Molecular cloning of a gene region from Bradyrhizobium japonicum essential for lipopolysaccharide synthesis

The gene region cloned from a lipopolysaccharide (LPS) mutant carrying the Tn5 and flanking DNA sequences was used as a probe to screen a gene bank prepared from wild-type Bradyrhizobium japonicum strain 61A101C and to isolate the corresponding wild-type LPS-gene region. By cross-hybridization experiments the LPS-gene region did not appear to be closely linked to previously cloned nodulation genes. A detailed restriction map of the LPS-gene region (5.5-kb EcoRI genomic fragment) was established and the mutation site was localized to be in a 300-bp PvuI/PstI restriction fragment. In genomic Southern-blot analysis of various rhizobia, the LPS-gene region was found to be conserved among all the slow-growing bradyrhizobia, but not the fast-growing rhizobia. The different groups of slow-growing bradyrhizobia are polymorphic for restriction-fragment length at the LPS-gene region.     

Conservation of symbiotic nitrogen fixation gene sequences in Rhizobium japonicum and Bradyrhizobium japonicum.

Southern hybridization with nif (nitrogen fixation) and nod (nodulation) DNA probes from Rhizobium meliloti against intact plasmid DNA of Rhizobium japonicum and Bradyrhizobium japonicum strains indicated that both nif and nod sequences are on plasmid DNA in most R. japonicum strains. An exception is found with R. japonicum strain USDA194 and all B. japonicum strains where nif and nod sequences are on the chromosome. In R. japonicum strains, with the exception of strain USDA205, both nif and nod sequences are on the same plasmid. In strain USDA205, the nif genes are on a 112-megadalton plasmid, and nod genes are on a 195-megadalton plasmid. Hybridization to EcoRI digests of total DNA to nif and nod probes from R. meliloti show that the nif and nod sequences are conserved in both R. japonicum and B. japonicum strains regardless of the plasmid or chromosomal location of these genes. In addition, nif DNA hybridization patterns were identical among all R. japonicum strains and with most of the B. japonicum strains examined. Similarly, many of the bands that hybridize to the nodulation probe isolated from R. meliloti were found to be common among R. japonicum strains. Under reduced hybridization stringency conditions, strong conservation of nodulation sequences was observed in strains of B. japonicum. We have also found that the plasmid pRjaUSDA193, which possess nif and nod sequences, does not possess sequence homology with any plasmid of USDA194, but is homologous to parts of the chromosome of USDA194. Strain USDA194 is unique, since nif and nod sequences are present on the chromosome instead of on a plasmid as observed with all other strains examined.     

Effect of iron availability on expression of the Bradyrhizobium japonicum hemA gene.

Bradyrhizobium japonicum produces delta-aminolevulinic acid, the universal precursor of tetrapyrroles, in a reaction catalyzed by the product of the hemA gene. Expression of the B. japonicum hemA gene is affected by iron availability. Activity of a hemA-lacZ fusion is increased approximately threefold by iron, and RNA analysis indicates that iron regulation is at the level of mRNA accumulation. To our knowledge, this is the first example of an iron-regulated heme biosynthetic gene in prokaryotes.     

Isolation and characterization of the DNA region encoding nodulation functions in Bradyrhizobium japonicum.

The DNA region encoding early nodulation functions of Bradyrhizobium japonicum 3I1b110 (I110) was isolated by its homology to the functionally similar region from Rhizobium meliloti. Isolation of a number of overlapping recombinant clones from this region allowed the construction of a restriction map of the region. The identified nodulation region of B. japonicum shows homology exclusively to those regions of R. meliloti and Rhizobium leguminosarum DNA known to encode early nodulation functions. The region of homology with these two fast-growing Rhizobium species was narrowed to an 11.7-kilobase segment. A nodulation-defective mutant of Rhizobium fredii USDA 201, strain A05B-2, was isolated and found to be defective in the ability to curl soybean root hairs. Some of the isolated recombinant DNA clones of B. japonicum were found to restore wild-type nodulation function to this mutant. Analysis of the complementation results allows the identification of a 1.8-kilobase region as essential for restoration of Hac function.     

Characterization of the Bradyrhizobium japonicum ftsH gene and its product

The Bradyrhizobium japonicum ftsH gene was cloned by using a set of widely applicable degenerated oligonucleotides. Western blot experiments indicated that the FtsH protein was produced under standard growth conditions and that it was not heat inducible. Attempts to delete the ftsH gene in B. japonicum failed, suggesting a pivotal cellular function of this gene. The expression of B. japonicum ftsH in an ftsH-negative Escherichia coli strain significantly enhanced the fitness of this mutant and reduced the steady-state level of sigma(32).     

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.     

Characterization of a Bradyrhizobium japonicum ferrochelatase mutant and isolation of the hemH gene.

A Tn5-induced mutant of Bradyrhizobium japonicum, strain LORBF1, was isolated on the basis of the formation of fluorescent colonies, and stable derivatives were constructed in backgrounds of strains LO and I110. The stable mutant strains LOek4 and I110ek4 were strictly dependent upon the addition of exogenous hemin for growth in liquid culture and formed fluorescent colonies. The fluorescent compound was identified as protoporphyrin IX, the immediate precursor of protoheme. Cell extracts of strains LOek4 and I110ek4 were deficient in ferrochelatase activity, the enzyme which catalyzes the incorporation of ferrous iron into protoporphyrin IX to produce protoheme. Mutant strain I110ek4 could take up 55Fe from the growth medium, but, unlike the parent strain, no significant incorporation of radiolabel into heme was found. This observation shows that heme was not synthesized in mutant strain I110ek4 and that the heme found in those cells was derived from exogenous hemin in the growth medium. The putative protein encoded by the gene disrupted in strain LORBF1 and its derivatives was homologous to ferrochelatases from eukaryotic organisms. This homology, along with the described mutant phenotype, provides strong evidence that the disrupted gene is hemH, that which encodes ferrochelatase. Mutant strain I110ek4 incited nodules on soybean that did not fix nitrogen, contained few viable bacteria, and did not express leghemoglobin heme or apoprotein. The data show that B. japonicum ferrochelatase is essential for normal nodule development.     

Analysis of the Bradyrhizobium japonicum hemH gene and its expression in Escherichia coli.

Complementation analysis showed that the Bradyrhizobium japonicum hemH gene was both necessary and sufficient to rescue mutant strains I110ek4 and I110bk2 in trans with respect to hemin auxotrophy, protoporphyrin accumulation, and the deficiency in ferrochelatase activity. The B. japonicum hemH gene was expressed in an Escherichia coli T7 expression system and yielded a 39-kDa protein, which was consistent with the predicted size of the deduced product. The overexpressed protein was purified and shown to contain ferrochelatase activity, thereby demonstrating that the hemH gene encodes ferrochelatase. When expressed from the lac promoter, the B. japonicum hemH gene was able to complement the enzyme activity of a ferrochelatase-defective E. coli mutant, and it also conferred hemin prototrophy on those cells. These latter findings confirm the identity of the hemH gene product and demonstrate that B. japonicum ferrochelatase can interact with the E. coli heme synthesis enzymes for heme formation in complemented cells.     

Peribacteroid membrane nodulin gene induction by Bradyrhizobium japonicum mutants

Seventeen translation products from Glycine max root mRNA precipitated with antiserum prepared against a peribacteroid membrane preparation from effective root nodules. Messenger RNA from fix ⁺ nodules coded for these 17 products plus 7 other nodule-specific polypeptides which bound to the antiserum. Of these 7 nodulins only 4 were present when nodules were infected with Bradyrhizobium japonicum 110 rif 15 2960, which induces the plant to produce empty peribacteroid membranes. In nodules infected with B. japonicum strains inducing either very short-lived or defective peribacteroid membrane, only 5 or 6, respectively, of these nodulins could be detected.From these results we hypothesize that the microsymbiont is responsible for the production of at least 4 different signals leading to peribacteriod membrane formation by the plant.