CSB452 LECTURE 2
Type III Secretion System
TTSS in gram-negative bacteria is needle like with long pilus similar to f lagella.
HrpA forms pilus via spontaneous polymerization. HrcN is ATPase that powers translocation.
with non-functional TTSS grows less.
Hrp = hypers ensitive reaction and pathogenecity genes. H rc = Hrp conserved genes.
Hrc Gene System
Exchangeable effector locus – Hrc gene cluster – conserved effector locus.
Hrc gene cluster is conserved core operon with HrcL regulatory gene in Pathogenicity Island.
Virulent genes only present in virulent strains. Different CG content occupying large chromosomal
regions. Flanked by direct repeats or bordered by tRNA or mobile elements.
Instability indicative of horizont al gene transfer.
HrcL is master TF: with signal, protease stop degrading HrcR. HrcR + Q
+ HrcS transcribe HrcL.
Shotgun Screen to Identify TTSE
Identified 1 factor that cause HR in soybeans.
pv glycinea and prepare shredded gDNA and transform gDNA into
Triparental mating: donor with Psg6DNA + Helper
+ recipient with Psg5 lacking Avr gene.
Grow Psg5 transconjugates and inoculate on plant leaf to assess HR. If fragment in donor contained
TTSE Avr gene then recipient should cause HR.
Bioinfor matic Screen for TTSE
All TTSE contain Secretion Signal + catalytic/binding domain with optional chaperone binding site.
Look for Hrp Box (
element bound by HrcL) upstream of ORF in complete genome of DC3000.
Look for ORF w ith characterisitic SS at N’ of TTSE.
TTSE but this is not a functional screen for translocation.
Transposon Screen for TTSE
AvrRpt2 is TTSE that cuase HR. without SS it does not induce HR.
Ttss in gram-negative bacteria is needle like with long pilus similar to flagella. hrp = hypersensitive reaction and pathogenecity genes. Exchangeable effector locus hrc gene cluster conserved effector locus. hrc gene cluster is conserved core operon with hrcl regulatory gene in pathogenicity island. virulent genes only present in virulent strains. Flanked by direct repeats or bordered by trna or mobile elements. hrcl is master tf: with signal, protease stop degrading hrcr. Identified 1 factor that cause hr in soybeans. grow p. syringae pv glycinea and prepare shredded gdna and transform gdna into e. coli colonies. Triparental mating: donor with psg6dna + helper e. coli + recipient with psg5 lacking avr gene. grow psg5 transconjugates and inoculate on plant leaf to assess hr. Ttse avr gene then recipient should cause hr. all ttse contain secretion signal + catalytic/binding domain with optional chaperone binding site.