Bacterial growth yields on EDTA,NTA, and their biodegradation intermediates

Ethylenediaminetetraacetic acid (EDTA) and nitrilotriacetic acid (NTA) are widely used anthropogenic chelating agents for control of metal speciation and are ubiquitous in natural waters and wastewaters. This is the first report of systematic measurement of the growth yields of a mixed culture (BNC1-BNC2) on EDTA and its biodegradation intermediates, and of Aminobacter aminovorans (aka Chelatobacter heintzii) ATCC 29600 on NTA and its biodegradation intermediates. The yields measured for BNC1-BNC2 co-culture were 75.0 g of cell dry weight (CDW) (mole of EDTA)⁻¹, 68.6 g of CDW (mole of ED3 A)⁻¹, 51.2 g of CDW (mole of N,N′-EDDA)⁻¹, 34.5 g of CDW (mole of ED)⁻¹, 26.3 g of CDW (mole of IDA)⁻¹, 12.2 g of CDW (mole of glycine)⁻¹, and 9.7 g of CDW (mole of glyoxylate)⁻¹. The yields measured for A. aminovorans were 44.3 g of CDW (mole of NTA)⁻¹, 37.9 g of CDW (mole of IDA)⁻¹, 15.2 g of CDW (mole of glycine)⁻¹, and 10.4 g of CDW (mole of glyoxylate)⁻¹. The biodegradation pathways of EDTA, NTA, and several of their metabolic intermediates include reactions catalyzed by oxygenase enzymes, which may reduce energy available for cell synthesis. Comparison of measured yields with predicted yields indicates that the effect of oxygenase reaction on cell yield can be quantified experimentally as well as modeled based on thermodynamics.     

Cloning, sequencing, and analysis of a gene cluster from Chelatobacter heintzii ATCC 29600 encoding nitrilotriacetate monooxygenase and NADH:flavin mononucleotide oxidoreductase.

Nitrilotriacetate (NTA) is an important chelating agent in detergents and has also been used extensively in processing radionuclides. In Chelatobacter heintzii ATCC 29600, biodegradation of NTA is initiated by NTA monooxygenase that oxidizes NTA to iminodiacetate and glyoxylate. The NTA monooxygenase activity requires two component proteins, component A and component B, but the function of each component is unclear. We have cloned and sequenced a gene cluster encoding components A and B (nmoA and nmoB) and two additional open reading frames, nmoR and nmoT, downstream of nmoA. Based on sequence similarities, nmoR and nmoT probably encode a regulatory protein and a transposase, respectively. The NmoA sequence was similar to a monooxygenase that uses reduced flavin mononucleotide (FMNH2) as reductant; NmoB was similar to an NADH:flavin mononucleotide (FMN) oxidoreductase. On the basis of this information, we tested the function of each component. Purified component B was shown to be an NADH:FMN oxidoreductase, and its activity could be separated from that of component A. When the Photobacterium fischeri NADH:FMN oxidoreductase was substituted for component B in the complete reaction, NTA was oxidized, showing that the substrate specificity of the reaction resides in component A. Component A is therefore an NTA monooxygenase that uses FMNH2 and O2 to oxidize NTA, and component B is an NADH:FMN oxidoreductase that provides FMNH2 for NTA oxidation.     

Modeling speciation effects on biodegradation in mixed metal/chelate systems

A new model, CCBATCH, comprehensively couples microbially catalyzed reactions to aqueous geochemistry. The effect of aqueous speciation on biodegradation reactions and the effect of biological reactions on the concentration of chemical species (e.g. H₂CO₃, NH ₄ ⁺ , O₂) are explicitly included in CCBATCH, allowing systematic investigation of kinetically controlled biological reactions. Bulk-phase chemical speciation reactions including acid/base and complexation are modeled as thermodynamically controlled, while biological reactions are modeled as kinetically controlled. A dual-Monod kinetic formulation for biological degradation reactions is coupled with stoichiometry for the degradation reaction to predict the rate of change of all biological and chemical species affected by the biological reactions. The capability of CCBATCH to capture pH and speciation effects on biological reactions is demonstrated by a series of modeling examples for the citrate/Fe(III) system. pH controls the concentration of potentially biologically available forms of citrate. When the percentage of the degradable substrate is low due to complexation or acid/base speciation, degradation rates may be slow despite high concentrations of substrate Complexation reactions that sequester substratein non-degradable forms may prevent degradation or stopdegradation reactions prior to complete substrate utilization. The capability of CCBATCH to couple aqueous speciation changes to biodegradation reaction kinetics and stoichiometry allows prediction of these key behaviors in mixed metal/chelate systems.     

The implications of trace metal-nitrilotriacetetic acid speciation on its environmental impact and toxicology

Substitution of nitrilotriacetic acid (NTA) for polyphosphates in detergents has brought questions concerning its potential toxicity and impact on trace metal distribution in the environment. A calculation based upon metal ligand equilibria, known environmental concentrations of NTA following extensive detergent usage, and the presence of competitive metal-binding ligands and trace elements demonstrates that NTA will be present almost completely as the calcium and magnesium chelates. An analogous estimate of the speciation of NTA in various toxicity studies demonstrates that the onset of chronic toxicity in feeding studies is coincident with the presence of significant concentrations of “free” NTA in the gastrointestinal tract. Massive doses of NTA over long periods of time cause reproducible renal tumors in rats, but dosages of 7500 ppm administered indefinitely are without measurable effect.     

A resin buffered method for controlling metal speciation in nutrient solutions for plant toxicity tests

Aims

Root elongation tests are sensitive bioassays for testing metal toxicity in nutrient solutions. The metal speciation and, hence, metal exposure conditions are little controlled in the traditional set-up. A resin buffered solution system was developed to overcome this issue.

Methods

Barley (Hordeum vulgare L.) root elongation was tested in aerated 140 mL solution batch systems supplied with 3.3 g Dowex resin for two plants. Copper toxicity was measured in presence or absence of the resin (+R/?R) and in presence or absence of a metal complexing ligand (+NTA; nitrilotriacetic acid/?NTA). In addition, the toxicity in the traditional set without resin and with daily solution replacement was included as a reference.

Results

Metal desorption from the resin is fast in these systems (k?=?0.82 h^?1). Total dissolved Cu roughly halved during 4 days in ?R/?NTA systems due to uptake, while it increased by 30 % in the +R/?NTA, probably due to complexation reactions by root-derived molecules. The toxicity (50 % reduction in root length, EC50) of the initial free Cu²⁺ was equal in all resin or chelate buffered systems and in the solutions with daily replacement, whereas this threshold was significantly larger in the ?R/?NTA due to Cu²⁺ uptake and complexation reactions.

Conclusion

The resin method is a convenient system for high throughput screening of metal toxicity and avoids uncertainties in metal speciation inherent to chelator buffered systems. Details are given how to prepare the resin to obtain a target metal ion activity.     

Chelate-Enhanced Phytoremediation of Soils Polluted with Heavy Metals

In general, hyperaccumulators are low biomass, slow-growing plants. High biomass non-hyperaccumulator plants by themselves are not a valid alternative for phytoextraction as they also have many limitations, such as small root uptake and little root-to-shoot translocation. In this context, chemically-induced phytoextraction (based on the fact that the application of certain chemicals, mostly chelating agents, to the soil significantly enhances metal accumulation by plants) has been proposed as an alternative for the cleaning up of metal polluted soils. But chelate-induced phytoextraction increases the risk of adverse environmental effects due to metal mobilization during extended periods of time. In order to minimize the phytotoxicity and environmental problems associated with the use of chelating agents, nowadays, research is being carried out on the gradual application of small doses of the chelating agent during the growth period. However, EDTA utilization in the future will most likely be limited to ex situconditions where control of the leachates can be achieved. There are other mobilizing agents which are much less harmful to the environment such as citric acid, NTA, and particularly EDDS. Research should also be aimed towards more innovative agronomic practices. Environmentally safe methods of chelate-induced phytoextraction must be developed before steps towards further development and commercialization of this remediation technology are taken. Most importantly, more applied projects in this field are needed to clarify the real potential and risks of this technology.     

Effects of three chelating agents,EDTA, NTA,and TPP,on the concentration of elements in rat tissues

Ethylene diamine tetraacetic acid (EDTA), nitrilotriacetic acid (NTA), and tripolyphosphate (TPP) sodium salts were given orally to rats at the dose of 1 mmol/kg/d for 35 d. The concentrations of Na, K, Ca, Mg, P, S, Fe, Sr, Cu, and Zn were determined in blood, plasma, brain, heart, muscle, liver, kidney, duodenum, and bone of control rats and of the rats receiving EDTA, NTA, and TPP. The main effect induced by EDTA, NTA, and TPP was a decrease of the concentrations of several elements Ca, Mg, Fe, P in the duodenum. Otherwise, EDTA induced an increase of Zn in the kidney (+ 20%), NTA, an increase of Fe in liver (+ 29%), and particularly an increase of Zn in bone (+ 44%). TPP induced a slight decrease of Zn and Cu in liver. In conclusion, EDTA, NTA, and TPP taken orally at the dose of 1 mmol/kg/d for 35 d induced moderate changes of the concentrations of some elements in rat tissues, but without signs of toxicity.     

Dynamics of Substrate Consumption and Enzyme Synthesis in Chelatobacter heintzii during Growth in Carbon-Limited Continuous Culture with Different Mixtures of Glucose and Nitrilotriacetate

Regulation of nitrilotriacetate (NTA) degradation and expression of NTA monooxygenase (NTA-MO) in the NTA-degrading strain Chelatobacter heintzii ATCC 29600 in continuous culture at a dilution rate of 0.06 h(sup-1) under transient growth conditions when the feed was switched between media containing NTA, glucose, or different mixtures thereof as the sole carbon and energy sources was investigated. A transition from NTA to glucose was accompanied by a rapid loss of NTA-MO. A transition from glucose to NTA resulted in a lag phase of some 25 h until NTA-MO expression started, and approximately 100 h was needed before a steady state for NTA-MO specific activity was reached. This transient lag phase was markedly shortened when mixtures of NTA plus glucose were supplied instead of NTA only; for example, when a mixture of 90% glucose and 10% NTA was used, induction of NTA-MO was detected after 30 min. This suggests a strong positive influence of alternative carbon substrates on the expression of other enzymes under natural environmental conditions. Regulation of NTA-MO expression and the fate of NTA-MO were also studied during starvation of both glucose-grown and NTA-grown cultures. Starvation of NTA-grown cells led to a loss of NTA-MO protein. No synthesis of NTA-MO (derepression) was observed when glucose-grown cells were starved.     

Cloning, sequencing, and characterization of a gene cluster involved in EDTA degradation from the bacterium BNC1

EDTA is a chelating agent, widely used in many industries. Because of its ability to mobilize heavy metals and radionuclides, it can be an environmental pollutant. The EDTA monooxygenases that initiate EDTA degradation have been purified and characterized in bacterial strains BNC1 and DSM 9103. However, the genes encoding the enzymes have not been reported. The EDTA monooxygenase gene was cloned by probing a genomic library of strain BNC1 with a probe generated from the N-terminal amino acid sequence of the monooxygenase. Sequencing of the cloned DNA fragment revealed a gene cluster containing eight genes. Two of the genes, emoA and emoB, were expressed in Escherichia coli, and the gene products, EmoA and EmoB, were purified and characterized. Both experimental data and sequence analysis showed that EmoA is a reduced flavin mononucleotide-utilizing monooxygenase and that EmoB is an NADH:flavin mononucleotide oxidoreductase. The two-enzyme system oxidized EDTA to ethylenediaminediacetate (EDDA) and nitrilotriacetate (NTA) to iminodiacetate (IDA) with the production of glyoxylate. The emoA and emoB genes were cotranscribed when BNC1 cells were grown on EDTA. Other genes in the cluster encoded a hypothetical transport system, a putative regulatory protein, and IDA oxidase that oxidizes IDA and EDDA. We concluded that this gene cluster is responsible for the initial steps of EDTA and NTA degradation.     

Chemical-assisted phytoremediation of CD-PAHs contaminated soils using Solanum nigrum L

A well-characterized cadmium (Cd) hyperaccumulating plant Solanum nigrum was grown in Cd and polycyclic aromatic hydrocarbons (PAHs) co-contaminated soil that was repeatedly amended with chemicals, including EDTA, cysteine (CY), salicylic acid (Sa), and Tween 80 (TW80), to test individual and combined treatment effects on phytoremediation of Cd-PAHs contaminated soils. Plant growth was negatively affected by exogenous chemicals except for EDTA. S. nigrum could accumulate Cd in tissues without assistant chemicals, while there was no visible effect on the degradation of PAHs. Cysteine had significant effects on phytoextraction of Cd and the highest metal extraction ratio (1.27%) was observed in 0.9 mmol/kg CY treatment. Both salicylic acid and Tween 80 had stimulative effects on the degradation of PAHs and there was the maximal degradation rate (52.6%) of total PAHs while 0.9 mmol/kg Sa was applied. Furthermore, the combined treatment T(0.1EDTA+0.9CY+0.5TW80) and T(0.5EDTA+0.9CY+03Sa) could not only increase the accumulation of Cd in plant tissues, but also promote the degradation of PAHs. These results indicated that S. nigrum might be effective in phytoextracting Cd and enhancing the biodegradation of PAHs in the co-contaminated soils with assistant chemicals.     

Prevention of toxicity of metal ions to Aeromonas and other bacteria in drinking-water samples using nitrilotriaceticacid (NTA) instead of ethylenediaminetetraaceticacid (EDTA)

The addition of chelating agent to drinking-water samples reduces die-off, due to the toxic effect of metal ions, of bacteria such as Aeromonas. The use of ethylenediaminetetraaceticacid (EDTA) is undesirable since it is a highly persistent chemical which contributes to environmental pollution. This study shows that the less persistent nitrilotriaceticacid (NTA) is a suitable alternative. No significant differences ( P < 0.001) were detected between EDTA and NTA in protecting bacteria.     

Identification, purification, and characterization of iminodiacetate oxidase from the EDTA-degrading bacterium BNC1

Microbial degradation of synthetic chelating agents, such as EDTA and nitrilotriacetate (NTA), may help immobilizing radionuclides and heavy metals in the environment. The EDTA- and NTA-degrading bacterium BNC1 uses EDTA monooxygenase to oxidize NTA to iminodiacetate (IDA) and EDTA to ethylenediaminediacetate (EDDA). IDA- and EDDA-degrading enzymes have not been purified and characterized to date. In this report, an IDA oxidase was purified to apparent homogeneity from strain BNC1 by using a combination of eight purification steps. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single protein band of 40 kDa, and by using size exclusion chromatography, we estimated the native enzyme to be a homodimer. Flavin adenine dinucleotide was determined as its prosthetic group. The purified enzyme oxidized IDA to glycine and glyoxylate with the consumption of O2. The temperature and pH optima for IDA oxidation were 35 degrees C and 8, respectively. The apparent Km for IDA was 4.0 mM with a kcat of 5.3 s(-1). When the N-terminal amino acid sequence was determined, it matched exactly with that encoded by a previously sequenced hypothetical oxidase gene of BNC1. The gene was expressed in Escherichia coli, and the gene product as a C-terminal fusion with a His tag was purified by a one-step nickel affinity chromatography. The purified fusion protein had essentially the same enzymatic activity and properties as the native IDA oxidase. IDA oxidase also oxidized EDDA to ethylenediamine and glyoxylate. Thus, IDA oxidase is likely the second enzyme in both NTA and EDTA degradation pathways in strain BNC1.     

Cloning, Sequencing, and Characterization of a Gene Cluster Involved in EDTA Degradation from the Bacterium BNC1

EDTA is a chelating agent, widely used in many industries. Because of its ability to mobilize heavy metals and radionuclides, it can be an environmental pollutant. The EDTA monooxygenases that initiate EDTA degradation have been purified and characterized in bacterial strains BNC1 and DSM 9103. However, the genes encoding the enzymes have not been reported. The EDTA monooxygenase gene was cloned by probing a genomic library of strain BNC1 with a probe generated from the N-terminal amino acid sequence of the monooxygenase. Sequencing of the cloned DNA fragment revealed a gene cluster containing eight genes. Two of the genes, emoA and emoB, were expressed in Escherichia coli, and the gene products, EmoA and EmoB, were purified and characterized. Both experimental data and sequence analysis showed that EmoA is a reduced flavin mononucleotide-utilizing monooxygenase and that EmoB is an NADH:flavin mononucleotide oxidoreductase. The two-enzyme system oxidized EDTA to ethylenediaminediacetate (EDDA) and nitrilotriacetate (NTA) to iminodiacetate (IDA) with the production of glyoxylate. The emoA and emoB genes were cotranscribed when BNC1 cells were grown on EDTA. Other genes in the cluster encoded a hypothetical transport system, a putative regulatory protein, and IDA oxidase that oxidizes IDA and EDDA. We concluded that this gene cluster is responsible for the initial steps of EDTA and NTA degradation.     

Mobilisation of recently absorbed Fe in ex vivo perfused rat duodena and the influence of iron status and subsequently absorbed chelators

To investigate the effect of subsequently absorbed metal chelators on recently absorbed ⁵⁹Fe, duodenal segments from iron-deficient and iron-adequate rats were perfused ex vivo until the ⁵⁹Fe tissue load had reached a steady state. Subsequently, the segments were perfused with 3 model chelators and their iron complexes: nitrilotriacetic acid (NTA), ethylenediaminetetraacetic acid (EDTA) and citrate. Of these, NTA and EDTA bind iron much tighter than citrate, and Fe–NTA complexes exchange iron within seconds while Fe-EDTA complexes need 48 h to reach equilibrium.

Duodenal mucosa-to-serosa transport rates were comparable for all 3 chelators and correlated linearly with luminal concentration. Subsequent perfusion with increasing NTA, Fe–NTA(1:2) and EDTA concentrations mobilised increasing amounts of ⁵⁹Fe from the duodenum. Mobilised ⁵⁹Fe moved preferentially back into the luminal perfusate in iron-adequate segments. In iron-deficient segments, ⁵⁹Fe preferentially continued the absorption process across the basolateral membrane. Fe–EDTA(1:1) hardly mobilised any ⁵⁹Fe back into the lumen, though basolateral transfer increased at high concentrations. Citrate and Fe–citrate(1:1) mobilised ⁵⁹Fe only at very high concentrations.

This behaviour is in accordance with the rules of complex chemistry: strong, fast reacting ligands like NTA show most impact. Slowly reacting complexes like Fe–EDTA(1:1) have little mobilising impact in spite of strong affinity between EDTA and iron. The low affinity between iron and citrate can be compensated by large concentration. Moreover, iron-deficient segments show stronger re-uptake of mobilised ⁵⁹Fe from the lumen and a stronger transfer of ⁵⁹Fe from the tissue across the basolateral membrane. Both are compatible with the more marked expression of divalent metal transporter 1 (DMT-1) and IREG-1 at the brushborder and basolateral membrane of iron-deficient enterocytes. The data suggest that iron ions interact with food ligands during their passage from the apical to the basolateral side of duodenal enterocytes.     

Biodegradabilities of Ethylenediamine-N,N′-disuccinic Acid (EDDS) and Other Chelating Agents

Biodegradabilities of chelating agents were tested with activated sludge. Ethylenediaminetetraacetic acid (EDTA) remained intact in the effluent even after acclimation for 100 days, but propanediamine-N,N′-disuccinic acid (PDDS) and nitrilotriacetic acid (NTA) were biodegraded after acclimation for 5 and 23 days, respectively. Optical isomers of ethylenediamine-N,N′-disuccinic acid (EDDS) had different biodegradabilities: SS- and RS-isomers were susceptible to biodegradation, but the RR-isomer was resistant. SS-isomer was degraded even by activated sludge without acclimation.     

Metal chelators react also with reactive oxygen and nitrogen species

Popular chelators (desferrioxamine, SIH, EDTA, EGTA, DTPA, and NTA) were demonstrated to have antioxidant properties, being able to reduce ABTS radical cation and react with peroxyl radicals, peroxynitrite, and hypochlorite. Desferrioxamine and SIH were most potent antioxidants in all cases. These results point to the necessity of a careful interpretation of experiments in which the inhibition of free radical reactions by antioxidants is used as a proof of involvement of metal ions in a reaction.     

Separation and identification of desferrioxamine and its iron chelating metabolites by high-performance liquid chromatography and fast atom bombardment mass spectrometry: choice of complexing agent and application to biological fluids

An HPLC-based method capable of separating desferrioxamine (DFO) and its iron chelating metabolites from uv-absorbing species present in biological fluids has been developed. This method relies on the use of nitrilotriacetic acid (NTA) as the complexing agent in the mobile phase, instead of EDTA, previously used in HPLC methods. The use of NTA ensures that iron contamination present in buffers and bound to the column does not interfere with analysis. The disadvantages of using EDTA are discussed. The identity of the iron chelating metabolites of DFO present in the urine of patients with beta-thalassemia major has been established using FAB mass spectrometry. The metabolism of DFO, reported in this study, takes place almost exclusively at the N-terminal region of the molecule and is in many respects similar to the degradation of the amino acid lysine. In addition, a metabolite which corresponds to N-hydroxylation of the terminal amino group has been identified.     

Effect of EDTA and Tannic Acid on the Removal of Cd,Ni, Pb and Cu from Artificially Contaminated Soil by Althaea rosea Cavan

In this study an ornamental plant of Althaea rosea Cavan was investigated for its potential use in the removal of Cd, Ni, Pb and Cu from an artificially contaminated soil. Effect of two different chelating agents on the removal has also been studied by using EDTA (ethylenediaminetetracetic acid) and TA (tannic acid). Both EDTA and TA have led to higher heavy metal concentration in shoots and leaves compared to control plants. However EDTA is generally known as an effective agent in metal solubilisation of soil, in this study, TA was found more effective to induce metal accumulation in Althaea rosea Cavan under the studied conditions. In addition to this, EDTA is toxic to some species and restraining the growth of the plants. The higher BCF (Bio Concentration Factor) and TF (Translocation Factor) values obtained from stems and leaves by the effects of the chemical enhancers (EDTA and TA) show that Althaea rosea Cavan is a hyper accumulator for the studied metals and may be cultivated to clean the contaminated soils.     

Identification, Purification, and Characterization of Iminodiacetate Oxidase from the EDTA-Degrading Bacterium BNC1

Microbial degradation of synthetic chelating agents, such as EDTA and nitrilotriacetate (NTA), may help immobilizing radionuclides and heavy metals in the environment. The EDTA- and NTA-degrading bacterium BNC1 uses EDTA monooxygenase to oxidize NTA to iminodiacetate (IDA) and EDTA to ethylenediaminediacetate (EDDA). IDA- and EDDA-degrading enzymes have not been purified and characterized to date. In this report, an IDA oxidase was purified to apparent homogeneity from strain BNC1 by using a combination of eight purification steps. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single protein band of 40 kDa, and by using size exclusion chromatography, we estimated the native enzyme to be a homodimer. Flavin adenine dinucleotide was determined as its prosthetic group. The purified enzyme oxidized IDA to glycine and glyoxylate with the consumption of O₂. The temperature and pH optima for IDA oxidation were 35°C and 8, respectively. The apparent K_m for IDA was 4.0 mM with a k_cat of 5.3 s⁻¹. When the N-terminal amino acid sequence was determined, it matched exactly with that encoded by a previously sequenced hypothetical oxidase gene of BNC1. The gene was expressed in Escherichia coli, and the gene product as a C-terminal fusion with a His tag was purified by a one-step nickel affinity chromatography. The purified fusion protein had essentially the same enzymatic activity and properties as the native IDA oxidase. IDA oxidase also oxidized EDDA to ethylenediamine and glyoxylate. Thus, IDA oxidase is likely the second enzyme in both NTA and EDTA degradation pathways in strain BNC1.     

The variable part of the dnaK gene as an alternative marker for phylogenetic studies of rhizobia and related alpha Proteobacteria

DnaK is the 70 kDa chaperone that prevents protein aggregation and supports the refolding of damaged proteins. Due to sequence conservation and its ubiquity this chaperone has been widely used in phylogenetic studies. In this study, we applied the less conserved part that encodes the so-called alpha-subdomain of the substrate-binding domain of DnaK for phylogenetic analysis of rhizobia and related non-symbiotic alpha-Proteobacteria. A single 330 bp DNA fragment was routinely amplified from DNA templates isolated from the species of the genera, Azorhizobium, Bradyrhizobium, Mesorhizobium, Rhizobium and Sinorhizobium, but also from some non-symbiotic alpha Proteobacteria such as Blastochloris, Chelatobacter and Chelatococcus. Phylogenetic analyses revealed high congruence between dnaK sequences and 16S rDNA trees, but they were not identical. In contrast, the partition homogeneity tests revealed that dnaK sequence data could be combined with other housekeeping genes such as recA, atpD or glnA. The dnaK trees exhibited good resolution in the cases of the genera Mesorhizobium, Sinorhizobium and Rhizobium, even better than usually shown by 16S rDNA phylogeny. The dnaK phylogeny supported the close phylogenetic relationship of Rhizobium galegae and Agrobacterium tumefaciens (R. radiobacter) C58, which together formed a separate branch within the fast-growing rhizobia, albeit closer to the genus Sinorhizobium. The Rhizobium and Sinorhizobium genera carried an insertion composed of two amino acids, which additionally supported the phylogenetic affinity of these two genera, as well as their distinctness from the Mesorhizobium genus. Consistently with the phylogeny shown by 16S-23S rDNA intergenic region sequences, the dnaK trees divided the genus Bradyrhizobium into three main lineages, corresponding to B. japonicum, B. elkanii, and photosynthetic Bradyrhizobium strains that infect Aeschynomene plants. Our results suggest that the 330 bp dnaK sequences could be used as an additional taxonomic marker for rhizobia and related species (alternatively to the 16S rRNA gene phylogeny).