Transcript Slide 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 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 Function of curli (thin aggregative fimbriae): Cell aggregation and clumping Ability to adhere to a solid surface Curli Cellulose Outer membrane Cytoplasmic membrane adrA CsgD Inducer of cellulose biosynthesis Curli subunits 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 AdrA cy-di-GMP acts as an allosteric activator of cellulose synthase machinery 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 csgDproficient 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 pyrBI gatYZAB ymdA yoaD yaiB (GMP biosynthesis) (pyrimidine metabolism) Involved in nucleotide metabolism (??) (transport of galactitol, possibly a signal for curli regulation?) (putative fimbrial gene, likely part of the csgBAC operon) (unkown, potential PDE-A)* (unknown function) * Enzymes involved in c-di-GMP breakdown Curli production (??) (??) The yoaD gene encodes a potential c-di-GMP phosphodiesterase IPTG - + - yoaD Plac + pGEMT 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? 30 1.4 25 1.2 1 20 0.8 15 0.6 10 0.4 5 0.2 0 0 0 2 Optical density (OD600nm) Relative expression ratio (csgD expression/no csgD Timing is everything….. 5 TIME (HOURS) =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 CsgD Adapted from Camilli and Bassler, Science 2006 “Global impact” by CsgD on protein expression Cytoplasm CsgD - + Outer membrane 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 Indeed, CsgD-dependent alteration in protein expression requires a functional rpoS gene WT CsgD WT rpoS 1 2 3 4 - + - + 1 CsgD - rpoS 2 + 3 4 - + How does CsgD affect sS-dependent expression? CsgD iraP iraP yaiB= unknown gene regulated by CsgD yaiB now annotated as iraP and identified as a factor for sS stabilization CsgD affects sS intracellular concentrations in a manner dependent on IraP WT 1 iraP 2 3 4 rpoS 5 6 50 KDa 6XHis-sS 35 KDa 30 KDa CsgD 7 - + - + - + CsgD induction of biofilm formation: part of a “general stress response”? Curli Cellulose Outer membrane Cytoplasmic membrane CsgD [EsS] Oxidative stress genes Acid resistance Resistance to desiccation Biofilm formation