Genomic Island-Encoded Diguanylate Cyclase from Vibrio alginolyticus Regulates Biofilm Formation and Motility in Pseudoalteromonas

Abstract

Many bacteria use the second messenger c-di-GMP to regulate exopolysaccharide production, biofilm formation, motility, virulence, and other phenotypes. The c-di-GMP level is controlled by the complex network of diguanylate cyclases (DGCs) and phosphodiesterases (PDEs) that synthesize and degrade c-di-GMP. In addition to chromosomally encoded DGCs, increasing numbers of DGCs were found to be located on mobile genetic elements. Whether these mobile genetic element-encoded DGCs can modulate the physiological phenotypes in recipient bacteria after horizontal gene transfer should be investigated. In our previous study, a genomic island encoding three DGC proteins (Dgc137, Dgc139, and Dgc140) was characterized in Vibrio alginolyticus isolated from the gastric cavity of the coral Galaxea fascicularis. Here, the effect of the three DGCs in four Pseudoalteromonas strains isolated from coral Galaxea fascicularis and other marine environments was explored. The results showed that when dgc137 is present rather than the three DGC genes, it obviously modulates biofilm formation and bacterial motility in these Pseudoalteromonas strains. Our findings implied that mobile genetic element-encoded DGC could regulate the physiological status of neighboring bacteria in a microbial community by modulating the c-di-GMP level after horizontal gene transfer.

Keywords: Pseudoalteromonas; Vibrio; biofilm; mobile genetic elements; motility.

Figure 1

The change in colony biofilm formation when DGC137 and three DGC proteins were expressed in Pseudoalteromonas strians. (a) Construction of pDgc137 and p3DGC. The DNA regions contained in pDgc137 and p3DGC are indicated as solid lines with the ORFs, and the end of the DNA region is indicated as dashed lines. (b) The change in colony biofilm in the indicated Pseudoalteromonas strains containing pDgc137 and p3DGC compared with the empty vector. “p” indicates the empty vector pBBR1Cm. At least three independent cultures were used, and only representative images are shown. The incubation days of the colony biofilm imaged are indicated under the strain name.

Figure 2

The change in biofilm formation and growth when the indicated Pseudoalteromonas strains contained pDgc137 and p3DGC compared with the empty vector pBBR1Cm. Biofilm formation and growth of Pp43201 (a), Pf43202 (b), Pl04301 (c), and Pr6842 (d) when pDgc137, p3DGC, and pBBR1Cm were expressed. “p” indicates pBBR1Cm. The host bacteria are indicated at the top of each panel. All the tested bacteria were cultured in 2216E medium for 0–8 h, and only the data from representative time points are shown. Individual data points are plotted with lines at the mean, and error bars represent the standard deviation. Statistical analysis: Unpaired t-test, asterisks represent statistically significant differences (ns, (not significant); * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001).

Figure 3

The change in pellicle formation when the indicated Pseudoalteromonas strains containing pDgc137 and p3DGC were compared with the empty vector. “p” indicates the empty vector pBBR1Cm. The white arrows indicate floating pellicles. The incubation days of the pellicle imaged are also shown on the left of the image. At least three independent cultures were used, and only representative images are shown.

Figure 4

The change in swimming ability when the indicated Pseudoalteromonas strians contained pDgc137 and p3DGC compared with the empty vector. Swimming diameter when the indicated Pseudoalteromonas strains containing pDgc137 (a), and p3DGC (b) were compared with the empty vector. “p” indicates the empty vector pBBR1Cm. The plates were imaged after culturing the strains in semisolid agar plates for 22 h (Pp43201), 43202 15 h (Pf43020), 22 h (Pl04301), and 52 h (Pr6842). At least three independent cultures were used, and only representative images are shown. Quantitative statistical data are shown under the representative images. Statistical analysis: Unpaired t-test, asterisks represent statistically significant differences (ns, (not significant); - *** p < 0.001; **** p < 0.0001).