Resource acquisition and allocation traits in symbiotic rhizobia with implications for life-history outside of legume hosts

Abstract

Resources that microbial symbionts obtain from hosts may enhance fitness during free-living stages when resources are comparatively scarce. For rhizobia in legume root nodules, diverting resources from nitrogen fixation to polyhydroxybutyrate (PHB) has been discussed as a source of host-symbiont conflict. Yet, little is known about natural variation in PHB storage and its implications for rhizobial evolution. We therefore measured phenotypic variation in natural rhizobia populations and investigated how PHB might contribute to fitness in the free-living stage. We found that natural populations of rhizobia from Glycine max and Chamaecrista fasciculata had substantial, heritable variation in PHB acquisition during symbiosis. A model simulating temperature-dependent metabolic activity showed that the observed range of stored PHB per cell could support survival for a few days, for active cells, or over a century for sufficiently dormant cells. Experiments with field-isolated Bradyrhizobium in starvation culture suggest PHB is partitioned asymmetrically in dividing cells, consistent with individual-level bet-hedging previously demonstrated in E. meliloti. High-PHB isolates used more PHB over the first month, yet still retained more PHB for potential long-term survival in a dormant state. These results suggest that stored resources like PHB may support both short-term and long-term functions that contribute to fitness in the free-living stage.

Keywords: dormancy; life-history; nitrogen fixation; plant–microbe interactions; polyhydroxyalkanoate; symbiosis.

Figure 1.

PHB accumulation varies widely among co-occurring rhizobia. Phenotypic distribution PHB per cell averaged within nodules collected from (a) G. max and C. fasciculata growing in the field, and (b) G. max inoculated with field-collected soil. The y-axis shows the probability density of PHB measurements estimated from a Gaussian kernel function in R [46]. (c) Variation in PHB accumulation due to environment only (horizontal axis) or genotype plus environment (vertical axis), measured in split-root G. max plants inoculated with a field-isolated strain (upper row of rug plot in (b)) paired with a common reference strain on the other half of the root system. Each point is mean of nine replicate nodules each of focal and reference strains, sampled from three plants. The grey box is the empirical 95% confidence interval for all nodules, which incorporates variability due to genotype, environment and measurement error.

Figure 2.

How long could PHB from nodules support survival in soil? (a) Measured distributions of PHB per cell for two field-isolated strains (approx. 3000 cells per strain pooled from six to eight senescent soya bean nodules from three host plants). (b) Model estimates for how long a rhizobia cell could support metabolism using only internal PHB, assuming starvation begins on 15 September. Oscillations within each curve represent changes in metabolic rate in response to seasonal changes in soil temperature. Estimated survival time decreases slightly if starvation begins in a warmer month (electronic supplementary material, appendix B). Solid curves represent full somatic maintenance and dormancy from Price & Sowers [22], while dotted curves are intermediate metabolic rates (10%, 1% and 0.1% of full somatic maintenance from bottom to top). Vertical lines show the geometric mean and 90th percentile PHB estimates for the low-PHB strain (grey) and the high-PHB strain (black) shown above in (a). (c) The top plot shows representative flow cytometry data from one of the senescent nodules used to obtain the distribution for the high-PHB isolate in (a), compared to data from the same isolate in a non-senescent nodule (bottom plot, not part of the dataset in (a)). Electronic supplementary material, appendix C contains further details on these data.

Figure 3.

Initial PHB only partially explains population trends in starvation culture. Each point represents a different rhizobia isolate in starvation culture (mean of two to three replicate aliquots). (a) Relative population of viable cells (from plate counts) after 4 and 15 months of starvation. Grey arrows indicate changes for the same strain. Only three isolates retained higher-than-initial population size after 15 months of starvation. (b) Net population growth (starting from approximately 10⁵ CFU ml⁻¹) versus PHB use during the first 29 days of starvation. The slope of the regression line (excluding two strains with net negative population growth) was not statistically significant from 0 (p = 0.25, F = 1.66 on 5 degrees of freedom).

Figure 4.

Segregation of high- and low-PHB subgroups in starvation cultures suggest asymmetric partitioning of PHB to old-pole cells. (a) Flow cytometry measurements (gated to exclude debris) for the same rhizobia population at different population densities (approx. 10⁵ or 10⁶ CFU ml⁻¹), showing segregation at low density into high- and low-PHB subgroups (above and below dotted-line) after 29 days of starvation. (b) Trends in the proportion of cells in the high-PHB subgroup (solid lines) compared with viable population size (dashed lines) for the same population (each point is the mean of two to three replicate aliquots). Rhizobia starved at low cell density (open symbols) showed a decrease in the proportion of high-PHB cells concurrent with approximately 10-fold population growth, whereas the same population showed little-to-no PHB segregation or population growth at high density (filled symbols; day 0 data not available, but was diluted from the same culture as the low-density populations).

Figure 5.

PHB use during starvation. (a) During the first month of starvation, the rate of PHB use slows in the high-PHB subgroup (i.e. ignoring low-PHB daughter cells, figure 4, and dividing the change in PHB in the high-PHB subgroup by the number of days elapsed, with final rates based on days 127–430). Two representative strains are shown: a high-PHB field isolate (triangles) and strain USDA110 (circles). Horizontal lines indicate theoretical PHB-use rates at 26°C (solid and dashed lines as in figure 2). (b) Rhizobia that acquired more PHB in nodules used more PHB during the first 29 days in starvation. Each point represents a different isolate in starvation culture (mean of two to three replicate aliquots).