Identification and characterization of the intracellular poly-3-hydroxybutyrate depolymerase enzyme PhaZ of Sinorhizobium meliloti

Abstract

BackgroundS. meliloti forms indeterminate nodules on the roots of its host plant alfalfa (Medicago sativa). Bacteroids of indeterminate nodules are terminally differentiated and, unlike their non-terminally differentiated counterparts in determinate nodules, do not accumulate large quantities of Poly-3-hydroxybutyrate (PHB) during symbiosis. PhaZ is in intracellular PHB depolymerase; it represents the first enzyme in the degradative arm of the PHB cycle in S. meliloti and is the only enzyme in this half of the PHB cycle that remains uncharacterized.ResultsThe S. meliloti phaZ gene was identified by in silico analysis, the ORF was cloned, and a S. meliloti phaZ mutant was constructed. This mutant exhibited increased PHB accumulation during free-living growth, even when grown under non-PHB-inducing conditions. The phaZ mutant demonstrated no reduction in symbiotic capacity; interestingly, analysis of the bacteroids showed that this mutant also accumulated PHB during symbiosis. This mutant also exhibited a decreased capacity to tolerate long-term carbon starvation, comparable to that of other PHB cycle mutants. In contrast to other PHB cycle mutants, the S. meliloti phaZ mutant did not exhibit any decrease in rhizosphere competitiveness; however, this mutant did exhibit a significant increase in succinoglycan biosynthesis.ConclusionsS. meliloti bacteroids retain the capacity to synthesize PHB during symbiosis; interestingly, accumulation does not occur at the expense of symbiotic performance. phaZ mutants are not compromised in their capacity to compete for nodulation in the rhizosphere, perhaps due to increased succinoglycan production resulting from upregulation of the succinoglycan biosynthetic pathway. The reduced survival capacity of free-living cells unable to access their accumulated stores of PHB suggests that PHB is a crucial metabolite under adverse conditions.

PhaP phasins play a principal role in poly-β-hydroxybutyrate accumulation in free-living Bradyrhizobium japonicum

“…We predicted that two putative phaZ genes from the B. japonicum genome [24] encode PHB depolymerases based on their similarities to the phaZ of S. meliloti (SMc02770 in S. meliloti 1021 genome) [25], which had previously been functionally characterized [20]. The results of amino acid sequence comparisons among the products of the phbC and phaZ paralogs are summarized in Figure 1.…”

A putative 3‐hydroxyisobutyryl‐CoA hydrolase is required for efficient symbiotic nitrogen fixation in Sinorhizobium meliloti and Sinorhizobium fredii NGR234

“…The ability to synthesize PHB is required for the catabolism of BHB as a carbon source by S. meliloti (Fig. ; Trainer and Charles, ; Trainer et al ., ). The BHB and l ‐valine utilization phenotypes of the smb20752 and ngr_b20860 mutants suggest a link in the metabolism of these compounds.…”

“…However, disrupting the phaZ gene that encodes the PHB depolymerase (Fig. ) had no effect on the smb20752 Fix phenotype (Supporting Information Table S3), and as phaZ mutants can synthesize but not catabolize PHB (Trainer et al ., ), any PHB‐related toxicity in the smb20752 mutant must arise from the PHB synthesis half of the PHB cycle (Fig. ).…”

The Class II Trehalose 6-phosphate Synthase Gene PvTPS9 Modulates Trehalose Metabolism in Phaseolus vulgaris Nodules

“…Furthermore, resident bacteroids of these organs were considerably larger than their equivalents in control nodules and contained a higher number of poly-β-hydroxybutyrate (PHB) granules in their cytoplasm (Figures 3H–J compared to Figures 3C, F, G). Although, the precise roles of PHB metabolism in the legume-rhizobium symbiosis are not completely understood, experimental evidence indicates that PHB is biosynthesized and turned over rapidly during active nitrogen fixation, and is extremely important for rhizobia to regulate plant carbon flux (Lodwig and Poole, 2003; Trainer et al, 2010). Even though the nitrogen fixation rate in PvTPS9 -RNAi nodules was not changed (Figure 2G), their altered morphology suggests that the metabolism of trehalose plays a pivotal role in nodule formation and development, and affects the carbon metabolism in rhizobia.…”