Mutations Suppressing the Lack of Prepilin Peptidase Provide Insights Into the Maturation of the Major Pilin Protein in Cyanobacteria

Abstract

Type IV pili are bacterial surface-exposed filaments that are built up by small monomers called pilin proteins. Pilins are synthesized as longer precursors (prepilins), the N-terminal signal peptide of which must be removed by the processing protease PilD. A mutant of the cyanobacterium Synechocystis sp. PCC 6803 lacking the PilD protease is not capable of photoautotrophic growth because of the impaired function of Sec translocons. Here, we isolated phototrophic suppressor strains of the original ΔpilD mutant and, by sequencing their genomes, identified secondary mutations in the SigF sigma factor, the γ subunit of RNA polymerase, the signal peptide of major pilin PilA1, and in the pilA1-pilA2 intergenic region. Characterization of suppressor strains suggests that, rather than the total prepilin level in the cell, the presence of non-glycosylated PilA1 prepilin is specifically harmful. We propose that the restricted lateral mobility of the non-glycosylated PilA1 prepilin causes its accumulation in the translocon-rich membrane domains, which attenuates the synthesis of membrane proteins.

Keywords: PilD peptidase; Synechocystis; Type IV pili; photosystem II; suppressor mutations.

FIGURE 1

Overview of mutations that restore photoautotrophic growth of the ΔpilD strain. (A) Sigma factor SigF contains three main domains (σ2, σ3, σ4); the suppressor mutation in rev1 is located in the σ2.1 region, which interacts with the TATA box of recognized genes. The SigF mutation found in the rev4 strain is located in domain σ4, which typically interacts with the -35 segment of the promoter (see a structural model bellow). Note that the rev4 strain contains another suppressor E95K mutation in the RNAP γ subunit. (B) The mutation identified in the rev2 strain causes the S3G amino-acid substitution in the signal peptide of the PilA1 protein. (C) The rev3 strain contains a point nucleotide substitution in the intergenic region between genes coding for the pilin proteins PilA1 and PilA2. Transcription unit TU1331 including its promoter (–10 and –35 elements; Kopf et al., 2014) is depicted together with the predicted pilA1 terminator (orange box; Hess, W. personal communication). According to Kopf et al. (2014), pilA2 and sll1696 genes do not have their own promoter, instead they seem to form one transcriptional unit with pilA1.

FIGURE 2

Characterization of spontaneous revertants derived from the ΔpilD strain. (A) Growth on agar plates was assessed for the WT, ΔpilD mutant and the four isolated ΔpilD revertants (rev1–4) at the 7th and 21st day of cultivation under mixotrophic conditions and (B) under photoautotrophic conditions. (C) Photoautotrophic growth of the WT and rev1-4 strains. Cells were grown in liquid culture and optical density was monitored spectroscopically. (D) Membrane protein complexes, isolated from the WT and mutant strains grown for 48 h in Glc^–, were solubilized and separated by CN-PAGE. The loading corresponds to the same number of cells per lane based on OD₇₃₀; 4 μg of Chl for WT. After separation, the gel was scanned (see Supplementary Figure 2) and PSII complexes were detected by Chl fluorescence after excitation by blue light in LAS 4000 (Fuji). PSII[1] and PSII[2] indicates the monomeric and dimeric PSII, respectively.

FIGURE 3

Aggregation of pilin-less cells and ΔpilD suppressor strains. Cell cultures were cultivated in Erlenmeyer flasks on a rotary shaker at 120 rpm in mixotrophic (Glc⁺) and photoautotrophic (Glc^–) conditions and then poured into Petri dishes and photographed at a similar optical density (730 nm).

FIGURE 4

Cellular levels of pilA1 mRNA in WT, ΔpilD mutant, and ΔpilD revertant strains. RNA was isolated from WT and mutant cells that were grown for 2 days in the presence (A) or absence (B) of glucose. RNA was blotted and hybridized sequentially with radiolabeled probes against pilA1 mRNA and 16S rRNA. An unknown large transcript, detected with the pilA1 probe, is indicated by an asterisk. As the signal of this long transcript is much weaker than the signal of pilA1, an enhanced image of the upper part of the membrane is also shown. ΔpilA1/2 and sigF^– mutants were included as negative controls; 3.75 μg of total isolated RNA was loaded in each lane. (C,D) Hybridization was repeated using independent cell cultures, the density of the signals were quantified using ImageQuant TL 10 (GE Healthcare), normalized to the signal of 16S RNA and plotted. The values represent means ± SD from three independent measurements. Asterisks indicate statistically significant differences in the pilA1 mRNA levels between WT and other strains as tested using a paired Student’s t-test (* P < 0.05).

FIGURE 5

Cellular levels of pPilA1^g and pPilA1ⁿ proteins and the stability of YidC and Ycf48 in the WT, ΔpilD mutant, and ΔpilD revertant strains. Membrane proteins were isolated from Glc⁺ (A) and Glc^– conditions (B), separated by SDS-PAGE and blotted; the loading corresponded to the same number of cells based on OD₇₃₀. Pilin and prepilin forms of PilA1, YidC insertase, and Ycf48 were immunodetected by specific antibodies. The gel stained with SYPRO Orange before blotting is shown below the blot as a loading control. pPilA1^g, pPilA1ⁿ designate glycosylated and non-glycosylated prepilins, respectively.

FIGURE 6

Synthesis of prepilin and PSII complexes in WT, ΔpilD mutant, and ΔpilD revertant strains. (A) WT and rev strains from Glc^– conditions and the ΔpilD (Glc⁺) were radiolabeled with a mixture of [³⁵S]-Met/Cys using a 20-min pulse. Isolated membrane proteins were separated by 16–20% SDS-PAGE; 1 μg of Chl was loaded for each strain. The gels were stained with Coomassie Blue (Supplementary Figure 5), dried, and the labeled proteins were detected by phosphorimaging. The signal of pPilA^g is indicated by a blue arrowhead and radiolabeled PilA1 by brown arrowhead. Asterisk indicates an intensively labeled band that has the same mobility in the gel as the pPilAⁿ. All three forms of radiolabeled D1 subunit are marked by green dashed lines; pD1 and iD1 indicate unprocessed and partially processed forms of the D1 subunit. D2 subunit is marked by a purple dashed line. (B) The radiolabeled rev1-4 samples (4 μg of Chl) were separated by 2D CN/SDS-PAGE, stained by SYPRO Orange stain (see Supplementary Figure 6 for the stained gels) and blotted onto a PVDF membrane. The labeled proteins were detected by phosphorimaging and the signal of prepilins was verified by immunodetection (Supplementary Figure 7). Dashed red lines highlight PSII assembly intermediates RCIIa and RCII* (Yu et al., 2018), green lines highlight labeled PSII subunits assembled into monomeric and dimeric PSII. The signals of pPilA^g and pPilAⁿ are indicated by blue and red arrowheads, respectively.

FIGURE 7

The role of pilA2-sll1696 operon in the PilA1 biogenesis and the co-immunoprecipitation of the SecY translocase with prepilin. (A) Immunodetection of PilA1 in membrane proteins isolated from WT and pilA2^– cells grown under Glc^– conditions. Membrane proteins were separated by SDS-PAGE and blotted; PilA1ⁿ indicates a putative non-glycosylated form of PilA1. (B) Immunodetection of pPilA1 in separated membrane proteins from ΔpilD and ΔpilD/pilA2^– cells. Both strains were grown in Glc⁺ conditions. (C) The anti-SecY antibody was incubated with membrane proteins isolated from the WT, ΔpilD mutant and rev2 and rev3 strains grown in Glc⁺ conditions and then immobilized on Protein A—Sepharose (Sigma, Germany), eluted in SDS buffer, and separated by SDS-PAGE. The gel was blotted and probed with anti-SecY and anti-PilA1 antibodies; the anti-SecE antibody was re-probed as a control. The level of PilA1 forms is also shown for the identical membrane samples to those used for the co-immunoprecipitation.

FIGURE 8

MD simulations of the Synechocystis pPilA1 and pPilA1-S3G proteins in a membrane bilayer. (A) Snapshots of the initial (0 ns) and the representative final (500 ns) conformation of pPilA1 in the TM (Supplementary Video 1). The signal peptide of pPilA1 is depicted in blue, the rest of the protein is in gray. Oxygen atoms in lipid molecules are shown as red spheres, sulfur (in sulfoquinovosyl diacylglycerol) as yellow spheres, phosphate atoms as orange spheres and nitrogen atoms (in POPC) as blue spheres. The position of Ser3 is highlighted by a magenta ball. Water molecules were omitted for clarity. (B) A snapshot of the initial (0 ns) and representative final (500 ns) positions of pPilA1 in the POPC bilayer (Supplementary Video 2). (C) Number of intermolecular hydrogen bonds between the pPilA1 signal peptide and TM or POPC lipid molecules for each frame (0–500 ns) of the MD simulation compared with the pPilA1-S3G. Numbers in parentheses indicate an average number of hydrogen bonds per frame formed during the simulation, numbers in bold show the number of hydrogen bonds for the period between 250–500 ns. See Supplementary Figures 8A,B for the description of hydrogen bonds between the lipid polar region and the pPilA1 signal peptide. (D) Representative final positions of PilA1-SG3 in the TM and POPC bilayer (Supplementary Videos 3, 4); see also Supplementary Figures 8C,D for the initial positions of the pPilA-S3G mutant protein.

FIGURE 9

Structure alignment of the Synechocystis SigF with group 2 and ECF sigma factors bound in RNAP holoenzymes. (A) The structure of Synechocystis SigF was predicted using I-TASSER (C-score = 0.98; Yang et al., 2015) and aligned with the crystal structure of the transcription initiation complex from E. coli containing RpoS (PDB code 5IPL; Liu et al., 2016) using Chimera software (Pettersen et al., 2004). RpoS is depicted in gold, SigF in blue, DNA in tan and the RNAP β’ subunit in white. Residues in SigF that are mutated in rev1 and rev4 strains are shown in dark blue; Q94 corresponds to the E95 residue in the Synechocystis RNAP γ subunit. (B) The predicted structure of SigF was aligned with the crystal structure of the RNAP-SigH transcription initiation complex from Mycobacterium tuberculosis (PDB code 6DV9; Li et al., 2019). ECF sigma factors lack the σ3 domain, however, the SigF R211 residue mutated in rev4 is highly conserved in ECF sigma factors (R153 in SigH; Li et al., 2019). SigH is depicted in magenta, SigF in blue, RNAP subunit β in white and the C-terminal flap-tip helix of the RNAP-β subunit (βFTH) in green (see text for details).