Synergy between S. cerevisiae and Rhizobium. etli in biofilm formation.
When S. cerevisiae Mat α Σ1278h and R.etli CE3 were grown in minimal medium with low glucose concentrations (0.1%), these species adhered to abiotic surfaces to form biofilms (Fig. 1). Interestingly, R. etli and S. cerevisiae formed a mixed biofilm whose biomass was ~ 3 times greater than that of either single-species biofilm (Fig. 1a). In addition, at 24 hr, the number of colony-forming units (CFU)/cm2 of R. etli CE3 in the mixed biofilm was higher than that in the pure biofilm (Supplementary Fig. 1). Confocal laser scanning microscopy of biofilms stained with the Live/Dead Kit (propidium iodide and SYTO9) showed that in the mixed biofilm, the yeast cells formed patches, and the bacterial cells covered most of the surface (Fig. 1b). In contrast, monospecies biofilms of R. etli and S. cerevisiae had lower structural complexity and contained a greater (80%) number of dead cells, and their individual densities were lower than their populations in the mixed biofilm (Fig. 1b). These results suggest that in mixed biofilms, S. cerevisiae promotes bacterial growth.
S. cerevisiae secretes dicarboxylic acids that promote R. etli growth and biofilm formation.
We found that the R. etli colonies that grew close to S. cerevisiae on solid glucose minimal medium were larger than those growing far from yeast colony (Fig. 2).
We used a visual growth promotion assay on solid medium to screen for S. cerevisiae knockout strains (YKO library) that influenced bacterial growth. 159 yeast mutants were unable to promote R. etli CE3 growth (Supplementary Table 3). In general, these mutants were defective in mitochondrial function. Interestingly, we found that 5 strains with mutations in genes coding for enzymes involved in the TCA cycle showed an enhanced ability to promote bacterial growth compared to that of the wild-type strain (Fig. 2a).
To determine how the S. cerevisiae mutants may affect the fungal-bacterial interaction, we analyzed factors that may be altered in mutants with mitochondrial function defects and a compromised TCA cycle.
We compared the production of TCA intermediates between the wild-type and mutant yeast strains. Mutants defective in mitochondrial function (mef1Δ, gep5Δ, sdh2Δ, ppa2Δ, imp1Δ, cox7Δ, cyc1 and cyc2Δ) produced low amounts of tricarboxylic acids (Supplementary Fig. 2a). In contrast, the aconitase mutant (aco1Δ) produced 60% more citrate and succinate; the fumarase mutant (fum1Δ) resulted in fumarate accumulation; the succinate dehydrogenase mutants (sdh1Δ and sdh4Δ) produced 80% more succinate; and the mitochondrial malate dehydrogenase mutant (mdh1Δ) produced 60% more malate and succinate (Supplementary Fig. 2b). These results suggested that the large quantities of tricarboxylic acids secreted by the mutant yeast played a role in promoting bacterial growth in the cocultures.
We analyzed the biomass of mixed biofilms formed by yeast cells defective in mitochondrial function (Σ1278B petit mutant). The ability of the wild-type and the petit mutant strains to form a monospecies biofilm was similar (Supplementary Fig. 3). In contrast, the mixed biofilm formed by yeast cells defective in mitochondrial function was significantly lower in biomass than that formed by the wild-type yeast strain (Fig. 2c). Also, Σ1278B petit mutant produced low amounts of tricarboxylic acids (Supplementary Fig. 2a).
We next measured the biomass of the mixed biofilm formed by S. cerevisiae and a Rhizobium mutant unable to take up C4-dicarboxylic acids (dctA-). This evaluation revealed that C4-dicarboxylate uptake by R. etli is necessary to form mixed biofilms with high biomass (Fig. 2d).
A symbiotic plasmid is involved in the phenotypic plasticity of R. etli.
The genome of Rhizobium etli CE3 is composed of a chromosome and 6 plasmids (pA, pB, pC, pD, PE and pF) [11]. To determine whether elements encoded by these replicons can participate in the establishment of commensalism, we evaluated the formation of biofilms by yeast and R. etli strains lacking these replicons [12]. We found that lack of pA, pB, pC or pF did not affect the ability of bacteria to coexist with yeast (Fig. 3a). Interestingly, a strain cured of plasmids pA-/pD- could not coexist with S. cerevisiae to form a mixed biofilm and obtain the benefits provided by the fungus (Fig. 3a).
To determine the genetic elements from the symbiotic plasmid involved in the interaction with yeast, we complemented the R. etli pA-/pD- strain with a cosmid library containing fragments of partial digestion (EcoRI) of the R. etli CE3 genome [13]. We found that a cosmid containing 9 ORFs from plasmid pD (GenBank: U80928.5) partially restored the ability of R. etli pA-/pD- to form a mixed biofilm (Fig. 3b). This cosmid contains 7 insertion sequences (IS) and a predicted operon encoding a secretory protein (RHE_PD00332) and an ABC transporter (RHE_PD00333) (Fig. 3b).
The complete operon or only the ABC transporter gene, including its endogenous promoter and terminator regions, was cloned into plasmid pBBR1MCS-3, and the resultant plasmids were named pDA1 and pDA2, respectively (Supplementary table 1 and 2). We found that complementation with either the complete operon or only the ABC transporter gene partially restored the ability of R. etli pA-/pD- to form a mixed biofilm with yeast (Fig. 3c). These results showed that the ABC transporter gene (RHE_PD00333) is involved in the fungal-bacteria interaction.
S. cerevisiae produces a small molecule that affects R. etli growth.
To determine how S. cerevisiae affects the growth of R. etli pA-/pD- (Fig. 4a), we evaluated the inhibitory activity of methanol extracts of S. cerevisiae culture supernatants. Interestingly, we found that the methanol extract inhibited R. etli pA-/pD- growth but had no activity against wild-type R. etli (Fig. 4b). We investigated the chemical constituents of the S. cerevisiae culture supernatants. As a result, a new sophoroside with bacteriostatic activity, named Sc2A, was isolated (Fig. 4c). The structure of Sc2A was elucidated by a combination of extensive spectroscopic analyses, including 2D NMR and HR-MS.
Sc2A was isolated as a crystalline powder with a positive optical rotation ([α]D25 +13.7°, c0.58, H2O). The molecular formula of Sc2A was determined to be C30H50O24 from its positive-mode FAB data (m/z 794.26 [M + H]+), which was consistent with the 13C NMR data. RMN1H (CD3OD, 400 MHz) data for Sc2A: δ 5.1 d (J = 3.6 Hz), 4.4 d (J = 8 Hz), 4.23 dd (J = 9, 4.8 Hz), 3.79 t (J = 10.8, 14.4 Hz), 3.73 m, 3.67 m, 3.639 m, 3.63 dd (J = 8, 9.2 Hz), 3.53 dd (J = 5.6, 5.2 Hz), 3.36 dd (J = 3.6, 4 Hz), 3.31 dd (J = 8, 8 Hz), 3.10 dd (J = 8, 7.6 Hz), 2.77 dd (J = 4.4, 6.8 Hz), 2.61 m, 2.46 m, 2.33 m, 2.12 m. RMN13C-DEPT (CD3OD, 400 MHz) data for Sc2A: δ 181.2 (C), 175.9 (C), 98.1(CH), 93.8 (CH), 78.05 (CH), 78.02 (CH), 76.30 (CH), 74.92 (CH), 73.80 (CH), 73.11 (CH), 71.78 (CH), 71.72 (CH), 64.37 (CH2), 62.87 (CH2),62.72 (CH2), 57.24 (CH), 30.70 (CH2), 26.19 (CH2), 28.21 (CH2).
The IR spectrum of Sc2A displayed characteristic absorptions of 3416.34 cm-1 (O-H), 1642.10 (C = O), 1405.44 (C-OH), 1242.93 (C-O-C), 1040.36 (C-H), and 598.48 (O-C-O).
Sc2A induces the expression of genes involved in symbiosis.
Expression from the nifH and fixA promoters was studied in R. etli monocultures and cocultures with yeast by monitoring GUS activity in living cells. Cells were grown on solid PY-D medium for 1 day, and monitoring of GUS expression showed that the nifH promoter was strongly induced when R. etli was grown with yeast in liquid medium and on solid medium (Fig. 5).