The mechanism of super biofilm formation caused by the deletion of a small fragment of the methylated chemotactic receptor protein WspA in Pseudomonas aeruginosa revealed by the Institute of Microbiology, Chinese Academy of Sciences

Biofilm is the main form of microbial survival
.

Biofilm-related research is one of the frontiers in the field of microbiology, and it has crucial research significance in basic life sciences and applied sciences such as medical treatment, industry, agriculture, and environmental governance
.


Pseudomonas aeruginosa has strong environmental adaptability and can survive in various environments such as water, soil, and crude oil
.

At the same time, Pseudomonas aeruginosa is also an opportunistic pathogen, which is easy to form biofilm, and is one of the model strains in biofilm research
.

Previous studies have found that Pseudomonas aeruginosa has a type of Rugose small colony variants (RSCVs) that can form dense and thick hyper-biofilms
.

The formation of super biofilms leads to greatly enhanced drug resistance of mutant strains, which are easy to escape the immune clearance of the host and have poor clinical prognosis
.


In addition, the formation of super biofilms also helps bacteria resist the survival pressure in the environment and can survive in a nutrient-poor environment
.

However, the formation mechanism of super biofilm strains remains to be elucidated
.


Ma Luyan's team from the Institute of Microbiology, Chinese Academy of Sciences carried out whole genome sequencing of two Pseudomonas aeruginosa strains isolated from petroleum.
The analysis found that the genomes of the two strains were highly homologous (identity>99.
99%), but one of them appeared to form a super biofilm The small colony wrinkle phenotype
.

SNP analysis combined with molecular biological verification revealed that the deletion of a small fragment of the methylated chemotactic receptor protein WspA in this strain was the cause of the formation of the strain's super biofilm
.


WspA protein is a signal-sensing receptor protein in the Wsp system, which determines the activation or closing of the Wsp system through methylation or demethylation
.

Deletion of amino acid residues 280-307 of WspA results in the protein locking the Wsp system in a persistently activated state
.


In Pseudomonas aeruginosa, the Wsp system coordinates bacterial movement and biofilm formation by regulating the synthesis of the intracellular second messenger cyclic-di-GMP.
The continuous activation of the Wsp system leads to the continuous accumulation of intracellular cyclic-di-GMP.
Thereby inhibiting bacterial movement, promoting the synthesis of extracellular polysaccharides, forming a super biofilm
.


In addition, Ma Luyan's team identified the methylation sites of WspA protein by Orbitrap, and found that there are two methylation sites E280 and E297 in the 280-307 amino acid region of WspA with a small fragment deletion
.

It is speculated that the absence of methylation sites prevents WspA protein from being modified by methylation and demethylation, resulting in the locked Wsp system in a persistently activated state
.

The homologous protein sequence alignment of WspA showed that there were three conserved repeat sequences in the region where amino acid fragment deletion (aa280-313) occurred, indicating that the spontaneous deletion in the corresponding DNA coding region may be the result of intragenic fragment recombination
.


This repeat is present in the WspA of several related genera and bacteria in similar habitats, suggesting the prevalence of similar mutations
.

Small deletions of WspA are also common in clinically isolated RSCV
.

The results of this study elucidate the mechanism of P.
aeruginosa super biofilm formation and the reasons why the bacteria gain a competitive advantage in special environments
.


In summary, this study revealed the mechanism by which the methylated chemotactic receptor protein WspA activates the synthesis of cyclic-di-GMP through the loss of small fragments, thereby leading to the formation of super biofilms in strains, which is related to the prevention and control of super biofilms.
The problem provides a theoretical basis and possible solutions
.


This research work, with Xu Anming, a doctoral student in Ma Luyan's group, as the first author, Associate Researcher Wang Di as the second author, and Researcher Ma Luyan as the corresponding author, will be published in the journal Environmental Microbiology in March 2022
.

This study was supported by the cooperation and assistance of Liu Shuangjiang's team and Li Defeng's team of the Institute of Microbiology
.

This research was supported by the National Natural Science Foundation of China Hydrosphere Major Research Program and the National Key Research and Development Program
.


Schematic diagram of the Wsp system.
Schematic diagram of the mechanism by which the deletion of amino acid residues 280-307 of WspA leads to an increase in the level of secondary messengers.
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