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.2022 Apr 29;18(4):e1010188.
doi: 10.1371/journal.pgen.1010188. eCollection 2022 Apr.

The differential expression of PilY1 proteins by the HsfBA phosphorelay allows twitching motility in the absence of exopolysaccharides

Affiliations

The differential expression of PilY1 proteins by the HsfBA phosphorelay allows twitching motility in the absence of exopolysaccharides

Shuanghong Xue et al. PLoS Genet..

Abstract

Type Four Pili (T4P) are extracellular appendages mediating several bacterial functions such as motility, biofilm formation and infection. The ability to adhere to substrates is essential for all these functions. In Myxococcus xanthus, during twitching motility, the binding of polar T4P to exopolysaccharides (EPS), induces pilus retraction and the forward cell movement. EPS are produced, secreted and weakly associated to the M. xanthus cell surface or deposited on the substrate. In this study, a genetic screen allowed us to identify two factors involved in EPS-independent T4P-dependent twitching motility: the PilY1.1 protein and the HsfBA phosphorelay. Transcriptomic analyses show that HsfBA differentially regulates the expression of PilY1 proteins and that the down-regulation of pilY1.1 together with the accumulation of its homologue pilY1.3, allows twitching motility in the absence of EPS. The genetic and bioinformatic dissection of the PilY1.1 domains shows that PilY1.1 might be a bi-functional protein with a role in priming T4P extension mediated by its conserved N-terminal domain and roles in EPS-dependent motility mediated by an N-terminal DUF4114 domain activated upon binding to Ca2+. We speculate that the differential transcriptional regulation of PilY1 homologs by HsfBA in response to unknown signals, might allow accessorizing T4P tips with different modules allowing twitching motility in the presence of alternative substrates and environmental conditions.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Seven suppressor mutants restore motility of aM.xanthus ΔepsW ΩcglB strain.
(A) Motility phenotypic assays of DZ2 (wild type), EM650 (ΔepsW), TM770 (ΩcglB), EM749 (ΔepsW ΩcglB), EM785 (ΔepsW ΩcglB sup4), EM788 (ΔepsW ΩcglB sup7), EM789 (ΔepsW ΩcglB sup8), EM790 (ΔepsW ΩcglB sup9), EM791 (ΔepsW ΩcglB sup10), EM792 (ΔepsW ΩcglB sup11), EM793 (ΔepsW ΩcglB sup12), on 0.5% agar and imaged at 48H (B) Motility phenotypic assays of seven suppressor mutants on 0.5% agar and imaged at 72H. (C) List of seven suppressor mutations. (D) Schematic representation of theM.xanthus genomic region containing themxan_5362–5366 genes andmxan_0359–0364 genes. Red arrows represent the mutation positions.
Fig 2
Fig 2. ΔepsW suppressors are loss-of-function mutants and their motility restoration is T4P dependent and EPS-independent.
(A) Motility assays of backcrossed suppressors and in-frame deletion mutants in ΔepsW, ΔepsWΩcglB and ΔepsWΩpilA backgrounds on 0.5% agar at 72H.hsfA* corresponds to suppressor C9,hsfB*C4 to C4,hsfB*C7 to C7,pilY1.1* to C10/C11/C12,pilW1* to C8. (B) Trypan Blue assays showing the relative EPS production of the indicated strains. All data represents the mean and standard deviation of at least three biological replicates. Raw data are available in S4 Table.
Fig 3
Fig 3.pilY1.1 andpilY1.3 are divergently regulated by the HsfBA phosphorelay.
(A) Schematic representation of HsfB and HsfA. Numbers indicate the aminoacid position; the response regulator domains are in blue; the histidine kinase domain in orange; the ATP hydrolysis domain in purple; the helix-turn-helix domain in green. The arrow indicates the phosphotransfer from the conserved histidine H in HsfB to the aspartate D in HsfA. (B) Motility phenotypes of DZ2 (wild type), EM817 (hsfA* from sup9), EM799 (hsfB*C4 from sup4), EM816 (hsfB*C7 from sup7), EM806 (ΔhsfA) and EM807 (ΔhsfB) on 0.5% agar at 48H. The star * represents suppressor mutations. (C) Relative expression of the indicated genes in the ΔhsfA mutant measured as log2-fold changes by RNA-Seq. Results are mean values from three biological replicates. The green and red colors indicate up- and down-regulation, respectively, as compared to wild type. (D) Relative expression of the indicated genes in the ΔhsfA mutant vs wild type measured as log10-transformed fold changes by qRT-PCR. Results are mean values from two biological replicates. Error bars indicate standard deviations. Raw data are available in S4 Table. (E) Representative Electrophoretic Mobility Shift Assays (EMSA) on 6.5% polyacrylamide. The indicated concentrations of purified His6-HsfA were incubated with the indicated DNA fragments. White and red arrowheads indicate free DNA and His6-HsfA-bound DNA, respectively. Putative HsfA binding in PhsfB, PpilY1.1, PpilY1.3 are aligned against the consensus. Conserved nucleotides are in red.
Fig 4
Fig 4. EPS-independent motility requires high levels of PilY1.3.
(A) Sheared pili of the indicated strains were diluted as indicated and probed with αPilA (top panel), αFLAG (middle panel) and αPilC antibodies (bottom panel) for dot blots. The graphic shows, in a biological duplicate, the relative intensity of the FLAG signal in ΔhsfA and ΔpilY1.1 vs wild type and normalized against the PilA signal. Error bars represent the standard deviation calculated on technical triplicates. Raw data are available in S4 Table. (B) Motility assays of EM810 (ΔepsW ΔhsfA), EM860 (ΔepW ΔhsfA ΔpilY1.3), EM813 (ΔepsW ΔpilY1.1) and EM864 (ΔepW ΔpilY1.1 ΔpilY1.3) on 0.5% agar at 72H.
Fig 5
Fig 5.pilY1.1 andpilY1.3 have functions in twitching motility.
(A) Motility phenotypes of DZ2 (wild type), TM389 (ΔpilA), EM808 (ΔpilY1.1), EM882 (ΔpilY1.2), EM856 (ΔpilY1.3), EM879 (ΔpilY1.1 ΔpilY1.2), EM880 (ΔpilY1.2 ΔpilY1.3), EM857 (ΔpilY1.1 ΔpilY1.3) and EM881 (ΔpilY1.1 ΔpilY1.2 ΔpilY1.3) on 0.5% agar and imaged at 48H. T4P-motile strains generate flares at the colony edge while non-motile strains form smooth-edged colonies. (B) Diameters of colonies of strains shown in (A). For each strain, the mean value of 3 biological replicates is plotted. Blue bar shows the diameters of the initial spots. Orange bar shows the swarm spreading. Raw data are available in S4 Table.
Fig 6
Fig 6. PilY1.1 and PilY1.3 are essential for T4P formation.
(A) Western blot detection of PilA and PilC in sheared-off T4P and total cell lysates of DZ2 (wild type), TM389 (ΔpilA), EM808 (ΔpilY1.1), EM882 (ΔpilY1.2), EM856 (ΔpilY1.3), EM857 (ΔpilY1.1 ΔpilY1.3), EM879 (ΔpilY1.1 ΔpilY1.2), EM880 (ΔpilY1.2 ΔpilY1.3), and EM881 (ΔpilY1.1 ΔpilY1.2 ΔpilY1.3). Membranes were probed with αPilA antibodies (top rows) and αPilC antibodies (bottom rows). The blue arrowhead indicates the band corresponding to the PilA protein and the black arrowhead indicates a cross-reacting band. (B) TEM micrographs of cells from the indicated strains. Arrowheads indicate T4P on the cell surface. (C) 5s time-lapse series, obtained by TIRF microscopy, of labeled T4P pilin filaments and polar cluster enrichment of the indicated strains bearing attmx8::PpilA-pilAD71C.
Fig 7
Fig 7. The C-terminal region of PilY1.1 and PilY1.3 is essential for T4P assembly.
(A) Schematic representation of PilY1 proteins. Numbers at each side of the grey bars indicates the length of protein fragments. CBS (purple) stays for Calcium Binding Sites; VWA for Von Willbrand factor A (blue); DUF (green) stays for DUF4114; the conserved PilY1 domain is indicated in orange. The letters indicate the conserved protein sequences. The underlined amino acids indicate a consensus for a given motif. The star* corresponds to the conserved active D498 of PilY1.1. (B) Motility phenotypes of DZ2 (wild type), EM901 (pilY1.1Δduf), EM898 (pilY1.3Δvwa), EM902 (ΔpilY1.3 pilY1.1Δduf), EM897 (ΔpilY1.1 pilY1.3Δvwa), EM909 (pilY1.1ΔdufpilY1.3Δvwa), EM937 (pilY1.1D498A) and EM938 (ΔpilY1.3 pilY1.1D498A) on 0.5% agar and pictured at 48H. (C) Western blot detection of PilA and PilC in sheared-off T4P and total cell lysate of the indicated strains and using DZ2 (wild type) and EM747 (ΔpilB) as positive and negative controls, respectively. Membranes were probed with αPilA antibodies (top rows) and αPilC antibodies (bottom rows). (D) TEM micrographs of the indicated strains. Arrowheads indicate T4P on the cell surface. (E) 5s time-lapse series, obtained by TIRF microscopy, of labeled T4P pilin filaments and polar cluster enrichment of the indicated strains bearing attmx8::PpilA-pilAD71C.
Fig 8
Fig 8. Proposed model on the regulation and role of PilY1 proteins.
(A) Unknown environmental or metabolic signals induce the HsfBA two-component system that regulates positively the expression of PilY1.1 and negatively that of PilY1.3. PilY1.1 and PilY1.3 might be bi-functional proteins involved i) in priming T4P extension via their conserved C-terminal domains and putative interactions with cytoplasmic Pil components; ii) via their variable N-terminal domains and upon their translocation to the pilus tips, in adhering to components of the extracellular matrix deposited on the substrate or on the surface of neighboring cells. PilY1.1 might bind to EPS while PilY1.3 might bind to a different component of the matrix such as a protein or a nucleic acid. (B) In the absence (ΔpilY1.1) or low amounts (ΔhsfA) of PilY1.1, PilY1.3 could be preferentially assembled on T4P allowing motility thanks to the binding to substrates different than EPS.
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