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

Maria A Trainer,Scott Rd Clark,Trevor C Charles,Alicja Zachertowska,Kathy N Lam,David Capstick

doi:10.1186/1471-2180-10-92

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.

Highlights

S. meliloti forms indeterminate nodules on the roots of its host plant alfalfa (Medicago sativa)
The phaZ::ΩSmSp was subsequently excised as an EcoRI fragment and subcloned into pK19mobsacB to give pAZ103. pAZ103 was introduced into S. meliloti Rm5000 by triparental mating using E. coli MT616 as a helper strain
Previous studies have demonstrated that the ability of certain bacteria to synthesize, accumulate and metabolize intracellular PHB stores is important in enhancing their capacity to survive unfavourable growth conditions [3437]

Summary

Introduction

S. meliloti forms indeterminate nodules on the roots of its host plant alfalfa (Medicago sativa). Several genera of soil bacteria can enter into nitrogen-fixing symbioses with leguminous plants. These genera, commonly referred to as the 'rhizobia', include Sinorhizobium, Rhizobium, Bradyrhizobium, and Azorhizobium. Microaerophilic nodules on the roots of the host plant are elicited by the bacteria. Following infection and colonization of the nodule tissue, the bacteria undergo differentiation into a mature state known as the bacteroid, which can reduce atmospheric dinitrogen to ammonia. Bacteroid metabolism is dominated by the production of fixed nitrogen, which is transferred directly to the host plant.

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