Screening and functional characterization of isocitrate lyase AceA in the biofilm formation of Vibrio alginolyticus.

Abstract

Biofilm is a well-known sessile lifestyle for bacterial pathogens, but a little is known about the mechanism on biofilm formation in Vibrio alginolyticus. In this study, we screened V. alginolyticus strains with strong biofilm formation ability from coastal seawater. The antibiotic resistance of the biofilm cells (BFs) was higher than that of the planktonic cells (PTs). To study the genes and pathways involved in biofilm formation, we performed transcriptome analysis of the BFs and PTs of V. alginolyticus R9. A total of 685 differentially expressed genes (DEGs) were upregulated, and 517 DEGs were downregulated in the BFs. The upregulated DEGs were significantly enriched in several pathways including glyoxylate and dicarboxylate metabolism, while the downregulated genes were significantly enriched in the flagellar assembly pathways. The key gene involved in glyoxylate shunt, aceA, was cloned, and ΔaceA mutant was constructed to determine the function of AceA in carbon source utilization, biofilm formation, and virulence. Real-time reverse transcription PCR showed that the expression of aceA was higher at the mature stage but lower at the disperse stage of biofilm formation, and the expression of the flagellar related genes was upregulated in ΔaceA. This is the first study to illustrate the global gene expression profile during the biofilm formation of V. alginolyticus, and isocitrate lyase AceA, the key enzyme involved in glyoxylate shunt, was shown to maintain biofilms accompanied by downregulation of flagellation but promoted dispersal of BFs at the late stage.IMPORTANCEBiofilms pose serious public problems, not only protecting the cells in it from environmental hazard but also affecting the composition and abundance of bacteria, algae, fungi, and protozoa. The important opportunistic pathogen Vibrio alginolyticus is extremely ubiquitously present in seawater, and it also exhibited a strong ability to form biofilm; thus, investigation on the biofilm formation of V. alginolyticus at molecular level is fundamental for the deeper exploration of the environmental concerns arose by biofilm. In this study, transcriptome analysis of biofilm cells (BFs) and planktonic cells (PTs) from V. alginolyticus was performed and AceA was screened to play an important role in biofilm formation. AceA was shown to maintain biofilms accompanied by downregulation of flagellation but promoted dispersal of BFs at the disperse stage. This method was helpful to further understand the ability and mechanism of V. alginolyticus biofilm formation and provide clues for prevention of V. alginolyticus infection.