Abstract
The acidity of soils significantly reduces the productivity of legumes mainly because of the detrimental effects of hydrogen ions on the legume plants, leading to the establishment of an inefficient symbiosis and poor biological nitrogen fixation. We recently reported the analysis of the fully sequenced genome of Rhizobium favelukesii LPU83, an alfalfa-nodulating rhizobium with a remarkable ability to grow, nodulate and compete in acidic conditions. To gain more insight into the genetic mechanisms leading to acid tolerance in R. favelukesii LPU83, we constructed a transposon mutant library and screened for mutants displaying a more acid-sensitive phenotype than the parental strain. We identified mutant Tn833 carrying a single-transposon insertion within LPU83_2531, an uncharacterized short ORF located immediately upstream from ubiF homolog. This gene encodes a protein with an enzymatic activity involved in the biosynthesis of ubiquinone. As the transposon was inserted near the 3′ end of LPU83_2531 and these genes are cotranscribed as a part of the same operon, we hypothesized that the phenotype in Tn833 is most likely due to a polar effect on ubiF transcription.
We found that a mutant in ubiF was impaired to grow at low pH and other abiotic stresses including 5 mM ascorbate and 0.500 mM Zn2+. Although the ubiF mutant retained the ability to nodulate alfalfa and Phaseolus vulgaris, it was unable to compete with the R. favelukesii LPU83 wild-type strain for nodulation in Medicago sativa and P. vulgaris, suggesting that ubiF is important for competitiveness. Here, we report for the first time an ubiF homolog being essential for nodulation competitiveness and tolerance to specific stresses in rhizobia.
Graphical abstract
Data Availability
Not applicable.
Code availability
Not applicable.
References
Cloutier J, Serge L, Yves C, Hani A (1996) Characterization and mutational analysis of nodHPQ genes of Rhizobium sp. strain N33. Mol Plant Microbe Interact Vol 9, 720–728. https://doi.org/10.1094/mpmi-9-0720, https://pubmed.ncbi.nlm.nih.gov/8870271/
Barran LR, Bromfield ESP, Brown DCW (2002) Identification and cloning of the bacterial nodulation specificity gene in the Sinorhizobium meliloti–Medicago laciniata symbiosis. Can J Microbiol 48:765–771. https://doi.org/10.1139/w02-072
Villegas MDC et al (2006) Nitrogen-fixing sinorhizobia with Medicago laciniata constitute a novel biovar (bv. medicaginis) of S. meliloti. Syst Appl Microbiol 29:526–538. https://doi.org/10.1016/j.syapm.2005.12.008
Tejerizo GT et al (2016) Rhizobium favelukesii sp. nov., isolated from the root nodules of alfalfa (Medicago sativa L). Int J System Evol Microbiol 66(11):4451–4457. https://doi.org/10.1099/ijsem.0.001373
Eardly BD, David B Hannaway, Bottomley P (1985) Characterization of Rhizobia from ineffective alfalfa nodules: ability to nodulate bean plants [Phaseolus vulgaris (L.) Savi.] t. 50, 1422–1427. https://doi.org/10.1128/aem.50.6.1422-1427.1985
Del Papa et al (1999) Isolation and characterization of alfalfa-nodulating rhizobia present in acidic soils of central Argentina and Uruguay. Appl Environ Microbiol 65:1420–1427. https://doi.org/10.1128/AEM.65.4.1420-1427.1999
Eardly B (1992) Phylogenetic position of Rhizobium sp. strain Or 191, a symbiont of both Medicagosativa and Phaseolus vulgaris, based on partial sequences of the 16S rRNA and nifH genes. Appl Environ Microbiol 58:1809–1815. https://doi.org/10.1128/aem.58.6.1809-1815.1992
Del Papa MF et al (2007) Identification and characterization of a nodH ortholog from the alfalfa-nodulating Or191-like rhizobia. Mol Plant Microbe Interact 20:138–145. https://doi.org/10.1094/MPMI-20-2-0138
Wegener C et al (2001) Genetic uniformity and symbiotic properties of acid-tolerant alfalfa-nodulating rhizobia isolated from dispersed locations throughout Argentina. Symbiosis 30, 141–162. https://dalspace.library.dal.ca/bitstream/handle/10222/77860/VOLUME%2030-NUMBERS%202-3-2001-PAGE%20141.pdf?sequence=1
Tabares-da Rosa S et al (2019) Rhizobia inoculants for alfalfa in acid soils: a proposal for Uruguay. Inoculantes rizobianos para alfalfa en suelos ácidos: una propuesta para Uruguay. Agrociencia Uruguay 23:1–13. https://doi.org/10.31285/agro.23.120
Castellani LG et al (2021) Exopolysaccharide characterization of Rhizobium favelukesii LPU83 and its role in the symbiosis with alfalfa. Front Plant Sci 12:1–17. https://doi.org/10.3389/fpls.2021.642576
Del Papa MF et al (2003) A microcosm study on the influence of pH and the host-plant on the soil persistence of two alfalfa-nodulating rhizobia with different saprophytic and symbiotic characteristics. Biol Fertil Soils 39:112–116. https://doi.org/10.1007/s00374-003-0690-6
Soto MJ, Pieter van Dillewijn F, Martínez-A, José I, Jiménez-Zurdo NT (2004) Attachment to plant roots and nod gene expression are not affected by pH or calcium in the acid-tolerant alfalfa-nodulating bacteria. FEMS Microbiol Ecol 48:71–77. https://doi.org/10.1016/j.femsec.2003.12.010
Brockwell J, Pilka A, Holliday RA (1991) Soil pH is a major determinant of the numbers of naturally occurring Rhizobium meliloti in non-cultivated soils in central New South Wales. Aust J Exp Agric 1. https://doi.org/10.1071/EA9910211
Graham PH (1992) Stress tolerance in Rhizobium and Bradyrhizobium, and nodulation under adverse soil conditions. Can J Microbiol 38:475–484. https://doi.org/10.1139/m92-079
Glenn AR, Dilworth MJ (1994) The life of root nodule bacteria in the acidic underground. FEMS Microbiol Lett 123:1–10. https://doi.org/10.1111/j.1574-6968.1994.tb07193.x
Ferguson BJ, Lin M, Gresshoff PM (2013) Regulation of legume nodulation by acidic growth conditions. Plant Signal Behav 8(3):1–5. https://doi.org/10.4161/psb.23426
Goss TJ, O’Hara GW, Dilworth MJ, Glenn AR (1990) Cloning, characterization, and complementation of lesions causing acid sensitivity in Tn5-induced mutants of Rhizobium meliloti WSM419. J Bacteriol 172:5173–5179. https://doi.org/10.1128/jb.172.9.5173-5179.1990
Tiwari RP, Reeve WG, Gleenn AR (1992) Mutations conferring acid sensitivity in the acid-tolerant strains Rhizobium meliloti WSM419 and Rhizobium leguminosarum biovar viciae WSM710. FEMS Microbiol Lett 100:107–112. https://doi.org/10.1111/j.1574-6968.1992.tb14027.x
Tiwari RP, Reeve WG, Dilworth MJ, Glenn AR (1996) Acid tolerance in Rhizobium meliloti strain WSM419 involves a two-component sensor-regulator system. Microbiology 142(Pt 7):1693–1704. https://doi.org/10.1099/13500872-142-7-1693
Tiwari RP, Reeve WG, Dilworth MJ, Glenn AR (1996) An essential role for actA in acid tolerance of Rhizobium meliloti. Microbiology 142:601–610. https://doi.org/10.1099/13500872-142-3-601
Kiss E, Huguet T, Poinsot V, Batut J (2004) The typA gene is required for stress adaptation as well as for symbiosis of Sinorhizobium meliloti 1021 with certain Medicago truncatula lines. Mol Plant Microbe Interact 17:235–244. https://doi.org/10.1094/MPMI.2004.17.3.235
Albicoro FJ et al (2021) The two-component system ActJK is involved in acid stress tolerance and symbiosis in Sinorhizobium meliloti. J Biotechnol 329:80–91. https://doi.org/10.1016/j.jbiotec.2021.01.006
Hawkins JP, Geddes BA, Oresnik IJ (2017) Succinoglycan production contributes to acidic pH tolerance in Sinorhizobium meliloti Rm1021. Mol Plant Microbe Interact 30:1009–1019. https://doi.org/10.1094/MPMI-07-17-0176-R
Primo ED et al (2019) Exopolysaccharide production in Ensifer meliloti laboratory and native strains and their effects on alfalfa inoculation. Arch Microbiol. https://doi.org/10.1007/s00203-019-01756-3
Nilsson JF et al (2019) Proteomic analysis of Rhizobium favelukesii LPU83 in response to acid stress. J Proteome Res. https://doi.org/10.1021/acs.jproteome.9b00275
Guerrero-Castro J, Lozano L, Sohlenkamp C (2018) Dissecting the acid stress response of Rhizobium tropici CIAT 899. Front Microbiol 9:846. https://doi.org/10.3389/fmicb.2018.00846
Guan N (2020) Microbial response to acid stress: mechanisms and applications. Appl Microbiol Biotechnol 51–65. https://doi.org/10.1007/s00253-019-10226-1
Simon R, Hotte B, Klauke B, Kosier B (1991) Isolation and characterization of insertion sequence elements from gram-negative bacteria by using new broad-host-range, positive selection vectors. J Bacteriol 173:1502–1508. https://doi.org/10.1128/jb.173.4.1502-1508.1991
Simon R, Quandt J, Klipp W (1989) New derivatives of transposon Tn5 suitable for mobilization of replicons, generation of operon fusions and induction of genes in Gram-negative bacteria. Gene 80:161–169. https://doi.org/10.1016/0378-1119(89)90262-X
Schäfer A et al (1994) Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19: selection of defined deletions in the chromosome of Corynebacterium glutamicum. Gene 145:69–73. https://doi.org/10.1016/0378-1119(94)90324-7
Kovach ME, Phillips RW, Elzer PH, Roop RM, Peterson KM (1994) pBBR1MCS: a broad-host-range cloning vector. Biotechniques 16, 800–802 . https://pubmed.ncbi.nlm.nih.gov/8068328/
Dombrecht B, Vanderleyden J, Michiels J (2001) Stable RK2-derived cloning vectors for the analysis of gene expression and gene function in gram-negative bacteria. Mol Plant-Microbe Interact 14:426–430. https://doi.org/10.1094/MPMI.2001.14.3.426
Hansen LH, Sørensen SJ, Jensen LB (1997) Chromosomal insertion of the entire Escherichia coli lactose operon, into two strains of Pseudomonas, using a modified mini-Tn5 delivery system. Gene 186:167–173. https://doi.org/10.1016/S0378-1119(96)00688-9
Behringer JE (1974) R factor transfer in Rhizobium leguminosarum. J Gen Microbiol 84:188–198. https://doi.org/10.1099/00221287-84-1-188
Vincent JM (1970) A manual for the practical study of root nodule bacteria. (Blackwell, Oxford and Edinburgh. https://doi.org/10.1002/jobm.19720120524
Sambrook J, Fritsch ER, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
Rossbach S, Kulpa DA, Rossbach U, de Bruijn FJ (1994) Molecular and genetic characterization of the rhizopine catabolism (mocABRC) genes of Rhizobium meliloti L5–30. MGG Mol Gen Genet 245:11–24. https://doi.org/10.1007/BF00279746
Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25:4876–4882. https://doi.org/10.1093/nar/25.24.4876
Lodeiro Aníbal R, López-García SL, Vázquez TEE, Favelukes G (2000) Stimulation of adhesiveness, infectivity, and competitiveness for nodulation of Bradyrhizobium japonicum by its pretreatment with soybean seed lectin. FEMS Microbiol Lett 188:177–184. https://doi.org/10.1111/j.1574-6968.2000.tb09190.x
Fähraeus G (1957) The infection of clover root hairs by nodule bacteria studied by a simple glass slide technique. J Gen Microbiol 16:374–381. https://doi.org/10.1099/00221287-16-2-374
Nakahigashi K, Miyamoto K, Nishimura K, Inokuchi H (1992) Isolation and characterization of a light-sensitive mutant of Escherichia coli K-12 with a mutation in a gene that is required for the biosynthesis of ubiquinone. J Bacteriol 174:7352–7359. https://doi.org/10.1128/jb.174.22.7352-7359.1992
Søballe B, Poole RK (1999) Microbial ubiquinones: multiple roles in respiration, gene regulation and oxidative stress management. Microbiology 145:1817–1830. https://doi.org/10.1099/13500872-145-8-1817
Nilsson JF et al (2021) Global transcriptome analysis of Rhizobium favelukesii LPU83 in response to acid stress. 1–16. https://doi.org/10.1093/femsec/fiaa235
Reeve WG, Tiwari RP, Wong CM, Dilworth MJ, Glenn AR (1998) The transcriptional regulator gene phrR in Sinorhizobium meliloti WSM419 is regulated by low pH and other stresses. Microbiology 144:3335–3342. https://doi.org/10.1099/00221287-144-12-3335
Shamseldin A, Werner D (2005) High salt and high pH tolerance of new isolated Rhizobium etli strains from Egyptian soils. Curr Microbiol 50:11–16. https://doi.org/10.1007/s00284-004-4391-7
Bahena MHR, Salazar S, Velázquez E, Laguerre G, Peix A (2015) Characterization of phosphate solubilizing rhizobacteria associated with pea (Pisum sativum L.) isolated from two agricultural soils. Symbiosis 67:33–41. https://doi.org/10.1007/s13199-015-0375-6
Oliveira DP et al (2017) Acid tolerant Rhizobium strains contribute to increasing the yield and profitability of common bean in tropical soils. J Soil Sci Plant Nutr 17:922–934. https://doi.org/10.4067/S0718-95162017000400007
Chen L, James LP, Helmann JD (1993) Metalloregulation in Bacillus subtilis: isolation and characterization of two genes differentially repressed by metal ions. J Bacteriol 175:5428–5437. https://doi.org/10.1128/jb.175.17.5428-5437.1993
Aussel L et al (2014) Biosynthesis and physiology of coenzyme Q in bacteria. Biochim Biophys Acta - Bioenerg 1837:1004–1011. https://doi.org/10.1016/j.bbabio.2014.01.015
Ma C et al (2010) Energy production genes sucB and ubiF are involved in persister survival and tolerance to multiple antibiotics and stresses in Escherichia coli. FEMS Microbiol Lett 303:33–40. https://doi.org/10.1111/j.1574-6968.2009.01857.x
Alexander K, Young IG (1978) Alternative hydroxylases for the aerobic and anaerobic biosynthesis of ubiquinone in Escherichia coli. Biochemistry 17:4750–4755. https://doi.org/10.1021/bi00615a024
Kwon O, Hudspeth ME, Meganathan R (1996) Anaerobic biosynthesis of enterobactin Escherichia coli: regulation of entC gene expression and evidence against its involvement in menaquinone (vitamin K2) biosynthesis. J Bacteriol 178:3252–3259. https://doi.org/10.1128/jb.178.11.3252-3259.1996
Riccillo PM et al (2000) Glutathione is involved in environmental stress responses in Rhizobium tropici, including acid tolerance. J Bacteriol 182:1748–1753. https://doi.org/10.1128/JB.182.6.1748-1753.2000
Meilhoc E, Cam Y, Skapski A, Bruand C (2010) The response to nitric oxide of the nitrogen-fixing symbiont Sinorhizobium meliloti. Mol Plant Microbe Interact 23:748–759. https://doi.org/10.1094/MPMI-23-6-0748
Santos R, Herouart D, Sigaud S, Touati D, Puppo A (2007) Oxidative burst in alfalfa-Sinorhizobium meliloti symbiotic interaction. Mol Plant Microbe Interact 14:86–89. https://doi.org/10.1094/MPMI.2001.14.1.86
Rubio MC et al (2004) Localization of superoxide dismutases and hydrogen peroxide in legume root nodules. Mol Plant Microbe Interact 17:1294–1305. https://doi.org/10.1094/MPMI.2004.17.12.1294
Chang C, Damiani I, Puppo A, Frendo P (2009) Redox changes during the legume–Rhizobium symbiosis. Mol Plant 2:370–377. https://doi.org/10.1093/mp/ssn090
Acknowledgements
The authors would like to honour Prof. Gabriel Favelukes and Prof. Oscar Grau who made valuable contributions to the development of the Institute of Biotechnology and Molecular Biology.
Funding
This work was supported by the National Scientific and Technical Research Council of Argentina (Consejo Nacional de Investigaciones Científicas y Técnicas – CONICET, Argentina) (Grant/Award PIP2015-0700) and the Ministry of Science, Technology and Productive Innovation (Ministerio de Ciencia, Tecnolología e Innovación Productiva – MinCyT, Argentina) (Grants/Awards PICT2017-2833 and PICT2017-2371). MCM and CV were supported by fellowships from CONICET. GTT, WOD, ML, MP, AL and MFDP are researchers at CONICET.
Author information
Authors and Affiliations
Contributions
MCM, WOD, MJL, MP and CV performed the experiments, and GATT carried out the molecular data analysis. AL and MFDP designed the experiments. MCM and MFDP wrote the manuscript. All authors revised and contributed to the manuscript.
Corresponding author
Ethics declarations
Ethics approval and consent to participate
Not applicable.
Consent for publication
Not applicable.
Conflict of interest
The authors declare no competing interests.
Additional information
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Rights and permissions
About this article
Cite this article
Martini, M.C., Vacca, C., Torres Tejerizo, G.A. et al. ubiF is involved in acid stress tolerance and symbiotic competitiveness in Rhizobium favelukesii LPU83. Braz J Microbiol 53, 1633–1643 (2022). https://doi.org/10.1007/s42770-022-00780-8
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s42770-022-00780-8