Biodegradation of 3-nitrotyrosine by Burkholderia sp. strain JS165 and Variovorax paradoxus JS171

The cascade of reactive nitrogen species generated from nitric oxide causes modification of proteins, lipids, and nucleic acids in a wide range of organisms. 3-Nitrotyrosine is one of the most common products of the action of reactive nitrogen species on proteins. Although a great deal is known about the formation of 3-nitrotyrosine, the subsequent metabolism of this compound is a mystery. Variovorax paradoxus JS171 and Burkholderia sp. strain JS165 were isolated from soil slurries when 3-nitrotyrosine was provided as the sole carbon, nitrogen, and energy source. During growth on 3-nitrotyrosine stoichiometric amounts of nitrite were released along with approximately one-half of the theoretically available ammonia. The catabolic pathway involving oxidative denitration is distinct from the pathway for tyrosine metabolism. The facile isolation and the specific, regulated pathway for 3-nitrotyrosine degradation in natural ecosystems suggest that there is a significant flux of 3-nitrotyrosine in such environments.     

Kinetics of 3-nitrotyrosine modification on exposure to hypochlorous acid

Abstract

The markers 3-nitrotyrosine and 3-chlorotyrosine are measured as surrogates for reactive nitrogen species and hypochlorous acid respectively, which are both elevated in inflamed human tissues. Previous studies reported a loss of 3-nitrotyrosine when exposed to hypochlorous acid, suggesting that observations of 3-nitrotyrosine underestimate the presence of reactive nitrogen species in diseased tissue (Whiteman and Halliwell, Biochemical and Biophysical Research Communications, 258, 168–172 (1999)). This report evaluates the significance of 3-nitrotyrosine loss by measuring the kinetics of the reaction between 3-nitrotyrosine and hypochlorous acid. The results demonstrate that 3-nitrotyrosine is chlorinated by hypochlorous acid or chloramines to form 3-chloro-5-nitrotyrosine. As 3-nitrotyrosine from in vivo samples is usually found within proteins rather than as free amino acid, we also examined the reaction of 3-nitrotyrosine modification in the context of peptides. The chlorination of 3-nitrotyrosine in peptides was observed to occur up to 700-fold faster than control reactions using equivalent amino acid mixtures. These results further advance our understanding of tyrosine chlorination and the use of 3-nitrotyrosine formed in vivo as a biomarker of reactive nitrogen species.     

Human atherosclerotic intima and blood of patients with established coronary artery disease contain high density lipoprotein damaged by reactive nitrogen species

High density lipoprotein (HDL) is the major carrier of lipid hydroperoxides in plasma, but it is not yet established whether HDL proteins are damaged by reactive nitrogen species in the circulation or artery wall. One pathway that generates such species involves myeloperoxidase (MPO), a major constituent of artery wall macrophages. Another pathway involves peroxynitrite, a potent oxidant generated in the reaction of nitric oxide with superoxide. Both MPO and peroxynitrite produce 3-nitrotyrosine in vitro. To investigate the involvement of reactive nitrogen species in atherogenesis, we quantified 3-nitrotyrosine levels in HDL in vivo. The mean level of 3-nitrotyrosine in HDL isolated from human aortic atherosclerotic intima was 6-fold higher (619 +/- 178 micromol/mol Tyr) than that in circulating HDL (104 +/- 11 micromol/mol Tyr; p < 0.01). Immunohistochemical studies demonstrated striking colocalization of MPO with epitopes reactive with an antibody to 3-nitrotyrosine. However, there was no significant correlation between the levels of 3-chlorotyrosine, a specific product of MPO, and those of 3-nitrotyrosine in lesion HDL. We also detected 3-nitrotyrosine in circulating HDL, and linear regression analysis demonstrated a strong correlation between the levels of 3-chlorotyrosine and levels of 3-nitrotyrosine. These observations suggest that MPO promotes the formation of 3-chlorotyrosine and 3-nitrotyrosine in circulating HDL but that other pathways also produce 3-nitrotyrosine in atherosclerotic tissue. Levels of HDL isolated from plasma of patients with established coronary artery disease contained twice as much 3-nitrotyrosine as HDL from plasma of healthy subjects, suggesting that nitrated HDL might be a marker for clinically significant vascular disease. The detection of 3-nitrotyrosine in HDL raises the possibility that reactive nitrogen species derived from nitric oxide might promote atherogenesis. Thus, nitrated HDL might represent a previously unsuspected link between nitrosative stress, atherosclerosis, and inflammation.     

Tyrosine nitration: Localisation,quantification, consequences for protein function and signal transduction

The nitration of free tyrosine or protein tyrosine residues generates 3-nitrotyrosine the detection of which has been utilised as a footprint for the in vivo formation of peroxynitrite and other reactive nitrogen species. The detection of 3-nitrotyrosine by analytical and immunological techniques has established that tyrosine nitration occurs under physiological conditions and levels increase in most disease states. This review provides an updated, comprehensive and detailed summary of the tissue, cellular and specific protein localisation of 3-nitrotyrosine and its quantification. The potential consequences of nitration to protein function and the pathogenesis of disease are also examined together with the possible effects of protein nitration on signal transduction pathways and on the metabolism of proteins.     

Metabolism of the Aliphatic Nitramine 4-Nitro-2,4-Diazabutanal by Methylobacterium sp. Strain JS178

The aliphatic nitramine 4-nitro-2,4-diazabutanal (NDAB; C₂H₅N₃O₃) is a ring cleavage metabolite that accumulates during the aerobic degradation of the energetic compound hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) by various Rhodococcus spp. NDAB is also produced during the alkaline hydrolysis of either RDX or octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) and during the photolysis of RDX. Traces of NDAB were observed in a soil sampled from an ammunition-manufacturing facility contaminated with both HMX and RDX, suggesting natural attenuation. In this study, we report the isolation of a soil bacterium that is able to degrade NDAB under aerobic conditions. The isolate is a pink-pigmented facultative methylotroph affiliated with the genus Methylobacterium. The strain, named Methylobacterium sp. strain JS178, degrades NDAB as a sole nitrogen source, with concomitant growth and formation of 1 molar equivalent of nitrous oxide (N₂O). Comparison of the growth yield of strain JS178 grown on NDAB, nitrite (NO₂⁻), or ammonium (NH₄⁺) as a nitrogen source revealed that 1 N equivalent is assimilated from each mole of NDAB, which completes the nitrogen mass balance. In radiotracer experiments, strain JS178 mineralized 1 C of the [¹⁴C]NDAB produced in situ from [¹⁴C]RDX by Rhodococcus sp. strain DN22. Studies on the regulation of NDAB degradation indicated that allantoin, an intermediate in the purine catabolic pathway and a central molecule in the storage and transport of nitrogen in plants, up-regulated the enzyme(s) involved in the degradation of the nitramine. The results reveal the potential for the sequential participation of rhodococci and methylobacteria to effect the complete degradation of RDX.     

Molecular Characterization and Substrate Specificity of Nitrobenzene Dioxygenase from Comamonas sp. Strain JS765

Comamonas sp. strain JS765 can grow with nitrobenzene as the sole source of carbon, nitrogen, and energy. We report here the sequence of the genes encoding nitrobenzene dioxygenase (NBDO), which catalyzes the first step in the degradation of nitrobenzene by strain JS765. The components of NBDO were designated Reductase_NBZ, Ferredoxin_NBZ, Oxygenase_NBZα, and Oxygenase_NBZβ, with the gene designations nbzAa, nbzAb, nbzAc, and nbzAd, respectively. Sequence analysis showed that the components of NBDO have a high level of homology with the naphthalene family of Rieske nonheme iron oxygenases, in particular, 2-nitrotoluene dioxygenase from Pseudomonas sp. strain JS42. The enzyme oxidizes a wide range of substrates, and relative reaction rates with partially purified Oxygenase_NBZ revealed a preference for 3-nitrotoluene, which was shown to be a growth substrate for JS765. NBDO is the first member of the naphthalene family of Rieske nonheme iron oxygenases reported to oxidize all of the isomers of mono- and dinitrotoluenes with the concomitant release of nitrite.     

Genome analysis of phage JS98 defines a fourth major subgroup of T4-like phages in Escherichia coli

Numerous T4-like Escherichia coli phages were isolated from human stool and environmental wastewater samples in Bangladesh and Switzerland. The sequences of the major head gene (g23) revealed that these coliphages could be placed into four subgroups, represented by the phages T4, RB69, RB49, and JS98. Thus, JS98 defines a new major subgroup of E. coli T4-like phages. We conducted an analysis of the 169-kb JS98 genome sequence. Overall, 198 of the 266 JS98 open reading frames (ORFs) shared amino acid sequence identity with the reference T4 phage, 41 shared identity with other T4-like phages, and 27 ORFs lacked any database matches. Genes on the plus strand encoded virion proteins, which showed moderate to high sequence identity with T4 proteins. The right genome half of JS98 showed a higher degree of sequence conservation with T4 and RB69, even for the nonstructural genes, than did the left genome half, containing exclusively nonstructural genes. Most of the JS98-specific genes were found in the left genome half. Two came as a hypervariability cluster, but most represented isolated genes, suggesting that they were acquired separately in multiple acquisition events. No evidence for DNA exchange between JS98 phage and the E. coli host genome or coliphages other than T4 was observed. No undesired genes which could compromise its medical use were detected in the JS98 genome sequence.     

Comparison of the downstream pathways for degradation of nitrobenzene by Pseudomonaspseudoalcaligenes JS45 (2-aminophenol pathway) and by Comamonas sp. JS765 (catechol pathway)

Nitrobenzene is degraded by Pseudomonas pseudoalcaligenes JS45 via 2-aminophenol to 2-aminomuconic semialdehyde, which is further degraded to pyruvate and acetaldehyde. Comamonas sp. JS765 degrades nitrobenzene via catechol to 2-hydroxymuconic semialdehyde. In this study we examined and compared the late steps of degradation of nitrobenzene by these two microorganisms in order to reveal the biochemical relationships of the two pathways and to provide insight for further investigation of their evolutionary history. Experiments showed that 2-hydroxymuconate, the product of the dehydrogenation of 2-hydroxymuconic semialdehyde, was degraded to pyruvate and acetaldehyde by crude extracts of Comamonas sp. JS765, which indicated the operation of a classical catechol meta-cleavage pathway. The semialdehyde dehydrogenases from Comamonas sp. JS765 and P. pseudoalcaligenes JS45 were able to metabolize both 2-amino- and 2-hydroxymuconic semialdehyde, with strong preference for the physiological substrate. 2-Aminomuconate was not a substrate for 4-oxalocrotonate decarboxylase from either bacterial strain. The close biochemical relationships among the classical catechol meta-cleavage pathway in Comamonas sp. JS765, 2-aminophenol meta-cleavage pathways in P. pseudoalcaligenes JS45, and an alternative 2-aminophenol meta-cleavage pathway in Pseudomonas sp. AP-3 suggest a common evolutionary origin. Received: 23 November 1998 / Accepted: 3 February 1999     

Tyrosine nitration: Localisation, quantification, consequences for protein function and signal transduction

The nitration of free tyrosine or protein tyrosine residues generates 3-nitrotyrosine the detection of which has been utilised as a footprint for the in vivo formation of peroxynitrite and other reactive nitrogen species. The detection of 3-nitrotyrosine by analytical and immunological techniques has established that tyrosine nitration occurs under physiological conditions and levels increase in most disease states. This review provides an updated, comprehensive and detailed summary of the tissue, cellular and specific protein localisation of 3-nitrotyrosine and its quantification. The potential consequences of nitration to protein function and the pathogenesis of disease are also examined together with the possible effects of protein nitration on signal transduction pathways and on the metabolism of proteins.     

Gaseous alkene biotransformation and enantioselective epoxyalkane formation by Nocardioides sp. strain JS614

Enantiopure epoxides are valuable intermediates in the synthesis of optically pure biologically active fine chemicals (e.g., pharmaceuticals) that are often difficult to produce by chemical approaches. An attractive alternative is biological synthesis by microorganisms expressing stereoselective enzymes. In this study, we investigated the ability of ethene-grown Nocardioides sp. strain JS614 to produce highly enantio-enriched epoxyalkanes via stereoselective monooxygenase-mediated alkene epoxidation. Ethene-grown JS614 cells transformed propene, 1-butene, and trans-2-butene to their corresponding epoxyalkanes at rates ranging from 27.1 to 44.0 nmol/min mg protein. Chiral gas chromatography analysis revealed that R-1,2-epoxypropane, R-1,2-epoxybutane, and trans-2R,3R-epoxybutane were produced in enantiomeric excess (e.e.) of 98%, 74%, and 82%, respectively. Ethene-grown JS614 cells also preferentially transformed trans-2S,3S-epoxybutane from a racemic mixture, but could not resolve racemic 1,2-epoxypropane. Glucose facilitated increased epoxyalkane production by ethene-grown JS614 cells. However, after 22 h of propene biotransformation with 20 mM glucose, 84% of ethene-grown JS614 cells lost membrane integrity and the remaining live cells were not viable. Propene biotransformation by JS614 was extended beyond 22 h and 54% more epoxypropane was produced when cells were resuspended in fresh buffer + glucose at 8-h intervals. We conclude that JS614 is a promising new biocatalyst for applications that involve enantiopure epoxide production.     

Species-Specific Oligonucleotides for Enumeration of Pseudomonas putida F1, Burkholderia sp. Strain JS150, and Bacillus subtilis ATCC 7003 in Biodegradation Experiments

Species-specific sequences were identified within the V4 variable region of 16S rRNA of two bacterial species capable of aromatic hydrocarbon metabolism, Pseudomonas putida F1 and Burkholderia sp. strain JS150, and a third, Bacillus subtilis ATCC 7003, that can function as a secondary degrader. Fluorescent in situ hybridization (FISH) with species-specific oligonucleotides was used for direct counting of these species throughout a phenol biodegradation experiment in batch culture. Traditional differential plate counting methods could not be used due to the similar metabolism and interactions of the primary degraders and difficulties in selecting secondary degraders in mixed culture. In contrast, the FISH method provided reliable quantitative results without interference from those factors.     

Loss of 3-nitrotyrosine on exposure to hypochlorous acid: implications for the use of 3-nitrotyrosine as a bio-marker in vivo

Peroxynitrite (ONOO-) is a reactive nitrogen species which in vivo is often assessed by the measurement of free or protein bound 3-nitrotyrosine. Indeed, 3-nitrotyrosine has been detected in many human diseases. However, at sites of inflammation there is also production of the powerful oxidant hypochlorous acid (HOCl) formed by the enzyme myeloperoxidase. Low concentrations of HOCl (<30 microM) caused significant and rapid loss (<10 minutes) of free and protein bound 3-nitrotyrosine. In contrast, no loss of 3-nitrotyrosine was observed with hydrogen peroxide, hydroxyl radical, or superoxide generating systems. Therefore, under conditions where there is concomitant peroxynitrite and hypochlorous acid formation, such as at sites of chronic inflammation, it is possible that HOCl removes 3-nitrotyrosine. This may have implications when assessing the role of reactive nitrogen species in disease conditions and could account for some of the discrepancies reported between 3-nitrotyrosine levels in tissues.     

Post-translational modifications of superoxide dismutase

Post-translational modifications of proteins control many biological processes through the activation, inactivation, or gain-of-function of the proteins. Recent developments in mass spectrometry have enabled detailed structural analyses of covalent modifications of proteins and also have shed light on the post-translational modification of superoxide dismutase. In this review, we introduce some covalent modifications of superoxide dismutase, nitration, phosphorylation, glutathionylaion, and glycation. Nitration has been the most extensively analyzed modification both in vitro and in vivo. Reaction of human Cu,Zn superoxide dismutase (SOD) with reactive nitrogen species resulted in nitration of a single tryptophan residue to 6-nitrotryptophan, which could be a new biomarker of a formation of reactive nitrogen species. On the other hand, tyrosine 34 of human MnSOD was exclusively nitrated to 3-nitrotyrosine and almost completely inactivated by the reaction with peroxynitrite. The nitrated MnSOD has been found in many diseases caused by ischemia/reperfusion, inflammation, and others and may have a pivotal role in the pathology of the diseases. Most of the post-translational modifications have given rise to a reduced activity of SOD. Since phosphorylation and nitration of SOD have been shown to have a possible reversible process, these modifications may be related to a redox signaling process in cells. Finally we briefly introduce a metal insertion system of SOD, focusing particularly on the iron misincorporation of nSOD, as a part of post-translational modifications.     

The Genome of Polaromonas sp. Strain JS666: Insights into the Evolution of a Hydrocarbon- and Xenobiotic-Degrading Bacterium, and Features of Relevance to Biotechnology

Polaromonas sp. strain JS666 can grow on cis-1,2-dichloroethene (cDCE) as a sole carbon and energy source and may be useful for bioremediation of chlorinated solvent-contaminated sites. Analysis of the genome sequence of JS666 (5.9 Mb) shows a bacterium well adapted to pollution that carries many genes likely to be involved in hydrocarbon and xenobiotic catabolism and metal resistance. Clusters of genes coding for haloalkane, haloalkanoate, n-alkane, alicyclic acid, cyclic alcohol, and aromatic catabolism were analyzed in detail, and growth on acetate, catechol, chloroacetate, cyclohexane carboxylate, cyclohexanol, ferulate, heptane, 3-hydroxybenzoate, hydroxyquinol, gentisate, octane, protocatechuate, and salicylate was confirmed experimentally. Strain JS666 also harbors diverse putative mobile genetic elements, including retrons, inteins, a miniature inverted-repeat transposable element, insertion sequence transposases from 14 families, eight genomic islands, a Mu family bacteriophage, and two large (338- and 360-kb) plasmids. Both plasmids are likely to be self-transferable and carry genes for alkane, alcohol, aromatic, and haloacid metabolism. Overall, the JS666 genome sequence provides insights into the evolution of pollutant-degrading bacteria and provides a toolbox of catabolic genes with utility for biotechnology.     

Reactions Involved in the Lower Pathway for Degradation of 4-Nitrotoluene by Mycobacterium Strain HL 4-NT-1

In spite of the variety of initial reactions, the aerobic biodegradation of aromatic compounds generally yields dihydroxy intermediates for ring cleavage. Recent investigation of the degradation of nitroaromatic compounds revealed that some nitroaromatic compounds are initially converted to 2-aminophenol rather than dihydroxy intermediates by a number of microorganisms. The complete pathway for the metabolism of 2-aminophenol during the degradation of nitrobenzene by Pseudomonas pseudoalcaligenes JS45 has been elucidated previously. The pathway is parallel to the catechol extradiol ring cleavage pathway, except that 2-aminophenol is the ring cleavage substrate. Here we report the elucidation of the pathway of 2-amino-4-methylphenol (6-amino-m-cresol) metabolism during the degradation of 4-nitrotoluene by Mycobacterium strain HL 4-NT-1 and the comparison of the substrate specificities of the relevant enzymes in strains JS45 and HL 4-NT-1. The results indicate that the 2-aminophenol ring cleavage pathway in strain JS45 is not unique but is representative of the pathways of metabolism of other o-aminophenolic compounds.     

Physiological and proteomic analysis of plant growth enhancement by the rhizobacteria <Emphasis Type="Italic">Bacillus</Emphasis> sp. JS

In this study, the effects of the plant growth-promoting rhizobacterium (PGPR), Bacillus sp. JS on the growth of tobacco (Nicotiana tabacum ‘Xanthi’) and lettuce (Lactuca sativa ‘Crispa’), were evaluated by comparing various growth parameters between plants treated with the bacterium and those exposed to water or nutrient broth as control. In both tobacco and lettuce, fresh weight and length of shoots were increased upon exposure to Bacillus sp. JS. To explain the overall de novo expression of plant proteins by bacterial volatiles, two-dimensional gel electrophoresis was performed on samples from PGPR-treated tobacco plants. Our results showed that chlorophyll a/b binding proteins were significantly up-regulated, and total chlorophyll content was also increased. Our findings indicate the potential benefits of using Bacillus sp. JS as a growth-promoting factor in agricultural practice, and highlight the need for further research to explore these benefits.     

A Novel 2-Aminomuconate Deaminase in the Nitrobenzene Degradation Pathway of Pseudomonas pseudoalcaligenes JS45

2-Aminomuconate, an intermediate in the metabolism of tryptophan in mammals, is also an intermediate in the biodegradation of nitrobenzene by Pseudomonas pseudoalcaligenes JS45. Strain JS45 hydrolyzes 2-aminomuconate to 4-oxalocrotonic acid, with the release of ammonia, which serves as the nitrogen source for growth of the microorganism. As an initial step in studying the novel deamination mechanism, we report here the purification and some properties of 2-aminomuconate deaminase. The purified enzyme migrates as a single band with a molecular mass of 16.6 kDa in 15% polyacrylamide gel electrophoresis under denaturing conditions. The estimated molecular mass of the native enzyme was 100 kDa by gel filtration and 4 to 20% gradient nondenaturing polyacrylamide gel electrophoresis, suggesting that the enzyme consists of six identical subunits. The enzyme was stable at room temperature and exhibited optimal activity at pH 6.6. The K_m for 2-aminomuconate was approximately 67 μM, and the V_max was 125 μmol · min⁻¹ · mg⁻¹. The N-terminal amino acid sequence of the enzyme did not show any significant similarity to any sequence in the databases. The purified enzyme converted 2-aminomuconate directly to 4-oxalocrotonate, rather than 2-hydroxymuconate, which suggests that the deamination was carried out via an imine intermediate.     

Biodegradation of 5-Nitroanthranilic Acid by Bradyrhizobium sp. Strain JS329

Biodegradation of synthetic compounds has been studied extensively, but the metabolic diversity required for catabolism of many natural compounds has not been addressed. 5-Nitroanthranilic acid (5NAA), produced in soil by Streptomyces scabies, is also the starting material for synthetic dyes and other nitroaromatic compounds. Bradyrhizobium JS329 was isolated from soil by selective enrichment with 5NAA. When grown on 5NAA, the isolate released stoichiometric amounts of nitrite and half of the stoichiometric amounts of ammonia. Enzyme assays indicate that the initial step in 5NAA degradation is an unusual hydrolytic deamination for formation of 5-nitrosalicylic acid (5NSA). Cloning and heterologous expression revealed the genes that encode 5NAA deaminase (naaA) and the 5NSA dioxygenase (naaB) that cleaves the aromatic ring of 5NSA without prior removal of the nitro group. The results provide the first clear evidence for the initial steps in biodegradation of amino-nitroaromatic compounds and reveal a novel deamination reaction for aromatic amines.The research on biodegradation/biotransformation of nitro compounds has focused on synthetic chemicals, but there are a substantial number of natural nitro-substituted compounds whose metabolism has not been explored. The biodegradation pathways for natural nitro compounds probably provided the metabolic diversity that enabled the rapid and recent evolution of pathways for degradation of synthetic nitro compounds.5-Nitroanthranilic acid (5-NAA), a natural nitroaromatic compound, is produced by Streptomyces scabies, but its physiological role is unclear (15). Synthetic 5NAA is used as the starting material for various nitroaromatic compounds and dyes (3). Substitution of the aromatic ring with amino, nitro, and carboxyl functional groups creates an interesting challenge for catabolic enzymes because any of the three groups could serve as a point of attack for dioxygenase enzymes prior to ring cleavage.Synthetic nitroanilines are toxic and used for the synthesis of pharmaceuticals, dyes, and pigments (27). In sewage, nitroanilines can be formed from the corresponding dinitroaromatic compounds under aerobic or anaerobic conditions (11). Early reports indicated that nitroanilines were resistant to biodegradation (1, 9, 11), but 4-nitroaniline was degraded by Pseudomonas sp. strain P6 (32) and Stenotrophomonas strain HPC 135 (26). Saupe reported that 3-nitroaniline could be degraded aerobically (27). The biodegradation pathways of nitroanilines are unknown, and they are typically classified as nondegradable or poorly degradable compounds (27).As part of a search for novel metabolic diversity and an effort to study the degradation pathway for recalcitrant nitroanilines, we report here the biodegradation of 5NAA as the sole carbon, nitrogen, and energy source by Bradyrhizobium JS329. The degradation pathway involves an unusual hydrolytic removal of the amino group and subsequent ring fission without prior removal of the nitro group.(A preliminary report of this work was presented at the 109th General Meeting of the American Society for Microbiology, 2009 [25].)     

Proteomic analysis of soybean nodule cytosol

An isolation procedure for soybean (Glycine max L. cv Williams 82) nodule cytosol proteins was developed which greatly improved protein resolution by two-dimensional polyacrylamide gel electrophoresis. The most abundant proteins were selected and analyzed by mass spectrometry. The identified proteins were categorized by function (% of total proteins analyzed): carbon metabolism (28%), nitrogen metabolism (12%), reactive oxygen metabolism (12%) and vesicular trafficking (11%). The first three categories were expected based on the known physiological functions of the symbiotic nitrogen fixation process. The number of proteins involved in vesicular trafficking suggests a very active exchange of macromolecules and membrane components. Among the 69 identified proteins were the enzymes of the three carbon portion of glycolysis, which were further characterized to support their roles in the sucrose synthase pathway to provide malate for the bacteroids. Proteomic analysis provides a functional tool by which to understand and further investigate nodule function.