Lecture 24: Bacterial Biofilms Pt. II
Today:
membrane potential
B. subtilis biofilm growth
agar substrate
objective
biofilm
MSgg media:
~1.3 cm by 60 hours
Biofilm cells divide labor by taking up different phenotypic states
B. subtilis can differentiate into different cell states, much like a developing organism!
Can swim
Can secrete matrix to form biofilm
Become dormant to survive extreme conditions
Can take up DNA
A series of kinases in B. subtilis detects the environment and activates gene expression
KinD
KinC
KinB
KinA
Detect the extracellular environment
Detect the intracellular environment
inside cell
outside cell
cell envelope
Activate transcription factors
Leads to different cell states depending on conditions!
Different conditions in time and space lead to different patterns of gene expression!
Not unlike development of a multicellular organism!
B. subtilis cell types form developmental patterns!
Biofilm section:
Produce costly matrix for everyone
Can escape and form a biofilm somewhere else?
Can survive extreme conditions
B. subtilis cell types form developmental patterns!
Extracellular matrix
Motility
B. subtilis biofilm heterogeneity
Extracellular matrix
Spore
B. subtilis biofilm heterogeneity
Extracellular matrix
Motility
Spore
B. subtilis biofilm heterogeneity
Extracellular matrix
Motility
Spore
There are many advantages to living in a biofilm community
There are many advantages to living in a biofilm community
tobramycin (antibiotic)
Enhanced antibiotic tolerance!
There are many advantages to living in a biofilm community
Matrix producer
Division of labor among differentiated cells!
But there are also inevitable group conflicts!
The Ugly
The Bad
The Good
Conflicts become even more complicated with large groups!
How can the simplest organisms resolve group conflicts?
Bacillus subtilis
?
Fundamental problem for cells in a biofilm
Biofilm
Competition for limited resources
glutamate
Interior cells have limited nutrient access
… a global problem !
Nutrient
Nutrient access
glutamate
Peripheral cells starve interior cells
Starvation
Growth
… a global problem !
Nutrient
Nutrient access
glutamate
Interior
Periphery
Competition requires a collective solution
glutamate
Long-range coordination
Interior
Periphery
Competition requires a collective solution
glutamate
… diffusion limited
Interior
Periphery
Familiar bacterial mechanisms are insufficient
Quorum sensing molecule
glutamate
Diffusion time increases non-linearly with distance!
Concentration also decays with diffusion!
Diffusion might not be the best way to couple behavior across a huge biofilm!
Diffusion-limited signaling is insufficient
cells
distance
signal
amplitude
diffusion
interior
periphery
An active relay mechanism?
distance
signal
amplitude
diffusion
interior
periphery
active relay
Question: how is such long-range coordination possible?
nutrient access
interior
periphery
biofilm
Action potentials enable long-range signaling
distance
signal
amplitude
diffusion
active relay
nervous system
Action potentials are propagated by ion channels
nervous system
potassium channel
(Doyle, et al. (1998))
Bacteria have ion channels . . .
nervous system
potassium channel
(Doyle, et al. (1998))
Courtesy of Steinar Stølen
B. subtilis Biofilm
Bacteria generate action potentials!
Bacteria generate action potentials!
objective
glass
PDMS
flow
Microfluidics
Thioflavin-T
(membrane
potential)
Bacteria generate action potentials!
+
N
N
CH3
CH3
H3C
H3C
+
N
N
+
N
N
-
-
+
+
+
N
N
-
-
-
in
out
Membrane potential
Bacteria generate action potentials!
Growth rate
Membrane potential
Thioflavin-T
(membrane
potential)
+
N
N
CH3
CH3
H3C
H3C
Periodic pausing of biofilm growth
alleviates starvation of interior cells
… which increases tolerance against antibiotics
Antibiotics
protected
What is the mechanism?
YugO
Mechanism for long-range signaling in biofilms
The trigger:
K+ ion channel
B. subtilis cell
Glutamate limitation
for interior cells
cell
YugO
Mechanism for long-range signaling in biofilms
The trigger:
Opening of YugO K+ channels
cell
YugO
Mechanism for long-range signaling in biofilms
The trigger:
Mechanism for long-range signaling in biofilms
Signal relay:
YugO
Depolarization of neighboring cells
YugO
Mechanism for long-range signaling in biofilms
Signal relay:
Reduced glutamate uptake
GltP
YugO
Mechanism for long-range signaling in biofilms
Signal relay:
Glutamate limitation
YugO
Mechanism for long-range signaling in biofilms
Signal relay:
Opening of YugO channels
YugO
Mechanism for long-range signaling in biofilms
Signal relay:
Bacteria propagate action potentials!?
Active long-range signal propagation
Diffusion
Signal amplitude
Active relay
Electrical signaling enables nutrient time-sharing!
nutrient access
periodic growth
Liu, et al. (2017)
Electrical signaling can couple distant biofilms
Liu, et al. (2017)
Electrical signaling can attract motile cells!
What have we learned?