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. 2010 Jan;75(2):376-93.
doi: 10.1111/j.1365-2958.2009.06973.x. Epub 2009 Nov 17.

C-ring requirement in flagellar type III secretion is bypassed by FlhDC upregulation

Marc Erhardt  1 , Kelly T Hughes
Affiliations

C-ring requirement in flagellar type III secretion is bypassed by FlhDC upregulation

Marc Erhardt et al. Mol Microbiol. 2010 Jan.

Abstract

The cytoplasmic C-ring of the flagellum consists of FliG, FliM and FliN and acts as an affinity cup to localize secretion substrates for protein translocation via the flagellar-specific type III secretion system. Random T-POP transposon mutagenesis was employed to screen for insertion mutants that allowed flagellar type III secretion in the absence of the C-ring using the flagellar type III secretion system-specific hook-beta-lactamase reporter (Lee and Hughes, 2006). Any condition resulting in at least a twofold increase in flhDC expression was sufficient to overcome the requirement for the C-ring and the ATPase complex FliHIJ in flagellar type III secretion. Insertions in known and unknown flagellar regulatory loci were isolated as well as chromosomal duplications of the flhDC region. The twofold increased flhDC mRNA level coincided in a twofold increase in the number of hook-basal bodies per cell as analysed by fluorescent microscopy. These results indicate that the C-ring functions as a nonessential affinity cup-like structure during flagellar type III secretion to enhance the specificity and efficiency of the secretion process.

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Figures

Figure 1
Figure 1
(A) Steps in the assembly of the bacterial flagellum. The self-assembly process of the flagellum initiates with formation of the MS-ring (FliF) in the cytoplasmic membrane. Afterwards, a flagellar-specific type III secretion (T3S) apparatus assembles within a central pore of the MS-ring and the C-ring is attached to the cytoplasmic face of the MS-ring. At this point, flagellar secretion substrates are now selectively secreted via the T3S apparatus and coupled to the proton-motive force (Paul et al., 2008). The hook polymerizes to an app. length of 55 nm that is determined by the molecular ruler FliK and this triggers a secretion specificity switch from rod-hook-type substrates to late-secretion substrates. Upon completion of the hook-basal-body complex, the negative regulator of late substrates gene expression, the anti-σ28 factor FlgM, is secreted thereby freeing σ28 to initiate transcription of late substrate genes, like fliC or the genes of the chemosensory system. (B) Hook-β-lactamase reporter system. Left panel: In a strain deleted for the proximal rod subunit genes, flgBC, hook-basal-body type substrates are secreted via the flagellar-specific T3S apparatus into the periplasm and subsequently degraded. β-lactamase (Bla) fused C-terminally to the hook protein FlgE is not degraded and confers resistance against lactam antibiotics, like ampicillin when secreted into the periplasm (ApR). Right panel: In a strain additionally deleted for two-thirds of the cytoplasmic C-ring (ΔfliMN), flagellar T3S is severely impaired and thus FlgE-Bla is not secreted into the periplasm and the strain is sensitive against ampicillin (ApS).
Figure 2
Figure 2. Locations of unstable Mud insertions in the Salmonella chromosome in strains duplicated for the flhDC region that conferred ampicillin resistance in the absence of the C-ring
The positions of eight ApR Mud insertions in the Salmonella chromosome were determined by DNA sequence analysis. Individual Mud insertions are indicated by a grey triangle and additionally the chromosomal loci of the insertions. These unstable ApR Mud insertions resulted from transposition into duplications of the flhDC region of the chromosome thus conferring ampicillin resistance in the absence of the C-ring by increased flhDC expression. The precise insertion points are given in Table 1.
Figure 3
Figure 3
(A) Overexpression of flhDC from the arabinose promoter confers ampicillin resistance in deletion mutants of the C-ring and ATPase complex. Strains harboring an additional, functional copy of flhDC under the control of the arabinose promoter were grown overnight in the absence of arabinose. Equal volumes of ten-fold serial dilutions were spotted on LB plates and PPBS Ap7.5 plates in the presence or absence of 0.2 % arabinose. Mutant strains missing two-thirds (ΔfliMN) or the complete C-ring (ΔfliG and ΔfliGMN), as well as a strain deleted for the ATPase complex FliHIJ, but not a mutant strain missing the MS-ring (ΔfliF) are able grow in the presence of excess FlhDC. WT = TH14902; ΔfliMN = TH15498; ΔfliG = TH15497; ΔfliGMN = TH14906; ΔfliF = TH14903; ΔfliHIJ = TH14905. (B) Effects of excess FlhDC on flagellar gene expression. Strain TH14156 (Para∷flhD+C+) was grown for 2.5 hours in LB media containing different concentrations of arabinose (0%, 0.05%, 0.2% and 0.6%) and total RNA was isolated of the pooled cultures of three biological replicates. Class 1 (flhDC), Class 2 (fliP and flgE), Class 3 (motAB) and rpoD transcript levels were analyzed by real-time qPCR as described in Experimental procedures. Relative gene expression was determined using the 2−ΔΔCT method (Livak & Schmittgen, 2001) and individual mRNA levels were normalized against multiple reference genes (rpoB, gyrB and gmk) and presented as fold change relative against the wildtype control (Vandesompele et al., 2002).
Figure 4
Figure 4. Schematic overview of T-POP insertions at seven different chromosomal loci that conferred ampicillin resistance in a C-ring deletion mutant
Individual T-POP insertions are shown by a triangle with an arrow indicating the direction of the tetA transcript. A summary of the precise insertion point of every T-POP insertion and the effect of the tetA transcript reading in adjacent chromosomal genes is given in Table 2.
Figure 5
Figure 5
(A) Relative flagellar Class 2 transcription levels of selected, ampicillin-resistance conferring, T-POP insertions. Relative Class 2 transcription from the fliL∷MudJ transcriptional reporter was measured by β-galactosidase assays as described in Experimental procedures. Class 2 transcription was measured in the presence or absence of tetracycline and normalized against the wildtype control. Addition of tetracycline induces transcription from the tetA promoter, which proceeds into chromosomal DNA adjacent to the T-POP insertion. The data shown are the mean +/− SD of at least three independent, biological replicates. (B) Relative ecnR transcription in a strain with a T-POP insertion in yiek near ecnR. Relative ecnR transcription from the ecnR∷MudJ transcriptional reporter was measured by β-galactosidase assays as described in Experimental procedures in strains harboring a T-POP insertion upstream of ecnR (PecnR∷T-POP) or in yiek near ecnR (ecnR7). ecnR transcription was measured in the presence or absence of tetracycline and normalized against the wildtype control. The data shown are the mean +/− SD of at least three independent, biological replicates. (C) Relative flagellar Class 1 transcription in strains overexpressing the transcriptional regulators SlyA and LrhA. Relative Class 1 transcription from the flhC∷MudJ transcriptional reporter was measured by β-galactosidase assays as described in Experimental procedures in strains harboring slyA or lrhA under control of the arabinose promoter. Class 1 transcription was measured in the presence or absence of 0.2 % arabinose, respectively and normalized against the wildtype control. The data shown are the mean +/− SD of at least three independent, biological replicates. (D) Relative flagellar Class 1 transcription in a strain overexpressing the flagellar-specific sigma factor σ28. Relative Class 1 transcription from the flhC∷MudJ transcriptional reporter was measured by β-galactosidase assays as described in Experimental procedures in a strain harboring fliA, the gene encoding for the flagellar-specific sigma factor σ28, under control of the arabinose promoter. Class 1 transcription was measured in the presence or absence of 0.2 % arabinose, respectively and normalized against the wildtype control. The data shown are the mean +/− SD of at least three independent, biological replicates.
Figure 6
Figure 6. Motility, flagellar Class 1 and Class transcription and FlgE-Bla protein levels of PflhD promoter mutants
(A) Motility of PflhD promoter mutants. A representative image shows the motility of different PflhD promoter mutants after 4 hours of incubation at 37°C. A dotted white line indicates the radius of the swarming circle. Consensus -10 box mutants of the P3, P5 and P6 flhD promoter display motility comparable to wildtype. The consensus -10 box mutant of the P1 flhD promoter and the combination of the consensus -10 box mutations of both the P1 and P4 flhD promoter showed increased swimming behavior, whereas the consensus -10 box mutation of the P2 flhD promoter displayed a decreased swimming speed. The PflhD promoter mutants were constructed in a strain harboring a functional fliM-gfp mutation (denoted here as WT). Motility was compared to S. enterica LT2 to show that the fliM-gfp mutation did not affect flagellar assembly or motility. (B) Quantitative motility assay of PflhD promoter mutants. The motility of different mutant strains was measured by determining the motility diameter after 4 hours incubation at 37 °C. For each strain the diameter of 10 independent biological replicates was measured and normalized to the motility diameter of the wildtype control (fliM-gfp) on the same motility plate. Motility was compared to S. enterica LT2 to show that the fliM-gfp mutation did not affect flagellar assembly or motility. The data is presented as box diagrams with whiskers according to the Tukey method. A horizontal line indicates the median and a black cross the mean. Outliners are represented by a black dot. (C) Relative Class 1, Class 2 and Class 3 mRNA levels as quantified by real-time qPCR. Class 1 (flhDC), Class 2 (flgE), Class 3 (motAB) and rpoD mRNA levels were analyzed by real-time qPCR as described in Experimental procedures. Relative gene expression was determined using the 2−ΔΔCT method (Livak & Schmittgen, 2001). Individual mRNA levels were normalized against rpoA, gyrB and gmk transcript levels and presented as fold change relative against the wildtype control (Vandesompele et al., 2002). The data shown represents the mean of three independent, biological samples ± SD. (D) Relative FlgE-Bla protein levels analyzed by quantitative Western blotting. Whole cell lysates were prepared of four independent biological replicates of wildtype or P1 + P4 consensus -10 box flhD promoter mutant cells respectively. The experiment was performed in a ΔfliP background in order to analyze total protein levels. The reporter protein FlgE-Bla was expressed from its native promoter and FlgE-Bla was detected using FlgE-specific antibodies and quantified as described in Experimental procedures. Data shown are the mean of four replicates ± SD.
Figure 7
Figure 7. Number of assembled hook-basal-body (HBB) complexes as analyzed by hook immunostaining
(A) Number of HBB complexes of the wildtype control. Left panel: distribution of HBBs per cell. Non-linear fitting of the Gaussian distribution was employed (red line) and the average HBB number per cell is 3.8 ± 2.8 (n = 144). Middle panel: hook immunostaining of exemplary cells expressing wildtype levels of flhDC. Green: HBB complexes (FlgE∷3×HA tag) labeled with anti-hemagglutinin antibodies coupled to Alexa Fluor 488. Red: cell membrane stained with FM-64. Blue: DNA stained with Hoechst. Right panel: distribution of HBBs per µM cell length. Cell length was measured based on the FM-64 membrane staining. Non-linear fitting of the Gaussian distribution was employed (red line) and the average HBB number per µM is 1.9 ± 1.2 (n = 144). Scale bar is 2 µM. (B) Number of HBB complexes of the PflhD P1 consensus -10 box mutant. Left panel: distribution of HBBs per cell. Non-linear fitting of the Gaussian distribution was employed (red line) and the average HBB number per cell is 9.0 ± 1.5 (n = 34). Middle panel: hook immunostaining of exemplary PflhD P1 consensus -10 box mutant cells with increased flhDC expression levels. Green: HBB complexes (FlgE∷3×HA tag) labeled with anti-hemagglutinin antibodies coupled to Alexa Fluor 488. Red: cell membrane stained with FM-64. Blue: DNA stained with Hoechst. Right panel: distribution of HBBs per µM cell length. Cell length was measured based on the FM-64 membrane staining. Non-linear fitting of the Gaussian distribution was employed (red line) and the average HBB number per µM is 3.9 ± 1.4 (n = 34). Scale bar is 2 µM. (C) Number of HBB complexes of the PflhD P1 + P4 consensus -10 box mutant. Left panel: distribution of HBBs per cell. Non-linear fitting of the Gaussian distribution was employed (red line) and the average HBB number per cell is 7.9 ± 1.9 (n = 53). Middle panel: hook immunostaining of exemplary PflhD P1 + P4 cinsensus -10 box mutant cells with increased flhDC expression levels. Green: HBB complexes (FlgE∷3×HA tag) labeled with anti-hemagglutinin antibodies coupled to Alexa Fluor 488. Red: cell membrane stained with FM-64. Blue: DNA stained with Hoechst. Right panel: distribution of HBBs per µM cell length. Cell length was measured based on the FM-64 membrane staining. Non-linear fitting of the Gaussian distribution was employed (red line) and the average HBB number per µM is 3.9 ± 0.6 (n = 53). Scale bar is 2 µM. (D) Number of HBB complexes of the PflhD P2 consensus -10 box mutant. Left panel: distribution of HBBs per cell. Middle panel: hook immunostaining of exemplary PflhD P2 consensus -10 box mutant cells with decreased flhDC expression levels. Green: HBB complexes (FlgE∷3×HA tag) labeled with anti-hemagglutinin antibodies coupled to Alexa Fluor 488. Red: cell membrane stained with FM-64. Blue: DNA stained with Hoechst. Right panel: distribution of HBBs per µM cell length. Cell length was measured based on the FM-64 membrane staining. Scale bar is 2 µM.
Figure 8
Figure 8. Regulatory network of flagellar Class 1 gene expression and model for flagellar type III secretion
(A) Schematic representation of the regulatory network of flagellar Class 1 gene expression of Salmonella enterica. Arrows represent interactions between different regulators of flagellar Class 1 transcription or post-class 1 transcription. LrhA (Lehnen et al., 2002) and SlyA (Spory et al., 2002) have been previously identified as regulators of flhDC and FliC respectively in Escherichia coli. Our T-POP mutagenesis identified LrhA and SlyA as negative regulators of flhDC expression in Salmonella enterica, as well as several known regulators like EcnR (Wozniak et al., 2008) or RcsB (Wang et al., 2007). We also identified several putative regulators of Class 2 transcription like STM1856 and STM2401/STM2402. (B) Model of the role of the C-ring affinity cup in flagellar type III secretion. Folded secretion substrates in complex with the cargo delivery complex FliHIJ dock to affinity sites of the C-ring awaiting secretion. Before PMF-dependent secretion, the secretion substrates are presumably unfolded using ATP-hydrolysis of the ATPase FliI thereby increasing the efficiency of the secretion process. The C-ring presumably acts as an affinity cup to enhance the specificity and efficiency of the flagellar type III secretion process under wildtype conditions by recruiting secretion substrates bound to the FliHIJ cargo delivery complex. Flagellar type III secretion is possible in the absence of the C-ring and ATPase complex if FlhD4C2 levels are increased. Increased FlhD4C2 levels result in elevated level of Class 2 secretion substrates and additionally in more potential secretion systems by doubling the number of available basal bodies. Accordingly, increased substrate levels and more potential secretion systems overcome the requirement for both the C-ring and the ATPase complex in flagellar T3S.
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