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 Citations (Scopus)

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
Pages (from-to)777-787
Number of pages11
JournalJournal of Bacteriology
Volume195
Issue number4
DOIs
StatePublished - Feb 1 2013

Fingerprint

Yersinia
Needles
Calcium
Yersinia pestis
Phenotype
Terminator Codon
Protein Transport
Missense Mutation
Proteins
Alleles
Amino Acids
Growth

Keywords

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

ASJC Scopus subject areas

  • Microbiology
  • Molecular Biology

Cite this

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

LcrV mutants that abolish Yersinia type III injectisome function. / Ligtenberg, Katherine Given; Miller, Nathan C.; Mitchell, Anthony; Plano, Gregory V; Schneewinda, Olaf.

In: Journal of Bacteriology, Vol. 195, No. 4, 01.02.2013, p. 777-787.

Research output: Contribution to journalArticle

Ligtenberg, KG, Miller, NC, Mitchell, A, Plano, GV & Schneewinda, O 2013, 'LcrV mutants that abolish Yersinia type III injectisome function', Journal of Bacteriology, vol. 195, no. 4, pp. 777-787. https://doi.org/10.1128/JB.02021-12
Ligtenberg, Katherine Given ; Miller, Nathan C. ; Mitchell, Anthony ; Plano, Gregory V ; Schneewinda, Olaf. / LcrV mutants that abolish Yersinia type III injectisome function. In: Journal of Bacteriology. 2013 ; Vol. 195, No. 4. pp. 777-787.
@article{da5e29c1eeea4264956bf7047a384d81,
title = "LcrV mutants that abolish Yersinia type III injectisome function",
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.",
keywords = "Biclustering, Constant row, Differential co-expression, Gene expression.",
author = "Ligtenberg, {Katherine Given} and Miller, {Nathan C.} and Anthony Mitchell and Plano, {Gregory V} and Olaf Schneewinda",
year = "2013",
month = "2",
day = "1",
doi = "10.1128/JB.02021-12",
language = "English",
volume = "195",
pages = "777--787",
journal = "Journal of Bacteriology",
issn = "0021-9193",
publisher = "American Society for Microbiology",
number = "4",

}

TY - JOUR

T1 - LcrV mutants that abolish Yersinia type III injectisome function

AU - Ligtenberg, Katherine Given

AU - Miller, Nathan C.

AU - Mitchell, Anthony

AU - Plano, Gregory V

AU - Schneewinda, Olaf

PY - 2013/2/1

Y1 - 2013/2/1

N2 - 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.

AB - 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.

KW - Biclustering

KW - Constant row

KW - Differential co-expression

KW - Gene expression.

UR - http://www.scopus.com/inward/record.url?scp=84873552875&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84873552875&partnerID=8YFLogxK

U2 - 10.1128/JB.02021-12

DO - 10.1128/JB.02021-12

M3 - Article

C2 - 23222719

AN - SCOPUS:84873552875

VL - 195

SP - 777

EP - 787

JO - Journal of Bacteriology

JF - Journal of Bacteriology

SN - 0021-9193

IS - 4

ER -