The Flagellar Gene Regulates Biofilm Formation and Mussel Larval Settlement and Metamorphosis

Abstract

Biofilms are critical components of most marine systems and provide biochemical cues that can significantly impact overall community composition. Although progress has been made in the bacteria–animal interaction, the molecular basis of modulation of settlement and metamorphosis in most marine animals by bacteria is poorly understood. Here, Pseudoalteromonas marina showing inducing activity on mussel settlement and metamorphosis was chosen as a model to clarify the mechanism that regulates the bacteria–mussel interaction. We constructed a flagellin synthetic protein gene fliP deletion mutant of P. marina and checked whether deficiency of fliP gene will impact inducing activity, motility, and extracellular polymeric substances of biofilms. Furthermore, we examined the effect of flagellar proteins extracted from bacteria on larval settlement and metamorphosis. The deletion of the fliP gene caused the loss of the flagella structure and motility of the ΔfliP strain. Deficiency of the fliP gene promoted the biofilm formation and changed biofilm matrix by reducing β-polysaccharides and increasing extracellular proteins and finally reduced biofilm-inducing activities. Flagellar protein extract promoted mussel metamorphosis, and ΔfliP biofilms combined with additional flagellar proteins induced similar settlement and metamorphosis rate compared to that of the wild-type strain. These findings provide novel insight on the molecular interactions between bacteria and mussels.

2(5H)-Furanone Disrupts Bacterial Biofilm Formation and Indirectly Reduces the Settlement of Plantigrades of the Mussel Mytilus coruscus

“…We found that flagellar assembly protein fliH (RF53_RS02940) participating in cell motility was significantly downregulated in FUR-exposed P. marina biofilms relative to the CTR biofilms. Our recent study also demonstrates that a flagellin synthetic protein gene fliP deletion of P. marina contributes to the loss of the flagella structure and motility (Liang et al, 2020). The flagellum is critical for the mobility of bacteria, which is crucial for the initial surface attachment, subsequent biofilm formation and virulence (O'Toole and Kolter, 1998;Watnick et al, 2001;Lemon et al, 2007;Liang et al, 2020).…”

“…The 10 −4 M FUR-treated biofilms were selected for biofilm staining due to the lowest plantigrade settlement rate for comparing the individual components in the biofilm matrix. The visualization of proteins, lipids, α-polysaccharides, and β-polysaccharides in the P. marina ECSMB14103 biofilms was carried out by following a previously described method (González-Machado et al, 2018;Liang et al, 2020). Four fluorescent dyes, including fluorescein isothiocyanate isomer I (FITC), DiIC 18 (5) oil, 1,1 -dioctadecyl-3,3,3 ,3tetramethylindodicarbocyanine perchlorate (DiD oil; Invitrogen D307), concanavalin A, tetramethylrhodamine conjugate (ConA-TmR; Invitrogen C860), and calcofluor white M2R (CFW; Sigma 18909) were used for staining the proteins, lipids, α-polysaccharides, and β-polysaccharides, respectively.…”

“…The glass slides were rinsed again with 150 mM NaCl solution and allowed to air dry. Image acquisition was performed by confocal laser scanning microscopy (Leica TCS SP8) using the 63 × objective with oil immersion (Liang et al, 2020). For image analysis, the biovolume of proteins, lipids, α-polysaccharides, and β-polysaccharides were calculated using FIJI software (Schindelin et al, 2012).…”

Deep-sea bacteria trigger settlement and metamorphosis of the mussel Mytilus coruscus larvae

“…Treated BFs (100H, 10A and FF) without alive bacteria showed low inducing activity, indicating that the living deep-sea bacteria is necessary to produce chemical signals to promote the settlement process in this species. Previous studies have also demonstrated that the settlement process of M. coruscus [10][11][12][13] larvae also required BFs of living offshore bacteria, as well as M. galloprovincialis 8 and H. elegans 20,24 larvae.…”

“…Confocal laser scanning microscopy (CLSM) and image analysis. Extracellular products were stained according to the method of Liang et al 12 . Virgibacillus sp.…”