3-Hydroxybutyrate Derived from Poly-3-Hydroxybutyrate Mobilization Alleviates Protein Aggregation in Heat-Stressed Herbaspirillum seropedicae SmR1

Abstract

Under conditions of carbon starvation or thermal, osmotic, or oxidative shock, mutants affected in the synthesis or mobilization of poly-3-hydroxybutyrate (PHB) are known to survive less well. It is still unclear if the synthesis and accumulation of PHB are sufficient to protect bacteria against stress conditions or if the stored PHB has to be mobilized. Here, we demonstrated that mobilization of PHB in Herbaspirillum seropedicae SmR1 was heat-shock activated at 45°C. In situ proton (¹H) nuclear magnetic resonance spectroscopy (i.e., ¹H-nuclear magnetic resonance) showed that heat shock increased amounts of 3-hydroxybutyrate (3HB) only in H. seropedicae strains able to synthesize and mobilize PHB. H. seropedicae SmR1 mutants unable to synthesize or mobilize PHB were more susceptible to heat shock and survived less well than the parental strain. When 100 mM 3-hydroxybutyrate was added to the medium, the ΔphaC1 strain (an H. seropedicae mutant unable to synthesize PHB) and the double mutant with deletion of both phaZ1 and phaZ2 (i.e., ΔphaZ1.2) (unable to mobilize PHB) showed partial rescue of heat adaptability (from 0% survival without 3HB to 40% of the initial viable population). Addition of 200 mM 3HB before the imposition of heat shock reduced protein aggregation to 15% in the ΔphaC1 mutant and 12% in the ΔphaZ1.2 mutant. We conclude that H. seropedicae SmR1 is naturally protected by 3HB released by PHB mobilization, while mutants unable to generate large amounts of 3HB under heat shock conditions are less able to cope with heat damage.IMPORTANCE Bacteria are subject to abrupt changes in environmental conditions affecting their growth, requiring rapid adaptation. Increasing the concentration of some metabolites can protect bacteria from hostile conditions that lead to protein denaturation and precipitation, as well as damage to plasma membranes. In this work, we demonstrated that under thermal shock, the bacterium Herbaspirillum seropedicae depolymerized its intracellular stock polymer known as poly-3-hydroxybutyrate (PHB), rapidly increasing the concentration of 3-hydroxybutyrate (3HB) and decreasing protein precipitation by thermal denaturation. Mutant H. seropedicae strains unable to produce or depolymerize PHB suffered irreparable damage during thermal shock, resulting in fast death when incubated at 45°C. Our results will contribute to the development of bacteria better adapted to high temperatures found either in natural conditions or in industrial processes. In the case of H. seropedicae and other bacteria that interact beneficially with plants, the understanding of PHB metabolism can be decisive for the development of more-competitive strains and their application as biofertilizers in agriculture.

Keywords: 3-hydroxybutyrate; PHA depolymerase; chiral acid R-3-hydroxybutyrate; in situ NMR; phasin; polyhydroxyalkanoate.

FIG 1

Effect of PHB accumulation on heat shock resistance of H. seropedicae. H. seropedicae strains were grown in NFb containing 20 mM NH₄Cl and 37 mM dl-malate at 30°C (orbital agitation at 120 rpm). (A) When the parental strain SmR1 and the ΔphaC1 mutant reached an OD₆₀₀ of 1.2, the cultures were transferred to a water bath at 30°C or 45°C. Bacterial survival was measured by counting CFU after 30 min incubation. Survival at 45°C was normalized to the number of CFU at 30°C. (B) Survival of the parental strain SmR1 and PHB mutants over 30 min of incubation at 45°C. The bacterial strains were grown and treated as described for panel A. (C) Survival of the ΔphaP1.2 mutant carrying the empty vector (pBBR1MCS-3), pLPA01 (wild-type copy of phaP1), or pLPA02 (wild-type copy of phaP2) over 30 min of incubation at 45°C. The symbols representing each strain are depicted in the bottom right corner. The bacterial strains were grown and treated as described for panel A. All experiments were performed in triplicate. Where appropriate, statistical significance is shown (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; independent two-sample t test). In panel B, the statistical significances refer to comparisons between SmR1 and the mutants. There was no statistical significance between SmR1 and ΔphaP2. In panel C, the statistical significances refer to comparisons between ΔphaP1.2 and ΔphaP1.2 complemented with phaP1 or phaP2 (P values of statistical tests are in Tables S1, S2, and S3 in the supplemental material).

FIG 2

Amounts of PHB accumulated interferes in heat-shock resistance by H. seropedicae. (A) SmR1 was grown in NFb with 37 mM dl-malate and 20 mM NH₄Cl at 30°C (orbital agitation at 120 rpm) (blue circles). When the OD₆₀₀ reached 0.2, the cultures were challenged at 45°C, ensuring that the level of PHB accumulated was small (≤1% PHB · cdw⁻¹). As a positive control, SmR1 was cultivated under high-PHB storage conditions (NFb with 25 mM d-glucose and 5 mM NH₄Cl) (blue triangles). When the OD₆₀₀ reached 1.0, the cultures were challenged at 45°C. Survival was measured by counting CFU. (B) As the ΔphaC1 strain cannot use d-glucose as the sole carbon source, it was grown in NFb with 37 mM dl-malate and 20 mM NH₄Cl (red circles). When ΔphaC1 cultures reached an OD₆₀₀ of 0.2, they were challenged at 45°C and survival was measured as described for panel A. For comparison, we plotted the survival of the ΔphaC1 mutant at OD₆₀₀ 1.2 from Fig. 1B (red triangles). Triplicate experiments were performed. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001 (independent two-sample t test). The statistical significances refer to comparisons between SmR1 with low PHB and SmR1 with high PHB levels in panel A and to comparisons between ΔphaC1 at low and high OD₆₀₀ (Table S4) in panel B.

FIG 3

Mobilization of PHB by PHA depolymerases contributes to heat shock resistance. (A) SmR1 and the ΔphaZ1, ΔphaZ2, and ΔphaZ1.2 mutants were grown in NFb (20 mM NH₄Cl and 37 mM dl-malate) at 30°C and subjected to heat shock as described in the legends to Fig. 1 and 2. (B) Mobilization of PHB was followed by Nile red staining and flow cytometry of SmR1 (black bars) and ΔphaZ1.2 (gray bars). The median values of fluorescence (in arbitrary units [a.u.]) were normalized to the number of cells. Experimental procedures were as described in panel A. (C) The samples used in panel B were also subjected to methanolysis and gas chromatography. PHB contents were normalized to cell dry weight. SmR1 (black bars) and ΔphaZ1.2 (gray bars). (D and E) Fluorescent microscope images of SmR1 before and after heat shock (30 min at 45°C), respectively. Replication of the experiments, statistical analyses, and so on were performed as described in the legends to Fig. 1 and 2. The statistical data are presented in Tables S5 and S6. *, P ≤0.05; **, P ≤ 0.01; ***, P ≤ 0.001 (independent two-sample t test). In panel B, the statistical significances refer to comparisons between the strains at different times of heat shock with the strains at time zero.

FIG 4

¹H-NMR monitoring of PHB mobilization and 3HB release. Strains were grown in NFb (containing 20 mM NH₄Cl and 37 mM dl-malate) as described in the legends to Fig. 1 and 2. (A) When the OD₆₀₀ of SmR1 reached 1.2, the cultures were centrifuged, resuspended in 1 ml D₂O, transferred to NMR tubes, and incubated inside an NMR spectrometer at 30°C (blue squares) or 45°C (red circles). The reference peak comprised the total area of the -CH₂ multiplet at δ 2.376. (B) The same procedure as in panel A was applied to SmR1 and mutant strains affected in PHB synthesis or mobilization. All strains were incubated at 45°C. Fig. S3 in the supplemental material shows the ¹H-NMR spectra acquired before and after heat shock. (C) A representative spectrum of ¹H-NMR for SmR1 and ΔphaC1 strains acquired after 60 min of incubation at 45°C. The -CH₂ multiplet at δ 2.376 is marked as 3HB. Note that the region around δ 2.376 in the ΔphaC1 spectrum shows only noise, indicating this mutant did not accumulate PHB and therefore was unable to secrete 3HB during heat shock.

FIG 5

3HB addition increases survival of H. seropedicae and protein solubility after heat shock. The SmR1 (A), ΔphaC1 (B), and ΔphaZ1.2 (C) strains were grown in NFb containing 20 mM NH₄Cl and 37 mM dl-malate at 30°C as listed in the legends to Fig. 1 and 2. The symbols representing each strain are depicted in the bottom, right-hand corner of panel C. Data for cultures treated with 200 mM 3HB are listed in Fig. S5 in the supplemental material. No significant death of any strain at 30°C within 30 min occurred. (D) SmR1 (black bars), ΔphaC1 (red bars), and ΔphaZ1.2 (green bars) strains were incubated with or without 200 mM 3HB at 45°C for 30 min. Replication and statistical analyses were performed as described in the legends to Fig. 1 and 2.