Effects of synthetic large-scale genome reduction on metabolism and metabolic preferences in a nutritionally complex environment
The soil bacterium Sinorhizobium meliloti forms nodules on the roots of leguminous plants, where N2 is reduced to ammonia. Its genome includes a 3.65 Mb chromosome, a 1.35 Mb pSymA megaplasmid, and a 1.68 Mb pSymB chromid. pSymA and pSymB constitute ~45 % of the genome and here a non-targeted approach was used to identify the metabolic consequences of the removal of these replicons. Polar and non-polar metabolites from wild-type, ∆pSymA, ∆pSymB, and ∆pSymAB cells and supernatants across a growth curve were analyzed by LC–HILIC–TOF–MS. 2008 metabolite features were identified in the extracellular metabolome of cells grown in LBmc containing yeast extract and casein hydrolysate. 1474 features were found from the intracellular metabolites of cells grown in minimal M9-sucrose medium. Analysis revealed both time and genotype influenced the metabolome, with the removal of pSymB having a much greater effect than the loss of pSymA. Strains lacking pSymB showed an increase in sugar, amino acid, and nucleotide metabolites in the intracellular metabolome, and the loss of pSymB clearly impaired the cell’s ability to catabolize exogenous amino acids. We conclude that despite the ability of wild-type, ∆pSymA, ∆pSymB, and ∆pSymAB strains to grow in both M9-sucrose and LBmc media, the removal of pSymA, and particularly pSymB, had clear and dramatic effects on the S. meliloti metabolome. The larger effect associated with the pSymB chromid is consistent with the large number of metabolic genes on this replicon and the greater genetic and metabolic integration of this replicon with the S. meliloti chromosome.
KeywordsSinorhizobium Metabolism Multipartite Microbial
This work is dedicated to the late Prof. Brian McCarry (1946–2013). Funding for this work was provided by NSERC to B. E. M. and T. M. F. The authors would like to thank the Center for Microbial Chemical Biology (CMCB) at McMaster for access to the LC–MS. F. F. is supported by an Ontario Graduate Scholarship and G. C. D by a NSERC CGS award.
Compliance with ethical standards
Conflict of interest
Authors Fan Fei, George C. diCenzo, Dawn M. E. Bowdish, Brian E. McCarry (deceased), and Turlough M. Finan declare that they have no conflict of interest.
This work did not involve human particpants and/or animals, and all authors have agreed to submission of the manuscript.
- Barsch, A., Tellström, V., Patschkowski, T., Küster, H., & Niehaus, K. (2006). Metabolite profiles of nodulated alfalfa plants indicate that distinct stages of nodule organogenesis are accompanied by global physiological adaptations. Molecular Plant-Microbe Interactions, 19(9), 998–1013. doi: 10.1094/MPMI-19-0998.CrossRefPubMedGoogle Scholar
- diCenzo, G., Milunovic, B., Cheng, J., & Finan, T. M. (2013). The tRNAarg gene and engA are essential genes on the 1.7-mb pSymB megaplasmid of Sinorhizobium meliloti and were translocated together from the chromosome in an ancestral strain. Journal of Bacteriology, 195(2), 202–212. doi: 10.1128/JB.01758-12.PubMedCentralCrossRefPubMedGoogle Scholar
- Dominguez-Ferreras, A., Perez-Arnedo, R., Becker, A., Olivares, J., Soto, M. J., & Sanjuan, J. (2006). transcriptome profiling reveals the importance of plasmid pSymB for Osmoadaptation of Sinorhizobium meliloti. Journal of Bacteriology, 188(21), 7617–7625. doi: 10.1128/JB.00719-06.PubMedCentralCrossRefPubMedGoogle Scholar
- Dunn, W. B., Broadhurst, D., Begley, P., Zelena, E., Francis-McIntyre, S., Anderson, N., et al. (2011). Procedures for large-scale metabolic profiling of serum and plasma using gas chromatography and liquid chromatography coupled to mass spectrometry. Nature Protocols, 6(7), 1060–1083. doi: 10.1038/nprot.2011.335.CrossRefPubMedGoogle Scholar
- Epstein, B., Branca, A., Mudge, J., Bharti, A. K., Briskine, R., Farmer, A. D., et al. (2012). Population genomics of the facultatively mutualistic bacteria Sinorhizobium meliloti and S. medicae. PLoS Genetics, 8(8), e1002868. doi: 10.1371/journal.pgen.1002868.PubMedCentralCrossRefPubMedGoogle Scholar
- Fei, F., Bowdish, D. M. E., & McCarry, B. E. (2014). Comprehensive and simultaneous coverage of lipid and polar metabolites for endogenous cellular metabolomics using HILIC-TOF-MS. Analytical and Bioanalytical Chemistry, 406, 3723–3733. doi: 10.1007/s00216-014-7797-5.PubMedCentralCrossRefPubMedGoogle Scholar
- Finan, T. M., O’Brian, M. R., Layzell, D. B., Vessey, J. K., & Newton, W. (Eds.). (2002). Nitrogen fixation: Global perspectives. Wallingford: CABI Publishing.Google Scholar
- Finan, T. M., Weidner, S., Wong, K., Buhrmester, J., Chain, P., Vorhölter, F. J., et al. (2001). The complete sequence of the 1683-kb pSymB megaplasmid from the N2-fixing endosymbiont Sinorhizobium meliloti. Proceedings of the National Academy of Sciences of the United States of America, 98(17), 9889–9894. doi: 10.1073/pnas.161294698.PubMedCentralCrossRefPubMedGoogle Scholar
- Gemperline, E., Jayaraman, D., Maeda, J., Ané, J.-M., & Li, L. (2015). Multifaceted investigation of metabolites during nitrogen fixation in Medicago via high resolution MALDI-MS imaging and ESI-MS. Journal of the American Society for Mass Spectrometry, 26(1), 149–158. doi: 10.1007/s13361-014-1010-0.PubMedCentralCrossRefPubMedGoogle Scholar
- Mauchline, T. H., Fowler, J. E., East, A. K., Sartor, A. L., Zaheer, R., Hosie, A. H. F., et al. (2006). Mapping the Sinorhizobium meliloti 1021 solute-binding protein-dependent transportome. Proceedings of the National Academy of Sciences of the United States of America, 103(47), 17933–17938. doi: 10.1073/pnas.0606673103.PubMedCentralCrossRefPubMedGoogle Scholar
- Mostafavi, M., Lewis, J. C., Saini, T., Bustamante, J. A., Gao, I. T., Tran, T. T., et al. (2014). Analysis of a taurine-dependent promoter in Sinorhizobium meliloti that offers tight modulation of gene expression. BMC Microbiology, 14(1), 295. doi: 10.1186/s12866-014-0295-2.PubMedCentralCrossRefPubMedGoogle Scholar
- Sallet, E., Roux, B., Sauviac, L., Jardinaud, M.-F., Carrère, S., Faraut, T., et al. (2013). Next-generation annotation of prokaryotic genomes with EuGene-P: application to Sinorhizobium meliloti 2011. DNA Research, 20(4), 339–354. doi: 10.1093/dnares/dst014.PubMedCentralCrossRefPubMedGoogle Scholar
- Ye, H., Gemperline, E., Venkateshwaran, M., Chen, R., Delaux, P.-M., Howes-Podoll, M., et al. (2013). MALDI mass spectrometry-assisted molecular imaging of metabolites during nitrogen fixation in the Medicago truncatula-Sinorhizobium meliloti symbiosis. The Plant Journal, 75(1), 130–145. doi: 10.1111/tpj.12191.CrossRefPubMedGoogle Scholar