Secretion systems in Bacteria

Question 1

Why do bacteria need secretion systems?

Answer 1

1. Export proteins and non-proteins to outer membrane and cell wall
2. formation of extracellular structures (pili and flagella)
3. injection of effector proteins which promote pathogenesis

Question 2

In gram negative what barriers must a protein need to cross?

Answer 2

• peptidoglycan and membranes, channels required

- selectivity

Question 3

Do proteins have to be folded when crossing out a Gram -

Answer 3

maybe, folded is bigger and harder to cross

Question 4

How might the energy be provided for a protein when secreted out of a cell

Answer 4

• The folding of a protein (entropy change)
• Electrochemical gradient
• ATP

Question 5

Explain a Type 1 secretion system

Answer 5

Transported substrates vary widely:
– Small (20kDa) right up to large (800kDa) proteins
– Polysaccharides
– Peptide signalling molecules
– Protein toxins (eg HlyA from E. coli)
• Characterized by repeats-in-toxin (RTX) exoproteins

The general model:
Forms a continuous channel from the cytoplasm to the extracellular space
Note how the peptide is secreted C-terminus first
and in an unfolded form

Question 6

RTX proteins

Answer 6

• Diverse sizes and functions
• First identified in toxins, but now known to cover enzymes,
S-layer proteins and specific functions such as nodulation
• Nonapeptide repeats in C-terminus of secreted protein
• consensus sequence:
X-(L/I/F)-X-G-G-X-G-(N/D)-D

Parallel β-helix motif

Ca2+ binding induces folding of the protein

Intracellular concentration much lower than extracellular- hence
extracellular folding is linked

Question 7

What are the 3 components of a Type 1 secretion system

Answer 7

ABC Transporter
– Inner membrane
– ATP hydrolysis powers secretion

• MFP
– Periplasmic
– Structural role; links ABC transporter to OM

• Outer Membrane Protein
– Outer membrane
– Forms a channel across the OM

Question 8

A related system to Type 1 - Biosynthesis of group 1 capsular polysaccharide

Answer 8

• Group 1 capsular polysaccharide is synthesized from
  oligosaccharides which are transported across the IM
  and then assembled into longer polymers in the
  periplasm

• Transit across the OM is then mediated by a membrane
protein called Wza

Wza forms a homo-octamer with a distinct banded structure.
The interior forms a large chamber through which the
oligosaccharide passes
Note that the transmembrane segment is made up of a helical
barrel, not a beta barrel

Question 9

Secretion by the Sec and TAT pathways

Answer 9

• Responsible for export of proteins from cytoplasm to periplasm
• First stage in the export of many proteins- feed into the type II and type V secretion systems
• Sec system is powered by ATP, TAT by proton motive force across the inner membrane

• SecYEG is a heterotrimeric complex of
three transmembrane proteins; they form
the secretion channel across the inner membrane

• Nascent peptides are directed to the Sec
complex by a signal peptide, which is
cleaved after translocation

• SecA provides the power for secretion- it is an ATPase and undergoes changes in
conformation on hydrolysis of ATP which power the export of polypeptide chain

• YidC plays a role in insertion of membrane proteins

Question 10

The TAT pathway

Answer 10

• TAT = twin-arginine translocation pathway

• Nomenclature comes from the consensus sequence within the signal peptide: [S/T]RRxFLK
(distinguished from Sec pathway signal sequence)

• Pathway widespread in Gram positive and Gram negative bacteria, as well as archea

• TAT pathway substrates include proteins that are folded before translocation eg proteins with
cofactors bound

General scheme for TAT translocation: TatB and C
recognise the ‘TAT’ signal, then associate with TatA,
which forms the channel across the membrane

Question 11

Type II secretion systems (T2SS)

Answer 11

• Proteins folded and assembled in the periplasm, then transported across the OM

• Related to type IV pilus biogenesis system

• Proteins secreted by T2SS include toxins, proteases,cellulases and lipases eg
– Pullulanase (starch-hydrolyzing lipoprotein; Klebsiella oxytoca)
– Cholera toxin (toxin; Vibrio cholerae)

Question 12

Whats the mechanisms of T2SS

Answer 12

• Powered by ATP hydrolysis (EpsE)

• An inner membrane complex directs secretion
(EpsE/F/L/M)

• Relies on pseudopilins (EpsG)- pilin-like proteins

• OM transition mediated by a secretin integral
membrane protein (EpsD)

Question 13

Pseudopillins

Answer 13

• Pseudopilins can form pilus-like fibres

• Several pseudopilins associated with each secretion system

• Proposed to function in a piston-like manner, to ‘push’
the toxin through the periplasm and across the OM
(controversial)