The Sensor Kinase BfimS Is A Global Regulator That Modulates Motility, Pigment And Virulence In Pseudomonas Aeruginosa

Pseudomonas aeruginosa, is one of the most common opportunistic pathogen in the clinical, which is widespread in nature and human skin, intestinal and respiratory tract. The bacteria can can cause severe infection of the immunocompromised patients, burn victims, and cystic fibrosis patients because it can produce a large number of virulence factors. The symptoms of such infections are generalized inflammation and sepsis.There are a large number of two-component systems in Pseudomonas aeruginosa. A two-component system often compromises a sensor histidine kinase and a response regulator. Histidine kinase sensor protein which locates on the cell membrane, is able to perceive a variety of stimuli in the environment, such as optical signal, a special small molecules, temperature, osmotic pressure and pH, etc. Once sensor protein is activated, the specific histidine in the protein can be phosphorylated. Then this phosphate group on the histidine is transferred to the aspartic acid residues on the response regulator protein. The conformation of phosphorylated response regulator protein change, which regulate the expression of different target genes. Response regulator protein can regulate its transcriptional activity through modulating its phosphorylation levels.In this study, BfmS (Biofilm maturation Sensor) is a sensor kinase in P. aeruginosa and with the cognate response regulator protein BfmR (Biofilm maturation Regulator), they constitute a two-component system. According to the research reports previously, two-component system BfmRS play a role in the development and maintenance of normal biofilm architecture of P. aeruginosa. At the same time, the two-component system BfmRS controls the process of phage-mediated cell lysis and DNA release during biofilm formation of P. aeruginosa. However, we found that this two-component system BfmRS, have a significant role in regulating the bacterial virulence factors or virulence-related process including the swarming motility, pyocyanin production, proteases and rhamnolipid secretion. Swarming motility can help the bacteria spread in the enviroment. Molitity is the first step before bacterial colonization and biofilm formation. Pyocyanin is a blue-green toxic metabolite secreted by P. aeruginosa. Becaused of its molecular structure, pyocyanin shows antibacterial activity, and can induce Caenorhabditis elegans death under oxidative stress. Pseudomonas aeruginosa can secrete a variety of proteases, which are able to degrade the hosts’ collagen to help microbe spreading and invading. Rhamnolipids are a class of glycolipid produced by Pseudomonas aeruginosa. They have a glycosyl head group, in this case a rhamnose moiety, and a3-(hydroxyalkanoyloxy)alkanoic acid (HAA) fatty acid tail. The most prominent feature is its surface tension activity, such as to significantly lower the surface tension of liquid, the interfacial tension between two liquids phase, or that between a liquid and a solid, changing the wettability of the solid surface. Because of their excellent characteristics, rhamnolipid is widely used in the oil industry, environmental control and the development of biological pesticides currently. In this paper, we constructed BfmS deletion mutants and BfmRS double mutants. We found that BfmS deletion mutants have attenuated swarming motility comparing with wild-type P. aeruginosa. Secondly the BfmS mutant secreted reduced pigments including pyocyanin, pyoverdine and pyorubin. In addition, the BfmS deletion mutant produces less rhamnolipid.The phenotypes are verified with the complementary strains. The complementary strains could restore fully or partially the phenotype of the mutants. Furthermore, reporter gene analysis revealed that BfmS regulates the transcription of rhlAB and bfmR during the early stationary phase consistent with the rhamnolipid plate assay. rhlA is the key gene in the rhamnolipid biosynthetic pathway. We conducted further research in the transcription of rhll and rhlR both belonging to rhl-quorum sensing system in P. aeruginosa and found that the BfmS mutants have lower transcription of rhll and rhlR compared with wildtype. As a result, BfmRS may regulate target genes independently or dependending on the rhl-quorum sensing system. The results of site-directed mutagenesis experiments show that BfmR may regulate target genes through the phosphorylation form. The transcription of bfmR in the BfmS deletion mutant became significantly higher than that of wldtype. Combining with the site-directed mutagenesis results, BfmS may regulate BfmR on the transcription level or post-translation level. And BfmS sensor protein within the bacterial cells functions in the form of the phosphatase.