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 ∆fliP 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 ∆fliP 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.

Keywords: Mytilus coruscus; biofilm; flagellar gene; larval settlement and metamorphosis; mussel.

Figure 1

Deletion of flagellin synthetic protein gene fliP. The deletion of the fliP gene was confirmed by PCR. The PCR product sizes of the wild-type strain were 1637 bp (line 1, fliP–SF/fliP–SR), 2558 bp (line 3, fliP–SF/fliP–LR), 2805 bp (line 5, fliP–LF/fliP–SR), and 3726 bp (line 7, fliP–LF/fliP–LR). The PCR product sizes of ΔfliP strain were 896 bp (line 2, fliP–SF/fliP–SR), 1817 bp (line 4, fliP–SF/fliP–LR), 2064 bp (line 6, fliP–LF/fliP–SR), and 2985 bp (line 7, fliP–LF/fliP–LR). M, marker.

Figure 2

Relative to the wild-type strain the ΔfliP strain reduced the inducing activity and increased bacterial density. (A) Inducing activities of biofilms on larval settlement and metamorphosis with different initial colony-forming unit (CFU); (B) biofilm density on glass slips with different initial CFU. Tested biofilms formed on the glass slips for 48 h.

Figure 3

Colony morphology of wild-type (A) and ΔfliP (B) strains and transmission electron microscopy images of wild-type (C) and ΔfliP (D) strains.

Figure 3

Colony morphology of wild-type (A) and ΔfliP (B) strains and transmission electron microscopy images of wild-type (C) and ΔfliP (D) strains.

Figure 4

Biofilm formation of wild-type and ΔfliP strains. (A) Swimming motility of wild-type and ΔfliP strains; (B) the confocal laser scanning microscopy (CLSM) images of biofilms formed by the wild-type strain; (C) the CLSM images of ΔfliP biofilms; (D) biofilm thickness statistical analysis of wild-type and ΔfliP strains.

Figure 5

The CLSM analysis of Pseudoalteromonas marina biofilm EPS and mussel settlement and metamorphosis after enzyme treatments. (A) Distribution of polysaccharides, proteins and lipids in biofilms of wild-type and ΔfliP strains; (B) Biovolume analysis of extracellular polysaccharides, proteins and lipids content in biofilms; (C) Mussel settlement and metamorphosis on biofilms treated by different proteases, lipases and glycohydrolases.

Figure 6

Effects of flagellar protein extracted on settlement and metamorphosis and survival rates. (A) Percentages of settlement and metamorphosis on flagellar protein tested; (B) percentages of settlement and metamorphosis on ΔfliP biofilms with addition of flagellar proteins; (C) effects of flagellar proteins extracted on larval survival rates; (D) effects of ΔfliP biofilms with addition of flagellar proteins extracted on larval survival rates.