Transcript Slide 1
“NucC – from phage regulator to bacterial regulator?” VWJ I H G fun(Z) FIFIIEE’ T U D C cox attP ogr RS orf-30 Q P O N M L XYK lysA lysB Bacteriophage P2 late gene transcription B A tin old ori Ogr-dependent transcription activation of bacteriophage P2 late promoters aCTD b s Ogr -55 ‘ b aNTD -35 -10 +1 BLAST search with P2 Ogr reveals numerous “hits” Fels-2 Comparison of members of the bacteriophage P2 Ogr family of transcription factors P2 Ogr Wphi Ogr L-413C Ogr 186 B PSP3 Pag NucC Fels-2 Ogr RlsB LsrS Highly conserved, small Zn-binding transcription activators Found among P2-related phages, where they regulate late gene expression 3 known homologs regulating bacterial genes NucC: S. marcescens extracellular nuclease, bacteriocin 28b RlsB: Erwinia amylovora levansucrase LsrA: Rahnella aquatilis levansucrase Serratia marcescens • Gram negative rod • Enterobacteriaceae • Found in various ecological niches soil water plants animals • Survives under extreme conditions disinfectants and antiseptics distilled water salt water • Produces characteristic red pigment, prodigiosin The Miracle of Bolsena 1263 S. marcescens as a human pathogen • “Operation Sea-Spray“ 1950 – – • US Army released S. marcescens as a biowarfare simulant over San Francisco Bay Followed by a significant outbreak of pneumonia and urinary tract infections between Oct 1950 and Feb 1951, including one fatality Opportunistic nosocomial pathogen implicated in wide range of infections – – – – – – Respiratory tract UTI Meningitis Wound (burn) infections Septicemia Infective endocarditis • Important pathogen of newborns and immunocompromised patients • Increasing incidence of antibiotic resistance S. marcescens as a plant pathogen • Etiologic agent of Cucurbit Yellow Vine Disease (CYVD) – Inhabits plant tissue called phloem – Causes chlorosis, rapid wilting and death of squash, pumpkin, watermelon and cantaloupe – Transmitted by an insect vector, the squash bug Anasa tristis Implication of Serratia marcescens in coral bleaching Patterson et al. (2002) PNAS 99:8725-8730 White pox is a lethal disease of the Caribbean elkhorn coral Acropora palmata. Looe Key Reef White pox disease lesion on A. palmata at Looe Key Reef, FL, August 1998. (The square pin (1.61 cm2) was for calibration of image tracing software) Scanning electron micrograph of the white pox pathogen (PDL100). The bacterium was identified as S. marcescens by 16S rRNA gene sequencing, carbon source utilization patterns, and standard microbiological testing Serratia marcescens in the headlines Extracellular proteins of S. marcescens • Serratia are unique among the Enterobacteriaceae in production of a large number of extracellular enzymes and other proteins – – – – – – – – • lipases chitinases proteases deoxynuclease gelatinase chloroperoxidase surfactant: “serrawettin” bacteriocins Many of these are growth phase regulated NucC, encoded on a cryptic prophage, is a common regulator of S. marcescens nuclease and bacteriocin 28b nucE nucD nucC + bss nucA Are there other NucC-regulated genes? Identification of genes in the NucC regulon Proteomic approach • • pH 7 Compare proteins (secreted, total) in NucC+ and NucC- cells on 2D gels Characterize NucC-regulated proteins by mass spectrometry NucC+ pH 4 pH 7 NucC- pH 4 Identification of Proteins by Mass Spectrometry Identification of genes in the NucC regulon • • • Genomic approach Isolate mRNA from NucC+ and NucC- cells Probe a spotted array of Serratia marcescens DNA fragments Characterize clones showing differential regulation For either of these approaches, a genome sequence is required http://www.sanger.ac.uk/Projects/S_marcescens/ Strain Db11 Is having one strain of S. marcescens good enough? Goal of this simulation: Does S. marcescens strain Db11 contain the nucC operon and the genes that we know it regulates?