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...
BackgroundBacterial nitrogen fixation is the biological process by which atmospheric nitrogen is uptaken by bacteroids located in plant root nodules and converted into ammonium through the enzymatic activity of nitrogenase. In practice, this biological process serves as a natural form of fertilization and its optimization has significant implications in sustainable agricultural programs. Currently, the advent of high-throughput technology supplies with valuable data that contribute to understanding the metabolic activity during bacterial nitrogen fixation. This undertaking is not trivial, and the development of computational methods useful in accomplishing an integrative, descriptive and predictive framework is a crucial issue to decoding the principles that regulated the metabolic activity of this biological process.ResultsIn this work we present a systems biology description of the metabolic activity in bacterial nitrogen fixation. This was accomplished by an integrative analysis involving high-throughput data and constraint-based modeling to characterize the metabolic activity in Rhizobium etli bacteroids located at the root nodules of Phaseolus vulgaris (bean plant). Proteome and transcriptome technologies led us to identify 415 proteins and 689 up-regulated genes that orchestrate this biological process. Taking into account these data, we: 1) extended the metabolic reconstruction reported for R. etli; 2) simulated the metabolic activity during symbiotic nitrogen fixation; and 3) evaluated the in silico results in terms of bacteria phenotype. Notably, constraint-based modeling simulated nitrogen fixation activity in such a way that 76.83% of the enzymes and 69.48% of the genes were experimentally justified. Finally, to further assess the predictive scope of the computational model, gene deletion analysis was carried out on nine metabolic enzymes. Our model concluded that an altered metabolic activity on these enzymes induced different effects in nitrogen fixation, all of these in qualitative agreement with observations made in R. etli and other Rhizobiaceas.ConclusionsIn this work we present a genome scale study of the metabolic activity in bacterial nitrogen fixation. This approach leads us to construct a computational model that serves as a guide for 1) integrating high-throughput data, 2) describing and predicting metabolic activity, and 3) designing experiments to explore the genotype-phenotype relationship in bacterial nitrogen fixation.
Pyruvate carboxylase (PYC), a biotin-dependent enzyme which catalyzes the conversion of pyruvate to oxaloacetate, was hypothesized to play an important anaplerotic role in the growth of Rhizobium etli during serial subcultivation in minimal media containing succinate (S. Encarnación, M. Dunn, K. Willms, and J. Mora, J. Bacteriol. 177:3058-3066, 1995). R. etli and R. tropici pyc::Tn5-mob mutants were selected for their inability to grow in minimal medium with pyruvate as a sole carbon source. During serial subcultivation in minimal medium containing 30 mM succinate, the R. etli parent and pyc mutant strains exhibited similar decreases in growth rate with each subculture. Supplementation of the medium with biotin prevented the growth decrease of the parent but not the mutant strain, indicating that PYC was necessary for the growth of R. etli under these conditions. The R. tropici pyc mutant grew normally in subcultures regardless of biotin supplementation. The symbiotic phenotypes of the pyc mutants from both species were similar to those of the parent strains. The R. etli pyc was cloned, sequenced, and found to encode a 126-kDa protein of 1,154 amino acids. The deduced amino acid sequence is highly homologous to other PYC sequences, and the catalytic domains involved in carboxylation, pyruvate binding, and biotinylation are conserved. The sequence and biochemical data show that the R. etli PYC is a member of the alpha4, homotetrameric, acetyl coenzyme A-activated class of PYCs.
The NifA-RpoN complex is a master regulator of the nitrogen fixation genes in alphaproteobacteria. Based on the complete Rhizobium etli genome sequence, we constructed an R. etli CFN42 oligonucleotide (70-mer) microarray and utilized this tool, reverse transcription (RT)-PCR analysis (transcriptomics), proteomics, and bioinformatics to decipher the NifA-RpoN regulon under microaerobic conditions (free life) and in symbiosis with bean plants. The R. etli NifA-RpoN regulon was determined to contain 78 genes, including the genes involved in nitrogen fixation, and the analyses revealed 42 new NifA-RpoN-dependent genes. More importantly, this study demonstrated that the NifA-RpoN regulon is composed of genes and proteins that have very diverse functions, that play fundamental and previously less appreciated roles in regulating the normal physiology of the cell, and that have important functions in providing adequate conditions for efficient nitrogen fixation in symbiosis. The R. etli NifA-RpoN regulon defined here has some components in common with other NifA-RpoN regulons described previously, but the vast majority of the components have been found only in the R. etli regulon, suggesting that they have a specific role in this bacterium and particular requirements during nitrogen fixation compared with other symbiotic bacterial models.
Previously, it was reported that the oxidative capacity and ability to grow on carbon sources such as pyruvate and glucose were severely diminished in the Rhizobium etli phaC::⍀Sm r /Sp r mutant CAR1, which is unable to synthesize poly--hydroxybutyric acid (PHB) (M. A. Cevallos, S. Encarnación, A. Leija, Y. Mora, and J. Mora, J. Bacteriol. 178: [1646][1647][1648][1649][1650][1651][1652][1653][1654] 1996). By random Tn5 mutagenesis of the phaC strain, we isolated the mutants VEM57 and VEM58, both of which contained single Tn5 insertions and had recovered the ability to grow on pyruvate or glucose. Nucleotide sequencing of the region surrounding the Tn5 insertions showed that they had interrupted an open reading frame designated aniA based on its high deduced amino acid sequence identity to the aniA gene product of Sinorhizobium meliloti. R. etli aniA was located adjacent to and divergently transcribed from genes encoding the PHB biosynthetic enzymes -ketothiolase (PhaA) and acetoacetyl coenzyme A reductase (PhaB). An aniA::Tn5 mutant (VEM5854) was constructed and found to synthesize only 40% of the wild type level of PHB. Both VEM58 and VEM5854 produced significantly more extracellular polysaccharide than the wild type. Organic acid excretion and levels of intracellular reduced nucleotides were lowered to wild-type levels in VEM58 and VEM5854, in contrast to those of strain CAR1, which were significantly elevated. Proteome analysis of VEM58 showed a drastic alteration of protein expression, including the absence of a protein identified as PhaB. We propose that the aniA gene product plays an important role in directing carbon flow in R. etli.Polyhydroxyalkanoic acids (PHAs) are a class of biodegradable plastics synthesized by many bacteria and thought to function as intracellular reserves of carbon and energy (2). Poly--hydroxybutyric acid (PHB) is a type of PHA composed of polymerized 3-hydroxybutyrate units. The pathways for the biosynthesis of PHA have been studied in various bacteria, and the genes encoding the biosynthetic enzymes have also been investigated (40). The most common PHB-biosynthetic pathway consists of three enzymes, the first of which is a -ketothiolase which condenses two molecules of acetyl coenzyme A (acetyl-CoA) to form acetoacetyl-CoA. The acetoacetyl-CoA is then reduced to D-(Ϫ)-3-hydroxybutyryl-CoA by an acetoacetyl-CoA reductase. Lastly, the D-(Ϫ)-3-hydroxybutyryl-CoA monomers are linked by PHB synthase. The genes encoding -ketothiolase, acetoacetyl-CoA reductase, and PHB synthase are designated phaA, phaB, and phaC, respectively.Rhizobium etli accumulates PHB both in symbiosis and in free life (7,14). PHB in rhizobia and other bacteria is thought to serve as a reserve of carbon and/or electrons to be utilized under suboptimal growth conditions (13,23). An R. etli PHBnegative mutant (CAR1) with an insertionally inactivated PHB synthase structural gene (phaC) was described previously. Physiological studies showed that CAR1 was unable to synthesize PHB and excreted more organic acids...
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