ubiF is involved in acid stress tolerance and symbiotic competitiveness in Rhizobium favelukesii LPU83

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.

Keywords: Acid tolerance; Competitiveness; Rhizobium favelukesii LPU83; Stress; ubiF.

Fig. 1

Genetic organization of the DNA region interrupted by transposon Tn5B20 insertion in the Tn833 mutant. The red arrow indicates the position where transposon was inserted (genome position 2,469,710), and the grey arrow shows the position of the predicted promoter of the operon containing LPU883_2531 (ORF1) and ubiF (genome position 2,473,610). The genome coordinates are relative to the reference genome of R. favelukesii LPU83. The genes in the region nearby are as follows: mdcF, malonate transport protein; ubiF, putative 2-polyprenyl-6-hydroxyphenol hydroxylase from the quinone biosynthetic pathway (UQ, coenzyme Q); LPU83_2531, open reading frame interrupted by Tn5B20; cysI, putative sulfite reductase (NADPH) protein from the cysteine biosynthetic pathway; cysG1, uroporphyrin III C-methyltransferase protein; LPU83_2535, unknown function

Fig. 2

Disruption of LPU83_2531/ubiF impairs growth in acid media. Growth kinetics in neutral (pH = 7.0) or acid (pH = 5.0) GS medium for the WT and mutants are shown. The proportion of growth was calculated as the CFU at each time point relative to time zero. Error bars represent the average of at least two independent experiments. The variations in the pH throughout each experiment were no greater than 0.6 units

Fig. 3

Lack of LPU83_2531/ubiF impacts on the survival of R. favelukesii LPU83 in stressing conditions. Survival of ubiF⁻ and LPU83_2531.⁻ mutants and the WT strain was evaluated in the presence of high concentrations of ZnSO₄ and ascorbic acid. CFU counting in medium at pH = 7.0 was used as a control. Log-phase cultures were diluted to a concentration of 10⁷ CFU/ml and incubated at 28 °C and 280 RPM. For each condition, the number of CFU was determined after 6 h or 24 h. nd, not detected

Fig. 4

Alfalfa nodulation kinetics of R. favelukesii LPU83 and its derivative strains. The nodules from the principal roots (pink), secondary roots (green) and total roots (purple) were determined at different time points as indicated in the “Materials and methods”. The results, expressed as the average number of nodules per plant, are taken from a representative experiment within a set of three. The standard deviations throughout were lower than 20% in all cases

Fig. 5

Loss of LPU83_2531/ubiF dramatically reduces the ability to compete with the WT strain. Nodulation competitiveness of LPU83_2531⁻ (A) or ubiF⁻ (B) mutants was evaluated against the R. favelukesii LPU83 WT strain in alfalfa plants. The results are presented as the mean value of the percentage of nodules occupied by each strain coinoculated from a 1:1 mixture. The occupancy of each strain in the nodules was determined by differential growth on TY solid plates supplemented with selective antibiotics