Two novel genes identified by large-scale transcriptomic analysis are essential for biofilm and rugose colony development of Vibrio vulnificus

Many pathogenic bacteria form biofilms to survive under environmental stresses and host immune defenses. Differential expression (DE) analysis of the genes in biofilm and planktonic cells under a single condition, however, has limitations to identify the genes essential for biofilm formation. Independent component analysis (ICA), a machine learning algorithm, was adopted to comprehensively identify the biofilm genes of Vibrio vulnificus, a fulminating human pathogen, in this study. ICA analyzed the large-scale transcriptome data of V. vulnificus cells under various biofilm and planktonic conditions and then identified a total of 72 sets of independently co-regulated genes, iModulons. Among the three iModulons specifically activated in biofilm cells, BrpT-iModulon mainly consisted of known genes of the regulon of BrpT, a transcriptional regulator controlling biofilm formation of V. vulnificus. Interestingly, the BrpT-iModulon additionally contained two novel genes, VV1_3061 and VV2_1694, designated as cabH and brpN, respectively. cabH and brpN were shared in other Vibrio species and not yet identified by DE analyses. Genetic and biochemical analyses revealed that cabH and brpN are directly up-regulated by BrpT. The deletion of cabH and brpN impaired the robust biofilm and rugose colony formation. CabH, structurally similar to the previously known calcium-binding matrix protein CabA, was essential for attachment to the surface. BrpN, carrying an acyltransferase-3 domain as observed in BrpL, played an important role in exopolysaccharide production. Altogether, ICA identified two novel genes, cabH and brpN, which are regulated by BrpT and essential for the development of robust biofilms and rugose colonies of V. vulnificus. Author summaryBiofilm formation is essential for pathogenic bacteria to survive under environmental stresses and host immune surveillances. Unlike conventional differential expression (DE) analysis, independent component analysis (ICA), a machine learning algorithm, could comprehensively analyze gene expression profiles under various conditions of biofilm and planktonic cells and identify the genes essential for biofilm formation. In this study, ICA decomposed large-scale transcriptome data of a devastating pathogen Vibrio vulnificus into the iModulons, sets of independently co-regulated genes. Among the iModulons activated in biofilm cells, BrpT-iModulon contained two novel genes not yet identified by DE analyses, cabH and brpN, in addition to the previously known genes of the BrpT regulon. Our results indicated that CabH and BrpN contribute to biofilm and rugose colony formation. CabH, carrying calcium-binding repeats, and BrpN, harboring an acyltransferase-3 domain, were identified to play a crucial role in surface attachment and exopolysaccharide production, respectively. Consequently, ICA newly identified cabH and brpN which are regulated by BrpT and are essential for robust biofilm and rugose colony development of V. vulnificus.

Automated summary

In this study, independent component analysis (ICA) was used to analyze the transcriptome data of Vibrio vulnificus and identified two novel genes, cabH and brpN, which are regulated by BrpT and essential for biofilm and rugose colony formation.