The hypBFCDE operon from Rhizobium leguminosarum biovar viciae is expressed from an Fnr-type promoter that escapes mutagenesis of the fnrN gene

Abstract

Pea (Pisum sativum L.) bacteroids produced by Rhizobium leguminosarum bv. viciae UPM791 synthesize a membrane-bound (NiFe) hydrogenase which oxidizes H 2 arising from the nitrogen fixation process in root nodules. Synthesis of the active enzyme requires the products of the structural genes hupSL and an array of accessory proteins from at least 15 additional genes, including the gene cluster hypABFCDE, likely involved in nickel metabolism. Unlike the hupSL genes, which are expressed only in symbiosis, the hypBFCDE operon was also activated in vegetative cells in response to low pO 2 in the culture medium. In microaerobic cells and in bacteroids, transcription of the hypBFCDE operon occurred from a promoter, P 5b , with a transcription initiation site located 190 bp upstream of the ATG start codon of hypB, within the coding sequence of hypA. Transcription start site 5b was preceded by an Fnr box (anaerobox), 5-TTGAgccatgTCAA-3, centered at position ؊39.5. Expression of the P 5b promoter in the heterologous Rhizobium meliloti bacterial host was dependent on the presence of an active fixK gene. A 2.6-kb EcoRI fragment was isolated from an R. leguminosarum bv. viciae UPM791 gene bank by complementing an R. meliloti FixK ؊ mutant. Sequencing of this DNA fragment identified an fnrN gene, and cassette insertion mutagenesis demonstrated that R. leguminosarum bv. viciae fnrN is able to replace the R. meliloti fixK gene for activation of both the R. leguminosarum bv. viciae hypBFCDE operon and the R. meliloti fix genes. However, bacteroids from a genomic FnrN ؊ mutant of R. leguminosarum bv. viciae exhibited wild-type levels of hydrogenase activity. Microaerobic expression of P 5b was reduced to ca. 50% of the wild-type level in the FnrN ؊ mutant. These results indicate that hyp gene expression escapes mutagenesis of the fnrN gene and suggest the existence of a second fnr-like gene in R. leguminosarum bv. viciae. Southern blot analysis with an fnrN internal probe revealed the presence of a second genomic region with homology to fnrN.A few strains of Rhizobium leguminosarum bv. viciae have been found to synthesize, in symbiosis with peas, an H 2 uptake (hup) system that recycles hydrogen evolved during the nitrogen reduction process in root nodules (2). In R. leguminosarum bv. viciae UPM791, the genetic determinants for this H 2 uptake system are clustered in a 20-kb DNA region which has been isolated in cosmid pAL618 (21). About 15 kb of this region have been sequenced, and 17 genes closely linked and oriented in the same direction were identified (14,15,31,33). The first two genes of the cluster, hupS and hupL, encode the structural polypeptides of a dimeric (NiFe)-type hydrogenase (14) and belong to an operon that also includes four additional genes, hupCDEF (15). The remaining genes, named hupG, H, I, J, and K and hypA, B, F, C, D, and E, are located downstream of hupF (31, 33). These genes encode hydrogenase accessory proteins that are required for H 2 oxidation in pea bacteroids, but their specific molecular functions...

Symbiotic Nitrogen Fixation

“…Furthermore, R. leguminosarum fnrN is able to complement an S. meliloti fixK mutant for fixNOQP expression (8,16), suggesting that the two proteins activate transcription in similar ways (8,15). Additional variations are found in R. leguminosarum bv viciae UPM791, in which two fnrN genes and no fixL, fixJ, or fixK homologs are present (15,16). Both fnrN genes are involved in activation of the fixNOQP operon (15,16).…”

“…R. leguminosarum bv. viciae VF39 contains FixK (30) and another transcriptional activator of the CRP-Fnr family, FnrN which activates the two fixNOQP copies (8,15,16,30). In contrast to FixK, FnrN has a region with a high level of similarity to a domain in the Escherichia coli Fnr protein involved in O 2 sensing, suggesting that the FnrN transcriptional activity may be negatively modulated by O 2 (8).…”

“…Additional variations are found in R. leguminosarum bv viciae UPM791, in which two fnrN genes and no fixL, fixJ, or fixK homologs are present (15,16). Both fnrN genes are involved in activation of the fixNOQP operon (15,16). In this strain, expression of both fnrN genes is autoregulated, thus ensuring equilibrated expression of fnrN in response to microaerobic conditions (7).…”

“…Both FixK and FnrN activate fixNOQP expression by binding to a DNA sequence located in the promoter region (TTGAT-N 4 -ATCAA) called the anaerobox (2,8,11). Furthermore, R. leguminosarum fnrN is able to complement an S. meliloti fixK mutant for fixNOQP expression (8,16), suggesting that the two proteins activate transcription in similar ways (8,15). Additional variations are found in R. leguminosarum bv viciae UPM791, in which two fnrN genes and no fixL, fixJ, or fixK homologs are present (15,16).…”

Molecular Biology of Microbial Hydrogenases

“…This promoter also binds the global regulator IHF and is transcribed by a σ 54 -RNA polymerase (Brito et al, 1997). Moreover, the Fnr-like protein, FnrN, indirectly activates H 2 ase expression by binding to the σ 70 -dependent promoter of hyp genes (Hernando et al, 1995), which are expressed in vegetative cells. There are two copies of fnrN genes, both possessing two anaeroboxes, TTGAT-N 4 -ATCAA (Colombo, et al, 2000) and expressed from a σ 54 -RNA polymerase (Clark et al, 2001).…”