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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

C-ring mutants phenocopy SwrB mutants for hook synthesis and SwrB overexpression bypasses C-ring defects.Fluorescence micrographs of the flagellar hook (FlgET123C) in the indicated genetic backgrounds. Ø column indicates no additional genetic modifications whereas (SwrB++) indicates the presence of an ectopically integrated Physpank-swrB construct induced with 1 mM IPTG. Membranes stained with FM4-64 and false colored red. Hooks stained with maleimide alexa fluor 488 and false colored green. The following strains were used to generate this panel: swrB (DK478), swrB++ (DK2089), fliM (DK1564), fliM SwrB++ (DK2087), fliG (DK2098), fliG SwrB++ (DK3041), fliF (DK3135), and fliF SwrB++ (DK3137). Scale bar is 4 μm.
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pgen.1005443.g009: C-ring mutants phenocopy SwrB mutants for hook synthesis and SwrB overexpression bypasses C-ring defects.Fluorescence micrographs of the flagellar hook (FlgET123C) in the indicated genetic backgrounds. Ø column indicates no additional genetic modifications whereas (SwrB++) indicates the presence of an ectopically integrated Physpank-swrB construct induced with 1 mM IPTG. Membranes stained with FM4-64 and false colored red. Hooks stained with maleimide alexa fluor 488 and false colored green. The following strains were used to generate this panel: swrB (DK478), swrB++ (DK2089), fliM (DK1564), fliM SwrB++ (DK2087), fliG (DK2098), fliG SwrB++ (DK3041), fliF (DK3135), and fliF SwrB++ (DK3137). Scale bar is 4 μm.

Mentions: Mutations in FliP and FliG that suppressed the absence of SwrB suggested that the mechanism by which SwrB enhanced hook assembly was related to flagellar secretion by way of basal body structure. Therefore, we further explored the relationship of SwrB to components of the basal body and C-ring for enhancing hook polymerization. To do so, strains were generated that contained the FlgET123C allele for fluorescent labeling of the flagellar hook in backgrounds mutated for SwrB, FliM, FliG, and FliF. Cells mutated for either FliM or FliG displayed a low frequency of flagellar hooks and resembled cells mutated for SwrB (Fig 9). By contrast, no hooks were detected in cells mutated for FliF (Fig 9). Next, the swrB gene was cloned downstream of an IPTG inducible Physpank promoter and integrated at an ectopic site in each of the strains tested (amyE::Physpank-swrB). In the presence of IPTG, overexpression of SwrB increased the frequency of hooks for the swrB, fliM and fliG mutants but did not restore hook formation to the fliF mutant (Fig 9). We conclude that the activation of hook assembly by SwrB does not require FliG or FliM, but that FliGQ132R nonetheless compensates for the absence of SwrB. We further conclude that the basal body structural component FliF is required for SwrB to activate hook assembly.


Functional Activation of the Flagellar Type III Secretion Export Apparatus.

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

C-ring mutants phenocopy SwrB mutants for hook synthesis and SwrB overexpression bypasses C-ring defects.Fluorescence micrographs of the flagellar hook (FlgET123C) in the indicated genetic backgrounds. Ø column indicates no additional genetic modifications whereas (SwrB++) indicates the presence of an ectopically integrated Physpank-swrB construct induced with 1 mM IPTG. Membranes stained with FM4-64 and false colored red. Hooks stained with maleimide alexa fluor 488 and false colored green. The following strains were used to generate this panel: swrB (DK478), swrB++ (DK2089), fliM (DK1564), fliM SwrB++ (DK2087), fliG (DK2098), fliG SwrB++ (DK3041), fliF (DK3135), and fliF SwrB++ (DK3137). Scale bar is 4 μm.
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Related In: Results  -  Collection

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pgen.1005443.g009: C-ring mutants phenocopy SwrB mutants for hook synthesis and SwrB overexpression bypasses C-ring defects.Fluorescence micrographs of the flagellar hook (FlgET123C) in the indicated genetic backgrounds. Ø column indicates no additional genetic modifications whereas (SwrB++) indicates the presence of an ectopically integrated Physpank-swrB construct induced with 1 mM IPTG. Membranes stained with FM4-64 and false colored red. Hooks stained with maleimide alexa fluor 488 and false colored green. The following strains were used to generate this panel: swrB (DK478), swrB++ (DK2089), fliM (DK1564), fliM SwrB++ (DK2087), fliG (DK2098), fliG SwrB++ (DK3041), fliF (DK3135), and fliF SwrB++ (DK3137). Scale bar is 4 μm.
Mentions: Mutations in FliP and FliG that suppressed the absence of SwrB suggested that the mechanism by which SwrB enhanced hook assembly was related to flagellar secretion by way of basal body structure. Therefore, we further explored the relationship of SwrB to components of the basal body and C-ring for enhancing hook polymerization. To do so, strains were generated that contained the FlgET123C allele for fluorescent labeling of the flagellar hook in backgrounds mutated for SwrB, FliM, FliG, and FliF. Cells mutated for either FliM or FliG displayed a low frequency of flagellar hooks and resembled cells mutated for SwrB (Fig 9). By contrast, no hooks were detected in cells mutated for FliF (Fig 9). Next, the swrB gene was cloned downstream of an IPTG inducible Physpank promoter and integrated at an ectopic site in each of the strains tested (amyE::Physpank-swrB). In the presence of IPTG, overexpression of SwrB increased the frequency of hooks for the swrB, fliM and fliG mutants but did not restore hook formation to the fliF mutant (Fig 9). We conclude that the activation of hook assembly by SwrB does not require FliG or FliM, but that FliGQ132R nonetheless compensates for the absence of SwrB. We further conclude that the basal body structural component FliF is required for SwrB to activate hook assembly.

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