____ to the cell; when treated with penicillin, the motion _____ which suggested it’s role in _____

perpendicular; stopped; PG synthesis

penicillin stops the _____ of the rod complex

motion

circumferential motion of MreB reflects _____ and _____ of glycan strands

polymerization; crosslinking

true or false: ALL bacteria elongated at the center

FALSE: some elongate from the tips

what are 2 examples of bacteria that elongate from the tip?

A. tumefaciens, S. venezuelae

the divisome is organized by a _____ of _____ filaments

ring; FtsZ

why do we only see the FtsZ labels in 2 spots here?

image is sliced in half → ring was sliced too!

first evidence for the formation of the divisome ring cam from _____

immunogold labeling

purified FtsZ protein will form a _____ and exerts a _____ on an artificial membrane

ring; constrictive force

the divisome will undergo _____ motions associated with _____ PG synthesis

circumferential; septal

how does the divisome close the septum for division?

starts at outer edge, as it circles the inside of the cell, it adds PG slowing working its way inlay (in a spiral motion), until it’s closed off

curved cells require _____ filaments

curvature-inducing

what is the name of the curvature-inducing filament found in C. crescentus?

CreS

CreS protein will align itself to the _____ which creates the curved shape

inner curved axis

Cells with a CreS knockout will be _____ instead of _____

straight rods; curved rods

what does necessary mean?

gene is required for a certain function/morphology (ko of gene will also ko the function/morphology)

what does sufficient mean?

the addition of a gene is enough to include a NEW function/morpholgoy not seen in WT

what are the 2 curvature-inducing filaments found in Vibrio cholerae?

CrvA and CrvB

is CrvA/CrvB necessary or sufficient for curvature?

BOTH!

if you remove CrvA, CrvB, or both in v. cholerae what would you expect to see?

absence of curvature! (necessary)

will V. cholerae still be curved if you only knock out ONE of the curvature-inducing filaments?

slightly, but dramatically reduced

if you modify E. coli (a straight rod) to express CrvA and CrvB, what would you expect to see?

formation of curvature! (sufficient)

PG hydrolases _____ the cell wall

cleave

what are the 3 types of PG hydrolases?

Lytic transglycosylase, endopeptidase, amidase

where does Lytic Transglycosylase cleave?

between glycans (between NAG-NAM)

where do Endopeptidases cleave?

between peptide linkage

where do amidases cleave?

between glycan and peptide

PG hydrolases are important for making _____ for newly incorporated PG so that a cell can _____

room; expand

PG hydolases are also required for cell _____ after division occurs

separation

PG hydrolases are secreted at HIGH levels during _____ to initiate cell _____

phage infection; lysis (releases the virus)

which PG hydrolase is also known as Lysozyme (which an antibacterial that we produce!)

Lytic Transglycosylase

PG hydrolysis is necessary for cell wall _____

expansion

because PG is a single cage-like molecule, old bonds must be _____ to make _____ for new material

cut; room

B. subtilis lacking 2 endopeptidases fail to _____ and will eventually _____

elongate; lyse

can you actually fully delete both endopeptidases in B. subitils?

NO! have to delete one and slowly deplete the other

3 _____ proteins are activated by cell _____ and separate daughter cells in E. coli

amidases; division

what are the 3 amidases activated by cell division in E. coli?

AmiA, AmiB, and AmiC

what happens if you delete all 3 amidases in E. coli? why?

all the cell are connected; can’t separate after division!

what proteins are knocked out in this organism?

all 3 aamidases

mutants in E. coli and other bacteria that fail to divide in the middle and form _____

minicells

minicell mutants have a defect in _____ causing _____ division and often resulting in a longer daughter cell and small minicell

Z-ring positioning; asymmetric

what are the cells called that do ASYMMETRICAL division?

“min” mutants

what is the group of genes that minicells are mutant for?

min genes

what are the 3 genes that compose the min system

MinC, MinD, MinE

what does MinC do?

inhibit Z-ring/divisome formation (prevents septum formation)