Poly-3-hydroxybutyrate (PHB) supports survival and reproduction in starving rhizobia

Abstract

The carbon that rhizobia in root nodules receive from their host powers both N(2) fixation, which mainly benefits the host, and rhizobium reproduction. Rhizobia also store energy in the lipid poly-3-hydroxybutyrate (PHB), which may enhance rhizobium survival when they are carbon limited, either in nodules or in the soil between hosts. There can be a conflict of interest between rhizobia and legumes over the rate of PHB accumulation, due to a metabolic tradeoff between N(2) fixation and PHB accumulation. To quantify the benefits of PHB to carbon-limited rhizobia, populations of genetically uniform rhizobia with high vs. low PHB (confirmed by flow cytometry) were generated by fractionating Sinorhizobium meliloti via density gradient centrifugation, and also by harvesting cells at early vs. late stationary phase. These rhizobia were starved for 165 days. PHB use during starvation was highly predictive of both initial reproduction and long-term population maintenance. Cultured S. meliloti accumulated enough PHB to triple their initial population size when starved, and to persist for c. 150 days before the population fell below its initial value. During the first 21 days of nodule growth, undifferentiated S. meliloti within alfalfa nodules accumulated enough PHB to support significant increases in reproduction and survival during starvation.

Rhizobitoxine producers gain more poly-3-hydroxybutyrate in symbiosis than do competing rhizobia, but reduce plant growth

“…Forward scatter (FSC) was linearly correlated with Nile Red fluorescence, and thus with PHB (Ratcliff et al, 2008) (Po0.0001, r 2 ¼ 0.87, Figure 1). The intercept was not significantly different from 0…”

“…Siratro), grown in pouches, were inoculated with either 10 5 Rtx( þ ) or Rtx(À) rhizobia, or with 5 Â 10 4 rhizobia of each strain. Plants were grown as described by Ratcliff et al (2008) for 12 weeks before harvesting. At harvest, nodules were randomly sampled; rhizobia per nodule and PHB per cell were determined flow cytometrically as described earlier (Ratcliff et al, 2008), but using forward scattering (FSC), calibrated against Nile Red fluorescence.…”

“…Plants were grown as described by Ratcliff et al (2008) for 12 weeks before harvesting. At harvest, nodules were randomly sampled; rhizobia per nodule and PHB per cell were determined flow cytometrically as described earlier (Ratcliff et al, 2008), but using forward scattering (FSC), calibrated against Nile Red fluorescence. Calibration data came from two preliminary experiments in which plant growth, nodule harvest and analysis of rhizobia were as described, but the plants were harvested after 4 or 6 weeks of growth.…”

“…Strains that divert resources from N 2 fixation to their own immediate reproduction, or to synthesis of poly-3-hydroxybutyrate (PHB) to support later reproduction (Ratcliff et al, 2008), could potentially outcompete more mutualistic strains in other nodules of the same plant (Denison, 2000;West et al, 2002). However, host sanctions against nodules that fix little N 2 reduce reproduction of cheating rhizobia by half (Kiers et al, 2003).…”

“…Increasing the number of nodules formed by a plant will not increase an Rtx strain's relative fitness (proportional representation in the population) unless Rtx rhizobia are disproportionately represented in the additional nodules, but other fitness benefits are possible. Rhizobia could benefit by using Rtx produced inside nodules (Ruan and Peters, 1992) to manipulate the host into forming larger nodules (allowing more rhizobial reproduction inside), or by increasing their synthesis of PHB to support later rhizobial reproduction (Ratcliff et al, 2008). However, the carbon cost of either excessive nodule growth or PHB synthesis could decrease the fitness of the legume host.…”