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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

Genetic maps indicating the location of the sob suppressors.A) Map of the location of the sob mutant classes as indicated by red dots. Gray boxes indicate the enlarged regions emphasized in panels B-E. For all panels, open arrows indicate open reading frames, bent arrows indicate promoters, lollipops indicate Rho-independent terminators, and “I” bars indicate the boundaries of genetic deletions. The map in panel A is not to scale. The maps in panels B-E share the 1 kb scale bar as indicated beneath panel E. B) Class I, II, III sob mutant locations. Left, cartoon of the Pfla/che promoter region indicating the location of the class II sob deletion boundaries. Right, sequence of the Pfla/che promoter region with sob Class I and III point mutant locations indicated with gray boxes and annotated in red letters. sob class II deletions annotated as red dotted lines. Important genetic landmarks including the codY terminator sequence, the PD3 promoter elements, the Pfla/che promoter elements, and the fla/che operon transcriptional start site are boxed and labeled. C) Class IV sob mutant locations. Left, cartoon of the cspB upstream intergenic region. Boundaries of deletions and the location of a single base pair mutation in red. Right, cartoon of the boundaries of the complementation constructs generated for the swrB sob24 allele. Results summarized and interpreted based on swarm assay presented in Fig 4H. D) Class V sob mutant location. Left, cartoon of the fliO/fliP genetic region and the location of the sob mutation indicated by a red asterisk. Right, sequence of the 3’ end of the fliO gene and the 5’ end of the fliP gene. Putative internal RBS sequence is boxed and labeled. The location of the sob class V mutation indicated by a red letter. E) Class VI sob mutant locations. Left, cartoon of the fliG genetic region and the location of the sob mutation indicated by a red asterisk. Right, structure of the Aquifex aeolicus FliG protein with the FliGQ132 residue which was mutated to an asparagine in the sob mutants [64]. The predicted locations of the FliF, MotA, and FliM proteins that interact with FliG are indicated in gray.
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pgen.1005443.g005: Genetic maps indicating the location of the sob suppressors.A) Map of the location of the sob mutant classes as indicated by red dots. Gray boxes indicate the enlarged regions emphasized in panels B-E. For all panels, open arrows indicate open reading frames, bent arrows indicate promoters, lollipops indicate Rho-independent terminators, and “I” bars indicate the boundaries of genetic deletions. The map in panel A is not to scale. The maps in panels B-E share the 1 kb scale bar as indicated beneath panel E. B) Class I, II, III sob mutant locations. Left, cartoon of the Pfla/che promoter region indicating the location of the class II sob deletion boundaries. Right, sequence of the Pfla/che promoter region with sob Class I and III point mutant locations indicated with gray boxes and annotated in red letters. sob class II deletions annotated as red dotted lines. Important genetic landmarks including the codY terminator sequence, the PD3 promoter elements, the Pfla/che promoter elements, and the fla/che operon transcriptional start site are boxed and labeled. C) Class IV sob mutant locations. Left, cartoon of the cspB upstream intergenic region. Boundaries of deletions and the location of a single base pair mutation in red. Right, cartoon of the boundaries of the complementation constructs generated for the swrB sob24 allele. Results summarized and interpreted based on swarm assay presented in Fig 4H. D) Class V sob mutant location. Left, cartoon of the fliO/fliP genetic region and the location of the sob mutation indicated by a red asterisk. Right, sequence of the 3’ end of the fliO gene and the 5’ end of the fliP gene. Putative internal RBS sequence is boxed and labeled. The location of the sob class V mutation indicated by a red letter. E) Class VI sob mutant locations. Left, cartoon of the fliG genetic region and the location of the sob mutation indicated by a red asterisk. Right, structure of the Aquifex aeolicus FliG protein with the FliGQ132 residue which was mutated to an asparagine in the sob mutants [64]. The predicted locations of the FliF, MotA, and FliM proteins that interact with FliG are indicated in gray.

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)

Genetic maps indicating the location of the sob suppressors.A) Map of the location of the sob mutant classes as indicated by red dots. Gray boxes indicate the enlarged regions emphasized in panels B-E. For all panels, open arrows indicate open reading frames, bent arrows indicate promoters, lollipops indicate Rho-independent terminators, and “I” bars indicate the boundaries of genetic deletions. The map in panel A is not to scale. The maps in panels B-E share the 1 kb scale bar as indicated beneath panel E. B) Class I, II, III sob mutant locations. Left, cartoon of the Pfla/che promoter region indicating the location of the class II sob deletion boundaries. Right, sequence of the Pfla/che promoter region with sob Class I and III point mutant locations indicated with gray boxes and annotated in red letters. sob class II deletions annotated as red dotted lines. Important genetic landmarks including the codY terminator sequence, the PD3 promoter elements, the Pfla/che promoter elements, and the fla/che operon transcriptional start site are boxed and labeled. C) Class IV sob mutant locations. Left, cartoon of the cspB upstream intergenic region. Boundaries of deletions and the location of a single base pair mutation in red. Right, cartoon of the boundaries of the complementation constructs generated for the swrB sob24 allele. Results summarized and interpreted based on swarm assay presented in Fig 4H. D) Class V sob mutant location. Left, cartoon of the fliO/fliP genetic region and the location of the sob mutation indicated by a red asterisk. Right, sequence of the 3’ end of the fliO gene and the 5’ end of the fliP gene. Putative internal RBS sequence is boxed and labeled. The location of the sob class V mutation indicated by a red letter. E) Class VI sob mutant locations. Left, cartoon of the fliG genetic region and the location of the sob mutation indicated by a red asterisk. Right, structure of the Aquifex aeolicus FliG protein with the FliGQ132 residue which was mutated to an asparagine in the sob mutants [64]. The predicted locations of the FliF, MotA, and FliM proteins that interact with FliG are indicated in gray.
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Related In: Results  -  Collection

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Show All Figures
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pgen.1005443.g005: Genetic maps indicating the location of the sob suppressors.A) Map of the location of the sob mutant classes as indicated by red dots. Gray boxes indicate the enlarged regions emphasized in panels B-E. For all panels, open arrows indicate open reading frames, bent arrows indicate promoters, lollipops indicate Rho-independent terminators, and “I” bars indicate the boundaries of genetic deletions. The map in panel A is not to scale. The maps in panels B-E share the 1 kb scale bar as indicated beneath panel E. B) Class I, II, III sob mutant locations. Left, cartoon of the Pfla/che promoter region indicating the location of the class II sob deletion boundaries. Right, sequence of the Pfla/che promoter region with sob Class I and III point mutant locations indicated with gray boxes and annotated in red letters. sob class II deletions annotated as red dotted lines. Important genetic landmarks including the codY terminator sequence, the PD3 promoter elements, the Pfla/che promoter elements, and the fla/che operon transcriptional start site are boxed and labeled. C) Class IV sob mutant locations. Left, cartoon of the cspB upstream intergenic region. Boundaries of deletions and the location of a single base pair mutation in red. Right, cartoon of the boundaries of the complementation constructs generated for the swrB sob24 allele. Results summarized and interpreted based on swarm assay presented in Fig 4H. D) Class V sob mutant location. Left, cartoon of the fliO/fliP genetic region and the location of the sob mutation indicated by a red asterisk. Right, sequence of the 3’ end of the fliO gene and the 5’ end of the fliP gene. Putative internal RBS sequence is boxed and labeled. The location of the sob class V mutation indicated by a red letter. E) Class VI sob mutant locations. Left, cartoon of the fliG genetic region and the location of the sob mutation indicated by a red asterisk. Right, structure of the Aquifex aeolicus FliG protein with the FliGQ132 residue which was mutated to an asparagine in the sob mutants [64]. The predicted locations of the FliF, MotA, and FliM proteins that interact with FliG are indicated in gray.
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