Mutations in a Rhizobium leguminosarum gene, rirA (rhizobial iron regulator), caused high-level, constitutive expression of at least eight operons whose transcription is normally Fe-responsive and whose products are involved in the synthesis or uptake of siderophores, or in the uptake of haem or of other iron sources. Close homologues of RirA exist in other rhizobia and in the pathogen Brucella ; many other bacteria have deduced proteins with more limited sequence similarity. None of these homologues had been implicated in Femediated gene regulation. Transcription of rirA itself is about twofold higher in cells grown in Fe-replete than in Fe-deficient growth media. Mutations in rirA reduced growth rates in Fe-replete and -depleted medium, but did not appear to affect symbiotic N 2 fixation.
SummaryIn the N 2 -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 s 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 s factor was identified, but this gene, rpoZ, did not affect the transcription of tonB Rl or hmuP-STUV. Mutations in tonB Rl , hmu genes and rpoZ did not affect symbiotic N 2 fixation in peas.
SummaryA 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 N 2 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 s 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-N 5 -hydroxyornithine via covalent attachment as an acyl thioester to a peptidyl carrier protein domain. Subsequent VbsA-catalysed acylation of the hydroxyornithine, followed by VbsLmediated 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.
The cycHJKL operon of Rhizobium leguminosarum has previously been shown to be involved in the maturation of cytochrome c, possibly by its involvement in the covalent attachment of haem to the apoprotein. Mutations in the University Of East cycHJKL genes abolish symbiotic nitrogen fixation. Here, we show that cyc mutants are pleiotropically defective. They have lost a high affinity iron acquisition system due to their failure to make or to export siderophores. They also accumulate protoporphyrin IX, the immediate precursor of haem. A model to account for these phenotypes is presented. Immediately upstream of cycH is a gene, /ipA, which is predicted to encode an outer-membrane lipoprotein.Further upstream of /@A, there are two other genes, whose products are similar in sequence to the widespread family of two-component transcriptional regulators. These two genes, feuP and feu0, did not affect the transcription of /@A, or of the cycHJKL operon. However, a mutation in feu0 also led to the loss of the high affinity iron uptake system, although siderophores were still produced.
John lnnes Centre, ColneyLane, Norwich NR4 7UH, UK 1
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