Engineering the Rhizobium leguminosarum bv. viciae hydrogenase system for expression in free-living microaerobic cells and increased symbiotic hydrogenase activity

Rhizobium leguminosarum bv. viciae UPM791 induces hydrogenase activity in pea (Pisum sativum L.) bacteroids but not in free-living cells. The symbiotic induction of hydrogenase structural genes (hupSL) is mediated by NifA, the general regulator of the nitrogen fixation process. So far, no culture conditions have been found to induce NifA-dependent promoters in vegetative cells of this bacterium. This hampers the study of the R. leguminosarum hydrogenase system. We have replaced the native NifA-dependent hupSL promoter with the FnrN-dependent fixN promoter, generating strain SPF25, which expresses the hup system in microaerobic free-living cells. SPF25 reaches levels of hydrogenase activity in microaerobiosis similar to those induced in UPM791 bacteroids. A sixfold increase in hydrogenase activity was detected in merodiploid strain SPF25(pALPF1). A time course induction of hydrogenase activity in microaerobic free-living cells of SPF25(pALPF1) shows that hydrogenase activity is detected after 3 h of microaerobic incubation. Maximal hydrogen uptake activity was observed after 10 h of microaerobiosis. Immunoblot analysis of microaerobically induced SPF25(pALPF1) cell fractions indicated that the HupL active form is located in the membrane, whereas the unprocessed protein remains in the soluble fraction. Symbiotic hydrogenase activity of strain SPF25 was not impaired by the promoter replacement. Moreover, bacteroids from pea plants grown in low-nickel concentrations induced higher levels of hydrogenase activity than the wild-type strain and were able to recycle all hydrogen evolved by nodules. This constitutes a new strategy to improve hydrogenase activity in symbiosis.     

Comparative strain typing of Rhizobium leguminosarum bv. viciae natural populations

372 natural isolates of Rhizobium leguminosarum bv. viciae, rescued from nodules of pea plants grown in an agricultural field in northern Italy, were analyzed by different methods. Three DNA-based fingerprinting techniques were lined up to compare their relative degree of resolution and possible advantages of each approach. The methods included (i) Eckhardt gel plasmid profiles, (ii) pulsed-field gel electrophoresis (PFGE) of genomic large fragment digests, and (iii) random amplified polymorphic DNA (RAPD) profiles, generated with arbitrary primers. The scheme also involved the isolation of a number of different isolates per nodule to estimate the level of intra-nodular variability. It was therefore possible to evaluate the frequency of double and multiple occupancies, and the proportion of the alternative profiles sharing the same nodule, generally resulting in a numerically dominant, main representative accompanied by a secondary one with a slightly different fingerprint. This finding revealed that the different profiles within a nodule are normally due to bacteria derived from the same single invader following genetic alterations possibly occurred during infection, e.g., by plasmid loss. The analysis of 31 nodules revealed 16 different patterns, representing the most frequently occurring nodulation-proficient isolates of the natural soil examined, five of which were found with frequencies around 15%. The sensitivity of the methods in differentiating isolates was compared. The relatedness of the different natural rhizobial isolates was investigated by densitometrical gel analysis of the fingerprints, allowing a comparison of the results. One of the most interesting conclusions was that the degree of information yielded by the plasmid gel profiling alone, carried out by simple visual inspection without software-aided analyses, was surprisingly high, as it enabled a placement of the isolates, whose accuracy, in terms of relatedness, was subsequently confirmed by each of the two genomic methods.     

Symbiotic plasmid rearrangement in Rhizobium leguminosarum bv. viciae VF39SM

A rearrangement between the symbiotic plasmid (pRleVF39d) and a nonsymbiotic plasmid (pRleVF39b) in Rhizobium leguminosarum bv. viciae VF39 was observed. The rearranged derivative showed the same plasmid profile as its parent strain, but hybridization to nod, fix, and nif genes indicated that most of the symbiotic genes were now present on a plasmid corresponding in size to pRleVF39b instead of pRleVF39d. On the other hand, some DNA fragments originating from pRleVF39b now hybridized to the plasmid band at the position of pRleVF39d. These results suggest that a reciprocal but unequal DNA exchange between the two plasmids had occurred.     

Visualization of symbiotic tissue in intact root nodules of Vicia tetrasperma using GFP-marked Rhizobium leguminosarum bv. viciae

In rhizobial symbiosis with legume plant hosts, the symbiotic tissue in the root nodules of indeterminate type is localized to the basal part of the nodule where the symbiotic zones contain infected cells (IC) interspersed with uninfected cells (UC) that are devoid of rhizobia. Although IC are easily distinguished in nodule sections using standard histochemical techniques, their observation in intact nodules is hampered by nodule tissue characteristics. Tagging of Rhizobium leguminosarum bv. viciae strain 128C30 with a constitutively expressed gene for green fluorescent protein (nonshifted mutant form cycle3) in combination with the advantages of the tiny nodules formed by Vicia tetrasperma (L.) SCHREB: . allowed for vital observation of symbiotic tissue using fluorescence microscopy. Separation of a red-shifted background channel and digital image stacking along z-axis enabled us to construct a nodule image in a classical fluorescence microscopy of nodules exceeding 1 mm in diameter. In parallel, visualization of nodule bacteria inside the symbiotic tissue by confocal microscopy at the excitation wavelength 488 nm clearly distinguished IC/UC pattern in the nodule virtual sections and revealed red-shifted fluorescence of nonrhizobial origin. This signal was located on the periphery of IC and increased with their degradation, thus suggesting accumulation of secondary metabolites, presumably flavonoids. The simultaneous detection of bacteria and secondary metabolites can be used for monitoring changes to intact nodule physiology in the model legumes. The advantage of V. tetrasperma as a suggested laboratory model for pea cross-inoculation group has been demonstrated.     

Hydrogenase genes are uncommon and highly conserved in Rhizobium leguminosarum bv. viciae

A screening for hydrogen uptake (hup) genes in Rhizobium leguminosarum bv. viciae isolates from different locations within Spain identified no Hup+ strains, confirming the scarcity of the Hup trait in R. leguminosarum. However, five new Hup+ strains were isolated from Ni-rich soils from Italy and Germany. The hup gene variability was studied in these strains and in six available strains isolated from North America. Sequence analysis of three regions within the hup cluster showed an unusually high conservation among strains, with only 0.5-0.6% polymorphic sites, suggesting that R. leguminosarum acquired hup genes de novo in a very recent event.     

Rhizobium leguminosarum bv. viciae genotypes interact with pea plants in developmental responses of nodules, roots and shoots

The variability of the developmental responses of two contrasting cultivars of pea (Pisum sativum) was studied in relation to the genetic diversity of their nitrogen-fixing symbiont Rhizobium leguminosarum bv. viciae. A sample of 42 strains of pea rhizobia was chosen to represent 17 genotypes predominating in indigenous rhizobial populations, the genotypes being defined by the combination of haplotypes characterized with rDNA intergenic spacer and nodD gene regions as markers. We found contrasting effects of the bacterial genotype, especially the nod gene type, on the development of nodules, roots and shoots. A bacterial nod gene type was identified that induced very large, branched nodules, smaller nodule numbers, high nodule biomass, but reduced root and aerial part development. The plants associated with this genotype accumulated less N in shoots, but N concentration in leaves was not affected. The results suggest that the plant could not control nodule development sustaining the energy demand for nodule functioning and its optimal growth. The molecular and physiological mechanisms that may be involved are discussed.     

Characterization of the urease gene cluster from Rhizobium leguminosarum bv. viciae

Moderate levels of urease activity (ca. 300 mU mg(-1)) were detected in Rhizobium leguminosarum bv. viciae UPM791 vegetative cells. This activity did not require urea for induction and was partially repressed by the addition of ammonium into the medium. Lower levels of urease activity (ca. 100 mU mg(-1)) were detected also in pea bacteroids. A DNA region of ca. 9 kb containing the urease structural genes ( ureA, ureB and ureC), accessory genes ( ureD, ureE, ureF, and ureG), and five additional ORFs ( orf83, orf135, orf207, orf223, and orf287) encoding proteins of unknown function was sequenced. Three of these ORFs ( orf83, orf135 and orf207) have a homologous counterpart in a gene cluster from Sinorhizobium meliloti, reported to be involved in urease and hydrogenase activities. R. leguminosarum mutant strains carrying Tn 5 insertions within this region exhibited a urease-negative phenotype, but induced wild-type levels of hydrogenase and nitrogenase activities in bacteroids. orf287 encodes a potential transmembrane protein with a C-terminal GGDEF domain. A mutant affected in orf287 exhibited normal levels of urease activity in culture cells. Experiments aimed at cross-complementing Ni-binding proteins required for urease and hydrogenase synthesis (UreE and HypB, respectively) indicated that these two proteins are not functionally interchangeable in R. leguminosarum.     

Chemical characterization of effective and ineffective strains of Rhizobium leguminosarum bv. viciae

Chemical composition of lipopolysaccharide (LPS) isolated from an effective (97) and ineffective (87) strains of R. l. viciae has been determined. LPS preparations from the two strains contained: glucose, galactose, mannose, fucose, arabinose, heptose, glucosamine, galactosamine, quinovosamine, and 3-N-methyl-3,6-dideoxyhexose, as well as glucuronic, galacturonic and 3-deoxyoctulosonic acid. The following fatty acids were identified: 3-OH 14:0, 3-OH 15:0, 3-OH 16:0, 3-OH 18:0 and 27-OH 28:0. The ratio of 3-OH 14:0 to other major fatty acids in LPS 87 was higher that in LPS 97. SDS/PAGE profiles of LPS indicated that, in lipopolysaccharides, relative content of S form LPS I to that of lower molecular mass (LPS II) was much higher in the effective strain 97 than in 87. All types of polysaccharides exo-, capsular-, lipo, (EPS, CPS, LPS, respectively) examined possessed the ability to bind faba bean lectin. The degree of affinity of the host lectin to LPS 87 was half that to LPS 97. Fatty acids (FA) composition from bacteroids and peribacteroid membrane (PBM) was determined. Palmitic, stearic and hexadecenoic acids were common components found in both strains. There was a high content of unsaturated fatty acids in bacteroids as well as in PBM lipids. The unsaturation index in the PBM formed by strain 87 was lower than in the case of strain 97. Higher ratio of 16:0 to 18:1 fatty acids was characteristic for PMB of the ineffective strain.     

Characterization of the quaternary amine transporters of Rhizobium leguminosarum bv. viciae 3841

Rhizobium leguminosarum bv. viciae 3841 contains six putative quaternary ammonium transporters (Qat), of the ABC family. Qat6 was strongly induced by hyperosmosis although the solute transported was not identified. All six systems were induced by the quaternary amines choline and glycine betaine. It was confirmed by microarray analysis of the genome that pRL100079-83 (qat6) is the most strongly upregulated transport system under osmotic stress, although other transporters and 104 genes are more than threefold upregulated. A range of quaternary ammonium compounds were tested but all failed to improve growth of strain 3841 under hyperosmotic stress. One Qat system (gbcXWV) was induced 20-fold by glycine betaine and choline and a Tn5::gbcW mutant was severely impaired for both transport and growth on these compounds, demonstrating that it is the principal system for their use as carbon and nitrogen sources. It transports glycine betaine and choline with a high affinity (apparent K(m), 168 and 294 nM, respectively).     

The major chemotaxis gene cluster of Rhizobium leguminosarum bv. viciae is essential for competitive nodulation

Rhizobium leguminosarum biovar viciae strain 3841 is a motile alpha-proteobacterium that can establish a nitrogen-fixing symbiosis within the roots of pea plants. In order to determine the contribution of chemotaxis to the lifestyle of R. leguminosarum, we have characterized the function of two chemotaxis gene clusters (che1 and che2) in controlling motility behaviour. We have found that both chemotaxis gene clusters modulate the motility swimming bias of R. leguminosarum cells and that the che1 cluster is the major pathway controlling swimming bias and chemotaxis. The che2 cluster also contributes to swimming bias, but has a minor effect on chemotaxis. Using competitive nodulation assays, we have demonstrated that a functional che1 cluster, but not the che2 cluster, promotes competitive nodulation of the peas. This finding implies that the environmental cue(s) triggering chemotaxis of R. leguminosarum bv. viciae cells towards the roots of pea and facilitating colonization are likely to be processed through the che1 cluster despite the contribution of both che clusters to swimming behaviour. A phylogenetic analysis of the distribution of che1 and che2 orthologues in the alpha-proteobacteria together with our results allow us to propose that che1 homologues are major controllers of chemotaxis and host association in the Rhizobiaceae.