Molecular characterization of a novel quorum sensing system of the diazotrophic grass endophyte Azoarcus sp. strain BH72

Abstract

Quorum sensing (QS) is a cell density dependent regulatory mechanism which is involved in regulation of several processes in bacteria. Azoarcus sp. BH72 communicates via QS and conditioned culture supernatant, obtained from a culture grown until the stationary growth phase, shows inducing ability on QS target gene expression.The presented study aimed to investigate the novel QS system of Azoarcus sp. BH72 by applying several methods: Comparative proteomic studies with Azoarcus wild type grown under QS conditions based on two-dimensional gel electrophoresis and mass spectrometry with an additional genome wide microarray approach were performed to detect QS regulated genes and proteins. Beside that, QS experiments were carried out with reporter gene studies by means of determination of beta-glucuronidase activity encoded by the gene uidA. While the QS dependent regulation of the pilAB operon, involved in the formation of type IV pili, could be validated by reporter gene studies, it could be shown for the first time that expression of azo3874, encoding a conserved hypothetical secreted protein, was as well induced under QS conditions. Additionally, the proteome experiments revealed that 18 % of the detected proteins were differentially synthesized under QS conditions. Furthermore, transcriptome studies showed that the expression of around 10 % of all Azoarcus sp. BH72 genes was under QS control. The computational analyses of regulated genes and proteins showed that several cellular processes such as type IV pili formation/regulation, oxidative stress response, transport pathways (iron, nickel, and phosphate) and energy metabolism were altered under QS conditions. The expression of several genes, encoding regulatory proteins were under QS control indicating that quorum sensing regulation is organized in a complex hierarchy in the studied beta-proteobacterium. Comparative proteomic studies with gel-based and gel-free methods with a pilR-mutant revealed that the responser regulator PilR is besides regulation of type IV pili biogenesis involved in activation as well as repression of a number of processes.QS experiments with different bacterial species could clearly show that conditioned culture supernatants from Azoarcus communis SWub3 or Azospira oryzae 6a3 induced the Azoarcus pilAB as well as azo3874 gene expression 2-fold. This observation indicates that Azoarcus sp. BH72 is capable of interspecies communication and that the novel quorum sensing system might be widespread in bacteria.Concluding, the presented study demonstrates that density dependent regulation is important for this grass endophyte.