“NucC – from phage regulator to bacterial regulator?”

Bacteriophage P2 late gene transcription

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Ogr-dependent transcription activation of bacteriophage P2 late promoters

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CTD

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BLAST search with P2 Ogr reveals numerous “hits”

Fels-2

Comparison of members of the bacteriophage P2 Ogr family of transcription factors

Highly conserved, small Zn-binding transcription activatorsFound among P2-related phages, where they regulate late gene expression

3 known homologs regulating bacterial genesNucC: S. marcescens extracellular nuclease, bacteriocin 28bRlsB: Erwinia amylovora levansucraseLsrA: Rahnella aquatilis levansucrase

P2 OgrWphi OgrL-413C Ogr186 BPSP3 PagNucCFels-2 OgrRlsBLsrS

Serratia marcescens

• Gram negative rod

• Enterobacteriaceae

• Found in various ecological niches soil

waterplantsanimals

• Survives under extreme conditionsdisinfectants and antiseptics

distilled water salt water

• Produces characteristic red pigment, prodigiosin

The Miracle of Bolsena1263

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

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

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.

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

nucAbss

+

Are there other NucC-regulated genes?

Identification of genes in the NucC regulon

• Compare proteins (secreted, total) in NucC+ and NucC- cells on 2D gels• Characterize NucC-regulated proteins by mass spectrometry

pH 7 pH 4 pH 7 pH 4NucC+ NucC-

Proteomic approach

Identification of Proteins by Mass Spectrometry

Identification of genes in the NucC regulon

• Isolate mRNA from NucC+ and NucC- cells• Probe a spotted array of Serratia marcescens DNA fragments• Characterize clones showing differential regulation

Genomic approach

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?

Does S. marcescens strain Db11 contain the nucC operon and the genes that we know it regulates?

Goal of this simulation: