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...

Strains of Rhizobium etli, Rhizobium meliloti, and Rhizobium tropici decreased their capacity to grow after successive subcultures in minimal medium, with a pattern characteristic for each species. During the growth of R. etli CE 3 in minimal medium (MM), a fermentation-like response was apparent: the O 2 content was reduced and, simultaneously, organic acids and amino acids were excreted and poly-␤-hydroxybutyrate (PHB) was accumulated. Some of the organic acids excreted into the medium were tricarboxylic acid (TCA) cycle intermediates, and, concomitantly, the activities of several TCA cycle and auxiliary enzymes decreased substantially or became undetectable. Optimal and sustained growth and a low PHB content were found in R. etli CE 3 when it was grown in MM inoculated at a low cell density with O 2 maintained at 20% or with the addition of supplements that have an effect on the supply of substrates for the TCA cycle. In the presence of supplements such as biotin or thiamine, no amino acids were excreted and the organic acids already excreted into the medium were later reutilized. Levels of enzyme activities in cells from supplemented cultures indicated that carbon flux through the TCA cycle was maintained, which did not happen in MM. It is proposed that the fermentative state in Rhizobium species is triggered by a cell density signal that results in the regulation of some of the enzymes responsible for the flux of carbon through the TCA cycle and that this in turn determines how much carbon is available for the synthesis and accumulation of PHB. The fermentative state of free-living Rhizobium species may be closely related to the metabolism that these bacteria express during symbiosis.In aerobic bacteria, the tricarboxylic acid (TCA) cycle functions to generate reduced nucleotides by the complete oxidation of pyruvate, which enters the cycle in the form of acetyl coenzyme A (acetyl-CoA). The reduced nucleotides are then used to generate ATP via the electron transport system. Another major function is to produce intermediates for anabolism, and several anaplerotic reactions serve to replenish TCA cycle intermediates which are consumed in these processes (32).The accumulation of the microbial reserve polyester poly-␤-hydroxybutyrate (PHB) in bacteria is well documented (1,6,45), as is the presence of PHB in several species of Rhizobium, both in the free state (44, 46) and in symbiosis (14,19,48). While several different pathways for the production of PHB in various groups of bacteria have been characterized (1), the most common pathway begins with the condensation of two molecules of acetyl-CoA to form acetoacetyl-CoA. Sequential reduction and polymerization reactions produce PHB. This product can be depolymerized and ultimately converted back to acetyl-CoA (Fig. 1). Like the TCA cycle, carbon flux through this pathway is greatly influenced by growth conditions, and the two cycles must compete for a common starting metabolite, acetyl-CoA. However, the function of PHB in cell metabolism in Rhizobium species has...

Evidence from in vitro and in vivo studies showed that in Rhizobium phaseoli ammonium is assimilated by the glutamine synthetase (GS)-glutamate synthase NADPH pathway. No glutamate dehydrogenase activity was detected. R. phaseoli has two GS enzymes, as do other rhizobia. The two GS activities are regulated on the basis of the requirement for low (GSI) or high (GSII) ammonium assimilation. When the 2-oxoglutarate/glutamine ratio decreases, GSI is adenylylated. When GSI is inactivated, GSII is induced. However, induction of GSH activity varied depending on the rate of change of this ratio. GSII was inactivated after the addition of high ammonium concentrations, when the 2-oxoglutarate/glutamine ratio decreased rapidly. Ammonium inactivation resulted in alteration of the catalytic and physical properties of GSII. GSII inactivation was not relieved by shifting of the cultures to glutamate. After GSH inactivation, ammonium was excreted into the medium. Glutamate synthase activity was inhibited by some organic acids and repressed when cells were grown with glutamate as the nitrogen source.It is generally accepted that ammonium assimilation in rhizobia proceeds mainly through the glutamine synthetase (GS)-glutamate synthase (GOGAT) pathway (6,13,15,(19)(20)(21). However, in contrast with other bacteria, rhizobium contains two forms of GS, and this presents additional complexity in the study of ammonium assimilation. The GSI enzyme is similar to the enzymes found in other gramnegative bacteria in regard to its monomeric and oligomeric structure, postranslational regulation, and amino acid sequence (5, 6, 25). The GSII enzyme has a lower molecular weight and is heat labile (6, 7, 10, 16). Recently, in Bradyrhizobium japonicum, the gene coding for GSII was cloned, the enzyme was purified, and it was found that its amino acid sequence more closely resembles that of plant GS than that of bacterial GS (4). The function of the two GS forms from rhizobia in the free-living state and during symbiosis remains controversial, since there are several reports about the regulation of GS in rhizobia, without distinction between the two GS activities (13,15,19,20,27).Other studies by Darrow (6) and Ludwig (16) showed that ammonium has a strong negative modulation effect on GSII and a minor effect on GSI. GSI from free-living rhizobia (6, 16) and bacteroids (3) was mainly regulated by adenylylation. However, Howitt et al. (11) reported that, although GSII was severely repressed when rhizobia were grown on ammonium, GSI had the same biosynthetic activity, regardless of the adenylylation state of the enzyme (11).Glutamine auxothrophs have been isolated in Rhizobium cowpea 32H1 and R. meliloti (13, 16), but the number of mutations responsible for these phenotypes remains uncertain.Carlson et al. (5) and Somerville and Kahn (25) have isolated the GSI genes from B. japonicum and R. meliloti, respectively. The GSI gene of B. japonicum was constitutively transcribed in different nitrogen conditions (5). The R. meliloti GSI gene has been inte...

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Strains of the same bacterial species often show considerable genomic variation. To examine the extent of such variation in Rhizobium etli, the complete genome sequence of R. etli CIAT652 and the partial genomic sequences of six additional R. etli strains having different geographical origins were determined. The sequences were compared with each other and with the previously reported genome sequence of R. etli CFN42. DNA sequences common to all strains constituted the greater part of these genomes and were localized in both the chromosome and large plasmids. About 700 to 1,000 kb of DNA that did not match sequences of the complete genomes of strains CIAT652 and CFN42 was unique to each R. etli strain. These sequences were distributed throughout the chromosome as individual genes or chromosomal islands and in plasmids, and they encoded accessory functions, such as transport of sugars and amino acids, or secondary metabolism; they also included mobile elements and hypothetical genes. Sequences corresponding to symbiotic plasmids showed high levels of nucleotide identity (about 98 to 99%), whereas chromosomal sequences and the sequences with matches to other plasmids showed lower levels of identity (on average, about 90 to 95%). We concluded that R. etli has a pangenomic structure with a core genome composed of both chromosomal and plasmid sequences, including a highly conserved symbiotic plasmid, despite the overall genomic divergence.

In this paper we report the cloning and sequence analysis of four genes, located on plasmid pb, which are involved in the synthesis of thiamin in Rhizobium etli (thiC, thiO, thiG, and thiE). Two precursors, 4-methyl-5-(␤-hydroxyethyl)thiazole monophosphate and 4-amino-5-hydroxymethylpyrimidine pyrophosphate, are coupled to form thiamin monophosphate, which is then phosphorylated to make thiamin pyrophosphate. The first open reading frame (ORF) product, of 610 residues, has significant homology (69% identity) with the product of thiC from Escherichia coli, which is involved in the synthesis of hydroxymethylpyrimidine. The second ORF product, of 327 residues, is the product of a novel gene denoted thiO. A protein motif involved in flavin adenine dinucleotide binding was found in the amino-terminal part of ThiO; also, residues involved in the catalytic site of D-amino acid oxidases are conserved in ThiO, suggesting that it catalyzes the oxidative deamination of some intermediate of thiamin biosynthesis. The third ORF product, of 323 residues, has significant homology (38% identity) with ThiG from E. coli, which is involved in the synthesis of the thiazole. The fourth ORF product, of 204 residues, has significant homology (47% identity) with the product of thiE from E. coli, which is involved in the condensation of hydroxymethylpyrimidine and thiazole. Strain CFN037 is an R. etli mutant induced by a single Tn5mob insertion in the promoter region of the thiCOGE gene cluster. The Tn5mob insertion in CFN037 occurred within a 39-bp region which is highly conserved in all of the thiC promoters analyzed and promotes constitutive expression of thiC. Primer extension analysis showed that thiC transcription in strain CFN037 originates within the Tn5 element. Analysis of c-type protein content and expression of the fixNOQP operon, which codes for the symbiotic terminal oxidase cbb 3 , revealed that CFN037 produces the cbb 3 terminal oxidase. These data show a direct relationship between expression of thiC and production of the cbb 3 terminal oxidase. This is consistent with the proposition that a purine-related metabolite, 5-aminoimidazole-4-carboxamide ribonucleotide, is a negative effector of the production of the symbiotic terminal oxidase cbb 3 in R. etli.

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...