Identification of active site residues in Bradyrhizobium japonicum acetyl-CoA synthetase.

Acetyl-CoA synthetase (ACS) catalyses the activation of acetate to acetyl-CoA in the presence of ATP and CoA. The gene encoding Bradyrhyzobium japonicum ACS has been cloned, sequenced, and expressed in Escherichia coli. The enzyme comprises 648 amino acid residues with a calculated molecular mass of 71,996 Da. The recombinant enzyme was also purified from the transformed E. coli. The enzyme was essentially indistinguishable from the ACS of B. japonicum bacteroids as to the criteria of polyacrylamide gel electrophoresis and biochemical properties. Based on the results of database analysis, Gly-263, Gly-266, Lys-269, and Glu-414 were selected for site-directed mutagenesis in order to identify amino acid residues essential for substrate binding and/or catalysis. Four different mutant enzymes (G263I, G266I, K269G, and E414Q) were prepared and then subjected to steady-state kinetic studies. The kinetic data obtained for the mutants suggest that Gly-266 and Lys-269 participate in the formation of acetyl-AMP, whereas Glu-414 may play a role in acetate binding.     

Identification of a Functional fur Gene in Bradyrhizobium japonicum

The recent identification of the iron response regulator (Irr) in Bradyrhizobium japonicum raised the question of whether the global regulator Fur is present in that organism. A fur gene homolog was isolated by the functional complementation of an Escherichia coli fur mutant. The B. japonicum Fur bound to a Fur box DNA element in vitro, and a fur mutant grown in iron-replete medium was derepressed for iron uptake activity. Thus, B. japonicum expresses at least two regulators of iron metabolism.     

Identification of extracytoplasmic proteins in Bradyrhizobium japonicum using phage display

A novel gene bank of Bradyrhizobium japonicum USDA110spc4 was constructed using pG3DSS, a phagemid vector designed for detecting genes encoding secreted proteins. In this phagemid, the phage protein III lacks its indigenous signal peptide required for protein secretion, thus recombinant fusion proteins are displayed on the phage surface only if a functional signal peptide is provided by an inserted DNA fragment. In addition, the N-terminal half of protein III has been replaced by a short linker region (the E-tag) that is recognized by a monoclonal antibody, which enables isolation of phages displaying a fusion protein. The expression library described here, therefore, provides a powerful means to affinity select for B. japonicum genes encoding extracytoplasmic proteins. In total, 182 DNA sequences were analyzed, among which 132 different putative extracytoplasmic proteins could be identified. The function of most proteins could be predicted and support an extracytoplasmic localization. In addition, genes encoding novel extracytoplasmic proteins were found. In particular, a novel family of small proteins has been identified that is characterized by a conserved pattern of four cysteine residues.     

A dominant-negative fur mutation in Bradyrhizobium japonicum

In many bacteria, the ferric uptake regulator (Fur) protein plays a central role in the regulation of iron uptake genes. Because iron figures prominently in the agriculturally important symbiosis between soybean and its nitrogen-fixing endosymbiont Bradyrhizobium japonicum, we wanted to assess the role of Fur in the interaction. We identified a fur mutant by selecting for manganese resistance. Manganese interacts with the Fur protein and represses iron uptake genes. In the presence of high levels of manganese, bacteria with a wild-type copy of the fur gene repress iron uptake systems and starve for iron, whereas fur mutants fail to repress iron uptake systems and survive. The B. japonicum fur mutant, as expected, fails to repress iron-regulated outer membrane proteins in the presence of iron. Unexpectedly, a wild-type copy of the fur gene cannot complement the fur mutant. Expression of the fur mutant allele in wild-type cells leads to a fur phenotype. Unlike a B. japonicum fur-null mutant, the strain carrying the dominant-negative fur mutation is unable to form functional, nitrogen-fixing nodules on soybean, mung bean, or cowpea, suggesting a role for a Fur-regulated protein or proteins in the symbiosis.     

Identification of genistein-inducible and type III-secreted proteins of Bradyrhizobium japonicum

Flagellin is the bulk protein secreted by Bradyrhizobium japonicum. For easier identification of minor protein fractions, the flagellin genes bll6865 and bll6866 were deleted. Extracellular proteins of the corresponding mutant were purified and separated by 2D gel electrophoresis. Several of the protein spots were detectable only after addition of genistein to the growth medium-genistein is an isoflavone secreted by soybean that activates the expression of genes encoding a type III secretion system. These secreted proteins were not present in supernatants of mutants in which conserved genes of the type III secretion system or the regulatory gene ttsI, which is essential for activation of the type III secretion system, are deleted. Out of 22 genistein-inducible protein spots 8 different proteins could be identified by mass spectrometry. One of the proteins, Blr1752, has similarity to NopP of Rhizobium sp. strain NGR234 that is known to be secreted. Another protein is Blr1656 (GunA2) that was shown previously to have endoglucanase activity. Three proteins have similarity to subunits of the flagellar apparatus. Some proteins appeared in several separate spots indicating posttranslational modification. A conserved tts box motif was found in the putative promoter region of six genes encoding secreted proteins.     

Identification of glyA as a symbiotically essential gene in Bradyrhizobium japonicum

A Bradyrhizobium japonicum Tn5 mutant (strain 3160) induced numerous, tiny, white nodules which were dispersed over the whole root system of its natural host plant, soybean (Glycine max). These ineffective, nitrogen non-fixing pseudonodules were disturbed at a very early step of bacteroid and nodule development. Subsequent cloning and sequencing of the DNA region mutated in strain 3160 revealed that the Tn5 insertion mapped in a gene that had 60% homology to the Escherichia coli glyA gene coding for serine hydroxymethyltransferase (SHMT; E.C.2.1.2.1.). SHMT catalyses the biosynthesis of glycine from serine and the transfer of a one-carbon unit to tetrahydrofolate. The B. japonicum glyA region was able to fully complement the glycine auxotrophy of an E. coli glyA deletion strain. Although the Tn5 insertion in B. japonicum mutant 3160 disrupted the glyA coding sequence, this strain was only a bradytroph (i.e. a leaky auxotroph). Thus, B. japonicum may have an additional pathway for glycine biosynthesis. Nevertheless, the glyA mutation was responsible for the drastic symbiotic phenotype visible on plants. It may be possible, therefore, that a sufficient supply with glycine and/or a functioning C1-metabolism are indispensable for the establishment of a fully effective, nitrogen-fixing root nodule symbiosis.     

Identification of Bradyrhizobium japonicum Nodule Isolates from Wisconsin Soybean Farms

One-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis was a more discriminating method than serotyping for identifying strains of Bradyrhizobium japonicum. Analysis of 543 nodule isolates from southeastern Wisconsin soybean farms revealed that none of the isolates were formed by any of the inoculant strains supplied by either of two inoculant companies. Twenty-nine indigenous strains and six inoculant strains were identified. Strain 61A76, the most competitive indigenous strain, formed 21% of the nodules. Indigenous strains 3030, 3058, 0336, and 3052 formed 15, 11, 9, and 9% of the nodules, respectively. These predominant strains were not associated with a particular soybean cultivar, soil type, or farm location.     

Identification of active-site residues of the pro-metastatic endoglycosidase heparanase

Heparanase is a beta-D-endoglucuronidase that cleaves heparan sulfate (HS) and has been implicated in many important physiological and pathological processes, including tumor cell metastasis, angiogenesis, and leukocyte migration. We report herein the identification of active-site residues of human heparanase. Using PSI-BLAST and PHI-BLAST searches of sequence databases, similarities were identified between heparanase and members of several of the glycosyl hydrolase families (10, 39, and 51) from glycosyl hydrolase clan A (GH-A), including strong local identities to regions containing the critical active-site catalytic proton donor and nucleophile residues that are conserved in this clan of enzymes. Furthermore, secondary structure predictions suggested that heparanase is likely to contain an (alpha/beta)(8) TIM-barrel fold, which is common to the GH-A families. On the basis of sequence alignments with a number of glycosyl hydrolases from GH-A, Glu(225) and Glu(343) of human heparanase were identified as the likely proton donor and nucleophile residues, respectively. The substitution of these residues with alanine and the subsequent expression of the mutant heparanases in COS-7 cells demonstrated that the HS-degrading capacity of both was abolished. In contrast, the alanine substitution of two other glutamic acid residues (Glu(378) and Glu(396)), both predicted to be outside the active site, did not affect heparanase activity. These data suggest that heparanase is a member of the clan A glycosyl hydrolases and has a common catalytic mechanism that involves two conserved acidic residues, a putative proton donor at Glu(225) and a nucleophile at Glu(343).     

Identification of active-site residues of sheep liver serine hydroxymethyltransferase.

Chemical modification of amino acid residues with phenylglyoxal, N-ethylmaleimide and diethyl pyrocarbonate indicated that at least one residue each of arginine, cysteine and histidine were essential for the activity of sheep liver serine hydroxymethyltransferase. The second-order rate constants for inactivation were calculated to be 0.016 mM-1 X min-1 for phenylglyoxal, 0.52 mM-1 X min-1 for N-ethylmaleimide and 0.06 mM-1 X min-1 for diethyl pyrocarbonate. Different rates of modification of these residues in the presence and in the absence of substrates and the cofactor pyridoxal 5'-phosphate as well as the spectra of the modified protein suggested that these residues might occur at the active site of the enzyme.     

Viability of Bradyrhizobium japonicum bacteriods

Homogenates from soybean nodules, formed by 12 strains of Bradyrhizobium japonicum, were plated into yeast-extract mannitol agar containing 3 or 37 g mannitol 1^-1. Viable counts ranged from 8.298 to 11.265 log₁₀ cells-gram nodule^-1. When monitored over the life cycle of the symbiosis, the viability of strains USDA 110 and USDA 123 increased with days after planting (DAP), and at 70 DAP was 95% and 81%, respectively. By contrast, the viability of USDA 38 bacteroids decreased with time, and at 70 DAP was only 1.9%. At 49 DAP, nodules induced by USDA 38 had significantly fewer bacteroids per peribacteroid membrane than those formed by USDA 110 or USDA 123, and at 70 DAP, 27% of the USDA 38 bacteroids showed some degree of degeneration. Viable counts of USDA 123 and USDA 110 bacteroids, isolated from the nodules of 12 different cultivars, ranged from 10.963 to 11.463 and from 10.683 to 11.117 log₁₀ viable cells-gram nodule^-1, respectively. Varying the osmolarity of the medium had no predictable effect on bacteroid viability. When surface-sterilized nodules of IPAGO 587 (high bacteroid viability) and USDA 38 (low bacteroid viability) were inoculated into a nonsterile silt loam soil, at rates equivalent to 5.0×10⁸ and 5.0×10⁶ viable bacteroids g^-1 soil, respectively, and then incubated at 28° C for 60 days, 4.3×10⁴ and 1.5×10⁴ surviving cells g^-1 soil, respectively, were recovered. Thus, despite differences due to host and strain variation, bacteroid viability appears to be unrelated to persistence of individual strains following an annual legume crop cycle.Journal paper No. 14930, Agricultural Experiment Station University of Minnesota, St. Paul, MN 55108, USA     

Carbon metabolism in Bradyrhizobium japonicum bacteroids

Abstract Carbon metabolism in Bradyrhizobium japonicum bacteroids is reviewed. Additionally, the bacteroid tricarboxylic acid (TCA) cycle and its regulation under oxygen-limited conditions is considered, with emphasis on possible sites of TCA cycle rate-limiting reactions. Furthermore, we consider other adaptive pathways that may be employed by these organisms while in symbiosis. These pathways include: (1) a poly-β-hydroxy-butyrate shunt, (2) a malate-aspartate shuttle, (3) an α-ketoglutarate-glutamate shunt, (4) a modified dicarboxylic acid cycle, and (5) fermentation pathways leading to lactate, acetaldehyde and ethanol. The effects of oxygen limitation on host carbon metabolism are also considered briefly.     

Characterization of the gene encoding glutamine synthetase I (glnA) from Bradyrhizobium japonicum.

We have isolated the Bradyrhizobium japonicum gene encoding glutamine synthetase I (glnA) from a phage lambda library by using a fragment of the Escherichia coli glnA gene as a hybridization probe. The rhizobial glnA gene has homology to the E. coli glnA gene throughout the entire length of the gene and can complement an E. coli glnA mutant when borne on an expression plasmid in the proper orientation to be transcribed from the E. coli lac promoter. High levels of glutamine synthetase activity can be detected in cell-free extracts of the complemented E. coli. The enzyme encoded by the rhizobial gene was identified as glutamine synthetase I on the basis of its sedimentation properties and resistance to heat inactivation. DNA sequence analysis predicts a high level of amino acid sequence homology among the amino termini of B. japonicum, E. coli, and Anabaena sp. strain 7120 glutamine synthetases. S1 nuclease protection mapping indicates that the rhizobial gene is transcribed from a single promoter 131 +/- 2 base pairs upstream from the initiation codon. This glnA promoter is active when B. japonicum is grown both symbiotically and in culture with a variety of nitrogen and carbon sources. There is no detectable sequence homology between the constitutively expressed glnA promoter and the differentially regulated nif promoters of the same B. japonicum strain.     

Nickel uptake in Bradyrhizobium japonicum.

Free-living Bradyrhizobium japonicum grown heterotrophically with 1 microM 63Ni2+ accumulated label. Strain SR470, a Hupc mutant, accumulated almost 10-fold more 63Ni2+ on a per-cell basis than did strain SR, the wild type. Nongrowing cells were also able to accumulate nickel over a 2-h period, with the Hupc mutant strain SR470 again accumulating significantly more 63Ni2+ than strain SR. These results suggest that this mutant is constitutive for nickel uptake as well as for hydrogenase expression. The apparent Kms for nickel uptake in strain SR and strain SR470 were found to be similar, approximately 26 and 50 microM, respectively. The Vmax values, however, were significantly different, 0.29 nmol of Ni/min per 10(8) cells for SR and 1.40 nmol of Ni/min per 10(8) cells for SR470. The uptake process was relatively specific for nickel; only Cu2+ and Zn2+ (10 microM) were found to appreciably inhibit the uptake of 1 microM Ni, while a 10-fold excess of Mg2+, Co2+, Fe3+, or Mn2+ did not affect Ni2+ uptake. The lack of inhibition by Mg2+ indicates that nickel is not transported by a magnesium uptake system. Nickel uptake was also inhibited by cold (53% inhibition at 4 degrees C) and slightly by the ionophores nigericin and carbonyl cyanide m-chlorophenylhydrazone. Other ionophores did not appreciably affect nickel uptake, even though they significantly stimulated O2 uptake. The cytochrome c oxidase inhibitors azide, cyanide, and hydroxylamine did not inhibit Ni2+ uptake, even at concentrations (of cyanide and hydroxylamine) that inhibited O2 uptake. The addition of oxidizable substrates such as succinate or gluconate did not increase nickel uptake, even though they increased respiratory activity. Nickel update showed a pH dependence with an optimum at 6.0. Most (approximately 85%) of the 63Ni2+ taken up in 1 min by strain SR470 was not exchangeable with cold nickel.     

Identification of potential active-site residues in the Escherichia coli leader peptidase.

Leader peptidase of Escherichia coli cleaves the leader sequence from the amino terminus of membrane and secreted proteins after these proteins insert across the membrane. Despite considerable research, the mechanism of catalysis of leader peptidase remains unknown. This peptidase cannot be classified using protease inhibitors to the serine, cysteine, aspartic acid, or metallo- classes of proteases (Zwizinski, C., Date, T., and Wickner, W. (1981) J. Biol. Chem. 256, 3593-3597). Using site-directed mutagenesis, we have attempted to place leader peptidase in one of these groups. We found that leader peptidase, lacking all of the cysteine residues, can cleave the leader peptide from procoat, the precursor to bacteriophage M13 coat protein. Replacement of each histidine residue with an alanyl residue was without effect on catalysis. Among all the serine and aspartic acid residues, serine 90 and serine 185 as well as aspartic acid 99, 153, 273, and 276 are necessary to cleave procoat in a detergent extract. However, only serine 90 and aspartic acid 153 were required for processing using a highly sensitive in vivo assay. In addition to the residues directly affecting catalysis, aspartic acid 99 plays a role in maintaining the structure of leader peptidase. Replacement of this residue with alanine results in a very unstable leader peptidase protein. This study thus defines two critical residues, serine 90 and aspartic acid 153, that may be directly involved in catalysis and provides evidence that leader peptidase belongs to a novel class of serine proteases.     

Autecology of Bradyrhizobium japonicum in soybean-rice rotations

The effect of rice culture on changes in the number of a strain of soybean root-nodule bacteria, (Bradyrhizobium japonicum CB1809), already established in the soil by growing inoculated soybean crops, was investigated in transitional red-brown earth soils at two sites in south-western New South Wales. At the first site, 5.5 years elapsed between the harvest of the last of four successive crops of soybean and the sowing of the next. In this period three crops of rice and one crop of triticale were sown and in the intervals between these crops, and after the crop of triticale, the land was fallowed. Before sowing the first rice crop, the number of Bradyrhizobium japonicum was 1.32×10⁵ g^–1 soil. The respective numbers of bradyrhizobia after the first, second and third rice crops were 4.52 ×10⁴, 1.26×10⁴ and 6.40×10² g^–1 soil. In the following two years the population remained constant. Thus sufficient bradyrhizobia survived in soil to nodulate and allow N₂-fixation by the succeeding soybean crop. At the second site, numbers of bradyrhizobia declined during a rice crop, but the decline was less than when the soil was fallowed (400-fold cf. 2200-fold). Multiplication of bradyrhizobia was rapid in the rhizosphere of soybean seedlings sown without inoculation in the rice bays. At 16 days after sowing, their numbers were not significantly different (p<0.05) from those in plots where rice had not been sown. Nodulation of soybeans was greatest in plots where rice had not been grown, but yield and grain nitrogen were not significantly different (p<0.05). Our results indicate that flooding soil has a deleterious effect on the survival of bradyrhizobia but, under the conditions of the experiments, sufficient B. japonicum strain CB 1809 survived to provide good nodulation after three crops of rice covering a total period of 5.5 years between crops of soybean.