Genetic and physiological characterization of a Rhizobium etli mutant strain unable to synthesize poly-beta-hydroxybutyrate

Abstract

Rhizobium etli accumulates poly-␤-hydroxybutyrate (PHB) in symbiosis and in free life. PHB is a reserve material that serves as a carbon and/or electron sink when optimal growth conditions are not met. It has been suggested that in symbiosis PHB can prolong nitrogen fixation until the last stages of seed development, but experiments to test this proposition have not been done until now. To address these questions in a direct way, we constructed an R. etli PHB-negative mutant by the insertion of an ⍀-Km interposon within the PHB synthase structural gene (phaC). The identification and sequence of the R. etli phaC gene are also reported here. Physiological studies showed that the PHB-negative mutant strain was unable to synthesize PHB and excreted more lactate, acetate, pyruvate, ␤-hydroxybutyrate, fumarate, and malate than the wild-type strain. The NAD ؉ /NADH ratio in the mutant strain was lower than that in the parent strain. The oxidative capacity of the PHB-negative mutant was reduced. Accordingly, the ability to grow in minimal medium supplemented with glucose or pyruvate was severely diminished in the mutant strain. We propose that in free life PHB synthesis sequesters reductive power, allowing the tricarboxylic acid cycle to proceed under conditions in which oxygen is a limiting factor. In symbiosis with Phaseolus vulgaris, the PHB-negative mutant induced nodules that prolonged the capacity to fix nitrogen.Poly-␤-hydroxybutyrate (PHB) and other polyhydroxyalkanoates (PHA) are accumulated by a wide range of bacteria as carbon and reductive-power storage compounds. Several species belonging to the genera Rhizobium, Bradyrhizobium, and Azorhizobium accumulate PHB in free life (40, 43) and in symbiosis (16,23,29,48). In contrast, in other species, such as Rhizobium meliloti, the accumulation of PHB is observed only in the free-living state or in the first steps of nodule development but never in nitrogen-fixing bacteroids (20). The physiological role of these compounds in symbiosis is not completely understood. It is known that bacteroids of Bradyrhizobium japonicum may accumulate PHB and fix nitrogen simultaneously, although both functions require large amounts of reductive power (48). Bergersen et al. (1) proposed that PHB reserves in bacteroids can support some nitrogen fixation during darkness and prolong the period of nitrogen fixation. Bacteroids can also use PHB as a source of energy and reductive power for nitrogen fixation when incubated, ex planta, at a low oxygen concentration (2). In Rhizobium etli, PHB is accumulated not only in the stationary phase, like in other bacteria, but also during exponential growth. Moreover, PHB is being synthesized and degraded continuously even under conditions in which none of the polymer accumulates (10). This suggests the presence of a very sensitive regulatory mechanism that controls the accumulation or degradation of PHB, thus allowing rapid modulation of the levels of reductive power and of oxidizable substrates. This situation is especially favorable in organism...

Polyester synthesis genes associated with stress resistance are involved in an insect–bacterium symbiosis

“…In the symbiotic context, PHA granules have been identified in rhizobial cells. Some species belonging to Rhizobium, Bradyrhizobium, and Azorhizobium accumulate PHA granules both in free life and in symbiosis, whereas in other species, such as Sinorhizobium meliloti, accumulation of PHA granules is observed only in free life but not in nitrogen-fixing bacteroids (30). Although advantages of PHA accumulation in free-living rhizobia, such as better reproduction and survival under low-nutrient conditions, have been clearly demonstrated (31), biological roles of PHAs in symbiotic rhizobia have been elusive and controversial (32,33).…”

Human selection and the relaxation of legume defences against ineffective rhizobia

“…For rhizobia, trade-offs exist between fixing nitrogen for the benefit of the plant versus hoarding resources like polyhydroxybutyrate (Hahn & Studer 1986;Cevallos et al 1996;Denison & Kiers 2004a,b;Akçay & Roughgarden 2007), which can enhance future survival or reproduction (Cai et al 2000).…”

Regulation of the TCA cycle and the general amino acid permease by overflow metabolism in Rhizobium leguminosarum

“…Amino acid biosynthesis and excretion may thus be part of an overflow pathway that removes excess carbon and reductant when the TCA cycle becomes limited or blocked. In addition, polyhydroxybutyrate (PHB) biosynthesis may function in a similar way in rhizobia (Bergersen & Turner, 1990;Povolo et al, 1994;Cevallos et al, 1996).…”