LcrV mutants that abolish Yersinia type III injectisome function

Katherine Given Ligtenberg, Nathan C. Miller, Anthony Mitchell, Gregory V. Plano, Olaf Schneewinda

Research output: Contribution to journalArticle

9 Scopus citations

Abstract

LcrV, the type III needle cap protein of pathogenic Yersinia, has been proposed to function as a tether between YscF, the needle protein, and YopB-YopD to constitute the injectisome, a conduit for the translocation of effector proteins into host cells. Further, insertion of LcrV-capped needles from a calcium-rich environment into host cells may trigger the low-calcium signal for effector translocation. Here, we used a genetic approach to test the hypothesis that the needle cap responds to the low-calcium signal by promotinginjectisome assembly. Growth restriction of Yersinia pestis in the absence of calcium (low-calcium response [LCR+] phenotype) was exploited to isolate dominant negative lcrV alleles with missense mutations in its amber stop codon (lcrV*). The addition of at leastfour amino acids or the eight-residue Strep tag to the C terminus was sufficient to generate an LCR- phenotype, with variant LcrV capping type III needles that cannot assemble the YopD injectisome component. The C-terminal Strep tag appears buried within the cap structure, blocking effector transport even in Y. pestis yscF variants that are otherwise calcium blind, a constitutive type III secretion phenotype. Thus, lcrV* mutants arrest the needle cap in a state in which it cannot respond to the low-calcium signal with eitherinjectisome assembly or the activation of type III secretion. Insertion of the Strep tag at other positions of LcrV produced variants with wild-type LCR+, LCR-, or dominant negative LCR- phenotypes, thereby allowing us to identify discrete sites within LcrV as essential for its attributes as a secretion substrate, needle cap, and injectisome assembly factor.

Original languageEnglish (US)
Pages (from-to)777-787
Number of pages11
JournalJournal of bacteriology
Volume195
Issue number4
DOIs
StatePublished - Feb 2013

Keywords

  • Biclustering
  • Constant row
  • Differential co-expression
  • Gene expression.

ASJC Scopus subject areas

  • Microbiology
  • Molecular Biology

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    Ligtenberg, K. G., Miller, N. C., Mitchell, A., Plano, G. V., & Schneewinda, O. (2013). LcrV mutants that abolish Yersinia type III injectisome function. Journal of bacteriology, 195(4), 777-787. https://doi.org/10.1128/JB.02021-12