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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

Class I but not Class III sob alleles in the Pfla/che promoter region increase Pfla/che expression.β-galactosidase assays of lacZ gene expression under the control of WT (open bars), Class I sob21 (gray bars), or Class III sob6 (black bars) Pfla/che promoter regions. Each reporter was expressed in either WT, swrB, or swrA mutant backgrounds as indicated. The following strains were used to generate this panel: amyE::PflacheWT-lacZ (DS793), amyE::Pflachesob21-lacZ (DS1426), amyE::Pflachesob6-lacZ (DS9120), swrB amyE::PflacheWT-lacZ (DK285), swrB amyE::Pflachesob21-lacZ (DK1130), swrB amyE::Pflachesob6-lacZ (DK1117), swrA amyE::PflacheWT-lacZ (DK284), swrA amyE::Pflachesob21-lacZ (DK1129), swrA amyE::Pflachesob6-lacZ (DK1116). Error bars are the standard deviations of three replicates. β-galactosidase values presented in S4 Table.
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pgen.1005443.g006: Class I but not Class III sob alleles in the Pfla/che promoter region increase Pfla/che expression.β-galactosidase assays of lacZ gene expression under the control of WT (open bars), Class I sob21 (gray bars), or Class III sob6 (black bars) Pfla/che promoter regions. Each reporter was expressed in either WT, swrB, or swrA mutant backgrounds as indicated. The following strains were used to generate this panel: amyE::PflacheWT-lacZ (DS793), amyE::Pflachesob21-lacZ (DS1426), amyE::Pflachesob6-lacZ (DS9120), swrB amyE::PflacheWT-lacZ (DK285), swrB amyE::Pflachesob21-lacZ (DK1130), swrB amyE::Pflachesob6-lacZ (DK1117), swrA amyE::PflacheWT-lacZ (DK284), swrA amyE::Pflachesob21-lacZ (DK1129), swrA amyE::Pflachesob6-lacZ (DK1116). Error bars are the standard deviations of three replicates. β-galactosidase values presented in S4 Table.

Mentions: To directly test the consequence of a sob class I allele in a swrB mutant background, the Pfla/che promoter region was cloned either from wild type or a class I sob21 allele upstream of a promoterless lacZ gene and inserted at an ectopic locus (amyE::Pfla/cheWT-lacZ and amyE::Pfla/chesobclassI-lacZ). Consistent with previous reports, the improved -10 promoter element of the sob class I allele increased expression of Pfla/che in wild type, swrB, and swrA mutant backgrounds relative to the wild type promoter (Fig 6, compare white and gray bars within strains). Unlike mutation of SwrA, however, mutation of SwrB did not reduce the expression of the Pfla/che wild type reporter in an otherwise wild type genetic background (Fig 6, compare white bars between strains). We conclude that although SwrB does not normally act to increase the expression of the Pfla/che promoter, increased transcriptional activity from the Pfla/che promoter nonetheless compensates for the absence of SwrB.


Functional Activation of the Flagellar Type III Secretion Export Apparatus.

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

Class I but not Class III sob alleles in the Pfla/che promoter region increase Pfla/che expression.β-galactosidase assays of lacZ gene expression under the control of WT (open bars), Class I sob21 (gray bars), or Class III sob6 (black bars) Pfla/che promoter regions. Each reporter was expressed in either WT, swrB, or swrA mutant backgrounds as indicated. The following strains were used to generate this panel: amyE::PflacheWT-lacZ (DS793), amyE::Pflachesob21-lacZ (DS1426), amyE::Pflachesob6-lacZ (DS9120), swrB amyE::PflacheWT-lacZ (DK285), swrB amyE::Pflachesob21-lacZ (DK1130), swrB amyE::Pflachesob6-lacZ (DK1117), swrA amyE::PflacheWT-lacZ (DK284), swrA amyE::Pflachesob21-lacZ (DK1129), swrA amyE::Pflachesob6-lacZ (DK1116). Error bars are the standard deviations of three replicates. β-galactosidase values presented in S4 Table.
© Copyright Policy
Related In: Results  -  Collection

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

pgen.1005443.g006: Class I but not Class III sob alleles in the Pfla/che promoter region increase Pfla/che expression.β-galactosidase assays of lacZ gene expression under the control of WT (open bars), Class I sob21 (gray bars), or Class III sob6 (black bars) Pfla/che promoter regions. Each reporter was expressed in either WT, swrB, or swrA mutant backgrounds as indicated. The following strains were used to generate this panel: amyE::PflacheWT-lacZ (DS793), amyE::Pflachesob21-lacZ (DS1426), amyE::Pflachesob6-lacZ (DS9120), swrB amyE::PflacheWT-lacZ (DK285), swrB amyE::Pflachesob21-lacZ (DK1130), swrB amyE::Pflachesob6-lacZ (DK1117), swrA amyE::PflacheWT-lacZ (DK284), swrA amyE::Pflachesob21-lacZ (DK1129), swrA amyE::Pflachesob6-lacZ (DK1116). Error bars are the standard deviations of three replicates. β-galactosidase values presented in S4 Table.
Mentions: To directly test the consequence of a sob class I allele in a swrB mutant background, the Pfla/che promoter region was cloned either from wild type or a class I sob21 allele upstream of a promoterless lacZ gene and inserted at an ectopic locus (amyE::Pfla/cheWT-lacZ and amyE::Pfla/chesobclassI-lacZ). Consistent with previous reports, the improved -10 promoter element of the sob class I allele increased expression of Pfla/che in wild type, swrB, and swrA mutant backgrounds relative to the wild type promoter (Fig 6, compare white and gray bars within strains). Unlike mutation of SwrA, however, mutation of SwrB did not reduce the expression of the Pfla/che wild type reporter in an otherwise wild type genetic background (Fig 6, compare white bars between strains). We conclude that although SwrB does not normally act to increase the expression of the Pfla/che promoter, increased transcriptional activity from the Pfla/che promoter nonetheless compensates for the absence of SwrB.

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