Gualdi, L., Brombacher, E.*, Bertagnoli, S. and Landini P.

1. The CsgD protein of Escherichia coli: arelay 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

2. 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

3. Function of curli (thin aggregative fimbriae): • Cell aggregation and clumping • Ability to adhere to a solid surface • Co-regulated with other adhesion genes

4. Locus Genetic structure and organization well conserved in Enterobacteria. However, silent in many E. coli lab strains. Curli: Long intertwined structure Proteinaceous Involved in adhesion to solid surfaces and in cell-cell aggregation 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

5. High osmolarity Curli expression regulation: Stationary phase (RpoS= alternative s factor) Osmolarity (EnvZ/OmpR and CpxA/CpxR TCRS) Low osmolarity Low temperature <32-34°C (Crl= accessory RNApol factor)

6. 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

7. What is the function of adrA? adrA “GGDEF MOTIF” PROTEIN Cyclic di-GMP bcsA, bcsB, bcsC, bcsZ (cellulose synthesis in E. coli)

8. Mechanism of cellulose biosynthesis activation by di-c-GMP AdrA cy-di-GMP acts as an allosteric activator of cellulose synthase machinery

9. 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

10. Role of cyclic di-GMP in the bacterial cell From Camilli and Bassler, Science 2006

11. 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

12. 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

13. Adhesion properties conferred by ectopic expression of CsgD A B pT7-7 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

14. Medium-dependent regulation of curli production takes place at the csgB promoter M9GLU LB Relative biofilm formation pT7 pT7CsgD pT7 pT7CsgD

15. 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)

16. IPTG + - + - Plac yoaD 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?

17. Timing is everything….. Relative expression ratio Optical density (OD600nm) TIME (HOURS) =yoaD expression (PDEA) =adrA expression (DGC)

18. 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

19. CsgD may act on intracellular cy-di-GMP pool CsgD Adapted from Camilli and Bassler, Science 2006

20. “Global impact” by the CsgD protein on gene expression Cytoplasm Outer membrane pT7-7 pT7-CsgD pT7-7 pT7-CsgD

21. rpoS regulon Hightly expressed in biofilm-forming strains CsgD seems to activate expression of rpoS-dependent proteins

22. 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

23. 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

24. 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

25. Presence of rpoS-dependent proteins in cell extracts correlate with sS cellular levels WT iraP rpoS 1 2 3 4 5 6 - + - + - + CsgD

26. CsgD iraP IraP csgDEFG [sS] [EsS] Genes belonging to rpoS regulon

27. Curli Cellulose Outer membrane Cytoplasmic membrane CsgD Activation Repression • - Fimbriae and cellulose csg, ymdA, adrA • Signalling system c-di-GMPadrA, (gsk), yoaD • sS-dependent genes - Metabolism pyrBI, gat, metA - Porin ompF, ompT - Iron-sensing fecR, fhuE - Cold-shock csp, infA Cell aggregation, Surface attachment Metabolic adaptation to biofilm growth conditions, activation of stress responses (via the rpoS regulon)