A simple, switchable pili‐labelling method by plasmid‐based replacement of pilin

Abstract

Summary Labelling of Type IV pili (TFP) can greatly improve our understanding of the pivotal roles of TFP in a variety of bacterial activities including motility, surface sensing and DNA‐uptake etc. Here we show a simple and switchable pili‐labelling method by plasmid‐based inducible replacement of PilA without genetic modification in bacterial genome employed by complicated methods. Using this method, we characterized pili morphology and twitching motility of Pseudomonas aeruginosa in details. More importantly, we demonstrate its application in studying the replenishment dynamics of pilin pool of P. aeruginosa.

Oscillation of type IV pili regulated by the circadian clock in cyanobacterium Synechococcus elongatus PCC7942

“…By directly tracking the movement of cells with fluorescent TFP, we found that Syn7942-PilAm cells can perform twitching by extension-retraction cycles of TFP. The extension and retraction rates of TFP were 0.69 ± 0.08 and 0.43 ± 0.03 μm/s, respectively, which are faster than those of P. aeruginosa ( 18 ). Although the mechanism may be similar to the twitching motility observed in other bacteria ( 19 , 20 ), Syn7942-PilAm displayed richer surface movement modes.…”

“…To directly visualize the behavior of TFP, we replaced an amino acid residue of the major pilin subunit PilA with a Cys for subsequent labeling with a thiol-reactive fluorescent maleimide dye ( 18 ). In detail, without changing the original pilA ( S ynpcc7942_0048 ) of Syn7942 wild type (hereafter Syn7942-WT), we overexpressed the mutated cassette encoding PilAm (PilA with S87C) with a strong promoter p cpc560 at the neutral site NSI (fig.…”

“…1A ). Compared with heterotrophic P. aeruginosa and V. cholerae ( 14 , 18 ), Syn7942 have more and longer TFP on average. In addition, we counted the number of dynamic TFP extension-retraction cycle events observed in single cells in a time period of 1 min, which we defined as the TFP dynamic activity.…”

“…Further investigation showed that TFP were involved in driving the phototaxis movement of Syn6803, whose presence or absence was regulated by the genes such as tax1 and tax3 related to phototaxis ( 20 ). In this study, with the TFP visualization techniques we developed previously ( 18 ), we are able to monitor the evolution of TFP morphology in Syn7942 in an in situ and real-time manner. Our results show that Syn7942 is more similar to P. aeruginosa cells in terms of TFP-driven surface motility: (i) Syn7942 twitches via extension and retraction of TFP; (ii) Syn7942 can also twitch via walking or parallel crawling mode and its walking is faster than crawling.…”

Factors Influencing Pseudomonas aeruginosa Initial Adhesion and Evolution at the Dodecane–Water Interface

“…The results (Figure ) show that Δ pilT has a larger area density of attached cells ((2.0 ± 0.2) × 10 –2 μm –2 ) than WT, suggesting that more and longer TFP are beneficial for cell attachment to the oil–water interface, whereas Δ pilA has a similar level ((1.2 ± 0.3) × 10 –2 μm –2 ) as WT, which seems to contradict the results of Δ pilT . This seemingly contradicting result of Δ pilA is probably caused by the degree of piliation of cells, as a WT cell typically has just one or two pili distributed at one pole of the cell, which is phenotypically not very different from that of Δ pilA in terms of the degree of piliation.…”