Involvement of ArlI, ArlJ, and CirA in Archaeal Type-IV Pilin-Mediated Motility Regulation

Abstract

Many prokaryotes use swimming motility to move toward favorable conditions and escape adverse surroundings. Regulatory mechanisms governing bacterial flagella-driven motility are well-established, however, little is yet known about the regulation underlying swimming motility propelled by the archaeal cell surface structure, the archaella. Previous research showed that deletion of the adhesion pilins (PilA1-6), subunits of the type IV pili cell surface structure, renders the model archaeon Haloferax volcanii non-motile. In this study, we used EMS mutagenesis and a motility assay to identify motile suppressors of the ΔpilA[1-6] strain. Of the eight suppressors identified, six contain missense mutations in archaella biosynthesis genes, arlI and arlJ. Overexpression of these arlI and arlJ mutant constructs in the respective multi-deletion strains ΔpilA[1-6]ΔarlI and ΔpilA[1-6]ΔarlJ confirmed their role in suppressing the ΔpilA[1-6] motility defect. Additionally, three suppressors harbor co-occurring disruptive missense and nonsense mutations in cirA, a gene encoding a proposed regulatory protein. A deletion of cirA resulted in hypermotility, while cirA overexpression in wild-type cells led to decreased motility. Moreover, qRT-PCR analysis revealed that in wild-type cells, higher expression levels of arlI, arlJ, and the archaellin gene arlA1 were observed in motile early-log phase rod-shaped cells compared to non-motile mid-log phase disk-shaped cells. Conversely, ΔcirA cells, which form rods during both early and mid-log phases, exhibited similar expression levels of arl genes in both growth phases. Our findings contribute to a deeper understanding of the mechanisms governing archaeal motility, highlighting the involvement of ArlI, ArlJ, and CirA in pilin-mediated motility regulation.

Figure 1.. EMS mutagenesis results in ΔpilA[1–6] suppressor mutants that regain swimming motility.

Two representative images of soft agar plates (0.3% w/v) that have been streaked with Hfx. volcanii ΔpilA[1–6] incubated with 0.05 M EMS for 20 minutes. White arrows point to outgrowths of motile suppressors after five days of incubation at 45°C.

Figure 2.. Residues of interest as sequence alignments, 3D structures, and genomic locations.

Sequence alignments of a) ArlI, b) ArlJ and e) CirA from Hfx. volcanii with homologs from four additional haloarchaeal species and five non-halophilic archaeal species of different phyla. Regions of the alignment surrounding the mutated residues from Table 1 are shown; other regions of the protein are either omitted or set to 25% transparency to ease visualization. Conserved residues across species are indicated in shades of blue, with darker blues corresponding to highly conserved residues. The ArlI conserved Aspartate Box motif and the CirA Walker A domain are indicated. c) Residues of ArlI and ArlJ corresponding to ΔpilA[1–6] suppressor mutations mapped to the AlphaFold structure predictions of the ArlI hexamer and ArlJ dimer, respectively. Yellow circles indicate the residues of interest. d) cirA is found between the archaellin genes (arlA1, arlA2) and the rest of the arl genes (arlC/E–arlJ). It is transcribed in the opposite direction as the arl genes. The genic locations of the residues corresponding to the mutations found in the ΔpilA[1–6] suppressor mutants as well as hypermotile mutants from a previous study are indicated, along with the conserved Walker A and Walker B motifs.

Figure 3.. Overexpression of mutant ArlI or ArlJ results in differential motility.

a) pTA963 expression vectors containing genes encoding either wild-type or mutant proteins were transformed into the indicated deletion strains. Empty vector was also transformed as control for all four deletion strains. Cells were stab-inoculated into 0.3% w/v agar plates, incubated at 45°C for 3 days and then at RT for 1 day before imaging. White bar represents 1 cm. b) Quantification of halo radius in millimeters across 7 to 10 replicates for each strain. Statistical analysis was performed using the unpaired nonparametric t-test. ** p<0.01, *** p<0.001 and **** p<0.0001.

Figure 4.. Overexpression of CirA results in reduced motility.

a) Empty pTA963 or vector containing cirA was transformed into wild type and ΔcirA strains. Cells were stab-inoculated onto 0.3% w/v agar plates, incubated at 45°C for 3 days, then at RT for 1 day before imaging. White bar represents 1 cm. b) Quantification of halo radius across 12 replicates of each stabbed strain. Statistical analysis was performed using the unpaired nonparametric t-test. * p<0.05 and **** p<0.0001.

Figure 5.. ΔcirA remains rod-shaped in mid-log phase.

Early-log (OD₆₀₀ 0.05) and mid-log (OD₆₀₀ 0.35) phase cultures of wild-type H53 and ΔcirA were harvested and cells were imaged using differential interference microscopy (DIC). The black bar represents 20 μm.

Figure 6.. ΔcirA displays a different transcription profile of arlA1, arlI, and arlJ than wild type.

qRT-PCR was conducted using primers for 16S rRNA, arlA1, arlI, and arlJ under the following conditions: 1) wild type at early-log; 2) wild type at mid-log; 3) ΔcirA at early-log; and 4) ΔcirA at mid-log. Values are shown as the negative difference in threshold crossing point −ΔC_t of arlA1, arlI, and arlJ relative to 16S rRNA. Error bars represent the standard deviation of five technical replicates.