In vivo nuclear magnetic resonance study of the osmoregulation of phosphocholine-substituted beta-1,3;1,6 cyclic glucan and its associated carbon metabolism in Bradyrhizobium japonicum USDA 110.

A phosphocholine-substituted beta-1,3;1,6 cyclic glucan (PCCG), an unusual cyclic oligosaccharide, has been isolated from Bradyrhizobium japonicum USDA 110 (D. B. Rolin, P. E. Pfeffer, S. F. Osman, B. S. Swergold, F. Kappler, and A. J. Benesi, Biochim. Biophys. Acta 1116:215-225, 1992). Data presented here suggest that PCCG synthesis is dependent on the carbon metabolism and that osmotic regulation of its biosynthesis parallels regulation of membrane-derived oligosaccharide biosynthesis observed in Escherichia coli (E. P. Kennedy, M. K. Rumley, H. Schulman, and L. M. G. van Golde, J. Biol. Chem. 251:4208-4213, 1976) and Agrobacterium tumefaciens (G. A. Cangelosi, G. Martinetti, and E. W. Nester, J. Bacteriol. 172:2172-2174, 1990). Growth of B. japonicum USDA 110 cells in the reference medium at relatively low osmotic pressures (LO) (65 mosmol/kg of H2O) caused a large accumulation of PCCG and unsubstituted beta-1,3;1,6 cyclic glucans (CG). Sucrose and polyethylene glycol, nonionic osmotica, reduce all growth rates and inhibit almost completely the production of PCCG at high osmotic pressures (HO) above 650 and 400 mosmol/kg of H2O), respectively. We used in vivo 13C nuclear magnetic resonance spectroscopy to identify the active osmolytes implicated in the osmoregulation process. The level of alpha,alpha-trehalose in B. japonicum cells grown in autoclaved or filter-sterilized solutions remained constant in HO (0.3 M sucrose or 250 g of polyethylene glycol 6000 per liter) medium. Significant amounts of glycogen and extracellular polysaccharides were produced only when glucose was present in the autoclaved HO 0.3 M sucrose media. The results of hypo- and hyperosmotic shocking of B. japonicum USDA 110 cells were monitored by using in vivo 31P and 13C nuclear magnetic resonance spectroscopy. The first observed osmoregulatory response of glycogen-containing cells undergoing hypoosmotic shock was release of P(i) into the medium. Within 7 h, reabsorption of P(i) was complete and production of PCCG was initiated. After 12 h, the PCCG content had increased by a factor of 7. Following the same treatment, cells containing little or no glycogen released trehalose and failed to produce PCCG. Thus the production of PCCG/CG in response to hypoosmotic shocking of stationary-phase cells was found to be directly linked to the interconversion of stored glycogen. Hyperosmotic shocking of LO-grown stationary-phase cells with sucrose had no effect on the content of previously synthesized CG/PCCG. The PCCG/CG content and its osmotically induced biosynthesis are discussed in terms of carbon metabolism and a possible role in hypoosmotic adaptation in B. japonicum USDA 110.     

Characterization of 1,2-dibromoethane-degrading haloalkane dehalogenase from Bradyrhizobium japonicum USDA110

Haloalkane dehalogenases (DHAs, E.C. 3.8.1.5) are very promising biocatalytic tools for the bioremediation of environmental pollutants which consists of haloalkanes. In the present work, we investigated the DHA from Bradyrhizobium japonicum USDA110 (BjDHA). The dehalogenase activity of B. japonicum USDA110 and RT-PCR analysis revealed that the BjDHA gene expression is induced by 1,2-dibromoethane (1,2-DBE) during the early exponential phase. The BjDHA gene was cloned, expressed in Escherichia coli BL21 (DE3) and characterized. The enzyme catalyzes the irreversible hydrolysis of a variety of haloalkanes to the corresponding alcohol, halide, and a hydrogen ion. The catalytic properties of the recombinant enzyme were investigated and the kinetic parameters (K_m, k_cat) for a number of substrates were determined. The results showed that the BjDHA displays wide substrate specificity towards haloalkanes and particular high activity towards 1,2-DBE. The enzyme has a different catalytic triad topology compared to the Xanthobacter haloalkane dehalogenase and is more similar to the Rhodococcus enzyme. In addition, consistent with its broad specificity, the BjDHA has a substantially larger and more polar active site cavity compared to the Xanthobacter and Rhodococcus enzymes and as a consequence, BjDHA is able to dehalogenate longer and polar compounds. These properties make this enzyme very promising bioremediation tool for environmental applications.     

Cloning of Protocatechuate 3,4-Dioxygenase Genes from Bradyrhizobium japonicum USDA110

A heterologous gene probe encoding the α and β subunits of the Pseudomonas cepacia protocatechuate 3,4-dioxygenase (PCD) was used to detect its homolog in the genome of Bradyrhizobium japonicum USDA110. Three cosmid clones carrying a 2.2-kb BamHI insert showed high levels of PCD activity. SacI digestion of one of the genomic clones, pBjG17, produced a 2.5-kb insert DNA that complemented a PCD mutant of P. cepacia.     

Structural and functional analysis of two different nodD genes in Bradyrhizobium japonicum USDA110.

Bradyrhizobium japonicum has two closely linked homologs of the nodulation regulatory gene, nodD; these homologs are located upstream of and in divergent orientation to the nodYABCSUIJ gene cluster. We report here the nucleotide sequence and mutational analyses of both nodD copies. The predicted NodD1 and NodD2 proteins shared 62% identical amino acid residues at corresponding positions and exhibited different degrees of homology with NodD proteins of other Bradyrhizobium, Azorhizobium, and Rhizobium strains. Induction of the nodYABCSUIJ operon, as measured by expression of a translational nodC'-'lacZ fusion, required the nodD1 gene, but not nodD2. A B. japonicum mutant deleted for both nodD copies (strain delta 1267) still showed residual nodulation activity; however, nodulation of soybean was significantly delayed, and nodulation of mung bean and siratro resulted in strongly reduced nodule numbers. Fully efficient nodulation of mung bean and siratro by strain delta 1267 was restored by genetic complementation with the nodD1 gene, but not with nodD2. We conclude from these data that nodD1 is the critical gene that contributes to maximal nodulation efficiency, whereas the nodD2 gene does not play any obvious role in nodulation of the host plants tested.     

Cyclic [beta]-1,6-1,3-Glucans of Bradyrhizobium japonicum USDA 110 Elicit Isoflavonoid Production in the Soybean (Glycine max) Host

High levels of cyclic [beta]-1,6-1,3-glucans (e.g. 0.1 mg mg-1 of total protein) are synthesized by free-living cells as well as by bacteroids of Bradyrhizobium japonicum USDA 110 (K.J. Miller, R.S. Gore, R. Johnson, A.J. Benesi, V.N. Reinhold [1990] J Bacteriol 172: 136-142; R.S. Gore and K.J. Miller [1993] Plant Physiol 102: 191-194). These molecules share structural features with glucan fragments isolated from the mycelial cell wall of the soybean (Glycine max) pathogen Phytophthora megasperma. These latter glucans have been shown to be potent elicitors (at nanogram levels) of the phytoalexin glyceollin in G. max. Using the well-characterized soybean cotyledon bioassay, we now show that the cyclic [beta]-1,6-1,3-glucans of B. japonicum USDA 110 are also biologically active elicitors of glyceollin production (but at microgram levels). We further show that both classes of [beta]-glucans elicit the production of the isoflavone daidzein within soybean cotyledon wound droplets.     

Characterization of a Mannitol-Utilizing, Nitrogen-Fixing Bradyrhizobium japonicum USDA 110 Derivative

We have isolated a colonial derivative of Bradyrhizobium japonicum USDA 110 (designated MN-110) that is both mannitol utilizing and N(2) fixing. Derivative MN-110 showed growth on mannitol and glucose similar to that of non-N(2)-fixing, mannitol-utilizing L2-110. Derivative MN-110 showed high constitutive and induced d-mannitol dehydrogenase activity (similar to L2-110) relative to N(2)-fixing, non-mannitol-utilizing I-110. Hybridization to EcoRI and HindIII total DNA digests with cloned USDA 110 nif DK and nif H genes revealed similar patterns for non-N(2)-fixing mannitol-utilizing derivative L1-110 and derivative MN-110. Symbiotic tests with soybean cultivars Ransom and Lee indicate MN-110 to be a superior N(2)-fixing derivative compared with derivative I-110 and the parent strain USDA 110. However, these differences were not revealed when comparing 28-day-old soybean-B. japonicum associations but were apparent in 49-day-old associations. It was apparent from this work that mannitol utilization was not necessarily correlated to symbiotic effectiveness in B. japonicum and that gene rearrangements were not responsible for differences in N(2) fixation between L1-110 or L2-110 and MN-110.     

Discovery of a mandelonitrile hydrolase from Bradyrhizobium japonicum USDA110 by rational genome mining

A mandelonitrile hydrolase bll6402 from Bradyrhizobium japonicum USDA110 was predicted by rational genome mining, i.e. combining traditional genome mining with functional analysis of the genetic organization of the putative nitrilase gene within the chromosome of microorganisms. This putative gene was cloned and over-expressed in Escherichia coli, and the encoded protein was purified to give a nitrilase with a molecular mass of about 37kDa. The molecular weight of the holoenzyme was about 455kDa, suggesting that nitrilase bll6402 self-aggregated to the active form with native structure being 12 subunits of identical size. This nitrilase was most active toward mandelonitrile with V(max) and K(m) for mandelonitrile being 44.7U/mg and 0.26mM, respectively. The k(cat) and overall catalytic efficiency k(cat)/K(m) were 27.0s(-1) and 1.04x10(5)M(-1)s(-1), indicating that nitrilase bll6402 is very active for the hydrolysis of mandelonitrile to mandelic acid. Nitrilase bll6402 also effectively hydrolyzed several mandelonitrile derivatives.     

Synthesis of a Low-Molecular-Weight Form of Exopolysaccharide by Bradyrhizobium japonicum USDA 110

A novel extracellular low-molecular-weight polysaccharide was detected as a contaminant within extracellular cyclic β-1,6-β-1,3-glucan preparations from Bradyrhizobium japonicum USDA 110 cultures. Compositional analysis, methylation analysis, and nuclear magnetic resonance analysis revealed that this low-molecular-weight polysaccharide was composed of the same pentasaccharide repeating unit previously described for the high-molecular-weight form of the exopolysaccharide (EPS) synthesized by B. japonicum strains. Mass spectrometry analysis indicated that the size of this low-molecular-weight form of EPS was consistent with a dimeric form of the pentasaccharide repeating unit.     

Complete genomic sequence of nitrogen-fixing symbiotic bacterium Bradyrhizobium japonicum USDA110.

The complete nucleotide sequence of the genome of a symbiotic bacterium Bradyrhizobium japonicum USDA110 was determined. The genome of B. japonicum was a single circular chromosome 9,105,828 bp in length with an average GC content of 64.1%. No plasmid was detected. The chromosome comprises 8317 potential protein-coding genes, one set of rRNA genes and 50 tRNA genes. Fifty-two percent of the potential protein genes showed sequence similarity to genes of known function and 30% to hypothetical genes. The remaining 18% had no apparent similarity to reported genes. Thirty-four percent of the B. japonicum genes showed significant sequence similarity to those of both Mesorhizobium loti and Sinorhizobium meliloti, while 23% were unique to this species. A presumptive symbiosis island 681 kb in length, which includes a 410-kb symbiotic region previously reported by G?ttfert et al., was identified. Six hundred fifty-five putative protein-coding genes were assigned in this region, and the functions of 301 genes, including those related to symbiotic nitrogen fixation and DNA transmission, were deduced. A total of 167 genes for transposases/104 copies of insertion sequences were identified in the genome. It was remarkable that 100 out of 167 transposase genes are located in the presumptive symbiotic island. DNA segments of 4 to 97 kb inserted into tRNA genes were found at 14 locations in the genome, which generates partial duplication of the target tRNA genes. These observations suggest plasticity of the B. japonicum genome, which is probably due to complex genome rearrangements such as horizontal transfer and insertion of various DNA elements, and to homologous recombination.     

Synthesis of a low-molecular-weight form of exopolysaccharide by Bradyrhizobium japonicum USDA 110

A novel extracellular low-molecular-weight polysaccharide was detected as a contaminant within extracellular cyclic beta-1,6-beta-1,3-glucan preparations from Bradyrhizobium japonicum USDA 110 cultures. Compositional analysis, methylation analysis, and nuclear magnetic resonance analysis revealed that this low-molecular-weight polysaccharide was composed of the same pentasaccharide repeating unit previously described for the high-molecular-weight form of the exopolysaccharide (EPS) synthesized by B. japonicum strains. Mass spectrometry analysis indicated that the size of this low-molecular-weight form of EPS was consistent with a dimeric form of the pentasaccharide repeating unit.     

Novel methylated triterpenoids of the gammacerane series from the nitrogen-fixing bacterium Bradyrhizobium japonicum USDA 110.

The nitrogen-fixing, symbiotic root-nodule forming bacterium Bradyrhizobium japonicum USDA 110 contained gammacerane derivatives next to triterpenoids of the hopane series. Diploptene, diplopterol, 2 beta-methyldiplopterol, aminobacteriohopanetriol and adenosylhopane were accompanied by tetrahymanol and the corresponding novel methylated homologues 2 beta-methyltetrahymanol, 20 alpha-methyltetrahymanol, and 2 beta,20 alpha-dimethyltetrahymanol. Incorporation of [(2)H(3)]methyl-L-methionine indicated that the additional methyl groups originated from methionine, probably with S-adenosylmethionine acting as methyl donor, with retention of the three deuterium atoms. The simultaneous presence of hopane and gammacerane derivatives seems a characteristic feature of the genus Bradyrhizobium and the phylogenetically closely related Rhodopseudomonas palustris.     

A new nitrilase from Bradyrhizobium japonicum USDA 110. Gene cloning, biochemical characterization and substrate specificity

A nitrilase gene blr3397 from Bradyrhizobium japonicum USDA110 was cloned and over-expressed in Escherichia coli, and the encoded protein was purified to give a nitrilase with a single band of about 34.5kD on SDS-PAGE. The molecular weight of the holoenzyme was about 340kD as determined by light scattering analysis, suggesting that nitrilase blr3397 self-aggregated to an active form with the native structure being a decamer. The V(max) and K(m) for phenylacetonitrile were 3.15U/mg and 4.36mM, respectively. The catalytic constant k(cat) and specificity constant k(cat)/K(m) were 111min(-1) and 2.6x10(4)min(-1)M(-1). This nitrilase is most active toward the hydrolysis of hydrocinnamonitrile among the tested substrates (4.3 times that of phenylacetonitrile). The nitrilase blr3397 shows higher activity towards the hydrolysis of aliphatic nitriles than that for the aromatic counterparts, and can be characterized as an aliphatic nitrilase in terms of activity. This nitrilase also possesses distinct features from the nitrilase bll6402 of the same microbe.     

Bradyrhizobium japonicum subspecies (USDA 110 and 26) characterized by fixed-nitrogen uptake and symbiotic indoleacetic acid production

Two dissimilar subspecies ofBradyrhizobium japonicum (USDA 110 and 26) differ in ammonia (NH₃) assimilation and symbiotic indoleacetic acid (IAA) production. Free-living cultures of type-strain USDA 26 grow on NH₃ as a sole N source and take up an NH₃ analog, methylamine, whereas USDA strain 110 does neither. Although both strains nodulate soybean effectively, root nodules infected with symbiont 26 contain 0.3–1.1 g IAA per gram fresh weight. Nodules infected by tryptophan catabolic variants 4b and 20d, derived from strain 26, also elicit an increased IAA content, two- to fourfold (2.0–3.9 g · g^–1). In contrast, nodules infected with the dissimilar subspecies (strains 110 and 123) contain significantly less IAA.     

Analysis of the Symbiotic Performance of Bradyrhizobium japonicum USDA 110 and Its Derivative I-110 and Discovery of a New Mannitol-Utilizing, Nitrogen-Fixing USDA 110 Derivative

Previously, Bradyrhizobium japonicum USDA 110 was shown to contain colony morphology variants which differed in nitrogen-fixing ability. Mannitol-utilizing derivatives L1-110 and L2-110 have been shown to be devoid of symbiotic nitrogen fixation ability, and non-mannitol-utilizing derivatives I-110 and S-110 have been shown to be efficient at nitrogen fixation. The objectives of this study were to determine the effect of media carbon sources on the symbiotic N₂-fixing ability of strain USDA 110 and to compare the effectiveness of strain USDA 110 and derivative I-110. Based on acetylene reduction activity and the nitrogen content of 41-day-old soybean plants, neither derivative I-110 nor cultures of USDA 110 grown in media favoring non-mannitol-using derivatives had symbiotic nitrogen fixation that was statistically superior to that of cultures of USDA 110 grown in media favoring mannitol-using derivatives. In another experiment 200 individual nodules formed by strain USDA 110 grown in yeast extract gluconate were screened for colony morphology of occupying variant(s) and acetylene reduction activity. Nodules occupied by mannitol-using derivatives (large colony type on 0.1% yeast extract-0.05% K₂HPO₄-0.08% MgSO₄ · 7H₂O-0.02% NaCl-0.001% FeCl₃ · 6H₂O [pH 6.7] with 1% mannitol [YEM] plates) had a mean acetylene reduction activity equal to that of nodules occupied by non-mannitol-using derivatives (small colony type on YEM plates). A total of 20 large colonial derivatives and 10 small colonial derivatives (I-110-like) were isolated and purified by repeated culture in YEM and YEG (same as YEM except 1% gluconate instead of 1% mannitol) media, respectively, followed by dilution in solutions containing 0.05% Tween 40. After 25 days of growth, soybean plants inoculated with the large colony isolates had mean whole-plant acetylene reduction activity, whole-plant dry weight, and whole-plant nitrogen contents equal to or better than those of plants inoculated with either the small colony isolates (I-110-like) or the I-110 (non-mannitol-using) derivative. Hence, the existence of a mannitol-utilizing derivative that fixes nitrogen in a culture of strain USDA 110 obtained from the U.S. Department of Agriculture, Beltsville, Md., was established. This new USDA 110 derivative was designated as MN-110 because it was a mannitol-utilizing nitrogen-fixing USDA 110 derivative. This derivative was morphologically indistinguishable from the non-nitrogen-fixing derivative L2-110 found in cultures obtained earlier from the U.S. Department of Agriculture, Beltsville. DNA-DNA homology and restriction enzyme analyses indicated that MN-110 is genetically related to other USDA 110 derivatives that have been characterized previously.     

Functional expression of Sinorhizobium meliloti BetS, a high-affinity betaine transporter, in Bradyrhizobium japonicum USDA110

Among the Rhizobiaceae, Bradyrhizobium japonicum strain USDA110 appears to be extremely salt sensitive, and the presence of glycine betaine cannot restore its growth in medium with an increased osmolarity (E. Boncompagni, M. Osteras, M. C. Poggi, and D. Le Rudulier, Appl. Environ. Microbiol. 65:2072-2077, 1999). In order to improve the salt tolerance of B. japonicum, cells were transformed with the betS gene of Sinorhizobium meliloti. This gene encodes a major glycine betaine/proline betaine transporter from the betaine choline carnitine transporter family and is required for early osmotic adjustment. Whereas betaine transport was absent in the USDA110 strain, such transformation induced glycine betaine and proline betaine uptake in an osmotically dependent manner. Salt-treated transformed cells accumulated large amounts of glycine betaine, which was not catabolized. However, the accumulation was reversed through rapid efflux during osmotic downshock. An increased tolerance of transformant cells to a moderate NaCl concentration (80 mM) was also observed in the presence of glycine betaine or proline betaine, whereas the growth of the wild-type strain was totally abolished at 80 mM NaCl. Surprisingly, the deleterious effect due to a higher salt concentration (100 mM) could not be overcome by glycine betaine, despite a significant accumulation of this compound. Cell viability was not significantly affected in the presence of 100 mM NaCl, whereas 75% cell death occurred at 150 mM NaCl. The absence of a potential gene encoding Na(+)/H(+) antiporters in B. japonicum could explain its very high Na(+) sensitivity.     

Functional Expression of Sinorhizobium meliloti BetS, a High-Affinity Betaine Transporter, in Bradyrhizobium japonicum USDA110

Among the Rhizobiaceae, Bradyrhizobium japonicum strain USDA110 appears to be extremely salt sensitive, and the presence of glycine betaine cannot restore its growth in medium with an increased osmolarity (E. Boncompagni, M. Østerås, M. C. Poggi, and D. Le Rudulier, Appl. Environ. Microbiol. 65:2072-2077, 1999). In order to improve the salt tolerance of B. japonicum, cells were transformed with the betS gene of Sinorhizobium meliloti. This gene encodes a major glycine betaine/proline betaine transporter from the betaine choline carnitine transporter family and is required for early osmotic adjustment. Whereas betaine transport was absent in the USDA110 strain, such transformation induced glycine betaine and proline betaine uptake in an osmotically dependent manner. Salt-treated transformed cells accumulated large amounts of glycine betaine, which was not catabolized. However, the accumulation was reversed through rapid efflux during osmotic downshock. An increased tolerance of transformant cells to a moderate NaCl concentration (80 mM) was also observed in the presence of glycine betaine or proline betaine, whereas the growth of the wild-type strain was totally abolished at 80 mM NaCl. Surprisingly, the deleterious effect due to a higher salt concentration (100 mM) could not be overcome by glycine betaine, despite a significant accumulation of this compound. Cell viability was not significantly affected in the presence of 100 mM NaCl, whereas 75% cell death occurred at 150 mM NaCl. The absence of a potential gene encoding Na⁺/H⁺ antiporters in B. japonicum could explain its very high Na⁺ sensitivity.     

An l-glucitol oxidizing dehydrogenase from Bradyrhizobium japonicum USDA 110 for production of d-sorbose with enzymatic or electrochemical cofactor regeneration

A gene in Bradyrhizobium japonicum USDA 110, annotated as a ribitol dehydrogenase (RDH), had 87 % sequence identity (97 % positives) to the N-terminal 31 amino acids of an l-glucitol dehydrogenase from Stenotrophomonas maltophilia DSMZ 14322. The 729-bp long RDH gene coded for a protein consisting of 242 amino acids with a molecular mass of 26.1 kDa. The heterologously expressed protein not only exhibited the main enantio selective activity with d-glucitol oxidation to d-fructose but also converted l-glucitol to d-sorbose with enzymatic cofactor regeneration and a yield of 90 %. The temperature stability and the apparent K _m value for l-glucitol oxidation let the enzyme appear as a promising subject for further improvement by enzyme evolution. We propose to rename the enzyme from the annotated RDH gene (locus tag bll6662) from B. japonicum USDA as a d-sorbitol dehydrogenase (EC 1.1.1.14).     

Stereoselectivity and conformational stability of haloalkane dehalogenase DbjA from Bradyrhizobium japonicum USDA110: the effect of pH and temperature

The effect of pH and temperature on structure, stability, activity and enantioselectivity of haloalkane dehalogenase DbjA from Bradyrhizobium japonicum USDA110 was investigated in this study. Conformational changes have been assessed by circular dichroism spectroscopy, functional changes by kinetic analysis, while quaternary structure was studied by gel filtration chromatography. Our study shows that the DbjA enzyme is highly tolerant to pH changes. Its secondary and tertiary structure was not affected by pH in the ranges 5.3-10.3 and 6.2-10.1, respectively. Oligomerization of DbjA was strongly pH-dependent: monomer, dimer, tetramer and a high molecular weight cluster of the enzyme were distinguished in solution at different pH conditions. Moreover, different oligomeric states of DbjA possessed different thermal stabilities. The highest melting temperature (T(m) = 49.1 ± 0.2 °C) was observed at pH 6.5, at which the enzyme occurs in dimeric form. Maximal activity was detected at 50 °C and in the pH interval 7.7-10.4. While pH did not have any effect on enantiodiscriminination of DbjA, temperature significantly altered DbjA enantioselectivity. A decrease in temperature results in significantly enhanced enantioselectivity. The temperature dependence of DbjA enantioselectivity was analysed with 2-bromobutane, 2-bromopentane, methyl 2-bromopropionate and ethyl 2-bromobutyrate, and differential activation parameters Δ(R-S)ΔH and Δ(R-S)ΔS were determined. The thermodynamic analysis revealed that the resolution of β-bromoalkanes was driven by both enthalpic and entropic terms, while the resolution of α-bromoesters was driven mainly by an enthalpic term. Unique catalytic activity and structural stability of DbjA in a broad pH range, combined with high enantioselectivity with particular substrates, make this enzyme a very versatile biocatalyst. Enzyme EC3.8.1.5 haloalkane dehalogenase.