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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.

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

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  1. 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

  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

  4. Curli Cellulose Outer membrane Cytoplasmic membrane adrA Inducer of cellulose biosynthesis CsgD Curli subunits

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

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

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

  8. 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

  9. 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

  10. 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?

  11. Timing is everything….. Relative expression ratio (csgD expression/no csgD Optical density (OD600nm) TIME (HOURS) =yoaD expression (PDEA) =adrA expression (DGC)

  12. 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

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

  14. “Global impact” by CsgD on protein expression Cytoplasm Outer membrane CsgD - + CsgD - +

  15. rpoS regulon CsgD seems to activate expression of rpoS-dependent proteins

  16. 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

  17. 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

  18. 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

  19. 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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