Kieran F. Scott

Sequence and organization of pobA, the gene coding for p-hydroxybenzoate hydroxylase, an inducible enzyme from Pseudomonas aeruginosa

The only recognized gene for the metabolism of p-hydroxybenzoate in Pseudomonads (pobA) has been isolated from Pseudomonas aeruginosa to provide the DNA for mutagenesis studies of the protein product, p-hydroxybenzoate hydroxylase. Since pobA is induced by p-hydroxybenzoate to produce large amounts of enzyme, its regulation in P. aeruginosa is significant. The nucleotide sequence of pobA is presented with the derived amino acid (aa) sequence, which has only two substitutions compared to the amino acid sequence obtained from the enzyme from P. fluorescens. The derived amino acid sequence predicts that the enzyme is a single polypeptide of 394 aa residues and contains one molecule of FAD. The complete structure of the protein from P. aeruginosa can be derived by analogy from the published structure of the protein from P. fluorescens. Transcription mapping was used to determine that there is one site for the initiation of mRNA synthesis in P. aeruginosa. The presence of a putative operator in the sequence suggests primary regulation by a repressor protein which binds p-hydroxybenzoate. The ribosome-binding site permits translation of the gene in Escherichia coli at levels comparable to its production in P. aeruginosa, but it produces no detectable product in E. coli under the influence of its own promoter sequence. The promoter does not conform to the common consensus sequence of E. coli promoters. The results have identified an apparent novel promoter for P. aeruginosa, which may reflect the presence of a sigma factor required for pobA induction. Repression of expression by glucose suggests a binding site in the sequence for catabolite repression.

Structural and functional analysis of nitrogenase genes from the broad-host-range Rhizobium strain ANU240

The genes encoding the structural components of nitrogenase, nifH, nifD and nifK, from the fast-growing, broad-host-range Rhizobium strain ANU240 have been identified and characterized. They are duplicated and linked in an operon nifHDK in both copies. Sequence analysis of the nifH gene from each copy, together with partial sequence analysis of the nifD and nifK genes, and restriction endonuclease analysis suggested that the duplication is precise. Comparison of the Fe-protein sequence from strain ANU240 with that from other nitrogen-fixing organisms revealed that, despite its broad host range and certain physiological properties characteristic of Bradyrhizobium strains, ANU240 is more closely related to the narrow-host-range Rhizobium strains than to the broad-host-range Bradyrhizobium strains. The promoter regions of both copies of the nif genes contain the consensus sequence characteristic of nif promoters, and functional analysis of the two promoters suggested that both nif operons are transcribed in nodules.

Organisation of nodulation and nitrogen fixation genes on a Rhizobium trifolii symbiotic plasmid

A Rhizobium trifolii symbiotic plasmid specific gene library was constructed and the physical organisation of regions homologous to nifHDK, nifA and nod genes was determined. These symbiotic gene regions were localised to u 25 kb region on the sym-plasmid, pPN1. In addition four copies of a reiterated sequence were identified on this plasmid, with one copy adjacent to nifH. No rearrangement of these reiterated sequences was observed between R. trifolii bacterial and bacteroid DNA. Analysis of a deletion derivative of pPN1 showed that these sequences were spread over a 110 kb region to the left of nifA.

Symbiotic Nitrogen Fixation Involving Rhizobium and the Non-Legume Parasponia

In classical terms Rhizobium is defined as that bacterial soil organism which can elicit nodule formation and if optimal, symbiotic nitrogen fixation on the roots (or at stems as in the case of Sesbania) of legumes. This definition may require alteration in view of the discovery 10 years ago that the tropical trees (or shrubs) belonging to the Parasponia genus are capable of nodulation and efficient nitrogen fixation in symbiosis with Rhizobium strains.

Conserved Nodulation Genes are Obligatory for Nonlegume Nodulation

The nodulation of the nonlegume plant Parasponia by bacterial strains occurs through a mechanism quite distinct from that used by these same strains to nodulate legume species (1,2,3). In contrast to the invasion via root hair curling, infection thread formation and bacterial release observed during legume nodulation (4), bacteria invade Parasponia roots through points of bacterium-induced meristematic activity which break the epidermal surface of the root allowing bacteria direct access to cortical cells. These cells become invaded via infection thread formation and nitrogen fixation occurs within these threads (5). the fully developed Parasponia nodule resembles a modified lateral root structure with a central vascular system (5). As with legume nodulation, there is also a host-specific component to nonlegume nodulation. Only certain strains of Rhizobium and Bradyrhizobium are capable of nodulating Parasponia and these strains do so with varying efficiency (6). One such organism, Bradyrhizobium (sp. Parasponia) strain ANU289 is not only capable of biological nitrogen fixation on Parasponia but also can effectively nodulate a broad range of tropical legumes. to determine the genetic basis of nonlegume nodulation, we have identified and characterised conserved nodulation genes from ANU289 and demonstrated that the expression of the genes nodKABC are obligatory for both legume and nonlegume nodulation. In addition we have identified cosmid clones carrying ANU289 DNA which can confer Parasponia nodulation on the narrow host-range strain Rhizobium trifolii ANU843.

Molecular Cloning and Organisation of Genes Involved in Symbiotic Nitrogen Fixation in Different Rhizobium Species

The symbiotic association between plants and bacteria of the genus Rhizobium is the result of a complex interaction between the bacterium and its host, requiring the expression of both bacterial and plant genes in a tightly coordinated manner. Bacteria bind to the emerging plant root hairs and invade the root tissue through the formation of an infection thread. The plant responds to this infection by the development of a highly differentiated root nodule. These nodules are the site of synthesis of the bacterial enzyme complex nitrogenase, which reduces atmospheric nitrogen to ammonia. The fixed nitrogen is then exported into the plant tissue and assimilated by plant-derived enzymes.

Mutants of Bradyrhizobium (Parasponia) sp. ANU 289 Affected in Assimilatory Nitrate Reduction also Show Lowered Symbiotic Effectiveness

Summary Bradyrhizobium ( Parasponia ) sp. ANU 289 possessed an assimilatory nitrate reductase which was induced by nitrate and repressed by ammonia. Nitrate reductase was rapidly removed or inactivated as nitrate was depleted from the growth medium. Strain ANU 289 was able to take up nitrate only after growth on nitrate-containing medium and nitrate uptake was dependent on assimilation of nitrate by nitrate reductase. Two mutants affected in nitrate assimilation were isolated after transposon mutagenesis. One of these retained the uninduced level of nitrate reductase under all growth conditions, while the other showed a delay in nitrate reductase induction. Both mutants had a reduced ability to fix nitrogen symbiotically and in vitro. It is suggested that factors involved in molybdenum metabolism or synthesis of electron transport components, which are required for activity of both nitrogenase and nitrate reductase, were affected in the mutants.

Nitrogenase Genes in the Fast-Growing Broad-Host Range Rhizobium Strain ANU240

The Rhizobium strain ANU240 (a derivative of strain NGR234 (Trinick, 1980) is a fast-growing strain capable of nitrogen fixation on a wide range of tropical legumes normally nodulated by slow-growing strains. It also nodulates the non-legume, Parasponia, but the symbiosis is ineffective (Trinick and Galbraith, 1980).

Organization and Primary Structure of Nitrogenase Genes in the Parasponia Rhizobium Strain ANU289

The Parasponia Rhizobium strain ANU289 effectively nodulates a wide range of tropical legumes and also the non-legume Parasponia (Trinick and Galbraith, 1980). In addition, nitrogenase activity in this strain can be induced in vitro (Mohapatra et al., 1982).