José M. Palacios

Gene Products of the hupGHIJ Operon Are Involved in Maturation of the Iron-Sulfur Subunit of the [NiFe] Hydrogenase from Rhizobium leguminosarum bv. viciae

In the present study, we investigate the functions of the hupGHIJ operon in the synthesis of an active [NiFe] hydrogenase in the legume endosymbiont Rhizobium leguminosarum bv. viciae. These genes are clustered with 14 other genes including the hydrogenase structural genes hupSL. A set of isogenic mutants with in-frame deletions (deltahupG, deltahupH, deltahupI, and deltahupJ) was generated and tested for hydrogenase activity in cultures grown at different oxygen concentrations (0.2 to 2.0%) and in symbiosis with peas. In free-living cultures, deletions in these genes severely reduced hydrogenase activity. The deltahupH mutant was totally devoid of hydrogenase activity at any of the O2 concentration tested, whereas the requirement of hupGIJ for hydrogenase activity varied with the O2 concentration, being more crucial at higher pO2. Pea bacteroids from the mutant strains affected in hupH, hupI, and hupJ exhibited reduced (20 to 50%) rates of hydrogenase activity compared to the wild type, whereas rates were not affected in the deltahupG mutant. Immunoblot experiments with HupL- and HupS-specific antisera showed that free-living cultures from deltahupH, deltahupI, and deltahupJ mutants synthesized a fully processed mature HupL protein and accumulated an unprocessed form of HupS (pre-HupS). Both the mature HupL and the pre-HupS forms were located in the cytoplasmic fraction of cultures from the deltahupH mutant. Affinity chromatography experiments revealed that cytoplasmic pre-HupS binds to the HupH protein before the pre-HupS-HupL complex is formed. From these results we propose that hupGHIJ gene products are involved in the maturation of the HupS hydrogenase subunit.

The twin-arginine translocation (Tat) system is essential for Rhizobium –legume symbiosis.

The Tat (twin‐arginine translocation) system mediates export of periplasmic proteins in folded conformation. Proteins transported via Tat contain a characteristic twin‐arginine motif in their signal peptide. Genetic determinants (tatABC genes) of the Tat system from Rhizobium leguminosarum bv. viciae were cloned and characterized, and a tatBC deletion mutant was constructed. The mutant lacked the ability for membrane targeting of hydrogenase, a known Tat substrate, and was impaired in hydrogenase activity. Interestingly, in the absence of a functional Tat system, only small, white nodules unable to fix nitrogen were induced in symbiosis with pea plants. Analysis of nodule structure and location of green fluorescent protein (GFP)‐tagged bacteria within nodules indicated that the symbiotic process was blocked in the tat mutant at a stage previous to bacteria release into cortical cells. The R. leguminosarum Tat‐deficient mutant lacked a functional cytochrome bc1 complex. This was consistent with the fact that R. leguminosarum Rieske protein, a key component of the symbiosis‐essential cytochrome bc1 complex, contained a typical twin‐arginine signal peptide. However, comparative analyses of nodule structure indicated that nodule development in the tat mutant was arrested at an earlier step than in a cytochrome bc1 mutant. These data indicate that the Tat pathway is also critical for proteins relevant to the initial stages of the symbiotic process.

Nucleotide sequence and organization of an H2-uptake gene cluster from Rhizobium leguminosarum bv. viciae containing a rubredoxin-like gene and four additional open reading frames

The nucleotide sequence of a 3.2 kb region following the hydrogenase structural operon (hupSLCDEF) in the H2-uptake gene cluster from Rhizobium leguminosarum by viciae strain 128C53 has been determined. Five closely linked genes encoding products of 16.3 (HupG), 30.5 (HupH), 8.0 (HupI), 18.4 (HupJ) and 38.7 (HupK) kDa were identified 166 bp downstream from hupF. Transposon insertions into hupG, hupH, hupJ and hupK suppress the H2-oxidizing capability of the wild-type strain. The amino acid sequence deduced from hupI contains two Cys-X-X-Cys motifs, characteristic of rubredoxins, separated by 29 amino acid residues showing strong sequence homology with other bacterial rubredoxins. The amino acid-derived sequence from hupG and hupH showed homology to products from genes hyaE and hyaF of the operon encoding hydrogenase 1 from Escherichia coli, and hupJ and hupK were related to open reading frames identified in Rhodobacter capsulatus and Azotobacter vinelandii hydrogenase gene clusters. An involvement of the hupGHIJK gene cluster in redox reactions related to hydrogenase synthesis or activity is predicted on the basis of the function as electron carrier attributed to rubredoxin.

Differential expression of hydrogen uptake (hup) genes in vegetative and symbiotic cells of Rhizobium leguminosarum

SummaryThe genetic determinants responsible for H2-uptake (hup genes) in Rhizobium leguminosarum are organized in six transcriptional units, designated regions hupI to hupVI, with region hupI coding for the hydrogenase structural genes (Leyva et al. 1990). Regulation of the expression of hup genes from R. leguminosarum was examined by using hup-lacZ fusions and mRNA dot-blot analysis. None of the six hup regions is transcribed in vegetative cells grown under normal aerobic conditions, whereas all six regions are transcribed in pea bacteroids. Additionally, exposure of cell cultures to low oxygen tensions specifically induces the expression of regions hupV and hupVI. By studying the expression of hupV- and hupVI-lacZ fusions in R. meliloti mutants it was determined that the microaerobic induction of these two regions is dependent on the regulatory fixLJ system, and that this control is exerted through fixK. Such expression was also shown to be nifA and ntrA independent. The functions of the hupV and hupVI gene products are unknown. The possibility that they play a regulatory role in hup gene expression is unlikely, since pea bacteroids from R. leguminosarum Hup− mutants carrying Tn5 insertions in regions hupV and hupVI contained normal levels of mRNA transcripts corresponding to the remaining hup regions.

The rkpU gene of Sinorhizobium fredii HH103 is required for bacterial K-antigen polysaccharide production and for efficient nodulation with soybean but not with cowpea.

In this work, the role of the rkpU and rkpJ genes in the production of the K-antigen polysaccharides (KPS) and in the symbiotic capacity of Sinorhizobium fredii HH103, a broad host-range rhizobial strain able to nodulate soybean and many other legumes, was studied. The rkpJ- and rkpU-encoded products are orthologous to Escherichia coli proteins involved in capsule export. S. fredii HH103 mutant derivatives were contructed in both genes. To our knowledge, this is the first time that the role of rkpU in KPS production has been studied in rhizobia. Both rkpJ and rkpU mutants were unable to produce KPS. The rkpU derivative also showed alterations in its lipopolysaccharide (LPS). Neither KPS production nor rkpJ and rkpU expression was affected by the presence of the flavonoid genistein. Soybean (Glycine max) plants inoculated with the S. fredii HH103 rkpU and rkpJ mutants showed reduced nodulation and clear symptoms of nitrogen starvation. However, neither the rkpJ nor the rkpU mutants were significantly impaired in their symbiotic interaction with cowpea (Vigna unguiculata). Thus, we demonstrate for the first time to our knowledge the involvement of the rkpU gene in rhizobial KPS production and also show that the symbiotic relevance of the S. fredii HH103 KPS depends on the specific bacterium-legume interaction.

Host-Dependent Expression of Rhizobium leguminosarum bv. viciae Hydrogenase Is Controlled at Transcriptional and Post-Transcriptional Levels in Legume Nodules

The legume host affects the expression of Rhizobium leguminosarum hydrogenase activity in root nodules. High levels of symbiotic hydrogenase activity were detected in R. leguminosarum bacteroids from different hosts, with the exception of lentil (Lens culinaris). Transcription analysis showed that the NifA-regulated R. leguminosarum hydrogenase structural gene promoter (P(1)) is poorly induced in lentil root nodules. Replacement of the P(1) promoter by the FnrN-dependent promoter of the fixN gene restored transcription of hup genes in lentil bacteroids, but not hydrogenase activity. In the P(fixN)-hupSL strain, additional copies of the hup gene cluster and nickel supplementation to lentil plants increased bacteroid hydrogenase activity. However, the level of activity in lentil still was significantly lower than in pea bacteroids, indicating that an additional factor is impairing hydrogenase expression inside lentil nodules. Immunological analysis revealed that lentil bacteroids contain reduced levels of both hydrogenase structural subunit HupL and nickel-binding protein HypB. Altogether, results indicate that hydrogenase expression is affected by the legume host at the level of both transcription of hydrogenase structural genes and biosynthesis or stability of nickel-related proteins HypB and HupL, and suggest the existence of a plant-dependent mechanism that affects hydrogenase activity during the symbiosis by limiting nickel availability to the bacteroid.

Symbiotic hydrogenase activity in Bradyrhizobium sp. (Vigna) increases nitrogen content in Vigna unguiculata plants

ABSTRACT Bradyrhizobium sp. (Lupinus) and Bradyrhizobium sp. (Vigna) mutants in which hydrogenase (hup) activity was affected were constructed and analyzed. Vigna unguiculata plants inoculated with the Bradyrhizobium sp. (Vigna) hup mutant showed reduced nitrogenase activity and also a significant decrease in nitrogen content, suggesting a relevant contribution of hydrogenase activity to plant yield.

Diversity of Bradyrhizobium strains nodulating Lupinus micranthus on both sides of the Western Mediterranean: Algeria and Spain.

Lupinus micranthus is a lupine distributed in the Mediterranean basin whose nitrogen fixing symbiosis has not been described in detail. In this study, 101 slow-growing nodule isolates were obtained from L. micranthus thriving in soils on both sides of the Western Mediterranean. The diversity of the isolates, 60 from Algeria and 41 from Spain, was addressed by multilocus sequence analysis of housekeeping genes (16S rRNA, atpD, glnII and recA) and one symbiotic gene (nodC). Using genomic fingerprints from BOX elements, 37 different profiles were obtained (22 from Algeria and 15 from Spain). Phylogenetic analysis based on 16S rRNA and concatenated atpD, glnII and recA sequences of a representative isolate of each BOX profile displayed a homogeneous distribution of profiles in six different phylogenetic clusters. All isolates were taxonomically ascribed to the genus Bradyrhizobium. Three clusters comprising 24, 6, and 4 isolates, respectively, accounted for most of the profiles. The largest cluster was close to the Bradyrhizobium canariense lineage, while the other two were related to B. cytisi/B. rifense. The three remaining clusters included only one isolate each, and were close to B. canariense, B. japonicum and B. elkanii species, respectively. In contrast, phylogenetic clustering of BOX profiles based on nodC sequences yielded only two phylogenetic groups. One of them included all the profiles except one, and belonged to symbiovar genistearum. The remaining profile, constituted by a strain related to B. elkanii, was not related to any well-defined symbiotic lineage, and may constitute both a new symbiovar and a new genospecies.

Symbiotic Autoregulation of nifA Expression in Rhizobium leguminosarum bv. viciae

NifA is the general transcriptional activator of nitrogen fixation genes in diazotrophic bacteria. In Rhizobium leguminosarum bv. viciae UPM791, the nifA gene is part of a gene cluster (orf71 orf79 fixW orf5 fixABCX nifAB) separated by 896 bp from an upstream and divergent truncated duplication of nifH (DeltanifH). Symbiotic expression analysis of genomic nifA::lacZ fusions revealed that in strain UPM791 nifA is expressed mainly from a sigma54-dependent promoter (P(nifA1)) located upstream of orf71. This promoter contains canonical NifA upstream activating sequences located 91 bp from the transcription initiation site. The transcript initiated in P(nifA1) spans 5.1 kb and includes nifA and nifB genes. NifA from Klebsiella pneumoniae was able to activate transcription from P(nifA1) in a heterologous Escherichia coli system. In R. leguminosarum, the P(nifA1) promoter is essential for effective nitrogen fixation in symbiosis with peas. In its absence, partially efficient nitrogen-fixing nodules were produced, and the corresponding bacteroids exhibited only low levels of nifA gene expression. The basal level of nifA expression resulted from a promoter activity originating upstream of the fixX-nifA intergenic region and probably from an incomplete duplication of P(nifA1) located immediately upstream of fixA.

The Tat pathway of plant pathogen Dickeya dadantii 3937 contributes to virulence and fitness

Protein secretion plays a very important role in the virulence of the bacterium Dickeya dadantii, the causative agent of soft rot disease, in a wide range of plant species. We studied the contribution of the twin-arginine translocation (Tat) protein system to the adaptation of D. dadantii 3937 to different growth conditions and to the interaction with the plant host. First, a list of 44 putative Tat substrates was obtained using bioinformatic programs taking advantage of the availability of the complete sequence of this bacterium. Second, a tatC mutant strain was constructed and analysed. The mutant displayed a pleiotropic phenotype, showing limited growth in an iron-depleted medium, higher sensitivity to copper, reduced motility on soft agar plates and attenuated virulence in witloof chicory leaves. Our results indicate the Tat system as an important determinant of the virulence and fitness of D. dadantii 3937. Potential Tat substrates related to the tatC mutant phenotype are discussed.