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Dissertation zugänglich unter
URN: urn:nbn:de:gbv:18-61687
URL: http://ediss.sub.uni-hamburg.de/volltexte/2013/6168/


The complete genome sequence of Rhizobium sp. NGR234 reveals a surprisingly large number of quorum quenching associated genes

Das Quorum Quenching Potential von Rhizobium sp. NGR234

Krysciak, Dagmar

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 Dokument 1.pdf (3.044 KB) 


SWD-Schlagwörter: Quorum Sensing , Zell-Zell Kommunikation , Quorum Quenching , Rhizobium sp. NGR234
Basisklassifikation: 35.70 , 58.30 , 42.13 , 42.30
Institut: Biologie
DDC-Sachgruppe: Biowissenschaften, Biologie
Dokumentart: Dissertation
Hauptberichter: Streit, Wolfgang R. (Prof. Dr.)
Sprache: Englisch
Tag der mündlichen Prüfung: 26.08.2011
Erstellungsjahr: 2011
Publikationsdatum: 07.06.2013
Kurzfassung auf Englisch: Rhizobium sp. NGR234 belongs to the α-proteobacteria and is a unique representative of rhizobia forming nitrogen-fixing nodules with more legumes than any other microsymbiont.
Many of the processes and genes necessary for an effective symbiosis were identified, but still there are significant gaps with respect to the bacterial interaction to fill and communication mechanisms to understand.
Within this research, the full genome sequence of Rhizobium sp. NGR234 was established, uncovering many striking features. The 6.9-Mbp genome is composed of the 3.93-Mbp chromosome (cNGR234), the 2.43-Mbp megaplasmid (pNGR234b) and the 0.54-Mbp symbiotic plasmid (pNGR234a). A total of 6,394 ORFs were assigned on the NGR234
genome, whereas 27% of the ORFs were related to genes with unknown function. Among many other remarkable features, a surprisingly high number of 132 proteins spread over the three replicons are linked to secretory processes, giving evidence that NGR234 encodes for
more different secretion systems than any other known Rhizobium. Additionally, systems linked to QS AI synthesis and quenching of such QSautoinducers could be discovered.Beside the AHL synthase TraI and the response regulator TraR present on the pNGR234b, a second possible QS system composed of NgrI/NgrR located on the chromosome was identified. Detailed sequence analyses uncovered not only a novel AHL synthase but also several putative AHL degrading enzymes spread in the genome of NGR234. Altogether 23 ORFs were found by similarity search against public databases being possibly involved in QQ processes. To confirm the surprisingly high number of genes linked to degradation of autoinducer 1 molecules, a function-based approach was implemented. A previously
published screening protocol using A. tumefaciens NTL4 was employed to verify candidate clones from a NGR234 genomic cosmid clone library. The genome wide functional analysis revealed the presence of five loci that consistently gave a positive result. Two of these loci were located on pNGR234b and three were encoded by cNGR234. The corresponding ORFs
of all cosmid clones could be localized by the combination of subcloning, transposon mutagenesis and NCBI BLAST analyses. The identified genes were designated dlhR, qsdR1, qsdR2, aldR and hitR-hydR. One main goal of the research was to verify the functional QQ activity of all genes and to characterize in detail the most promising genes present on
pNGR234b. Consequently, heterologous expression and purification were realized for DlhR and QsdR1. DlhR resembles a bacterial dienelactone hydrolase while QsdR1 shows high similarities to a metallo-β-lactamase, comprising two conserved motifs attributed to AHLases. The QQ impact of both purified proteins was investigated using biosensor strains A. tumefaciens NTL4, P. aeruginosa PAO1 and C. violaceum CV026. In all strains, QS-dependent processes like swarming and violacein production as well as biofilm formation were reproducibly inhibited by both enzymes. Recombinant DlhR and QsdR1 investigated with the ONPG assay confirmed the ability to hydrolytically degrade 3-oxo-C8-HSL. In general, less than 73% of the employed AHL were detected for DlhR and a more pronounced degradation of AHLs (down to 40%) was measured for QsdR1. Furthermore, experimental data from competitive colonization of roots in the rhizosphere of cowpeas showed that extra copies of dlhR and the qsdR1 gene strongly contributed to plant root colonization fitness of NGR234, emphasizing the ecological importance of QQ during root colonization of seedlings (i.e. biofilm formation). Finally, to uncover the underlying cleaving mechanism of AHL degradation by both proteins, HPLC-MS analysis was employed. The HPLC-UV as well as mass spectra for DlhR and QsdR1 confirmed lactonolytic activity, giving evidence that both proteins act as true lactonases that had not being described for NGR234 or in earlier QQ
studies.

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