|Titel:||Symbiotic plasmid (pNGR234a) copy number in the broad host range bacterium Sinorhizobium fredii NGR234 depends on quorum sensing and phenotypic heterogeneity||Sprache:||Englisch||Autor*in:||Duin, Hilke Johanna||Schlagwörter:||Sinorhizobium fredii NGR234; Plant-Microbe-Interaction; Symbiosis||GND-Schlagwörter:||Quorum sensingGND
|Erscheinungsdatum:||2021-07||Tag der mündlichen Prüfung:||2021-10-28||Zusammenfassung:||
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 different NGR234 strains observed by confocal laser scanning microscopy (CLSM). Subsequently, an automated cell counting pipeline and the identification of fluorescence intensity with the CellProfiler 3.1.8 software gave the opportunity to analyse CLSM images comparing NGR234 WT and the NGR234 mutant strains. All promoters showed heterogeneous expression in NGR234 WT and the studied mutant strains, ANU265, NGR234 ΔrepX and NGR234 ΔNGR_a01725+. Analysing single cells by CLSM and using an automated analysis revealed how population-wide expression patterns are shaped by single cell expression. However, the total range of the heterogeneous expression of each promoter was influenced in each mutant. Interestingly, ANU265 was observed to have three times higher median fluorescence intensities for all measured promoters after 24 h, had similar levels after 48 h and equal or higher levels of median fluorescence intensities after 72 h compared to the WT. The level of the overall phenotypic heterogeneity for the measured promoters was increased after 48 h. Examining the effect of the small ORFs repX and NGR_a01725, revealed an effect on phenotypic heterogeneity of the observed promoters as well. Even tough deletion of repX leads to a total loss of the symbiotic plasmid comparable to the ANU265 strain, results for the NGR234 ΔrepX mutant were different. Here median fluorescent intensities were either on WT level or below for all promoters. The range of expression intensities decreased, resulting in less phenotypic heterogeneity after 48 h. The deletion of the small ORF NGR_a01725 on the other hand increased the promoter activities after 48 h compared to the NGR234 WT. Additionally, the degree of phenotypic heterogeneity for the measured promoter activities was enhanced as well. These results show that QS and QQ are not as homogeneous as expected and that expression of these genes is influenced by the symbiotic plasmid copy number and small ORFs encoded thereon.
|Enthalten in den Sammlungen:||Elektronische Dissertationen und Habilitationen|
geprüft am 27.01.2022
geprüft am 27.01.2022