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
N1 - Copyright:
Copyright 2013 Elsevier B.V., All rights reserved.
PY - 2013/2
Y1 - 2013/2
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.
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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 -