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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 is required for assembly of hooks but not basal bodies.Panels A and B) Fluorescence micrographs of the flagellar filament (HagT209C) in the indicated genetic backgrounds. Membranes were stained with FM4-64 and false colored red. Flagella were stained with maleimide alexa fluor 488 and false colored green. The following strains were used to generate these panels: wild type (DS1916), swrA (DS9515), swrB (DS9319), flgM (DK486), swrA flgM (DK487), swrB flgM (DS488). Panel C) Fluorescence micrographs of the flagellar hook (FlgET123C) in the indicated genetic backgrounds. Membranes were stained with FM4-64 and false colored red. Hooks were stained with maleimide alexa fluor 488 and false colored green. The following strains were used to generate this panel: wild type (DS7673), swrA (DK480), swrB (DK478). Panel D) Fluorescence micrographs of flagellar basal bodies (FliM-GFP) in the indicated genetic backgrounds. Membranes were stained with the membrane stain FM4-64 and false colored red. FliM-GFP puncta were false colored green. The following strains were used to generate this panel: wild type (DS8521), swrA (DS8600), swrB (DK479). Scale bar is 4 μm.
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pgen.1005443.g001: SwrB is required for assembly of hooks but not basal bodies.Panels A and B) Fluorescence micrographs of the flagellar filament (HagT209C) in the indicated genetic backgrounds. Membranes were stained with FM4-64 and false colored red. Flagella were stained with maleimide alexa fluor 488 and false colored green. The following strains were used to generate these panels: wild type (DS1916), swrA (DS9515), swrB (DS9319), flgM (DK486), swrA flgM (DK487), swrB flgM (DS488). Panel C) Fluorescence micrographs of the flagellar hook (FlgET123C) in the indicated genetic backgrounds. Membranes were stained with FM4-64 and false colored red. Hooks were stained with maleimide alexa fluor 488 and false colored green. The following strains were used to generate this panel: wild type (DS7673), swrA (DK480), swrB (DK478). Panel D) Fluorescence micrographs of flagellar basal bodies (FliM-GFP) in the indicated genetic backgrounds. Membranes were stained with the membrane stain FM4-64 and false colored red. FliM-GFP puncta were false colored green. The following strains were used to generate this panel: wild type (DS8521), swrA (DS8600), swrB (DK479). Scale bar is 4 μm.

Mentions: SwrB is a single-pass transmembrane protein of unknown function that is required for swarming motility in B. subtilis [38,39]. Swarming motility over a solid surface requires an increase in the number of flagella relative to swimming in liquid, and mutants defective in the master regulator of flagellar biosynthesis, SwrA, have reduced flagellar number and are unable to swarm [40–42]. To determine whether cells mutated for SwrB have a defect in flagellar filament number, a variant of the flagellar filament protein Hag that could be labeled with a fluorescent dye (HagT209C) was introduced to the wild type, swrB, and swrA mutant backgrounds [43]. Cells mutated for SwrB appeared to have fewer filaments than wild type and resembled cells mutated for SwrA (Fig 1A). We conclude that SwrB is required for the assembly of wild type numbers of flagellar filaments and we infer that the reduction in filament number accounts for the swarming defect of a swrB mutant.


Functional Activation of the Flagellar Type III Secretion Export Apparatus.

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

SwrB is required for assembly of hooks but not basal bodies.Panels A and B) Fluorescence micrographs of the flagellar filament (HagT209C) in the indicated genetic backgrounds. Membranes were stained with FM4-64 and false colored red. Flagella were stained with maleimide alexa fluor 488 and false colored green. The following strains were used to generate these panels: wild type (DS1916), swrA (DS9515), swrB (DS9319), flgM (DK486), swrA flgM (DK487), swrB flgM (DS488). Panel C) Fluorescence micrographs of the flagellar hook (FlgET123C) in the indicated genetic backgrounds. Membranes were stained with FM4-64 and false colored red. Hooks were stained with maleimide alexa fluor 488 and false colored green. The following strains were used to generate this panel: wild type (DS7673), swrA (DK480), swrB (DK478). Panel D) Fluorescence micrographs of flagellar basal bodies (FliM-GFP) in the indicated genetic backgrounds. Membranes were stained with the membrane stain FM4-64 and false colored red. FliM-GFP puncta were false colored green. The following strains were used to generate this panel: wild type (DS8521), swrA (DS8600), swrB (DK479). Scale bar is 4 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4526659&req=5

pgen.1005443.g001: SwrB is required for assembly of hooks but not basal bodies.Panels A and B) Fluorescence micrographs of the flagellar filament (HagT209C) in the indicated genetic backgrounds. Membranes were stained with FM4-64 and false colored red. Flagella were stained with maleimide alexa fluor 488 and false colored green. The following strains were used to generate these panels: wild type (DS1916), swrA (DS9515), swrB (DS9319), flgM (DK486), swrA flgM (DK487), swrB flgM (DS488). Panel C) Fluorescence micrographs of the flagellar hook (FlgET123C) in the indicated genetic backgrounds. Membranes were stained with FM4-64 and false colored red. Hooks were stained with maleimide alexa fluor 488 and false colored green. The following strains were used to generate this panel: wild type (DS7673), swrA (DK480), swrB (DK478). Panel D) Fluorescence micrographs of flagellar basal bodies (FliM-GFP) in the indicated genetic backgrounds. Membranes were stained with the membrane stain FM4-64 and false colored red. FliM-GFP puncta were false colored green. The following strains were used to generate this panel: wild type (DS8521), swrA (DS8600), swrB (DK479). Scale bar is 4 μm.
Mentions: SwrB is a single-pass transmembrane protein of unknown function that is required for swarming motility in B. subtilis [38,39]. Swarming motility over a solid surface requires an increase in the number of flagella relative to swimming in liquid, and mutants defective in the master regulator of flagellar biosynthesis, SwrA, have reduced flagellar number and are unable to swarm [40–42]. To determine whether cells mutated for SwrB have a defect in flagellar filament number, a variant of the flagellar filament protein Hag that could be labeled with a fluorescent dye (HagT209C) was introduced to the wild type, swrB, and swrA mutant backgrounds [43]. Cells mutated for SwrB appeared to have fewer filaments than wild type and resembled cells mutated for SwrA (Fig 1A). We conclude that SwrB is required for the assembly of wild type numbers of flagellar filaments and we infer that the reduction in filament number accounts for the swarming defect of a swrB mutant.

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