Transition from planktonic cells to biofilm in E. coli: the CsgD regulon.
Gualdi, L., Brombacher, E.*, and Landini P.
Dipartimento di Scienze Biomolecolari e Biotecnologie, Università degli Studi di Milano;
*Swiss Federal Institute of Environmental Technology (EAWAG), Switzerland
Biofilm formationBiofilm formation:
• 1. Adhesion factors: e.g. flagella in Escherichia coli, Pseudomonas
aeruginosa;
• 2. Extracellular polysaccharides
• 3. Cell density; e.g. “Quorum sensing”
In Enterobacteria such as Escherichia coli, Salmonella enterica
presence of a specific adhesion factor
CURLI FIBERS
Adhesion Microcolony Maturation
Function of curli (thin aggregative fimbriae):
Cell aggregation and clumping
Ability to adhere to a solid surface
CsgD
Outer membrane
Cytoplasmic membrane
Curli
Cellulose
adrA
Curli subunitsCurli subunits
Inducer of celluloseInducer of cellulosebiosynthesisbiosynthesis
What is the function of AdrA?
adrA
“GGDEF MOTIF” PROTEIN
Cyclic di-GMP
bcsA, bcsB, bcsC, bcsZ
(cellulose synthesis in E. coli)
Mechanism of cellulose biosynthesis activation by c-di-GMP
cy-di-GMP acts as an allosteric activatorof cellulose synthase machinery
AdrA
Role of cyclic di-GMP in the bacterial cell
From Camilli and Bassler, Science 2006
CsgD-dependent regulation: is there more to the curli/cellulose matrix?
• At least two genes are directly controlled by CsgD: csgBA (curli subunits) and adrA (cellulose)
• Genomic and proteomic approach comparing csgD-proficient to strains in which no csgD expression is detectable to identify novel csgD-dependent genes
CsgD+
No CsgD
CsgD as “global regulator?”According to the GA experiments, CsgD also controls the
following genes/operons (among others):
gsk (GMP biosynthesis) pyrBI (pyrimidine metabolism) gatYZAB (transport of galactitol, possibly
a signal for curli regulation?) ymdA (putative fimbrial gene, likely
part of the csgBAC operon)
yoaD (unkown, potential PDE-A)* yaiB (unknown function)
Involved in nucleotideInvolved in nucleotidemetabolism (??)metabolism (??)
Curli productionCurli production (??)(??)
(??)(??)
* Enzymes involved in c-di-GMP breakdown
IPTG + - + -
pGEMT pGEMTyoaD
The yoaD gene encodes a potential c-di-GMP phosphodiesterase
Inhibition of biofilm formation by yoaD expression would be consistent with a PDE role for the YoaD protein (inhibition of cellulose biosynthesis), but….
Why would both postitive (csgBA, adrA) and negative (yoaD) factors for bacterial cell aggregation be regulated by the same mechanism?
yoaDPlac
Timing is everything…..
0
5
10
15
20
25
30
0 2 5
0
0.2
0.4
0.6
0.8
1
1.2
1.4
TIME (HOURS)
Rel
ativ
e ex
pre
ssio
n r
atio
(csg
D e
xpre
ssio
n/n
o c
sgD
Op
tica
l den
sity
(O
D60
0nm
)
=adrA expression (DGC) =yoaD expression (PDEA)
A feedback control for cellulose biosynthesis
• CsgD activates the adrA gene, resulting in c-di-GMP accumulation and cellulose biosynthesis
• At the onset of stationary phase, the yoaD gene is also activated to counteract the effect of AdrA and reduce cellulose biosynthesis, possibly to reduce glucose consumption
CsgD may act on intracellular cy-di-GMP pool
Adapted from Camilli and Bassler, Science 2006
CsgD
“Global impact” by CsgD on protein expression
Cytoplasm Outer membrane
CsgD - + CsgD - +
Protein Function Regulation
WrbA Enhancer of trp repressor protein binding to DNA
rpoS-dependent
PflB Pyruvate formate lyase I
(anaerobic metabolism)
Induced anaerobically
GadA Glutamate decarboxylase
(resistance to acid)
rpoS-dependent
CsgG Involved in assembly or transport protein for curli; novel lipoprotein
rpoS-dependent
Dps Unspecifically binds and protects DNA from oxidative damage mediated by hydrogen peroxide
rpoS-dependent
rpoS regulon
CsgD seems to activate expression of rpoS-dependent proteins
- + - + CsgD
1 2 3 4
WT rpoS
1 2 3 4
WT rpoS
- + - + CsgD
Indeed, CsgD-dependent alteration in protein expression requires a functional rpoS gene
How does CsgD affect S-dependent expression?
iraP
CsgD
iraP
yaiB= unknown gene regulated by CsgD
yaiB now annotated as iraP and identified as a factor for S stabilization
CsgD affects S intracellular concentrations in a manner dependent on IraP
1 2 3 4 5 6 7
35 KDa
50 KDa
- + - + - + CsgD
WT iraP rpoS
30 KDa
6XHis-S
Biofilm formation
CsgD
Curli
Cellulose
Outer membrane
Cytoplasmic membrane
[ES]Oxidative stress genesAcid resistanceResistance to desiccation
CsgD induction of biofilm formation: part of a “general stress response”?