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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.

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Transition from planktonic cells to biofilm in E. coli : the CsgD regulon .

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Transition from planktonic cells to biofilm in e coli the csgd regulon

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


Transition from planktonic cells to biofilm in e coli the csgd regulon

  • 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


Transition from planktonic cells to biofilm in e coli the csgd regulon

  • Function of curli (thin aggregative fimbriae):

  • Cell aggregation and clumping

  • Ability to adhere to a solid surface


Transition from planktonic cells to biofilm in e coli the csgd regulon

Curli

Cellulose

Outer membrane

Cytoplasmic membrane

adrA

Inducer of cellulose

biosynthesis

CsgD

Curli subunits


Transition from planktonic cells to biofilm in e coli the csgd regulon

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

Mechanism of cellulose biosynthesis activation by c-di-GMP

AdrA

cy-di-GMP acts as an allosteric activator

of cellulose synthase machinery


Role of cyclic di gmp in the bacterial cell

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

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

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 nucleotide

metabolism (??)

Curli production

(??)

(??)

* Enzymes involved in c-di-GMP breakdown


Transition from planktonic cells to biofilm in e coli the csgd regulon

Plac

yoaD

pGEMT

The yoaD gene encodes a potential

c-di-GMP phosphodiesterase

IPTG+ - + -

pGEMTyoaD

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?


Timing is everything

Timing is everything…..

Relative expression ratio

(csgD expression/no csgD

Optical density (OD600nm)

TIME (HOURS)

=yoaD expression (PDEA)

=adrA expression (DGC)


A feedback control for cellulose biosynthesis

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

CsgD may act on intracellular cy-di-GMP pool

CsgD

Adapted from Camilli and Bassler, Science 2006


Global impact by csgd on protein expression

“Global impact” by CsgD on protein expression

Cytoplasm

Outer membrane

CsgD - +

CsgD - +


Transition from planktonic cells to biofilm in e coli the csgd regulon

rpoS regulon

CsgD seems to activate expression of rpoS-dependent proteins


Transition from planktonic cells to biofilm in e coli the csgd regulon

WT

WT

rpoS

rpoS

1 2 3 4

1 2 3 4

- + - +

CsgD

Indeed, CsgD-dependent alteration in protein expression

requires a functional rpoS gene

- + - +

CsgD


Transition from planktonic cells to biofilm in e coli the csgd regulon

CsgD

iraP

iraP

How does CsgD affect sS-dependent expression?

yaiB= unknown gene

regulated by CsgD

yaiB now annotated as iraP and identified as a factor for sS stabilization


Transition from planktonic cells to biofilm in e coli the csgd regulon

CsgD affects sS intracellular concentrations

in a manner dependent on IraP

WT

iraP

rpoS

1 2 3 4 5 6 7

50 KDa

6XHis-sS

35 KDa

30 KDa

- + - + - +

CsgD


Transition from planktonic cells to biofilm in e coli the csgd regulon

CsgD induction of biofilm formation:

part of a “general stress response”?

Biofilm

formation

Curli

Cellulose

Outer membrane

Cytoplasmic membrane

CsgD

Oxidative stress genes

Acid resistance

Resistance to desiccation

[EsS]


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