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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

Some sob classes increase fla/che operon expression.QRT-PCR analysis of fla/che operon transcript levels at various positions in the operon. At the top of the graphs is a schematic of the fla/che operon where individual genes are drawn to scale and represented as boxes. Shaded boxes correspond to the locations of data points immediately below. Data points express the mRNA concentration corresponding to the gene probed by QRT-PCR at the kb distance fla/che operon transcriptional start site. Each data point is the average of three independently harvested RNA samples from whole cell lysates normalized to the transcript abundance of the constitutively expressed sigA gene. For each graph, the same wild type dataset from strain 3610 (closed circles) was used as a comparator. The following strains were used to generate this panel: A) swrA (DS2415), swrB (DS234), B) swrB sob21 (Class I, DS9148), C) swrB sob37 (Class II, DS9831), D) swrB sob27 (Class III, DS9154), and E) swrB sob24 (Class IV, DS9151).
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pgen.1005443.g007: Some sob classes increase fla/che operon expression.QRT-PCR analysis of fla/che operon transcript levels at various positions in the operon. At the top of the graphs is a schematic of the fla/che operon where individual genes are drawn to scale and represented as boxes. Shaded boxes correspond to the locations of data points immediately below. Data points express the mRNA concentration corresponding to the gene probed by QRT-PCR at the kb distance fla/che operon transcriptional start site. Each data point is the average of three independently harvested RNA samples from whole cell lysates normalized to the transcript abundance of the constitutively expressed sigA gene. For each graph, the same wild type dataset from strain 3610 (closed circles) was used as a comparator. The following strains were used to generate this panel: A) swrA (DS2415), swrB (DS234), B) swrB sob21 (Class I, DS9148), C) swrB sob37 (Class II, DS9831), D) swrB sob27 (Class III, DS9154), and E) swrB sob24 (Class IV, DS9151).

Mentions: Expression from the Pfla/che promoter is thought to control transcript abundance of the enitre fla/che operon [49,50,55,56]. To determine fla/che transcript levels in the absence of SwrB, QRT-PCR was conducted at various positions along the length of operon. Consistent with results observed with the Pfla/che-lacZ reporter assay, mutation of SwrA resulted in a reduction in fla/che operon transcript abundance but mutation of SwrB did not (Fig 7A). When the sob21-improved Pfla/che -10 element class I allele was present at the native site in the chromosome, transcript levels increased from 2-to-8 fold depending on the genetic position in the fla/che operon (Fig 7B). We conclude that although SwrB does not normally act to increase fla/che operon transcript levels, increased fla/che transcript levels 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)

Some sob classes increase fla/che operon expression.QRT-PCR analysis of fla/che operon transcript levels at various positions in the operon. At the top of the graphs is a schematic of the fla/che operon where individual genes are drawn to scale and represented as boxes. Shaded boxes correspond to the locations of data points immediately below. Data points express the mRNA concentration corresponding to the gene probed by QRT-PCR at the kb distance fla/che operon transcriptional start site. Each data point is the average of three independently harvested RNA samples from whole cell lysates normalized to the transcript abundance of the constitutively expressed sigA gene. For each graph, the same wild type dataset from strain 3610 (closed circles) was used as a comparator. The following strains were used to generate this panel: A) swrA (DS2415), swrB (DS234), B) swrB sob21 (Class I, DS9148), C) swrB sob37 (Class II, DS9831), D) swrB sob27 (Class III, DS9154), and E) swrB sob24 (Class IV, DS9151).
© Copyright Policy
Related In: Results  -  Collection

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

pgen.1005443.g007: Some sob classes increase fla/che operon expression.QRT-PCR analysis of fla/che operon transcript levels at various positions in the operon. At the top of the graphs is a schematic of the fla/che operon where individual genes are drawn to scale and represented as boxes. Shaded boxes correspond to the locations of data points immediately below. Data points express the mRNA concentration corresponding to the gene probed by QRT-PCR at the kb distance fla/che operon transcriptional start site. Each data point is the average of three independently harvested RNA samples from whole cell lysates normalized to the transcript abundance of the constitutively expressed sigA gene. For each graph, the same wild type dataset from strain 3610 (closed circles) was used as a comparator. The following strains were used to generate this panel: A) swrA (DS2415), swrB (DS234), B) swrB sob21 (Class I, DS9148), C) swrB sob37 (Class II, DS9831), D) swrB sob27 (Class III, DS9154), and E) swrB sob24 (Class IV, DS9151).
Mentions: Expression from the Pfla/che promoter is thought to control transcript abundance of the enitre fla/che operon [49,50,55,56]. To determine fla/che transcript levels in the absence of SwrB, QRT-PCR was conducted at various positions along the length of operon. Consistent with results observed with the Pfla/che-lacZ reporter assay, mutation of SwrA resulted in a reduction in fla/che operon transcript abundance but mutation of SwrB did not (Fig 7A). When the sob21-improved Pfla/che -10 element class I allele was present at the native site in the chromosome, transcript levels increased from 2-to-8 fold depending on the genetic position in the fla/che operon (Fig 7B). We conclude that although SwrB does not normally act to increase fla/che operon transcript levels, increased fla/che transcript levels 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