The CsgD protein of Escherichia coli: a relay between bacterial biofilm formation and gene...
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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 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
Co-regulated with other adhesion genes
CurliCurli:: Long intertwined structureProteinaceousInvolved 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
Low
osmolarity
Curli expression regulationCurli expression regulation:
Stationary phase(RpoS= alternative factor)
Osmolarity(EnvZ/OmpR and CpxA/CpxR TCRS)
Low temperature
<32-34°C(Crl= accessory RNApol factor)
High osmolarity
CsgD directly activates at least another promoter: adrA
PcsgB::luxAB PadrA::luxAB
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
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
cy-di-GMP acts as an allosteric activatorof cellulose synthase machinery
AdrA
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
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)
X
XuidA
-glucur.
CsgD+
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
X
XuidA
-glucur.
CsgD+
No CsgD
Protein pattern in SDS-PAGE
MG1655W (WT)
EB12 (csgA mutant)No curli production
LG04 (bcsA mutant)No cellulose production
A pT7-7 pT7-CsgD pT7-7 pT7-CsgD
B
A: Biofilm formation assay usin gcrystal violet staining in microtiter plates
B. Spots on Congo Red-supplemented plates
Adhesion properties conferred by ectopic expression of CsgD
Medium-dependent regulation of curli production takes place at the csgB promoter
M9GLULB
pT7 pT7CsgD pT7 pT7CsgD
Rel
ativ
e b
iofi
lm f
orm
atio
n
CsgD as “global regulator?”In addition to the genes already shown, according
to the GA experiments, CsgD also controls the following genes/operons:
gsk (GMP biosynthesis) pyrBI (pyrimidine metabolism) gat (transport of galactitol) ymdA (putative fimbrial gene)
yoaD (potential di-cyclic-GMP hydrolase) yaiB (unknown function)
IPTG + - + -
pGEMT pGEMTyoaD
Overexpression of the yoaD gene inhibits cell aggregation in a curli-proficient E. coli strain
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?
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
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 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
“Global impact” by the CsgD protein on gene expression
Cytoplasm Outer membrane
pT7-7 pT7-7pT7-CsgD 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
rpoS regulonHightly expressed in
biofilm-forming strains
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
1 2 3 4 5 6 7
6xHis-S
35 KDa
50 KDa
- + - + - + CsgD
WT iraP rpoS
30 KDa
CsgD affects S intracellular concentrations in a manner dependent on IraP
Presence of rpoS-dependent proteins in cell extracts correlate with S cellular levels
- + - + - + CsgD
1 2 3 4 5 6
WT iraP rpoS
CsgD
- Fimbriae and cellulose csg, ymdA, adrA
-Signalling system c-di-GMPadrA, (gsk), yoaD
S-dependent genes
Curli
- Metabolism pyrBI, gat, metA
- Porin ompF, ompT
- Iron-sensing fecR, fhuE
- Cold-shock csp, infA
Activation Repression
Cellulose
Cell aggregation, Surface attachmentMetabolic adaptation to biofilm growth conditions, activation of stress responses(via the rpoS regulon)
Outer membrane
Cytoplasmic membrane