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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

SwrB increases σD activity but not σD protein levels.A) Western blot analysis of whole cell lysates from the indicated genetic backgrounds probed with anti-Hag, anti-SigD, and anti-SigA primary antibodies. Black carets indicate antibody specific targets. Gray caret indicates a non-specific cross-reacting band recognized by the anti-SigD antibody. Strains used to generate this panel: wild type (DS908), swrA (DS4015), swrB, (DS4040), flgM (DS4264), swrA flgM (DS4034), swrB flgM (DS4090). B) β-galactosidase assays of Phag-lacZ transcriptional activity expressed in Miller Units in the indicated genetic backgrounds. The Ø symbol indicates that no further genetic modification was included whereas “flgM” indicates the introduction of a flgM mutation to the indicated genetic background. β-galactosidase values presented in S3 Table. The following strains were used to generate this panel: wild type (DS9461), flgM (DK313), swrA (DK288), swrA flgM (DK318), swrB (DK289), swrB flgM (DK319). C) The frequency of fluorescent “ON” cells expressing Phag-GFP in the indicated genetic backgrounds. Over 600 cells were counted per strain in triplicate to obtain average percentages and standard deviations. The Ø symbol indicates that no further genetic modification was included whereas “flgM” indicates the introduction of a flgM mutation to the indicated genetic background. Sample raw data images used to generate the percentages are presented in S1 Fig. Strains used to generate this panel: wild type (DS908), swrA (DS4015), swrB (DS4040), flgM (DS4264), swrA flgM (DS4034), and swrB flgM (DS4090).
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pgen.1005443.g002: SwrB increases σD activity but not σD protein levels.A) Western blot analysis of whole cell lysates from the indicated genetic backgrounds probed with anti-Hag, anti-SigD, and anti-SigA primary antibodies. Black carets indicate antibody specific targets. Gray caret indicates a non-specific cross-reacting band recognized by the anti-SigD antibody. Strains used to generate this panel: wild type (DS908), swrA (DS4015), swrB, (DS4040), flgM (DS4264), swrA flgM (DS4034), swrB flgM (DS4090). B) β-galactosidase assays of Phag-lacZ transcriptional activity expressed in Miller Units in the indicated genetic backgrounds. The Ø symbol indicates that no further genetic modification was included whereas “flgM” indicates the introduction of a flgM mutation to the indicated genetic background. β-galactosidase values presented in S3 Table. The following strains were used to generate this panel: wild type (DS9461), flgM (DK313), swrA (DK288), swrA flgM (DK318), swrB (DK289), swrB flgM (DK319). C) The frequency of fluorescent “ON” cells expressing Phag-GFP in the indicated genetic backgrounds. Over 600 cells were counted per strain in triplicate to obtain average percentages and standard deviations. The Ø symbol indicates that no further genetic modification was included whereas “flgM” indicates the introduction of a flgM mutation to the indicated genetic background. Sample raw data images used to generate the percentages are presented in S1 Fig. Strains used to generate this panel: wild type (DS908), swrA (DS4015), swrB (DS4040), flgM (DS4264), swrA flgM (DS4034), and swrB flgM (DS4090).

Mentions: Consistent with a reduction in flagellar filament assembly, cells lacking either SwrB or SwrA showed a reduced level of the flagellar filament protein, Hag, by Western blot analysis (Fig 2A). The reduction in Hag protein was likely due to reduced transcription of the hag gene as cells mutated for either SwrB or SwrA showed lower expression from a reporter in which the hag promoter (Phag) was fused to the lacZ gene encoding β-galactosidase (Phag-lacZ) (Fig 2B, white bars), and a reduced frequency of cells expressing a reporter in which the Phag promoter was fused to green fluorescent protein (GFP) (Phag-gfp) (Fig 2C, white bars, and S1 Fig) [39,41]. The Phag promoter is transcribed by RNA polymerase and the alternative sigma factor, σD [44]. Whereas cells mutated for SwrA exhibited a reduced level of σD protein, cells mutated for SwrB exhibited a level of σD protein comparable to the wild type (Fig 2A). We conclude that the absence of SwrB resulted in reduced expression of the hag gene but unlike the absence of SwrA, the defect occurred downstream of σD protein levels.


Functional Activation of the Flagellar Type III Secretion Export Apparatus.

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

SwrB increases σD activity but not σD protein levels.A) Western blot analysis of whole cell lysates from the indicated genetic backgrounds probed with anti-Hag, anti-SigD, and anti-SigA primary antibodies. Black carets indicate antibody specific targets. Gray caret indicates a non-specific cross-reacting band recognized by the anti-SigD antibody. Strains used to generate this panel: wild type (DS908), swrA (DS4015), swrB, (DS4040), flgM (DS4264), swrA flgM (DS4034), swrB flgM (DS4090). B) β-galactosidase assays of Phag-lacZ transcriptional activity expressed in Miller Units in the indicated genetic backgrounds. The Ø symbol indicates that no further genetic modification was included whereas “flgM” indicates the introduction of a flgM mutation to the indicated genetic background. β-galactosidase values presented in S3 Table. The following strains were used to generate this panel: wild type (DS9461), flgM (DK313), swrA (DK288), swrA flgM (DK318), swrB (DK289), swrB flgM (DK319). C) The frequency of fluorescent “ON” cells expressing Phag-GFP in the indicated genetic backgrounds. Over 600 cells were counted per strain in triplicate to obtain average percentages and standard deviations. The Ø symbol indicates that no further genetic modification was included whereas “flgM” indicates the introduction of a flgM mutation to the indicated genetic background. Sample raw data images used to generate the percentages are presented in S1 Fig. Strains used to generate this panel: wild type (DS908), swrA (DS4015), swrB (DS4040), flgM (DS4264), swrA flgM (DS4034), and swrB flgM (DS4090).
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Related In: Results  -  Collection

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pgen.1005443.g002: SwrB increases σD activity but not σD protein levels.A) Western blot analysis of whole cell lysates from the indicated genetic backgrounds probed with anti-Hag, anti-SigD, and anti-SigA primary antibodies. Black carets indicate antibody specific targets. Gray caret indicates a non-specific cross-reacting band recognized by the anti-SigD antibody. Strains used to generate this panel: wild type (DS908), swrA (DS4015), swrB, (DS4040), flgM (DS4264), swrA flgM (DS4034), swrB flgM (DS4090). B) β-galactosidase assays of Phag-lacZ transcriptional activity expressed in Miller Units in the indicated genetic backgrounds. The Ø symbol indicates that no further genetic modification was included whereas “flgM” indicates the introduction of a flgM mutation to the indicated genetic background. β-galactosidase values presented in S3 Table. The following strains were used to generate this panel: wild type (DS9461), flgM (DK313), swrA (DK288), swrA flgM (DK318), swrB (DK289), swrB flgM (DK319). C) The frequency of fluorescent “ON” cells expressing Phag-GFP in the indicated genetic backgrounds. Over 600 cells were counted per strain in triplicate to obtain average percentages and standard deviations. The Ø symbol indicates that no further genetic modification was included whereas “flgM” indicates the introduction of a flgM mutation to the indicated genetic background. Sample raw data images used to generate the percentages are presented in S1 Fig. Strains used to generate this panel: wild type (DS908), swrA (DS4015), swrB (DS4040), flgM (DS4264), swrA flgM (DS4034), and swrB flgM (DS4090).
Mentions: Consistent with a reduction in flagellar filament assembly, cells lacking either SwrB or SwrA showed a reduced level of the flagellar filament protein, Hag, by Western blot analysis (Fig 2A). The reduction in Hag protein was likely due to reduced transcription of the hag gene as cells mutated for either SwrB or SwrA showed lower expression from a reporter in which the hag promoter (Phag) was fused to the lacZ gene encoding β-galactosidase (Phag-lacZ) (Fig 2B, white bars), and a reduced frequency of cells expressing a reporter in which the Phag promoter was fused to green fluorescent protein (GFP) (Phag-gfp) (Fig 2C, white bars, and S1 Fig) [39,41]. The Phag promoter is transcribed by RNA polymerase and the alternative sigma factor, σD [44]. Whereas cells mutated for SwrA exhibited a reduced level of σD protein, cells mutated for SwrB exhibited a level of σD protein comparable to the wild type (Fig 2A). We conclude that the absence of SwrB resulted in reduced expression of the hag gene but unlike the absence of SwrA, the defect occurred downstream of σD protein levels.

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