Symbiotic plasmid (pNGR234a) copy number in the broad host range bacterium Sinorhizobium fredii NGR234 depends on quorum sensing and phenotypic heterogeneity
Sinorhizobium fredii NGR234 is an Alphaproteobacterium of the Rhizobiaceae family. This Gram-negative bacterium is characterised by its remarkably broad host range. It can induce root nodules and fix atmospheric Nitrogen with over 120 legume plant genera and even with one non-legume. S. fredii NGR234’s genome consists of three replicons, the chromosome and two megaplasmids, pNGR234b and pNGR234a. The chromosome and the pNGR234a plasmid encode each for one quorum sensing (QS) system, consisting of an autoinducer synthase, ngrI and traI respectively, and their regulators, ngrR and traR. Recently, studies indicated that deleting both QS system autoinducer synthases, traI and ngrI, the plasmid copy number of the pNGR234a plasmid increased and nearly all genes encoded on the symbiotic plasmid were highly transcribed leading to the identification of new open reading frames (ORFs). Analysis of these data showed a regulatory function of some of these newly discovered small ORFs, like NGR_a01725 or repX on plasmid copy number. This study provides further evidence that the plasmid copy number of the symbiotic plasmid in the rhizobia species S. fredii NGR234, besides multiple other factors, like non-coding RNA, small ORFs and some still unknown factors, is influenced by QS. To develop a deeper understanding and provide further information how the plasmid copy number is regulated in S. fredii NGR234, effects of copy number changes were investigated by real-time quantitative PCR. Cultivation of NGR234 wild type (WT) and QS autoinducer synthase mutants using spent medium of different Gram-negative bacteria confirmed that the copy number of the symbiotic plasmid of NGR234 is regulated by externally provided autoinducers. Another main aspect studied here is phenotypic heterogeneity in S. fredii NGR234. The promoters of the autoinducer synthases, traI and ngrI, as well as the quorum quenching (QQ) gene qsdR1 promoter were linked to individual fluorescent proteins on one broad host range plasmid and after transformation of ...