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

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)

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

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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…..

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=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

Adapted from Camilli and Bassler, Science 2006

CsgD

“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 iraP

csgDEFG

[S][ES]

Genes belonging to rpoS regulon

IraP

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