TFP-based Characterization Of Cell Movement And Its Role In Biofilm Formation

Bacterial biofilm is the main factor of iatrogenic bacterial infection,and its formation process is closely related to the movement of bacteria.The bacterial movement is also crucial for the survival of bacteria in nature.Over the years,researchers have used a variety of macroscopic methods to explore cell movement and biofilms,giving us a macroscopic understanding of bacterial movement and biofilms.In recent years,with the development of bacterial microscopic tracking technology and image processing methods,more and more studies have focused on bacterial movement and biofilm formation at the single-cell level,especially on Type IV pili(TFP)-mediated cell movement and its impact on biofilm biofilm formation has become a research hotspot in the field of microorganisms.TFP is a protein appendage protruding from the cell surface plays an important role in bacterial movement and biofilm formation.Real-time visualization of TFP helps us to further understand the mechanism of bacterial movement and biofilm formation.In this paper,based on the cysteine replacement method,we first developed a method for labeling pili by arabinose-induced plasmids that can be turned on when needed and turned off when not needed.This method is simple and maintains the integrity of the bacterial genome,and the biological properties such as normal growth and movement of cells were not affected.We also successfully applied this method to Pseudomonas aeruginosa PAO1,Synechococcus elongatus PCC 7942.Next,we analyzed the movement and pili changes of Synechococcus elongatus PCC 7942 at the single-cell level by visualization of the pili.Synechococcus elongatus PCC 7942 has three movement modes: perpendicular crawling,parallel crawling and walking,and these movements are pulled by the retraction of the TFP.TFP also shows a completely different characteristic in the day-night circle,TFP length and number reach a maximum at dusk and a minimum at dawn;this change is related to energy supply,but rhythm regulation is the main factor of this change.At the same time,we analyzed the location and number distribution of Pseudomonas aeruginos PAO1 TFP at the single-cell level.TFP are present in the majority(～76%)at the poles,and the distribution of pili is dynamic,constantly changing as bacteria move across the surface.We used the controllability of TFP visualization to measure the supplementation of pil A in the pili pool of Pseudomonas aeruginosa.We explored the reversal behavior of bacteria in the biofilm microcolony,which is an important part of population regulation and domain expansion of biofilms.Based on the visualization of P.aeruginosa TFP,we further explored the relationship between TFP-based bacterial movement and c-di-GMP accumulation during P.aeruginosa PAO1 biofilm formation using the p Cdr A::gfp reporter.We found that the place where c-di-GMP accumulated the most was the highest point of bacterial visits;and its movement speed was negatively correlated with c-di-GMP in both single cell growth and cell sorting progeny proliferation.When agar was used to control cell movement,c-di-GMP was more likely to accumulate,but this regulation was no longer present when the sia D and sad C genes were knocked out.Finally,we explored the effects of psl G on Pseudomonas aeruginosa PAO1 bacterial movement,cell fate and biofilm development at the single-cell level.The deletion of psl G resulted in long chains of cells connected by PSL,which increased the probability of ―Two-bright‖ events in the progeny,increased the possibility of cell adhesion,and accelerated the formation of biofilm microcolonies.In conclusion,we have developed a pili labeling method without disrupting bacterial genomes,and based on bacterial microscopic tracking technology,enabling long-term in situ real-time observation of living cells of Synechococcus elongatus and Pseudomonas aeruginosa and their labeled pili.We not only characterized the TFP-based bacterial movement of the two bacteria,but also explored the roles of TFP,c-di-GMP and psl G genes in the biofilm formation of Pseudomonas aeruginosa.This provides us with more perspectives to explore various physiological processes such as bacterial movement mechanism,rhythm regulation,natural transformation and biofilm formation at the single-cell level,thereby providing a technical basis and new ideas for people to better utilize microorganisms to serve human beings.