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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

Cytological quantification of flagellar hooks and basal bodies.3-D SIM microscopy and Imaris software was used count puncta per cell for either basal bodies (red dots, FliM-GFP) or hooks (green dots/maleimide stained FlgET123C) relative to cell length on 30 individual cells each of the wild type (DS8521/DS7673), swrB (DK479/DK478) and swrA (DS8600/DK480) backgrounds. Open circles represent the puncta number and length averages of the matching color, and vertical and horizontal bars indicate the standard deviations respectively.
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pgen.1005443.g003: Cytological quantification of flagellar hooks and basal bodies.3-D SIM microscopy and Imaris software was used count puncta per cell for either basal bodies (red dots, FliM-GFP) or hooks (green dots/maleimide stained FlgET123C) relative to cell length on 30 individual cells each of the wild type (DS8521/DS7673), swrB (DK479/DK478) and swrA (DS8600/DK480) backgrounds. Open circles represent the puncta number and length averages of the matching color, and vertical and horizontal bars indicate the standard deviations respectively.

Mentions: FlgM inhibition of σD activity is enhanced when cells are defective in flagellar hook synthesis, and a defect in flagellar hook synthesis would also account for the reduction in flagellar filaments observed in the SwrB mutant [51,52]. To measure flagellar hook numbers, a variant of the flagellar hook protein FlgE that could be labeled with a fluorescent dye (FlgET123C) was introduced to the wild type, swrB, and swrA mutant backgrounds [51]. Cells mutated for swrB appeared to have fewer hooks than the wild type and instead resembled the reduced numbers of hooks in cells mutated for swrA (Fig 1C). To count flagellar hooks, 3D structured illumination microscopy (3D-SIM) was conducted on hook-stained cells of each genetic background and each individual cell was expressed as a point on a scatter plot representing the number of hooks versus cell length (Fig 3, green symbols). Whereas wild type cells had an average of 15 hooks, swrB and swrA mutants had an average of 5 and 4 hooks per cell, respectively. We conclude that the swrB mutant was defective in hook assembly and as a result exhibited enhanced inhibition of σD by FlgM.


Functional Activation of the Flagellar Type III Secretion Export Apparatus.

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

Cytological quantification of flagellar hooks and basal bodies.3-D SIM microscopy and Imaris software was used count puncta per cell for either basal bodies (red dots, FliM-GFP) or hooks (green dots/maleimide stained FlgET123C) relative to cell length on 30 individual cells each of the wild type (DS8521/DS7673), swrB (DK479/DK478) and swrA (DS8600/DK480) backgrounds. Open circles represent the puncta number and length averages of the matching color, and vertical and horizontal bars indicate the standard deviations respectively.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4526659&req=5

pgen.1005443.g003: Cytological quantification of flagellar hooks and basal bodies.3-D SIM microscopy and Imaris software was used count puncta per cell for either basal bodies (red dots, FliM-GFP) or hooks (green dots/maleimide stained FlgET123C) relative to cell length on 30 individual cells each of the wild type (DS8521/DS7673), swrB (DK479/DK478) and swrA (DS8600/DK480) backgrounds. Open circles represent the puncta number and length averages of the matching color, and vertical and horizontal bars indicate the standard deviations respectively.
Mentions: FlgM inhibition of σD activity is enhanced when cells are defective in flagellar hook synthesis, and a defect in flagellar hook synthesis would also account for the reduction in flagellar filaments observed in the SwrB mutant [51,52]. To measure flagellar hook numbers, a variant of the flagellar hook protein FlgE that could be labeled with a fluorescent dye (FlgET123C) was introduced to the wild type, swrB, and swrA mutant backgrounds [51]. Cells mutated for swrB appeared to have fewer hooks than the wild type and instead resembled the reduced numbers of hooks in cells mutated for swrA (Fig 1C). To count flagellar hooks, 3D structured illumination microscopy (3D-SIM) was conducted on hook-stained cells of each genetic background and each individual cell was expressed as a point on a scatter plot representing the number of hooks versus cell length (Fig 3, green symbols). Whereas wild type cells had an average of 15 hooks, swrB and swrA mutants had an average of 5 and 4 hooks per cell, respectively. We conclude that the swrB mutant was defective in hook assembly and as a result exhibited enhanced inhibition of σD by FlgM.

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