Analysis of DNA sequences transcribed at high levels in Bradyrhizobium japonicum bacteroids but not necessary for symbiotic effectiveness.

Five regions of the Bradyrhizobium japonicum genome, which are transcribed at high levels in nitrogen-fixing soybean (Glycine max) nodules, were identified. None of these regions contained previously identified genes (e.g., nif, nod, and fix genes) that are known to be essential for development of functional nitrogen-fixing nodules. To assess the role of these regions in the development of the B. japonicum-soybean symbiosis, we cloned and used them to construct B. japonicum strains, in which large DNA segments (2.0-6.8 kilobases) containing the highly transcribed regions were deleted. The deletion strains were examined for symbiotic effectiveness and were found to be indistinguishable from the wild-type strain. Transcription of the cloned regions under a variety of physiological conditions and in several defined mutant B. japonicum strains was also examined. The transcriptional start sites for one pair of divergent transcripts were determined; the promoters do not contain any of the conserved sequences found in B. japonicum genes involved in symbiosis or nitrogen metabolism.

Isolation of Bradyrhizobium japonicum DNA sequences that are transcribed at high levels in bacteroids.

DNA sequences have been isolated that are expressed at high levels in bacteroids, the differentiated form of the soybean microsymbiont, Bradyrhizobium japonicum. Random-primed cDNA was synthesized using total RNA isolated from purified B. japonicum bacteroids or from cells grown in culture. When used directly to screen bacteriophage lambda libraries, these cDNA probes produced a high background hybridization signal due to sequence similarity between B. japonicum and E. coli ribosomal DNA (rDNA) operons. To reduce this background signal, the rDNA operon of B. japonicum was cloned and the rDNA plasmid DNA used in subtractive hybridization with the cDNA probes and as a competitor in hybridization solutions. This method greatly reduced the background signal in screening of genomic libraries and thus permitted the identification of twelve unique recombinant phage which contained sequences that are expressed at higher levels in B. japonicum bacteroids than in cells grown in culture.

Bradyrhizobium japonicum glnB, a putative nitrogen-regulatory gene, is regulated by NtrC at tandem promoters.

The glnB gene from Bradyrhizobium japonicum, the endosymbiont of soybeans (Glycine max), was isolated and sequenced, and its expression was examined under various culture conditions and in soybean nodules. The B. japonicum glnB gene encodes a 12,237-dalton polypeptide that is highly homologous to the glnB gene products from Klebsiella pneumoniae and Escherichia coli. The gene is located directly upstream from glnA (encoding glutamine synthetase), a linkage not observed in enteric bacteria. The glnB gene from B. japonicum is expressed from tandem promoters, which are differentially regulated in response to the nitrogen status of the medium. Expression from the downstream promoter involves the B. japonicum ntrC gene product (NtrC) in both free-living and symbiotic cells. Thus, glnB, a putative nitrogen-regulatory gene in B. japonicum, is itself Ntr regulated, and NtrC is active in B. japonicum cells in their symbiotic state.

Isolation, nucleotide sequence, and preliminary characterization of the Escherichia coli K-12 hemA gene.

The Escherichia coli hemA gene, essential for the synthesis of 5-aminolevulinic acid (ALA), was isolated and sequenced. The following criteria identified the cloned gene as hemA. (i) The gene complemented a hemA mutation of E. coli. (ii) The gene was localized to approximately 26.7 min on the E. coli chromosomal linkage map, consistent with the location of the mapped hemA locus. Furthermore, DNA sequence analysis established that the cloned gene lay directly upstream of prfA, which encodes polypeptide chain release factor 1. (iii) Deletion of the gene resulted in a concomitant requirement for ALA. The hemA gene directed the synthesis of a 46-kilodalton polypeptide in maxicell experiments, as predicted by the coding sequence. The DNA and deduced amino acid sequences of the E. coli hemA gene displayed no detectable similarity to the ALA synthase sequences which have been characterized from a variety of organisms, but are very similar to the cloned Salmonella typhimurium hemA sequences (T. Elliott, personal communication). Results of S1 nuclease protection experiments showed that the hemA mRNA appeared to have two different 5' ends and that a nonoverlapping divergent transcript was present upstream of the putative distal hemA transcriptional start site.

Role of the Bradyrhizobium japonicum ntrC gene product in differential regulation of the glutamine synthetase II gene (glnII).

We isolated the ntrC gene from Bradyrhizobium japonicum, the endosymbiont of soybean (Glycine max), and examined its role in regulating nitrogen assimilation. Two independent ntrC mutants were constructed by gene replacement techniques. One mutant was unable to produce NtrC protein, while the other constitutively produced a stable, truncated NtrC protein. Both ntrC mutants were unable to utilize potassium nitrate as a sole nitrogen source. In contrast to wild-type B. japonicum, the NtrC null mutant lacked glnII transcripts in aerobic, nitrogen-starved cultures. However, the truncated-NtrC mutant expressed glnII in both nitrogen-starved and nitrogen-excess cultures. Both mutants expressed glnII under oxygen-limited culture conditions and in symbiotic cells. These results suggest that nitrogen assimilation in B. japonicum is regulated in response to both nitrogen limitation and oxygen limitation and that separate regulatory networks exist in free-living and symbiotic cells.

Differential transcription of the two glutamine synthetase genes of Bradyrhizobium japonicum.

Bradyrhizobium japonicum induces the formation of nitrogen-fixing symbiotic root nodules on soybean plants. The B. japonicum genome encodes two isoforms of glutamine synthetase (GS). One form, GSI, encoded by the gene glnA, is similar in structure and activity to the enzyme found in all other bacteria. The second form, GSII, encoded by glnII, is structurally related to the eucaryotic enzyme. Genetic analyses indicate that glnA or glnII alone is sufficient to provide glutamine prototrophy, whereas the double mutation glnA glnII produces glutamine auxotrophy. The glnA gene is transcribed from a single promoter that has a structure most similar to that of the bacterial consensus promoter. The level of transcription of glnA is not specifically affected by nitrogen limitation of growth. The glnII gene is also transcribed from a single promoter; however, this promoter has structural features characteristic of promoters controlled by the nitrogen regulation system. In contrast to glnA, physiological studies indicate that glnII transcription is regulated in response to nitrogen source availability. Under aerobic growth conditions, expression of glnII is induced when growth is limited by nitrogen source depletion as expected for regulation by the nitrogen regulation system.

A genetic locus essential for formate-dependent growth of Bradyrhizobium japonicum.

A genetic locus essential for the formate-dependent growth of Bradyrhizobium japonicum was isolated by complementation of ethyl methanesulfonate-induced mutants with a cosmid gene library of B. japonicum DNA. Three related cosmids containing 18.7 kilobase pairs of B. japonicum DNA in common were identified as being able to restore formate-dependent growth capability to mutants lacking either ribulosebisphosphate carboxylase or both ribulosebisphosphate carboxylase and phosphoribulokinase activities. To further localize the complementing gene(s), a series of four deletions spanning a total of 16.1 kilobase pairs were introduced into the B. japonicum chromosome. Each resulting deletion mutant lacked formate dehydrogenase activity and lacked ribulosebisphosphate carboxylase activity and immunologically detectable protein. Three of the four also lacked phosphoribulokinase activity. Two other mutants in which the deletion-bearing recombinant plasmid had integrated into the chromosome also lacked ribulosebisphosphate carboxylase activity and protein and phosphoribulokinase activities. The genetic locus defined by these mutants could contain the structural genes for these enzymes or a regulatory gene(s) controlling their expression or both.

Structure of the Bradyrhizobium japonicum gene hemA encoding 5-aminolevulinic acid synthase.

The nucleotide (nt) sequence of the hemA gene, which encodes 5-aminolevulinic acid synthase (ALAS) from the bacterium Bradyrhizobium japonicum, is presented. This sequence predicts a protein of 408 amino acids (aa) with an Mr of 44,599. This predicted amino acid sequence is highly homologous to that of the chicken embryonic liver ALAS, exhibiting a 48.8% identical amino acid sequence over the entire length of the bacterial protein. A single mRNA start point was demonstrated by S1 protection analysis for the B. japonicum hemA. The 5' end of the transcript is 100 nt upstream from the start codon. The sequence of the promoter region has some sequence homology to the -35 nt region of the Escherichia coli consensus promoter sequence but not to the -10 nt region. There is also one block of 9 nt found in both the B. japonicum glnA and hemA promoter regions.

Bacterial delta-aminolevulinic acid synthase activity is not essential for leghemoglobin formation in the soybean/Bradyrhizobium japonicum symbiosis.

Previous studies of legume nodules have indicated that formation of the heme moiety of leghemoglobin is a function of the bacterial symbiont. We now show that a hemA mutant of Bradyrhizobium japonicum that cannot carry out the first step in heme biosynthesis forms fully effective nodules on soybeans. The bacterial mutant strain was constructed by first isolating the wild-type hemA gene encoding delta-aminolevulinic acid synthase (EC 2.3.1.37) from a cosmid library, using a fragment of the Rhizobium meliloti hemA gene as a hybridization probe. A deletion of the hemA gene region, generated in vitro, then was used to construct the analogous chromosomal mutation by gene-directed mutagenesis. The mutant strain had no delta-aminolevulinic acid synthase activity and was unable to grow in minimal medium unless delta-aminolevulinic acid was added. Despite its auxotrophy, the mutant strain incited nodules that appeared normal, contained heme, and were capable of high levels of acetylene reduction. These results rule out bacterial delta-aminolevulinic acid synthase activity as the exclusive source of delta-aminolevulinic acid for heme formation in soybean nodules.

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.

Isolation and expression of the Bradyrhizobium japonicum adenylate cyclase gene (cya) in Escherichia coli.

A 5.0-kilobase-pair HindIII fragment of Bradyrhizobium japonicum DNA containing the cya gene which encodes adenylate cyclase was isolated as an insert in pBR322, using marker rescue of the maltose-negative phenotype of an Escherichia coli cya mutant for identification. The isolated B. japonicum DNA fragment was capable of reversing the pleiotropic phenotype of cya mutations when inserted in either orientation in the HindIII site of pBR322. The complemented E. coli strains produced high levels of cyclic AMP. No sequence homology between the B. japonicum cya gene and that of E. coli was detected by hybridization analysis.

Physical organization of the Bradyrhizobium japonicum nitrogenase gene region.

In Bradyrhizobium japonicum USDA 110 the three genes that encode the nitrogenase enzyme complex are separated into two transcription units, nifH and nifDK. We have physically mapped a 33-kilobase-pair region of the B. japonicum genome that contains both nifH and nifDK. The nifDK operon is located transcriptionally upstream from nifH, and all three genes are transcribed in the same direction. Within the 20-kilobase-pair region that separates the promoters for these two transcription units, we have identified a region homologous to the Klebsiella pneumoniae nifA gene. This nifA homology is situated about 6 kilobase pairs upstream from the nifH transcriptional initiation signal, in an analogous position to the nifA-like locus previously described for Rhizobium meliloti. In addition, we have characterized a second distinct nifA homology in B. japonicum that is not directly linked to the nitrogenase structural gene region.

The nifH and nifDK promoter regions from Rhizobium japonicum share structural homologies with each other and with nitrogen-regulated promoters from other organisms.

In several species of Rhizobium the three genes which encode the nitrogenase complex are separated into two operons, nifH and nifDK. We have mapped the transcriptional promoter sites for these two operons from R. japonicum USDA strain 110 by S1 protection analyses using bacterial RNA isolated from soybean nodules. Transcription of the nifDK operon is initiated at a site located 46 nucleotides upstream of the proposed translation initiation codon. nifH transcription initiates predominantly at a site 152 nucleotides upstream of the proposed translation initiation codon. An additional minor start for nifH is found 35 nucleotides downstream of the major initiation site. The nucleotide sequences of these promoter sites are presented. Comparison of the major R. japonicum nif promoter sequences reveals a high degree of sequence homology with the conserved sequences clustered in the -11 to -15, -21 to -25, -27 to -31, and -38 to -41 regions. Conservation is also observed for the -11 to -15 and -21 to -25 regions with the R. meliloti nifHDK, a cowpea Rhizobium nifH, and even Klebsiella pneumoniae nif promoters. The -27 to -31 and -38 to -42 region homologies are not conserved with the other known nif promoters. These results are discussed in light of models for nif promoter activation.

Interaction of Bacillus subtilis RNA polymerase core with two specificity-determining subunits. Competition between sigma and the SPO1 gene 28 protein.

Gene activity in the development of phage SPO1 is transcriptionally regulated. Early viral genes are transcribed by the major vegetative cell RNA polymerase (E. sigma). Middle viral genes are transcribed by RNA polymerase core (E) bearing the SPO1 gene 28 protein (gp28) instead of sigma. This paper deals with the competitive interactions of sigma and gp28 with E which must, at least in part, be involved in the ability of viral middle gene expression to succeed early gene expression. An in vitro assay has been developed for determining the proportions of early (E. sigma) and SPO1 middle (E.gp28) Bacillus subtilis RNA polymerase in mixtures. The assay, which is based on the transcription of two well studied SPO1 restriction fragments followed by gel electrophoresis and quantitation of the E. sigma and E.gp28 transcription products, has been used to study the interactions of sigma and gp28 with RNA polymerase core. These subunits are found to be capable of competitively excluding each other. The binding competition is sensitive to ionic strength, with sigma being more effective below 0.2 total ionic strength and gp28 being more effective at higher values. The outcome of competition is also dependent on the order of addition of subunits and the reconstitution kinetics have been studied. The subunit competition is rather insensitive, particularly at higher ionic strength, to temperature. Gp28 can convert E. sigma to E.gp28 but the reverse reaction occurs much less efficiently. The B. subtilis delta protein biases the sigma-gp28 competition in favor of the sigma subunit. the implications of these results for the physiological transition of transcriptional specificity during SPO1 development are discussed.

SP01 gene 27 is required for viral late transcription.

The SP01 mutant sus HA20 (gene 27) was found to be defective for synthesis of viral late RNA. It is known that gene 27 is also required for viral DNA replication. The SP01 gene 27 product resembles the T4 gene 45 product, which also has a dual role in viral DNA replication and late transcription.

Changes in the association between Bacillus subtilis RNA polymerase core and two specificity-determining subunits during transcription.

The Bacillus subtilis RNA polymerase sigma subunit and the phage SPO1-coded gene 28 protein are responsible for selective binding of RNA polymerase to early and middle SPO1 promoters, respectively. The association of the RNA polymerase core with each of these subunits weakens during the elongation of RNA chains. Similar changes are known to be an essential part of the Escherichia coli RNA polymerase sigma cycle.

A transcriptional map of the bacteriophage SP01 genome. I. The major early promoters.

Bacteriophage SP01 early transcripts come predominantly from the terminally repeated 12-kilobase-pair segments of its genome. Much less early RNA comes from a relatively large region of the genome which surrounds the early regulatory gene 28. (The gene 28 protein positively regulates viral middle gene expression.) The major sites at which Bacillus subtilis RNA polymerase holoenzyme binds to SP01 DNA and initiates RNA synthesis have been mapped and the direction of transcription from each of these promoter sites has been determined. There are 12 such sites spanning 11.1 kilobase pairs at each end of the SP01 genome and each set of 12 sites defines a complex set of convergent transcription units.

A transcriptional map of the bacteriophage SP01 genome: II. The major early transcription units.

The genome of the Bacillus subtilis phage SP01 contains a 12.6-kilobase-pair terminal redundancy. The transcription units in this region, which are utilized in vitro by B. subtilis RNA polymerase holoenzme, have been mapped. In vitro transcripts were separated and characterized by gel electrophoresis. Since there is such a large number of promoters in this region, it was necessary to employ conditions of transcription under which only subsets of all the transcripts would be made. Selective synthesis was achieved by transcription of restriction fragments containing a small number of promoters and by dinucleotide priming. Transcription units were mapped by observing the shortening of transcripts that resulted from restriction enzyme cleavage of the template. The mapping indicates the presence of two blocks of overlapping transcription units, oriented so as to yield convergent transcription toward a bidirectional termination region. The frequency of readthrough at the bidirectional terminator is low. Several partial termination sites of differing efficiencies have also been identified, including two that are active only at low ribonucleoside triphosphate concentrations. We also describe an effect of NaCl concentration on the migration of RNA in gels, which is specific for only two of the SP01 early transcripts. This effect may indicate unusual secondary structures for these two RNA molecules.

Gel electrophoretic separation of transcription complexes: an assay for RNA polymerase selectivity and a method for promoter mapping.

We describe a method for analyzing ternary transcription complexes, of RNA polymerase, DNA and nascent RNA32 chains, by agarose gel electrophoresis. When the RNA of such complexes is 32P-labelled, a simple comparison of the DNA fluorogram with an autoradiogram identifies transcriptionally active DNA molecules and restriction fragments in any mixture. Two limitations on the method are described: 1) retardation during electrophoresis of polymerase-DNA complexes relative to their conjugate bare NA fragments; 2) failure of very large ternary complexes to enter gels. The following potential applications of the method are surveyed: transcription unit (elongation) mapping, separation of RNA molecules in a mixture of transcripts, dinucleotide primer mapping and identification of preferred template conformations.