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

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15

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30

0 2 5

0

0.2

0.4

0.6

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1.2

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TIME (HOURS)

Rel

ativ

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atio

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o c

sgD

Op

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(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”?