The Queuosine pathway is not essential for Ensifer medicae WSM419 symbiosis and certain components of this pathway modulate lipid biosynthesis

Jaco Zandberg

The Queuosine pathway (Q-pathway) is a complex biosynthetic pathway responsible for the production of Q-modified tRNA (Q-tRNA). Q-tRNA alters the codon–anticodon interactions for the Q-family of tRNA molecules which are charged with the amino acids asparagine, aspartate, histidine or tyrosine, improving the efficiency and stringency of translation. Q-tRNA has been shown to be essential for cell survival in certain stressful conditions, bacterial virulence, and recently for the establishment of an effective symbiotic relationship between the RNB Ensifer meliloti 1021 and its legume host Medicago truncatula. This thesis has shown that the Q-pathway is ubiquitous in 139 root-nodule bacteria (RNB) characterized in the Genomic Encyclopaedia-Root Nodule Bacteria (GEBA-RNB) project. Access to the GEBA-RNB genomes provided an essential resource to identify, categorise and catalogue a total of 1,245 que genes in a comprehensive Q-pathway RNB database. The constructed database contains all Q-pathway genes for each strain, IMG unique accession numbers, protein domains and predicted protein functions. The database enabled specific genes to be targeted for inactivation in the narrow host range RNB E. medicae WSM419 and in the broad host range RNB E. fredii NGR234. Using this information, four inactivation vectors were successfully created and verified to inactivate queD, queE and queG in E. medicae WSM419 and queG in E. fredii NGR234. The inactivation vectors were successfully used to create six double cross-over mutants of E. medicae WSM419 (two independent mutations in each of the targeted que genes) and two queG single cross-over mutants of E. fredii NGR234. All of the mutations in E. medicae WSM419 were verified by PCR amplification. These mutants were then used to investigate the role of the Q-pathway in WSM419 by extensively phenotyping free-living and symbiotic forms. Inactivation of queD or queE was found to decrease the growth rate of these mutants in free-living conditions. These mutations were shown to significantly reduce the final cell density (P-value = <0.05) of cultures exposed to ZnSO4, CuSO4, NaCl and at 20oC in comparison to the wild-type and queG mutant. However, no significant difference in the final cell density (P-value = >0.05) was observed for cultures exposed to pH 5.7, pH 7.0, EtOH, Sucrose, H2O2, and at 37oC. However, all mutant cultures treated with SDS showed a significant reduction in final cell density (P-value = <0.05) compared to the wild-type. Furthermore, this study revealed that the queD and queE mutants, but not the queG mutant, were affected in their ability to produce succinoglycan. Succinoglycan is essential for the symbiotic proficiency of E. meliloti 1021 with Medicago spp. The symbiotic proficiency of the E. medicae WSM419 mutant derivatives were therefore investigated. This study revealed that the Q-pathway is not required for the establishment of a successful E. medicae-M. truncatula symbiosis. These results are contrary to the findings of the published study by Marchetti et al. (2013) and calls into question the role of the Q-pathway in symbiotic function. Finally, this study revealed that QueE modulates the production of long chain fatty acids, most likely through an affect via FadR or FabH. There was an increase in the long chain fatty acid content of the queE mutant compared to the wild-type as measured by a headspace gas-chromatography mass-spectrometry. A model has now been constructed and presented in this thesis to explain the relationship between the findings of this study and the components of the Q-pathway. This model presents new avenues for future research into the role of que genes not only in the NHR E. medicae WSM419 but also in the BHR microsymbiont E. fredii NGR234.

The Queuosine pathway is not essential for Ensifer medicae WSM419 symbiosis and certain components of this pathway modulate lipid biosynthesis

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