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Functional Activation of the Flagellar Type III Secretion Export Apparatus.

Phillips AM, Calvo RA, Kearns DB - PLoS Genet. (2015)

Bottom Line: Flagella are assembled sequentially from the inside-out with morphogenetic checkpoints that enforce the temporal order of subunit addition.Genetic suppressor analysis indicates that SwrB activates the flagellar type III secretion export apparatus by the membrane protein FliP.We conclude that SwrB enhances the probability that the flagellar basal body adopts a conformation proficient for secretion to ensure that rod and hook subunits are not secreted in the absence of a suitable platform on which to polymerize.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Indiana University, Bloomington, Indiana, United States of America.

ABSTRACT
Flagella are assembled sequentially from the inside-out with morphogenetic checkpoints that enforce the temporal order of subunit addition. Here we show that flagellar basal bodies fail to proceed to hook assembly at high frequency in the absence of the monotopic protein SwrB of Bacillus subtilis. Genetic suppressor analysis indicates that SwrB activates the flagellar type III secretion export apparatus by the membrane protein FliP. Furthermore, mutants defective in the flagellar C-ring phenocopy the absence of SwrB for reduced hook frequency and C-ring defects may be bypassed either by SwrB overexpression or by a gain-of-function allele in the polymerization domain of FliG. We conclude that SwrB enhances the probability that the flagellar basal body adopts a conformation proficient for secretion to ensure that rod and hook subunits are not secreted in the absence of a suitable platform on which to polymerize.

No MeSH data available.


Related in: MedlinePlus

SwrB overexpression bypasses the hook protein secretion hook in cells mutated for FliM and FliG but not cells mutated for FliF.A) FlgE secretion assay in a hook cap (flgD) mutant background. Mutation of the hook cap FlgD has previously been shown to abolish FlgE polymerization and allow constitutive secretion of FlgE into the extracellular environment [51,65]. Cell pellet (pellet) and cell supernatant (supe) samples were harvested from B. subtilis cultures of the indicated genetic background and probed with anti-FlgE primary antibody. The following strains were used to generate the panel: ΔflgD (DK2102), ΔflgD ΔfliM (DK2103), ΔflgD ΔfliG (DK2201), ΔflgD ΔfliF (DS2229), ΔflgD ΔfliP (DK2199), ΔflgD ΔswrB (DK2214), and ΔflgD ΔflhA (DK2198). B) FlgE secretion assay in a hook cap (flgD) mutant background and in which swrB is artificially expressed from the IPTG-inducible Physpank promoter (swrB++). Strains contain the thrC::Physpank-swrB construct and were grown in the presence of 1 mM IPTG. The following strains were used to generate the panel: ΔflgD swrB++ (DK2282), ΔflgD ΔfliM swrB++ (DK2283), ΔflgD ΔfliG swrB++ (DK2284), ΔflgD ΔfliF swrB++ (DK2285), ΔflgD ΔfliP swrB++ (DK2286), ΔflgD ΔswrB swrB++ (DK2287), and ΔflgD ΔflhA swrB++ (DK2288). C) FlgE secretion assay in a hook cap (flgD) mutant background and in which the fliPsob22 allele was ectopically expressed from the native fla/che promoter. The following strains were used to generate the panel: ΔflgD fliPsob22 (DK2289), ΔflgD ΔfliM fliPsob22 (DK2290), ΔflgD ΔfliG fliPsob22 (DK2291), ΔflgD ΔfliF fliPsob22 (DK2292), ΔflgD ΔfliP fliPsob22 (DK2293), ΔflgD ΔswrB fliPsob22 (DK2294), ΔflgD ΔflhA fliPsob22 (DK2295).
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pgen.1005443.g010: SwrB overexpression bypasses the hook protein secretion hook in cells mutated for FliM and FliG but not cells mutated for FliF.A) FlgE secretion assay in a hook cap (flgD) mutant background. Mutation of the hook cap FlgD has previously been shown to abolish FlgE polymerization and allow constitutive secretion of FlgE into the extracellular environment [51,65]. Cell pellet (pellet) and cell supernatant (supe) samples were harvested from B. subtilis cultures of the indicated genetic background and probed with anti-FlgE primary antibody. The following strains were used to generate the panel: ΔflgD (DK2102), ΔflgD ΔfliM (DK2103), ΔflgD ΔfliG (DK2201), ΔflgD ΔfliF (DS2229), ΔflgD ΔfliP (DK2199), ΔflgD ΔswrB (DK2214), and ΔflgD ΔflhA (DK2198). B) FlgE secretion assay in a hook cap (flgD) mutant background and in which swrB is artificially expressed from the IPTG-inducible Physpank promoter (swrB++). Strains contain the thrC::Physpank-swrB construct and were grown in the presence of 1 mM IPTG. The following strains were used to generate the panel: ΔflgD swrB++ (DK2282), ΔflgD ΔfliM swrB++ (DK2283), ΔflgD ΔfliG swrB++ (DK2284), ΔflgD ΔfliF swrB++ (DK2285), ΔflgD ΔfliP swrB++ (DK2286), ΔflgD ΔswrB swrB++ (DK2287), and ΔflgD ΔflhA swrB++ (DK2288). C) FlgE secretion assay in a hook cap (flgD) mutant background and in which the fliPsob22 allele was ectopically expressed from the native fla/che promoter. The following strains were used to generate the panel: ΔflgD fliPsob22 (DK2289), ΔflgD ΔfliM fliPsob22 (DK2290), ΔflgD ΔfliG fliPsob22 (DK2291), ΔflgD ΔfliF fliPsob22 (DK2292), ΔflgD ΔfliP fliPsob22 (DK2293), ΔflgD ΔswrB fliPsob22 (DK2294), ΔflgD ΔflhA fliPsob22 (DK2295).

Mentions: FliF could be required for SwrB activation of hook assembly because FliF also serves as the polymerization platform for the flagellar rod and hook. Thus, SwrB could stimulate flagellar secretion in the absence of FliF but the hook subunits would accumulate in the supernatant due to an inability to polymerize. To determine the effect SwrB has on hook protein secretion, strains were generated that were mutated for the extracellular hook chaperone protein FlgD such that all strains would fail to polymerize FlgE, and FlgE would thus be secreted into the supernatant [51,65]. Cells mutated for FliM and FliG reduced the amount of secreted hook protein whereas cells mutated for FliF, FliP, and FlhA abolished hook secretion (Fig 10A). Next, the IPTG-inducible Physpank-swrB construct was added to each strain to determine the effect that over-expression of SwrB would have on hook secretion. When SwrB was over-expressed, the amount of hook protein in the supernatant appeared to increase in the fliM, fliG, and swrB mutants, but not in the fliF, fliP, or flhA mutants (Fig 10B). Finally, addition of the Pfla/che-fliPsob22 complementation construct provided in trans appeared to increase FlgE secretion in fliM, fliG, swrB, and fliP mutants but not in the fliF or flhA mutant (Fig 10C). We conclude FliF, FliP, and FlhA are downstream of SwrB for hook protein secretion. In sum, we conclude that SwrB activates an early flagellar morphogenetic checkpoint by catalyzing a FliF-basal body conformation that activates the type III secretion export apparatus for hook subunit secretion.


Functional Activation of the Flagellar Type III Secretion Export Apparatus.

Phillips AM, Calvo RA, Kearns DB - PLoS Genet. (2015)

SwrB overexpression bypasses the hook protein secretion hook in cells mutated for FliM and FliG but not cells mutated for FliF.A) FlgE secretion assay in a hook cap (flgD) mutant background. Mutation of the hook cap FlgD has previously been shown to abolish FlgE polymerization and allow constitutive secretion of FlgE into the extracellular environment [51,65]. Cell pellet (pellet) and cell supernatant (supe) samples were harvested from B. subtilis cultures of the indicated genetic background and probed with anti-FlgE primary antibody. The following strains were used to generate the panel: ΔflgD (DK2102), ΔflgD ΔfliM (DK2103), ΔflgD ΔfliG (DK2201), ΔflgD ΔfliF (DS2229), ΔflgD ΔfliP (DK2199), ΔflgD ΔswrB (DK2214), and ΔflgD ΔflhA (DK2198). B) FlgE secretion assay in a hook cap (flgD) mutant background and in which swrB is artificially expressed from the IPTG-inducible Physpank promoter (swrB++). Strains contain the thrC::Physpank-swrB construct and were grown in the presence of 1 mM IPTG. The following strains were used to generate the panel: ΔflgD swrB++ (DK2282), ΔflgD ΔfliM swrB++ (DK2283), ΔflgD ΔfliG swrB++ (DK2284), ΔflgD ΔfliF swrB++ (DK2285), ΔflgD ΔfliP swrB++ (DK2286), ΔflgD ΔswrB swrB++ (DK2287), and ΔflgD ΔflhA swrB++ (DK2288). C) FlgE secretion assay in a hook cap (flgD) mutant background and in which the fliPsob22 allele was ectopically expressed from the native fla/che promoter. The following strains were used to generate the panel: ΔflgD fliPsob22 (DK2289), ΔflgD ΔfliM fliPsob22 (DK2290), ΔflgD ΔfliG fliPsob22 (DK2291), ΔflgD ΔfliF fliPsob22 (DK2292), ΔflgD ΔfliP fliPsob22 (DK2293), ΔflgD ΔswrB fliPsob22 (DK2294), ΔflgD ΔflhA fliPsob22 (DK2295).
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pgen.1005443.g010: SwrB overexpression bypasses the hook protein secretion hook in cells mutated for FliM and FliG but not cells mutated for FliF.A) FlgE secretion assay in a hook cap (flgD) mutant background. Mutation of the hook cap FlgD has previously been shown to abolish FlgE polymerization and allow constitutive secretion of FlgE into the extracellular environment [51,65]. Cell pellet (pellet) and cell supernatant (supe) samples were harvested from B. subtilis cultures of the indicated genetic background and probed with anti-FlgE primary antibody. The following strains were used to generate the panel: ΔflgD (DK2102), ΔflgD ΔfliM (DK2103), ΔflgD ΔfliG (DK2201), ΔflgD ΔfliF (DS2229), ΔflgD ΔfliP (DK2199), ΔflgD ΔswrB (DK2214), and ΔflgD ΔflhA (DK2198). B) FlgE secretion assay in a hook cap (flgD) mutant background and in which swrB is artificially expressed from the IPTG-inducible Physpank promoter (swrB++). Strains contain the thrC::Physpank-swrB construct and were grown in the presence of 1 mM IPTG. The following strains were used to generate the panel: ΔflgD swrB++ (DK2282), ΔflgD ΔfliM swrB++ (DK2283), ΔflgD ΔfliG swrB++ (DK2284), ΔflgD ΔfliF swrB++ (DK2285), ΔflgD ΔfliP swrB++ (DK2286), ΔflgD ΔswrB swrB++ (DK2287), and ΔflgD ΔflhA swrB++ (DK2288). C) FlgE secretion assay in a hook cap (flgD) mutant background and in which the fliPsob22 allele was ectopically expressed from the native fla/che promoter. The following strains were used to generate the panel: ΔflgD fliPsob22 (DK2289), ΔflgD ΔfliM fliPsob22 (DK2290), ΔflgD ΔfliG fliPsob22 (DK2291), ΔflgD ΔfliF fliPsob22 (DK2292), ΔflgD ΔfliP fliPsob22 (DK2293), ΔflgD ΔswrB fliPsob22 (DK2294), ΔflgD ΔflhA fliPsob22 (DK2295).
Mentions: FliF could be required for SwrB activation of hook assembly because FliF also serves as the polymerization platform for the flagellar rod and hook. Thus, SwrB could stimulate flagellar secretion in the absence of FliF but the hook subunits would accumulate in the supernatant due to an inability to polymerize. To determine the effect SwrB has on hook protein secretion, strains were generated that were mutated for the extracellular hook chaperone protein FlgD such that all strains would fail to polymerize FlgE, and FlgE would thus be secreted into the supernatant [51,65]. Cells mutated for FliM and FliG reduced the amount of secreted hook protein whereas cells mutated for FliF, FliP, and FlhA abolished hook secretion (Fig 10A). Next, the IPTG-inducible Physpank-swrB construct was added to each strain to determine the effect that over-expression of SwrB would have on hook secretion. When SwrB was over-expressed, the amount of hook protein in the supernatant appeared to increase in the fliM, fliG, and swrB mutants, but not in the fliF, fliP, or flhA mutants (Fig 10B). Finally, addition of the Pfla/che-fliPsob22 complementation construct provided in trans appeared to increase FlgE secretion in fliM, fliG, swrB, and fliP mutants but not in the fliF or flhA mutant (Fig 10C). We conclude FliF, FliP, and FlhA are downstream of SwrB for hook protein secretion. In sum, we conclude that SwrB activates an early flagellar morphogenetic checkpoint by catalyzing a FliF-basal body conformation that activates the type III secretion export apparatus for hook subunit secretion.

Bottom Line: Flagella are assembled sequentially from the inside-out with morphogenetic checkpoints that enforce the temporal order of subunit addition.Genetic suppressor analysis indicates that SwrB activates the flagellar type III secretion export apparatus by the membrane protein FliP.We conclude that SwrB enhances the probability that the flagellar basal body adopts a conformation proficient for secretion to ensure that rod and hook subunits are not secreted in the absence of a suitable platform on which to polymerize.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Indiana University, Bloomington, Indiana, United States of America.

ABSTRACT
Flagella are assembled sequentially from the inside-out with morphogenetic checkpoints that enforce the temporal order of subunit addition. Here we show that flagellar basal bodies fail to proceed to hook assembly at high frequency in the absence of the monotopic protein SwrB of Bacillus subtilis. Genetic suppressor analysis indicates that SwrB activates the flagellar type III secretion export apparatus by the membrane protein FliP. Furthermore, mutants defective in the flagellar C-ring phenocopy the absence of SwrB for reduced hook frequency and C-ring defects may be bypassed either by SwrB overexpression or by a gain-of-function allele in the polymerization domain of FliG. We conclude that SwrB enhances the probability that the flagellar basal body adopts a conformation proficient for secretion to ensure that rod and hook subunits are not secreted in the absence of a suitable platform on which to polymerize.

No MeSH data available.


Related in: MedlinePlus