Alanine 32 in PilA is important for PilA stability and type IV pili function in Myxococcus xanthus

Abstract

Type IV pili (TFP) are membrane-anchored filaments with a number of important biological functions. In the model organism Myxococcus xanthus, TFP act as molecular engines that power social (S) motility through cycles of extension and retraction. TFP filaments consist of several thousand copies of a protein called PilA or pilin. PilA contains an N-terminal α-helix essential for TFP assembly and a C-terminal globular domain important for its activity. The role of the PilA sequence and its structure-function relationship in TFP-dependent S motility remain active areas of research. In this study, we identified an M. xanthus PilA mutant carrying an alanine to valine substitution at position 32 in the α-helix, which produced structurally intact but retraction-defective TFP. Characterization of this mutant and additional single-residue variants at this position in PilA demonstrated the critical role of alanine 32 in PilA stability, TFP assembly and retraction.

Fig. 1.

Phenotype summary of the PilA mutant A32V. (a) PilA amino acid sequence in the M. xanthus wild-type strain. Grey type shows the signal peptide of PilA; underlined sequences correspond to the N-terminal α-helix predicted using the scratch protein predictor (http://scratch.proteomics.ics.uci.edu/). The residue marked with a dot underneath is alanine 32. (b) Motility, surface properties and social phenotypes of mutant A32V. Top to bottom rows show swarming on 0.3 % agar surfaces, swarming on 1.5 % agar surfaces, EPS production and fruiting body (FB) formation. Strains from left to right are wild-type strain DK1622 (WT), ΔpilA, mutant A32V and ΔpilT. All experiments were repeated three times and representative images are shown. (c) Whole-cell PilA production and surface pili were tested using Western blots. The same four strains were used as in panel (b). (d) Electron microscopy images showing wild-type and A32V cell surface pili (indicated by black arrows). Bar, 200 nm.

Fig. 2.

TFP in A32V have binding ability but are unable to retract. (a) TFP binding and retraction abilities were tested using precipitation and mixing assays. For precipitation, TFP were sheared off from the cell surface of difA and the pilA-A32V, difA double mutant and precipitated with either chitin or wild-type EPS. Lanes: 1, total sheared pili; 2, precipitated by chitin; 3, precipitated by EPS. Mixing/retraction assays were conducted by pre-incubating cells with MOPS buffer, chitin or EPS for 30 min, followed by vigorous shearing to measure the remaining surface pili. Lanes: 4, cells pre-incubated with MOPS buffer; 5, cells pre-incubated with chitin; 6, cells pre-incubated with EPS. (b) Visual evidence that A32V TFP can tether to polystyrene surfaces. M. xanthus cells were deposited into 24-well cell culture plates containing 1 % methylcellulose in MOPS buffer, and individual cells were analysed for motility and tethering behaviour on solid surfaces. Tethered cells appear as dots in the image, indicated by black arrows. Top to bottom photos show the wild-type strain DK1622 (WT), A32V and ΔpilA. Left to right images were taken at 30 s intervals, showing that cells with TFP can change between tethering and ‘lying down’ status.

Fig. 3.

Effects of PilA A32 mutations on TFP production and function. Eight M. xanthus mutants with different site-specific mutations at PilA A32 were tested for their effects on TFP-related phenotypes. Top to bottom rows show swarming ability on a 0.3 % agar surface, EPS production, fruiting body (FB) formation, whole-cell PilA production and surface pili production. Western blot data for the whole-cell PilA and surface pili were reorganized from two original blots. WT, wild-type.

Fig. 4.

Proline mutations in the α-helix of PilA result in mutants with unstable PilA. Four M. xanthus mutants with proline mutations in the α-helix of PilA were tested for their PilA-related phenotypes. Top to bottom rows show swarming ability on a 0.3 % agar surface, EPS production, fruiting body (FB) formation, whole-cell PilA production and surface pili production. Western blot data for the whole-cell PilA and surface pili were reorganized from two original blots. WT, wild-type.