Evidence that the Rhizobium regulatory protein RirA binds to cis-acting iron-responsive operators (IROs) at promoters of some Fe-regulated genes.

Mutations in rirA of Rhizobium have been shown to deregulate expression of several genes that are normally repressed by iron. A conserved sequence, the iron-responsive operator (IRO), was identified near promoters of vbsC (involved in the synthesis of the siderophore vicibactin), rpoI (specifies an ECF sigma factor needed for vicibactin synthesis) and the two fhuA genes (encoding vicibactin receptor). Removal of these IRO sequences abolished Fe-responsive repression. Most of these genes were constitutively expressed in the heterologous host, Paracoccus denitrificans, but introduction of the cloned rirA gene repressed expression of these Rhizobium genes in this heterologous host if the corresponding IRO sequences were also intact. These observations are the first to examine the mechanisms of RirA, which has no sequence similarity to well-known iron-responsive regulators such as Fur or DtxR. They provide strong circumstantial evidence that RirA is a transcriptional regulator that binds to cis-acting regulatory sequences near the promoters of at least some of the genes whose expression it controls in response to Fe availability.

The vbs genes that direct synthesis of the siderophore vicibactin in Rhizobium leguminosarum: their expression in other genera requires ECF sigma factor RpoI.

A cluster of eight genes, vbsGSO, vbsADL, vbsC and vbsP, are involved in the synthesis of vicibactin, a cyclic, trihydroxamate siderophore made by the symbiotic bacterium Rhizobium leguminosarum. None of these vbs genes was required for symbiotic N2 fixation on peas or Vicia. Transcription of vbsC, vbsGSO and vbsADL (but not vbsP) was enhanced by growth in low levels of Fe. Transcription of vbsGSO and vbsADL, but not vbsP or vbsC, required the closely linked gene rpoI, which encodes an ECF sigma factor of RNA polymerase. Transfer of the cloned vbs genes, plus rpoI, to Rhodobacter, Paracoccus and Sinorhizobium conferred the ability to make vicibactin on these other genera. We present a biochemical genetic model of vicibactin synthesis, which accommodates the phenotypes of different vbs mutants and the homologies of the vbs gene products. In this model, VbsS, which is similar to many non-ribosomal peptide synthetase multienzymes, has a central role. It is proposed that VbsS activates L-N5-hydroxyornithine via covalent attachment as an acyl thioester to a peptidyl carrier protein domain. Subsequent VbsA-catalysed acylation of the hydroxyornithine, followed by VbsL-mediated epimerization and acetylation catalysed by VbsC, yields the vicibactin subunit, which is then trimerized and cyclized by the thioesterase domain of VbsS to give the completed siderophore.

dpp genes of Rhizobium leguminosarum specify uptake of delta-aminolevulinic acid.

An operon with homology to the dppABCDF genes required to transport dipeptides in bacteria was identified in the N2-fixing symbiont, Rhizobium leguminosarum. As in other bacteria, dpp mutants were severely affected in the import of delta-aminolevulinic acid (ALA), a heme precursor. ALA uptake was antagonized by adding dipeptides, indicating that these two classes of molecule share the same transporter. Mutations in dppABCDF did not affect symbiotic N2 fixation on peas, suggesting that the ALA needed for heme synthesis is not supplied by the plant or that another uptake system functions in the bacteroids. The dppABCDF operon of R. leguminosarum resembles that in other bacteria, with a gap between dppA and dppB containing inverted repeats that may stabilize mRNA and may explain why transcription of dppA alone was higher than that of dppBCDF. The dppABCDF promoter was mapped and is most likely recognized by sigma70.

The Rhizobium leguminosarum tonB gene is required for the uptake of siderophore and haem as sources of iron.

In the N2-fixing bacterium Rhizobium leguminosarum, mutations in a homologue of tonB (tonB(Rl)) block the import of vicibactin and haem as iron sources in free-living bacteria. TonB(Rl) mutants were normal for growth with ferric dicitrate and slightly reduced for growth with haemoglobin as sole iron sources. The deduced TonB(Rl) product is larger than that of (for example) Escherichia coli, on account of an extended N-terminal domain. Transcription of tonB(Rl) was enhanced in low-Fe growth conditions; this was not controlled by Fur, nor RpoI, an Fe-regulated extracytoplasmic sigma factor. Upstream of tonB(Rl) and transcribed divergently is an operon, hmuPSTUV, whose products are homologous to ABC transporters involved in haem uptake in pathogenic bacteria. Expression of hmuPSTUV was enhanced in low-Fe conditions, and hmu mutants show slightly diminished growth on haem as sole Fe source, suggesting that there is more than one system for the uptake of this molecule. hmuPSTUV expression appears to be from three closely linked promoters. Downstream of hmuPSTUV, a gene that may encode an extracytoplasmic sigma factor was identified, but this gene, rpoZ, did not affect the transcription of tonB(Rl) or hmuPSTUV. Mutations in tonB(Rl), hmu genes and rpoZ did not affect symbiotic N(2) fixation in peas.

Metals and the rhizobial-legume symbiosis--uptake, utilization and signalling.

In this review, we consider how the nitrogen-fixing root nodule bacteria, the 'rhizobia', acquire various metals, paying particular attention to the uptake of iron. We also review the literature pertaining to the roles of molybdenum and nickel in the symbiosis with legumes. We highlight some gaps in our knowledge, for example the lack of information on how rhizobia acquire molybdenum. We examine the means whereby different metals affect rhizobial physiology and the role of metals as signals for gene regulation. We describe the ways in which genetics has shown (or not) if, and how, particular metal uptake and/or metal-mediated signalling pathways are required for the symbiotic interaction with legumes.

The purMN genes of Rhizobium leguminosarum and a superficial link with siderophore production.

We isolated a mutant of R. leguminosarum initially on the basis of reduced production of the siderophore vicibactin on chrome azurol sulfonate (CAS)/agar indicator plates. The mutation was in the purMN operon and the mutant was shown to be an adenine auxotroph and defective for nodulation of peas. The siderophore defect appears to be trivial, being due to diminished growth of the auxotroph on agar-based minimal medium, which contains unknown contaminant(s) that allow it grow poorly. Transcriptional fusions showed that purMN was transcribed at relatively high levels in media containing purines. Expression was enhanced, approximately twofold, if purines were omitted.

A putative ECF sigma factor gene, rpol, regulates siderophore production in Rhizobium leguminosarum.

A cloned Rhizobium leguminosarum gene, termed rpoI, when transferred to wild-type strains, caused overproduction of the siderophore vicibactin. An rpoI mutant was defective in Fe uptake but was unaffected in symbiotic N2 fixation. The RpoI gene product was similar in sequence to extra-cytoplasmic sigma factors of RNA polymerase. Transcription of rpoI was reduced in cells grown in medium that was replete with Fe.

Is the fur gene of Rhizobium leguminosarum essential?

Using primers corresponding to conserved regions of the bacterial regulatory gene fur, a homologue of this gene from the genome of Rhizobium leguminosarum biovar viciae, the nitrogen-fixing symbiont of peas, was isolated and sequenced. The fur gene is normally expressed constitutively, independent of the presence of Fe in the medium, but in one Rhizobium strain it was transcribed at a low level. Attempts to isolate a fur knockout mutant failed, suggesting that the gene is essential for free-living growth. In other bacteria, certain fur mutations confer manganese resistance; however, none of the manganese-resistant mutants of R. leguminosarum which we isolated was corrected by the cloned fur gene. When the cloned R. leguminosarum fur gene was introduced into a fur mutant of Escherichia coli, it caused some Fe-dependent reduction in the amount of siderophore, indicating that it can function heterologously.

Purification and characterization of the protease enzymes of Streptomyces thermovulgaris grown in rapemeal-derived media.

When grown in a particulate-free, protein-rich medium derived from rapemeal (termed medium B), Streptomyces thermovulgaris produced multiple protease enzymes. The main protease activity was attributed to two types of serine protease, denoted as SV1 and SV2. A metallo protease component (SV3) and an azocaseinase component (SV4) were also present. Protease SV1 had a molecular weight of 30 kDa and a pI of 5.8. Protease SV2 was characterized by a high thermostability in the presence of calcium ions and had a pI of 8.4. This enzyme had a molecular weight of 60 kDa, but we suggest that this is the dimeric form, with 30 kDa being the monomer unit. The method chosen for initial downstream processing influenced both the yield and type of protease purified. When cell-free supernatant fluid was concentrated using ultrafiltration, rather than acetone precipitation, a higher percentage and a greater range of proteases were recovered. The medium used for the growth of Strep. thermovulgaris also appeared to affect the type of protease produced. A more diverse range of proteases were produced on rapemeal-derived medium when compared to yeast extract medium.

A region of a Sym plasmid of Rhizobium leguminosarum biovar phaseoli has similarity to prokaryotic insertion sequences and to eukaryotic integrases.

Near the nod and nif genes of the Sym plasmid pRP2JI of Rhizobium leguminosarum biovar phaseoli are three open reading frames whose deduced polypeptide products have similarities to those of genes in bacterial insertion sequences. The similarity of one of these ORFs was significantly greater to that of the integrase region of pol proteins of eukaryotic retroviruses and transposable elements in animals and plants than it was to the transposases of prokaryotic insertion sequences. In the noncoding region of the IS-like element, there was a sequence similar to that which had been identified close to nod genes in Azorhizobium caulinodans.

Pleiotropic effects of regulatory ros mutants of Agrobacterium radiobacter and their interaction with Fe and glucose.

Four exo mutants of Agrobacterium radiobacter, defective in the synthesis of acidic exopolysaccharide were complemented by a gene from that species, which is similar to the transcriptional regulator, ros, of A. tumefaciens. It was confirmed that this A. radiobacter gene, which we term rosAR, like ros, repressed its own transcription as well as that of virC and virD, two loci involved in tumorigenesis. The sequence of RosAR suggested that it might bind to a transition metal and its repressor abilities were shown to require Fe in the medium; repression was also enhanced with increasing levels of glucose. Certain rosAR mutants, in which its 3' end was removed were dominant; i.e., when plasmids containing such mutant forms of the gene were introduced into wild-type A. radiobacter, the transconjugants were nonmucoid. Such effects were also seen in a wide range of bacteria, including Escherichia coli and Xanthomonas. Several mutants that were complementd by rosAR also accumulated protoporphyrin, suggesting a defect in haem synthesis.

Characterization of the cycHJKL genes involved in cytochrome c biogenesis and symbiotic nitrogen fixation in Rhizobium leguminosarum.

Mutants of Rhizobium leguminosarum bv. viciae unable to respire via the cytochrome aa3 pathway were identified by the inability to oxidize N,N'-dimethyl-p-phenylenediamine. Two mutants which were complemented by cosmid pIJ1942 from an R. leguminosarum clone bank were identified. Although pea nodules induced by these mutants contained many bacteroids, no symbiotic nitrogen fixation was detected. Heme staining of cellular proteins revealed that all cytochrome c-type heme proteins were absent. These mutants lacked spectroscopically detectable cytochrome c, but cytochromes aa3 and d were present, the latter at a higher-than-normal level. DNA sequence analysis of complementing plasmids revealed four apparently cotranscribed open reading frames (cycH, cycJ, cycK, and cycL). CycH, CycJ, CycK, and CycL are homologous to Bradyrhizobium japonicum and Rhizobium meliloti proteins thought to be involved in the attachment of heme to cytochrome c apoproteins; CycK and CycL are also homologous to the Rhodobacter capsulatus ccl1 and ccl2 gene products and the Escherichia coli nrfE and nrfF gene products involved in the assembly of c-type cytochromes. The absence of cytochrome c heme proteins in these R. leguminosarum mutants is consistent with the view that the cycHJKL operon could be involved in the attachment of heme to apocytochrome c.

Protease production by Streptomyces thermovulgaris grown on rapemeal-derived media.

A range of actinomycete species was tested for their ability to grow on particulate and particle-free rapeseed meal-derived media. Streptomycetes grew on both types of medium and produced a number of extracellular enzymes. Highest activities of protease were produced by Streptomyces thermovulgaris and reflected the high available protein content of rapemeal. Enzyme production and growth were analysed in fermentor-grown batch cultures of S. thermovulgaris using the particle-free rapemeal broth termed medium B. Growth was biphasic and the majority of the protease was produced during the second slower phase. Analysis of the protease as azocaseinase activity revealed a high degree of thermostability in the presence of calcium such that approximately 20% of the activity remained after incubation at 70 degrees C for 24 h. Gel filtration suggested that S. thermovulgaris synthesized more than one kind of protease and this was confirmed by using specific peptide substrates and inhibitors which revealed the presence of distinct serine and metallo-type enzymes.