* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download + - + - + CsgD
Cancer epigenetics wikipedia , lookup
Epigenetics in stem-cell differentiation wikipedia , lookup
Epigenetics in learning and memory wikipedia , lookup
History of genetic engineering wikipedia , lookup
No-SCAR (Scarless Cas9 Assisted Recombineering) Genome Editing wikipedia , lookup
Genome evolution wikipedia , lookup
Microevolution wikipedia , lookup
Point mutation wikipedia , lookup
Long non-coding RNA wikipedia , lookup
Gene nomenclature wikipedia , lookup
Epigenetics of neurodegenerative diseases wikipedia , lookup
Site-specific recombinase technology wikipedia , lookup
Epigenetics of diabetes Type 2 wikipedia , lookup
Designer baby wikipedia , lookup
Vectors in gene therapy wikipedia , lookup
Protein moonlighting wikipedia , lookup
Polycomb Group Proteins and Cancer wikipedia , lookup
Epigenetics of human development wikipedia , lookup
Gene therapy of the human retina wikipedia , lookup
Gene expression programming wikipedia , lookup
Nutriepigenomics wikipedia , lookup
Gene expression profiling wikipedia , lookup
Therapeutic gene modulation wikipedia , lookup
Artificial gene synthesis wikipedia , lookup
The CsgD protein of Escherichia coli: a relay 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 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 Co-regulated with other adhesion genes Curli: Long intertwined structure Proteinaceous Involved in adhesion to solid surfaces and in cell-cell aggregation Locus Genetic structure and organization well conserved in Enterobacteria. However, silent in many E. coli lab strains. 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 Curli expression regulation: Stationary phase (RpoS= alternative s factor) Osmolarity (EnvZ/OmpR and CpxA/CpxR TCRS) Low temperature <32-34°C (Crl= accessory RNApol factor) Low osmolarity High osmolarity 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 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 di-c-GMP AdrA cy-di-GMP acts as an allosteric activator of cellulose synthase machinery 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 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 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 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 Adhesion properties conferred by ectopic expression of CsgD A pT7-7 B 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 Medium-dependent regulation of curli production takes place at the csgB promoter %Relative biofilm formation of MG1655pT7 biofilm formation 600 M9GLU LB 500 400 300 200 100 0 pT7 M9/sup pT7CsgD pT7 pT7CsgD CsgD as “global regulator?” In addition to the genes already shown, according to the GA experiments, CsgD also controls the following genes/operons: gsk pyrBI gat ymdA (GMP biosynthesis) (pyrimidine metabolism) (transport of galactitol) (putative fimbrial gene) yoaD yaiB (potential di-cyclic-GMP hydrolase) (unknown function) Overexpression of the yoaD gene inhibits cell aggregation in a curli-proficient E. coli strain IPTG - + yoaD Plac + pGEMT pGEMTyoaD 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? - 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 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 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 CsgD may act on intracellular cy-di-GMP pool CsgD Adapted from Camilli and Bassler, Science 2006 “Global impact” by the CsgD protein on gene expression Cytoplasm pT7-7 pT7-CsgD Outer membrane pT7-7 pT7-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 Hightly expressed in biofilm-forming strains 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 2 iraP 3 4 rpoS 5 6 7 50 KDa 6xHis-sS 35 KDa 30 KDa CsgD - + - + - + Presence of rpoS-dependent proteins in cell extracts correlate with sS cellular levels WT 1 CsgD - iraP rpoS 2 3 4 5 6 + - + - + CsgD iraP IraP csgDEFG [EsS] Genes belonging to rpoS regulon [sS] Curli Cellulose Outer membrane Cytoplasmic membrane CsgD Activation Repression - Fimbriae and cellulose csg, ymdA, adrA - Metabolism pyrBI, gat, metA -Signalling system c-di-GMPadrA, (gsk), yoaD - Porin ompF, ompT sS-dependent genes - Iron-sensing fecR, fhuE Cell aggregation, Surface attachment Metabolic adaptation to biofilm growth conditions, activation of stress responses (via the rpoS regulon) - Cold-shock csp, infA