Transcript + - + - + CsgD

The CsgD protein of Escherichia coli: a
relay between bacterial biofilm formation
and gene expression.
Gualdi, L., Brombacher, E.*, Bertagnoli, S.
and Landini P.
Dipartimento di Scienze Biomolecolari e Biotecnologie, Università
degli Studi di Milano;
*Swiss Federal Institute of Environmental Technology (EAWAG),
Switzerland
Biofilm formation:
•
1. Adhesion factors: e.g. flagella in Escherichia coli, Pseudomonas aeruginosa;
•
2. Extracellular polysaccharides
•
3. Cell density; e.g. “Quorum sensing”
Adhesion
Microcolony
Maturation
In Enterobacteria such as Escherichia coli, Salmonella enterica
presence of a specific adhesion factor
CURLI FIBERS
Function of curli (thin aggregative fimbriae):
Cell aggregation and clumping
Ability to adhere to a solid surface
Co-regulated with other adhesion genes
Curli:
Long intertwined structure
Proteinaceous
Involved in adhesion to solid surfaces and in cell-cell
aggregation
Locus
Genetic structure and organization well conserved in
Enterobacteria.
However, silent in many E. coli lab strains.
csgBA Operon: encodes curli structural subunits
CsgA  major subuint
CsgB  nucleator protein
csgDEFG Operon:
CsgD  transcription activator of csgBA operon
CsgEFG  assembly and transport of curli subunit
Curli expression regulation:
Stationary phase
(RpoS= alternative s factor)
Osmolarity
(EnvZ/OmpR and CpxA/CpxR
TCRS)
Low temperature
<32-34°C
(Crl= accessory RNApol factor)
Low
osmolarity
High
osmolarity
CsgD directly activates at least another
promoter: adrA
The csgBA and the adrA promoters display a common sequence upstream
of the -35 promoter element necessary for CsgD-dependent expression:
CGGGTGAGTTA (PcsgB)
CGGGTGAGCTA (PadrA)
=MG1655
=PHL628
PcsgB::luxAB
PadrA::luxAB
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 di-c-GMP
AdrA
cy-di-GMP acts as an allosteric activator
of cellulose synthase machinery
Cyclic-di-GMP is a “fashionable”
second messenger in bacteria
• Originally identified as allosteric inducer of
cellulose biosynthesis in G. xylinum
• Involved in exopolysaccharide production
in many bacteria
• Cell cycle and differentiation in C.
crescentus
• Biofilm vs. virulence gene expression in P.
aeruginosa
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 promoters (csgBA and adrA) are directly
controlled by CsgD
• However, in many lab strains the csgD operon is
cryptic
High-level expression in BL21(DE3)
(for protein purification)
Low-level expression in “normal” E. coli strains
(for measurement of csg gene expression)
CsgD+
X
uidA
X
b-glucur.
No CsgD
CsgD-dependent regulation: is there
more to the curli/cellulose matrix?
• At least two promoters (csgBA and adrA) are directly
controlled by CsgD
• However, in many lab strains the csgD operon is
cryptic
Protein pattern in SDS-PAGE
CsgD+
X
uidA
X
b-glucur.
No CsgD
Adhesion properties conferred by ectopic expression of CsgD
A
pT7-7
B
pT7-CsgD
pT7-7
pT7-CsgD
MG1655W (WT)
EB12 (csgA mutant)
No curli production
LG04 (bcsA mutant)
No cellulose production
A: Biofilm formation assay usin gcrystal violet staining in microtiter plates
B. Spots on Congo Red-supplemented plates
Medium-dependent regulation of curli
production takes place at the csgB promoter
%Relative
biofilm formation
of MG1655pT7
biofilm formation
600
M9GLU
LB
500
400
300
200
100
0
pT7
M9/sup
pT7CsgD
pT7
pT7CsgD
CsgD as “global regulator?”
In addition to the genes already shown, according
to the GA experiments, CsgD also controls the
following genes/operons:
gsk
pyrBI
gat
ymdA
(GMP biosynthesis)
(pyrimidine metabolism)
(transport of galactitol)
(putative fimbrial gene)
yoaD
yaiB
(potential di-cyclic-GMP hydrolase)
(unknown function)
Overexpression of the yoaD gene inhibits cell aggregation
in a curli-proficient E. coli strain
IPTG
-
+
yoaD
Plac
+
pGEMT
pGEMTyoaD
This result 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?
-
30
1.4
25
1.2
1
20
0.8
15
0.6
10
0.4
5
0.2
0
0
0
2
Optical density (OD600nm)
Relative expression ratio
Timing is everything…..
5
TIME (HOURS)
=adrA expression (DGC)
=yoaD expression (PDEA)
A feedback control for cellulose
biosynthesis
• CsgD activates the adrA gene, resulting in
di-c-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
CsgD
Adapted from Camilli and Bassler, Science 2006
“Global impact” by the CsgD
protein on gene expression
Cytoplasm
pT7-7
pT7-CsgD
Outer membrane
pT7-7
pT7-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
Hightly expressed in
biofilm-forming strains
rpoS regulon
CsgD seems to activate expression of rpoS-dependent proteins
Indeed, CsgD-dependent alteration in protein expression
requires a functional rpoS gene
WT
CsgD
WT
rpoS
1
2
3
4
-
+
-
+
1
CsgD
-
rpoS
2
+
3
4
-
+
How does CsgD affect sS-dependent expression?
CsgD
iraP
iraP
yaiB= unknown gene
regulated by CsgD
yaiB now annotated as
iraP and identified as a
factor for sS stabilization
CsgD affects sS intracellular concentrations
in a manner dependent on IraP
WT
1
2
iraP
3
4
rpoS
5
6
7
50 KDa
6xHis-sS
35 KDa
30 KDa
CsgD
-
+
-
+
-
+
Presence of rpoS-dependent proteins in cell
extracts correlate with sS cellular levels
WT
1
CsgD
-
iraP
rpoS
2
3
4
5
6
+
-
+
-
+
CsgD
iraP
IraP
csgDEFG
[EsS]
Genes belonging to
rpoS regulon
[sS]
Curli
Cellulose
Outer membrane
Cytoplasmic membrane
CsgD
Activation
Repression
- Fimbriae and cellulose csg, ymdA, adrA
- Metabolism pyrBI, gat, metA
-Signalling system c-di-GMPadrA, (gsk), yoaD
- Porin ompF, ompT
sS-dependent genes
- Iron-sensing fecR, fhuE
Cell aggregation, Surface attachment
Metabolic adaptation to biofilm growth
conditions, activation of stress responses
(via the rpoS regulon)
- Cold-shock csp, infA