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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

Suppressors of swrB (sob) mutants restore swarming motility in the absence of SwrB.Quantitative swarm expansion assays of the indicated genetic backgrounds. Genotypes in parentheses indicate complementation constructs. The following strains were used the generate panels: A) wild type (3610), swrB (DS234), B) swrB sob21 (DS9148), swrB sob23 (DS9150), swrB sob29 (DS9156), swrB sob31 (DS9158), swrB sob34 (DS9817), C) swrB sob4 (DS4633), swrB sob30 (DS9157), swrB sob36 (DS9819), swrB sob37 (DS9831), swrB sob39 (DS9846), D) swrB sob6 (DS7063), swrB sob27 (DS9154), swrB sob33 (DS9816), swrB sob35 (DS9818), swrB sob38 (DS9845), E) swrB sob7 (DS7064) swrB sob24 (DS9151), swrB sob25 (DS9152), swrB sob26 (DS9153), F) swrB sob22 (DS9149), G) swrB sob20 (DS9147), swrB sob28 (DS9155), H) swrB (DS234), swrB sob24 (comp1) (DK2015), swrB sob24 (comp2) (DK2017), swrB sob24 (comp3) (DK2016), swrB sob24 (comp4) (DK2018), swrB sob24 (DS9151) I) cspB (DK1905), swrB sob24 (DS9151), swrB cspB (DK1937), J) fliO (DS6468), fliO (fliO+) (DK52), fliO (fliOsob22) (DK53), K) swrB fliO (DK54), swrB fliO (fliO+) (DK65), swrB fliO (fliOsob22) (DK66), L) fliP (DS7119), fliP (fliP+) (DK577), fliP (fliPsob22) (DK579), M) swrB fliP (DK519), swrB fliP (fliP+) (DK572), swrB fliP (fliPsob22) (DK574), N) swrB (DS234), swrB amyE::Physpank-fliO (DK1230), swrB amyE::Physpank-fliP (DK601) grown in the presence of 1 mM IPTG, O) fliG (DS7357), fliG (fliG+) (DK737), fliG (fliGsob28) (DK568), P) swrB fliG (DK584), swrB fliG (fliG+) (DK738), swrB fliG (fliGsob28) (DK598). Each data point is the average of three replicates.
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pgen.1005443.g004: Suppressors of swrB (sob) mutants restore swarming motility in the absence of SwrB.Quantitative swarm expansion assays of the indicated genetic backgrounds. Genotypes in parentheses indicate complementation constructs. The following strains were used the generate panels: A) wild type (3610), swrB (DS234), B) swrB sob21 (DS9148), swrB sob23 (DS9150), swrB sob29 (DS9156), swrB sob31 (DS9158), swrB sob34 (DS9817), C) swrB sob4 (DS4633), swrB sob30 (DS9157), swrB sob36 (DS9819), swrB sob37 (DS9831), swrB sob39 (DS9846), D) swrB sob6 (DS7063), swrB sob27 (DS9154), swrB sob33 (DS9816), swrB sob35 (DS9818), swrB sob38 (DS9845), E) swrB sob7 (DS7064) swrB sob24 (DS9151), swrB sob25 (DS9152), swrB sob26 (DS9153), F) swrB sob22 (DS9149), G) swrB sob20 (DS9147), swrB sob28 (DS9155), H) swrB (DS234), swrB sob24 (comp1) (DK2015), swrB sob24 (comp2) (DK2017), swrB sob24 (comp3) (DK2016), swrB sob24 (comp4) (DK2018), swrB sob24 (DS9151) I) cspB (DK1905), swrB sob24 (DS9151), swrB cspB (DK1937), J) fliO (DS6468), fliO (fliO+) (DK52), fliO (fliOsob22) (DK53), K) swrB fliO (DK54), swrB fliO (fliO+) (DK65), swrB fliO (fliOsob22) (DK66), L) fliP (DS7119), fliP (fliP+) (DK577), fliP (fliPsob22) (DK579), M) swrB fliP (DK519), swrB fliP (fliP+) (DK572), swrB fliP (fliPsob22) (DK574), N) swrB (DS234), swrB amyE::Physpank-fliO (DK1230), swrB amyE::Physpank-fliP (DK601) grown in the presence of 1 mM IPTG, O) fliG (DS7357), fliG (fliG+) (DK737), fliG (fliGsob28) (DK568), P) swrB fliG (DK584), swrB fliG (fliG+) (DK738), swrB fliG (fliGsob28) (DK598). Each data point is the average of three replicates.

Mentions: To determine how SwrB regulates hook assembly, spontaneous suppressor mutations were isolated that restored swarming motility to a swrB mutant. When a swrB mutant was inoculated in the center of a swarm agar plate, cells initially grew as a tight central colony and, unlike the wild type, failed to spread from the inoculum origin (Fig 4A). However, after 24 hours of incubation, flares of cells that had regained the ability to swarm emerged from the central colony and cells from these flares were clonally isolated. Twenty-four spontaneous sob (suppressor of swrB) mutants were independently isolated. Each suppressor was validated by PCR length polymorphism to confirm the presence of the swrB mutant allele. A combination of candidate gene sequencing, phage transduction linkage mapping, and Illumina whole-genome sequencing was used to identify the location of each of the sob suppressor mutations. Based on the swarm behavior and location of the suppressor mutations, the sob alleles were divided into six classes and analyzed separately (Table 1 and Figs 4B–4G and 5A).


Functional Activation of the Flagellar Type III Secretion Export Apparatus.

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

Suppressors of swrB (sob) mutants restore swarming motility in the absence of SwrB.Quantitative swarm expansion assays of the indicated genetic backgrounds. Genotypes in parentheses indicate complementation constructs. The following strains were used the generate panels: A) wild type (3610), swrB (DS234), B) swrB sob21 (DS9148), swrB sob23 (DS9150), swrB sob29 (DS9156), swrB sob31 (DS9158), swrB sob34 (DS9817), C) swrB sob4 (DS4633), swrB sob30 (DS9157), swrB sob36 (DS9819), swrB sob37 (DS9831), swrB sob39 (DS9846), D) swrB sob6 (DS7063), swrB sob27 (DS9154), swrB sob33 (DS9816), swrB sob35 (DS9818), swrB sob38 (DS9845), E) swrB sob7 (DS7064) swrB sob24 (DS9151), swrB sob25 (DS9152), swrB sob26 (DS9153), F) swrB sob22 (DS9149), G) swrB sob20 (DS9147), swrB sob28 (DS9155), H) swrB (DS234), swrB sob24 (comp1) (DK2015), swrB sob24 (comp2) (DK2017), swrB sob24 (comp3) (DK2016), swrB sob24 (comp4) (DK2018), swrB sob24 (DS9151) I) cspB (DK1905), swrB sob24 (DS9151), swrB cspB (DK1937), J) fliO (DS6468), fliO (fliO+) (DK52), fliO (fliOsob22) (DK53), K) swrB fliO (DK54), swrB fliO (fliO+) (DK65), swrB fliO (fliOsob22) (DK66), L) fliP (DS7119), fliP (fliP+) (DK577), fliP (fliPsob22) (DK579), M) swrB fliP (DK519), swrB fliP (fliP+) (DK572), swrB fliP (fliPsob22) (DK574), N) swrB (DS234), swrB amyE::Physpank-fliO (DK1230), swrB amyE::Physpank-fliP (DK601) grown in the presence of 1 mM IPTG, O) fliG (DS7357), fliG (fliG+) (DK737), fliG (fliGsob28) (DK568), P) swrB fliG (DK584), swrB fliG (fliG+) (DK738), swrB fliG (fliGsob28) (DK598). Each data point is the average of three replicates.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4526659&req=5

pgen.1005443.g004: Suppressors of swrB (sob) mutants restore swarming motility in the absence of SwrB.Quantitative swarm expansion assays of the indicated genetic backgrounds. Genotypes in parentheses indicate complementation constructs. The following strains were used the generate panels: A) wild type (3610), swrB (DS234), B) swrB sob21 (DS9148), swrB sob23 (DS9150), swrB sob29 (DS9156), swrB sob31 (DS9158), swrB sob34 (DS9817), C) swrB sob4 (DS4633), swrB sob30 (DS9157), swrB sob36 (DS9819), swrB sob37 (DS9831), swrB sob39 (DS9846), D) swrB sob6 (DS7063), swrB sob27 (DS9154), swrB sob33 (DS9816), swrB sob35 (DS9818), swrB sob38 (DS9845), E) swrB sob7 (DS7064) swrB sob24 (DS9151), swrB sob25 (DS9152), swrB sob26 (DS9153), F) swrB sob22 (DS9149), G) swrB sob20 (DS9147), swrB sob28 (DS9155), H) swrB (DS234), swrB sob24 (comp1) (DK2015), swrB sob24 (comp2) (DK2017), swrB sob24 (comp3) (DK2016), swrB sob24 (comp4) (DK2018), swrB sob24 (DS9151) I) cspB (DK1905), swrB sob24 (DS9151), swrB cspB (DK1937), J) fliO (DS6468), fliO (fliO+) (DK52), fliO (fliOsob22) (DK53), K) swrB fliO (DK54), swrB fliO (fliO+) (DK65), swrB fliO (fliOsob22) (DK66), L) fliP (DS7119), fliP (fliP+) (DK577), fliP (fliPsob22) (DK579), M) swrB fliP (DK519), swrB fliP (fliP+) (DK572), swrB fliP (fliPsob22) (DK574), N) swrB (DS234), swrB amyE::Physpank-fliO (DK1230), swrB amyE::Physpank-fliP (DK601) grown in the presence of 1 mM IPTG, O) fliG (DS7357), fliG (fliG+) (DK737), fliG (fliGsob28) (DK568), P) swrB fliG (DK584), swrB fliG (fliG+) (DK738), swrB fliG (fliGsob28) (DK598). Each data point is the average of three replicates.
Mentions: To determine how SwrB regulates hook assembly, spontaneous suppressor mutations were isolated that restored swarming motility to a swrB mutant. When a swrB mutant was inoculated in the center of a swarm agar plate, cells initially grew as a tight central colony and, unlike the wild type, failed to spread from the inoculum origin (Fig 4A). However, after 24 hours of incubation, flares of cells that had regained the ability to swarm emerged from the central colony and cells from these flares were clonally isolated. Twenty-four spontaneous sob (suppressor of swrB) mutants were independently isolated. Each suppressor was validated by PCR length polymorphism to confirm the presence of the swrB mutant allele. A combination of candidate gene sequencing, phage transduction linkage mapping, and Illumina whole-genome sequencing was used to identify the location of each of the sob suppressor mutations. Based on the swarm behavior and location of the suppressor mutations, the sob alleles were divided into six classes and analyzed separately (Table 1 and Figs 4B–4G and 5A).

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