Isolation and characterization of mutants of <i>Rhizobium meliloti</i> unable to synthesize poly-β-hydroxybutyrate

Povolo, Silvana; Tombolini, Riccardo; Morea, Anna; Anderson, Angelika; Casella, Sergio; Nuti, Marco

doi:10.1139/m94-131

Cited by 51 publications

(40 citation statements)

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“…The demonstration that symbiotic N 2 fixation ability is not affected in any of the PHB cycle mutant strains is consistent with reports indicating that neither PHB synthesis nor degradation is required for effective symbiosis (3,38,51), although PHB synthesis has been shown to be important for symbiotic competition (51). PHB is accumulated as an endogenous source of carbon and energy, and fluorescence microscopy provided visual evidence of PHB utilization during carbon-free starvation in Legionella pneumophila (26).…”

Section: Discussionsupporting

confidence: 88%

Requirement for the Enzymes Acetoacetyl Coenzyme A Synthetase and Poly-3-Hydroxybutyrate (PHB) Synthase for Growth of Sinorhizobium meliloti on PHB Cycle Intermediates

2000

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We have identified two Sinorhizobium meliloti chromosomal loci affecting the poly-3-hydroxybutyrate degradation pathway. One locus was identified as the gene acsA, encoding acetoacetyl coenzyme A (acetoacetyl-CoA) synthetase. Analysis of the acsA nucleotide sequence revealed that this gene encodes a putative protein with a molecular weight of 72,000 that shows similarity to acetyl-CoA synthetase in other organisms. Acetyl-CoA synthetase activity was not affected in cell extracts of glucose-grown acsA::Tn5 mutants; instead, acetoacetyl-CoA synthetase activity was drastically reduced. These findings suggest that acetoacetyl-CoA synthetase, rather than CoA transferase, activates acetoacetate to acetoacetyl-CoA in the S. meliloti poly-3-hydroxybutyrate cycle. The second locus was identified as phbC, encoding poly-3-hydroxybutyrate synthase, and was found to be required for synthesis of poly-3-hydroxybutyrate deposits.The catabolism of intracellular carbon stores is a strategy employed by many bacterial species to survive nutritionally suboptimal conditions. Polyhydroxyalkanoates (PHA), such as poly-3-hydroxybutyrate (PHB), accumulate in cells when growth is limited but carbon availability is not (2). This stored carbon can then be utilized during conditions of otherwise limiting carbon availability (45). PHB synthesis and degradation are collectively referred to as the PHB cycle. The extent of PHB accumulation is dependent on the relative rates of synthesis and degradation, which in turn are controlled by growth conditions (36,41).PHA have attracted substantial industrial interest for their use as high-quality, biodegradable plastics (2). This interest has driven research efforts directed at PHA biosynthesis. The genes encoding the enzymes responsible for PHA synthesis have been isolated and characterized from a number of bacterial species (33, 43), including Sinorhizobium meliloti strains Rm41 (49) and Rm1021 (51). The degradative portion of the cycle has not been subjected to similar attention, and it is less well defined both genetically and biochemically.We have recently isolated a number of S. meliloti mutants that are affected in the ability to utilize PHB cycle intermediates as sole carbon sources to support growth (11). The mutations mapped to loci on both the chromosome and the pRmeSU47b (pEXO) megaplasmid. Two loci on the megaplasmid have been identified via enzymatic and nucleotide sequence analyses. One encodes the enzyme 3-hydroxybutyrate dehydrogenase (3), and the other encodes methylmalonyl coenzyme A (methylmalonyl-CoA) mutase (10). In this paper, we further characterize and identify two of the chromosomal loci. MATERIALS AND METHODSStrains, plasmids, and culture conditions. Bacterial strains and plasmids are listed in Table 1. The construction of new strains is described in the text. Bacterial culture in Luria-Bertani (LB) and TY complex media and modified M9 minimal salts medium and antibiotic selection were carried out as previously described (12). Modified M9 minimal salts medium was supplemented...

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Section: Discussionsupporting

confidence: 88%

Requirement for the Enzymes Acetoacetyl Coenzyme A Synthetase and Poly-3-Hydroxybutyrate (PHB) Synthase for Growth of Sinorhizobium meliloti on PHB Cycle Intermediates

2000

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“…Mutants unable to synthesize PHB have been isolated from several bacterial species, i.e., A. eutrophus, Rhodospirillum rubrum, Rhodobacter sphaeroides, Pseudomonas oleovorans, Pseudomonas putida, and R. meliloti (21,22,32,37). In all of them, the defect was located on the PHB synthase gene.…”

Section: Discussionmentioning

confidence: 99%

“…Recently, a PHB-negative mutant of R. meliloti was described (32). Its physiological characterization was not reported, but this mutant is not affected in nitrogen fixation.…”

Section: Discussionmentioning

confidence: 99%

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

et al. 1996

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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...

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“…Polyhydroxybutyrate (PHB) granules, produced by S. meliloti during the invasion process, are degraded during bacteroid differentiation and appear to be used preferentially as a carbon source at this stage 109 . Surprisingly, mutants in PHB synthesis (phbC) or degradation (bdhA) are symbiotically competent, indicating that alternate carbon sources are available to the bacteria during differentiation [109][110][111] . In a mixed infection, phbC and bdhA mutants are less competitive for nodule occupancy than the wild type, suggesting that the ability to synthesize and use PHB might allow the bacteria to compete in situations of reduced carbon availability 109,111 .…”

Section: Nih-pa Author Manuscriptmentioning

confidence: 99%

How rhizobial symbionts invade plants: the Sinorhizobium–Medicago model

et al. 2007

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Nitrogen-fixing rhizobial bacteria and leguminous plants have evolved complex signal exchange mechanisms that allow a specific bacterial species to induce its host plant to form invasion structures through which the bacteria can enter the plant root. Once the bacteria have been endocytosed within a host-membrane-bound compartment by root cells, the bacteria differentiate into a new form that can convert atmospheric nitrogen into ammonia. Bacterial differentiation and nitrogen fixation are dependent on the microaerobic environment and other support factors provided by the plant. In return, the plant receives nitrogen from the bacteria, which allows it to grow in the absence of an external nitrogen source. Here, we review recent discoveries about the mutual recognition process that allows the model rhizobial symbiont Sinorhizobium meliloti to invade and differentiate inside its host plant alfalfa (Medicago sativa) and the model host plant barrel medic (Medicago truncatula).The recent completion of the Sinorhizobium meliloti genome sequence, and the progress towards the completion of the Medicago truncatula genome sequence, have led to a surge in the molecular characterization of the determinants that are involved in the development of the symbiosis between rhizobial bacteria and leguminous plants. Aromatic compounds from legumes called flavonoids first signal the rhizobial bacteria to produce lipochitooligosaccharide compounds called Nod factors 1 . Nod factors that are secreted by the bacteria activate multiple responses in the host plant that prepare the plant to receive the invading bacteria. Nod factors and symbiotic exopolysaccharides induce the plant to form infection threads, which are thin tubules filled with bacteria that penetrate into the plant cortical tissue and deliver the bacteria to their target cells. Plant cells in the inner cortex internalize the invading bacteria in host-membrane-bound compartments that mature into structures known Correspondence to G.C.W. gwalker@mit.edu. Competing interests statementThe authors declare no competing financial interests. DATABASES Invasion of plant rootsAlthough plant roots are exposed to various micro-organisms in the soil, their cell walls form a strong protective barrier against most harmful species. The early steps in the invasion of barrel medic (M. truncatula) and alfalfa (Medicago sativa) roots by S. meliloti are characterized by the reciprocal exchange of signals that allow the bacteria to use the plant root hair cells as a means of entry. Initial signal exchangeFlavonoid compounds (2-phenyl-1,4-benzopyrone derivatives) produced by leguminous plants are the first signals to be exchanged by host-rhizobial symbiont pairs 1 (FIG. 1). Flavonoids bind bacterial NodD proteins, which are members of the LysR family of transcriptional regulators, and activate these proteins to induce the transcription of rhizobial genes 1,2 . For example, the M. sativa-derived flavonoid luteolin stimulates binding of an active form of NodD1 to an S. meliloti 'nod-box' p...

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Isolation and characterization of mutants of Rhizobium meliloti unable to synthesize poly-β-hydroxybutyrate

Cited by 51 publications

References 0 publications

Requirement for the Enzymes Acetoacetyl Coenzyme A Synthetase and Poly-3-Hydroxybutyrate (PHB) Synthase for Growth of Sinorhizobium meliloti on PHB Cycle Intermediates

Requirement for the Enzymes Acetoacetyl Coenzyme A Synthetase and Poly-3-Hydroxybutyrate (PHB) Synthase for Growth of Sinorhizobium meliloti on PHB Cycle Intermediates

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

How rhizobial symbionts invade plants: the Sinorhizobium–Medicago model

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