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Deciphering regulatory mechanisms involved in phenotypic heterogeneity of the autoinducer synthase genes traI and ngrI in Sinorhizobium fredii NGR234 using single cell technologies and RNA-seq
Determinanten der phänotypischen Heterogenität der Autoinducer Synthase Gene traI und ngrI in Sinorhizobium fredii NGR234 unter Verwendung von Einzelzelltechnologien und RNA-Seq
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Freie Schlagwörter (Deutsch):
Mikrobiologie , Transkriptomik , Phänotypische Heterogenität , Rhizobium
Streit, Wolfgang R. (Prof. Dr.)
Tag der mündlichen Prüfung:
Kurzfassung auf Englisch:
The Gram-negative, symbiotic and nitrogen-fixing bacterium Sinorhizobium fredii NGR234 (NGR234) is able to nodulate over 120 different legume plant genera (Pueppke, Broughton 1999a). No other bacterium is currently known with such a broad host range. In recent years, notable achievements in deciphering the mechanisms responsible for this exceptional host range were accomplished. However there are still significant gaps in understanding the involved inter- and intraspecific communication mechanisms and their role in host range. To further identify possible molecular mechanisms linked to the biological phenomenon of broad host range two lines of work were followed:
First, population-wide and global transcriptome studies indicated that NGR234 has two major quorum sensing regulons each controlling several hundred genes. The traI/R regulon exclusively controls 130 genes, whereby the ngrI/R system regulates 280 genes. Additionally, 186 genes are co-regulated by both quorum sensing systems. To obtain these data a NGR234-ΔtraI and a NGR234-ΔngrI deletion mutant were constructed, and their transcriptomes compared with the corresponding transcriptome of the wild type strain. In general, flagellar and Type 4 pilus genes were regulated as well as genes for the biosynthesis of exopolysaccharides. Astonishingly, the second T3SS seemed to be regulated in both mutants, although no function could be identified for this secretion system so far. Further, we analyzed the transcriptome of a newly constructed NGR234-ΔtraI-ΔngrI double deletion mutant to better understand the processes in the absence of any autoinducer. The surprising finding in the background of this mutant was that 98 % of the genes encoded on the symbiotic plasmid pNGR234a were upregulated. Among the highest upregulated genes were the repABC genes and this probably resulted in a general increase of the copy number of the pNGR234a replicon. The increase in copy number and repABC transcription was linked to the high level expression of two hitherto not identified small open reading frames (ORFs), located in the intergenic region between repA and traI. These novel ORFs were designated repX, probably coding for a 58 aa protein and repA0, probably encoding a 143 aa protein. Both ORFs were highly transcribed in the absence of autoinducer in NGR234-ΔtraI-ΔngrI but not in the presence of autoinducers or in wild type. In line with this finding, it was observed that symbiotic proteins such as the nodulation outer membrane proteins NopABCLPX were produced in the NGR234-ΔtraI-ΔngrI but not in the wild type and in the absence of plant-released flavonoids. Similar, the NGR234-ΔtraI-ΔngrI produced sufficient levels of nodulation factor to induce root hair curling in the absence of plant-released flavonoids. In addition to NGR234-ΔtraI-ΔngrI, NGR234-ΔtraI and NGR234-ΔngrI, the deletion mutants NGR234-ΔtraM and NGR234-ΔtraR were constructed and tested for the copy number of their symbiotic plasmid pNGR234a.
Second, and in addition to the population-wide studies, single cell analyses were performed. Therefore, seven promoter fusions were constructed and mobilized into NGR234 wild type cells and some of those additionally into a NGR234-ΔtraI and a NGR234-ΔngrI mutant, using the DsRed2 protein as marker. Our tests implied that under laboratory conditions high levels of phenotypic heterogeneous gene expression exist. Tests indicated that heterogeneity was highest after 24 h growth for the PtraI and PngrI and after 48 h growth in case of the PdlhR and PqsdR1 promoter fusions. The percentage of fluorescing cells in case of PtraI and PngrI were nearly 31 % for PtraI and nearly 40 % for PngrI after 24 h and nearly 40 % for PdlhR and nearly 48 % for PqsdR1 after 48 h growth.
Thereby, we provided strong evidence that under laboratory conditions surprisingly many genes in NGR234 are heterogeneously regulated especially the two autoinducer synthase genes traI and ngrI. However, the addition of external autoinducer and the addition of plant exudates led to a homogenization of the culture. These results imply that there are mechanisms that can overwrite NGR234’s heterogeneous gene regulation and that heterogeneity may perhaps be not of high relevance in rhizospheres.
Most results of this work were published in Krysciak et al. 2014, Appl Environ Microbiol, Grote et al. 2014, Appl Environ Microbiol, and Grote & Krysciak et al. 2016, unpublished manuscript.