Generation and characterization of a novel Rhizobium NGR234 mutant

This work focuses on the genetic regulation of the nodulation genes found in the symbiotic bacterium Bradyrhizobium japonicum. This work specifically addresses the question of what regulates the important transcriptional regulator gene nolA. The first part of this work uses nolA-luxCDABE fusions to show nolA induction and that the nolA promoter region can drive the lux operon. The plasmid-encoded fusion in Escherichia coli was used to show that the NolA protein induces the nolA promoter to drive the lux operon. The level of nolA induction over the course of the E. coli growth curve was determined by measuring the amount of light produced by the protein products of the lux operon. Without NolA, there was an initial spike of light production which tapered off as the culture density increased. In the presence of NolA, there was a second peak of light production at an optical density of 0.4 (measured at A600). A Tn5 with a nolA-luxCDBE transcriptional fusion was introduced into the B. japonicum chromosome, but the B. japonicum cells did not provide sufficient amounts of the fatty acid substrate (i.e., myristolate) for light production. The B. japonicum cells gave off light when an aldehyde substrate (decanal) was added to the culture. The need for exogenous substrate limited the usefulness of the noiA-luxCDABE fusion as we had intended to use it to view nolA induction in growing, intact host plant nodules. The second part of this work focuses on the generation of a Rhizobium species NGR234 mutant unable to induce a plasmid-encoded noiA-lacZ fusion in response to chitin or high cell population density. The mutants were generated by mating a plasmid (pJQ15Sp) bearing a Tn5 with antibiotic resistance markers into JNR1 ceils (R. NGR234 with a plasmid-encoded noiA-lacZ fusion, pBGAIac4). The transposase was encoded on pJQ15Sp outside the insertion sequences. Some mutants (JNR7-9) were selected based on their lack of nolA induction in response to higher culture densities, and other mutants (JNR1Sp1-45) were selected based on their lack of noiA induction in response to chitin. The lack of response was confirmed by β-galactosidase activity assays. The plasmids were isolated from the mutants and checked for Tn5 insertions before Southern blots were done to determine the number of Tn5 chromosomal insertions. Southern blot analysis revealed that these mutants were interrupted in the same gene. Since interrupting a single gene removes R. NGR234\u27s ability to induce the nolA promoter in response to chitin or high culture density, it seems likely that the single interrupted gene\u27s product passes both signals to nolA. One mutant, JNR9, was chosen for further analysis and plant nodulation assays. The interrupted gene of JNR9 was cloned into the cosmid vector pHC79 using the Promega Packagene kit (results confirmed by Southern blot). The resulting cosmid (pJNR9A) was found to have multiple copies of pHC79. It underwent subcloning to make the cosmid pD32A, which has only one copy of pHC79. The cosmid pD32A will be sequenced at a later time. R. NGR234, JNR1, and JNR9 were used in 28-day nodulation assays on soybean, cowpea, mungbean, and siratro plants. On soybean, R. NGR234 causes large lumpy growths on the roots. The mutant JNR9 caused twice as many growths as the R. NGR234 and JNR1. On cowpea and mungbean plants, JNR9 did not nodulate as well as the controls. On siratro plants, it did nodulate as well as the wild-type bacteria. The differences in JNR9\u27s nodulation ability in different plant hosts may indicate differences in the importance of NoiA in the different hosts

Deletion of rRNA Operons of Sinorhizobium fredii Strain NGR234 and Impact on Symbiosis With Legumes

During their lifecycle, from free-living soil bacteria to endosymbiotic nitrogen-fixing bacteroids of legumes, rhizobia must colonize, and cope with environments where nutrient concentrations and compositions vary greatly. Bacterial colonization of legume rhizospheres and of root surfaces is subject to a fierce competition for plant exudates. By contrast root nodules offer to rhizobia sheltered nutrient-rich environments within which the cells that successfully propagated via infection threads can rapidly multiply. To explore the effects on symbiosis of a slower rhizobia growth and metabolism, we deleted one or two copies of the three functional rRNA operons of the promiscuous Sinorhizobium fredii strain NGR234 and examined the impact of these mutations on free-living and symbiotic lifestyles. Strains with two functional rRNA operons (NGRΔrRNA1 and NGRΔrRNA3) grew almost as rapidly as NGR234, and NGRΔrRNA1 was as proficient as the parent strain on all of the five legume species tested. By contrast, the NGRΔrRNA1,3 double mutant, which carried a single rRNA operon and grew significantly slower than NGR234, had a reduced symbiotic proficiency on Cajanus cajan, Macroptilium atropurpureum, Tephrosia vogelii, and Vigna unguiculata. In addition, while NGRΔrRNA1 and NGR234 equally competed for nodulation of V. unguiculata, strain NGRΔrRNA1,3 was clearly outcompeted by wild-type. Surprisingly, on Leucaena leucocephala, NGRΔrRNA1,3 was the most proficient strain and competed equally NGR234 for nodule occupation. Together, these results indicate that for strains with otherwise identical repertoires of symbiotic genes, a faster growth on roots and/or inside plant tissues may contribute to secure access to nodules of some hosts. By contrast, other legumes such as L. leucocephala appear as less selective and capable of providing symbiotic environments susceptible to accommodate strains with a broader spectrum of competences

Trehalose and the nitrogen fixing nodule symbiosis of legumes : studies on rhizobia deficient in the trehalose-6-phosphate synthase gene "ots"A

The non-reducing disaccharide trehalose (a-D-glucopyranosyl-1,1-a-D-glucopyranoside) is widespread in nature, but is normally not present in higher plants. With respect to plant-microbe interactions, it is interesting that trehalose is regularly found in plant roots interacting with antagonistic fungi, mycorrhizal fungi, and in nitrogen-fixing root nodules, probably as a microbial substance. The impact of trehalose on plant metabolism and its role in nitrogen fixing symbiosis is unclear. This work focuses on the nodule symbiosis. It represents a genetic approach to study the role of trehalose synthesis by the microsymbiont. One pathway for trehalose synthesis is the OtsA/B pathway. Trehalose is synthesized from UDP-glucose and glucose-6-phosphate in a two-step process by the action of trehalose-6-phosphate synthase (OtsA) and trehalose-6-phosphate phosphatase (OtsB). Homologues of the genes coding for these two enzymes in Escherichia coli, otsA and otsB, have been localized on the symbiotic plasmid of Rhizobium sp. NGR234 (pNGR234a). To study the significance of rhizobial trehalose synthesis in free living and symbiotic rhizobia, an Ω-cassette was inserted into the otsA homologue. Phenotypically, the deletion of the rhizobial otsA-homologue strongly reduced trehalose synthesis under microaerobic growth conditions. Thus, there are strong indications that the rhizobial trehalose synthesis induced under hypoxic conditions is directed by the symbiotic plasmid encoded otsA-homologue in conjunction with otsB. The functionality of otsA has therefore indirectly been demonstrated in Rhizobium sp. NGR234, which is the first time in aproteobacteria in general. In addition, the induction of otsA and its homologues by low oxygen conditions has not been previously reported. The natural environment inside nodules is characterized by low oxygen. In contrast, trehalose synthesis under salt stress was not influenced by the mutation of otsA. This indicates that Rhizobium sp. NGR234 exhibits a second trehalose pathway. Activities of maltooligosyltrehalose synthase and maltooligosyl trehalohydrolase (MOS – pathway) had been demonstrated in Rhizobium sp. NGR234 in previous work. To study the role of rhizobial otsA in symbiosis, various host plants were infected with Rhizobium sp. NGRWotsA. In a number of hosts, average nodule size was reduced, nodule number was increased (up to 30 %) and nitrogen fixation was reduced compared to control plants infected with the wildtype strain NGR234. Analysis of the carbohydrate content of these nodules revealed significant increases in the levels of sucrose, hexoses and starch. Thus the deletion of the potential rhizobial otsA-homologue has a severe impact on rhizobium-legume symbiosis, and a signal function of trehalose in carbohydrate partitioning and root nodule development is proposed

Genetic snapshots of the Rhizobium species NGR234 genome

BACKGROUND: In nitrate-poor soils, many leguminous plants form nitrogen-fixing symbioses with members of the bacterial family Rhizobiaceae. We selected Rhizobium sp. NGR234 for its exceptionally broad host range, which includes more than I 12 genera of legumes. Unlike the genome of Bradyrhizobium japonicum, which is composed of a single 8.7 Mb chromosome, that of NGR234 is partitioned into three replicons: a chromosome of about 3.5 Mb, a megaplasmid of more than 2 Mb (pNGR234b) and pNGR234a, a 536,165 bp plasmid that carries most of the genes required for symbioses with legumes. Symbiotic loci represent only a small portion of all the genes coded by rhizobial genomes, however. To rapidly characterize the two largest replicons of NGR234, the genome of strain ANU265 (a derivative strain cured of pNGR234a) was analyzed by shotgun sequencing. RESULTS: Homology searches of public databases with 2,275 random sequences of strain ANU265 resulted in the identification of 1,130 putative protein-coding sequences, of which 922 (41%) could be classified into functional groups. In contrast to the 18% of insertion-like sequences (ISs) found on the symbiotic plasmid pNGR234a, only 2.2% of the shotgun sequences represent known ISs, suggesting that pNGR234a is enriched in such elements. Hybridization data also indicate that the density of known transposable elements is higher in pNGR234b (the megaplasmid) than on the chromosome. Rhizobium-specific intergenic mosaic elements (RIMEs) were found in 35 shotgun sequences, 6 of which carry RIME2 repeats previously thought to be present only in Rhizobium meliloti. As non-overlapping shotgun sequences together represent approximately 10% of ANU265 genome, the chromosome and megaplasmid may carry a total of over 200 RIMEs. CONCLUSIONS: 'Skimming' the genome of Rhizobium sp. NGR234 sheds new light on the fine structure and evolution of its replicons, as well as on the integration of symbiotic functions in the genome of a soil bacterium. Although most putative coding sequences could be distributed into functional classes similar to those in Bacillus subtilis, functions related to transposable elements were more abundant in NGR234. In contrast to ISs that accumulated in pNGR234a and pNGR234b, the hundreds of RIME elements seem mostly attributes of the chromosome

ß-Glucuronidase (GUS) transposons for ecological and genetic studies of rhizobia and other Gram-negative bacteria.

A series of transposons are described which contain the gusA gene, encoding β-glucuronidase (GUS), expressed from a variety of promoters, both regulated and constitutive. The regulated promoters include the tac promoter which can be induced by IPTG, and nifH promoters which are symbiotically activated in legume nodules. One transposon contains gusA with a strong Shine-Dalgarno translation initiation context, but no promoter, and thus acts as a promoter-probe transposon. In addition, a gus operon deletion strain of Escherichia coli, and a transposon designed for use in chromosomal mapping using PFGE, are described. The GUS transposons are constructed in a mini-Tn5 system which can be transferred to Gram-negative bacteria by conjugation, and will form stable genomic insertions. Due to the absence of GUS activity in plants and many bacteria of economic importance, these transposons constitute powerful new tools for studying the ecology and population biology of bacteria in the environment and in association with plants, as well as for studies of the fundamental molecular basis of such interactions. The variety of assays available for GUS enable both quantitative assays and spatial localization of marked bacteria to be carried out

Characterization of an agrobacterial plasmid inducible for transfer by mannopine and evolution of the core replication and transfer functions of repabc plasmids with class i quorum-sensing and transfer systems

repABC plasmids are ubiquitous in the α-proteobacteria and are important to the biology of the bacteria that harbor them for several reasons. First, they can carry large amounts of DNA, thereby conferring a wide variety of important characteristics. Some of these traits are important for the biology of the bacteria that harbor them. For example the repABC plasmids in species of Agrobacterium can encode virtually all of the genes responsible for inducing crown gall tumors and hairy roots on susceptible plant hosts. These plasmids also encode the genes required for production by the plant, and utilization by the bacteria of unique carbon conjugates called opines. Similarly, repABC plasmids in species of Rhizobium confer nodulation and nitrogen fixation when the bacteria are in symbiosis with a suitable plant host. Second, the repABC replicons have a broad host-range, and a subset of these plasmids encode a conjugative transfer system allowing these biologically relevant elements to transfer between and among species of bacteria. Perhaps the best-studied transfer system of the repABC plasmids is the Class I system composed of a type four secretion system encoded by the traI/trb operon and a DNA metabolism system encoded by the two tra operons. These operons are regulated by a quorum-sensing system involving three proteins: TraR, TraI, and TraM. TraR directly activates the transfer regulon but needs its ligand, an acyl-homoserine lacone quorum-sensing signal synthesized by TraI, to be active. One additional component, TraM, binds to TraR directly and inactivates the quorum-sensing protein when the signal for transfer is absent. The octopine-type Ti plasmids in A. tumefaciens strains 15955 and R10 are inducible for conjugative transfer by octopine because traR is the distal member of an operon inducible by the conjugative opine. However, a second non-functional allele of traR, called trlR, is present in the mannopine transport operon, an operon that is inducible by the opine mannopine. Based on the location and inducibility of trlR by mannopine, we hypothesized that there would be a functional allele of traR that is similarly located in a mannopine-inducible operon and that mannopine would induce transfer of a plasmid in a wild-type isolate of Agrobacterium. To this end we characterized and analyzed a collection of mannopine-utilizing field isolates for the ability of mannopine to induce transfer. We found five such isolates. Further characterization of the mannopine-utilizing plasmids in these strains indicated that these plasmids all are highly related. We analyzed and sequenced one such element, pAoF64/95. First, pAoF64/95 is not a virulence element; it does not contain the genes for virulence or a T-region. Instead pAoF64/95 is an opine-catabolic plasmid and encodes all of the genes for utilization of three of the four mannityl opines- mannopine, mannopinic acid and agropinic acid- as well as the agrocinopines. Indeed, strains harboring pAoF64/95 can utilize these three mannityl opines and are also sensitive to agrocin 84, an indication that the strain can utilize the agrocinopine opines. Second, an otherwise plasmid-less strain harboring pAoF64/95 transfers the mannopine-utilizing trait to a recipient when grown with mannopine. Moreover, mutational analysis of traR and traM encoded by pAoF64/95 suggests that the functions of TraR and TraM as activator and antiactivator are conserved. Finally, the genes involved in Class I transfer of pAoF64/95 are not organized as they are in Ti plasmids. For all repABC plasmids with Class I transfer systems, the traI/trb operon is always adjacent and divergently oriented to the repABC operon. In the Ti plasmids, the tra region along with traR and traM are located distantly from the trb-repABC region and more often are located near the genes for uptake and catabolism of the conjugative opine. Additionally, in the Ti plasmids known to be conjugative, traR is invariably located in an operon inducible by the conjugative opine. This organization of the genes for conjugative transfer we call Group I organization. However, in pAoF64/95, like the Ri plasmids of A. rhizogenes and many plasmids in species of Rhizobium, the location of the tra genes is contiguous with the trb-repABC region and traR is monocistronic, an organization we name Group II. Based upon these two modes of organization of plasmids with Class I transfer systems (Group I and Group II), we hypothesized that the component gene systems represent divergent evolutionary lineages. We assessed the evolution of the transfer, quorum-sensing, and replication and partition proteins and found that the quorum-sensing and transfer proteins form two clades that are consistent with the two modes of plasmid organization, indicating that the two organizational groups of plasmids are evolving divergently. Despite the obligatory linkage of the repABC operon with the traI/trb operon, the repABC proteins evolve independently of the transfer and quorum-sensing proteins. Moreover, while RepA and RepB coevolve, RepC evolves independently. Functional analysis indicates that TraR can dimerize and activate tra box-containing promoters of members within a clade, but not between clades. This is further evidence that proteins within, but not between clades are cross-functional. In contrast, the oriT regions are highly conserved and do not form two major clades. Consistent with the phylogeny, cloned oriT regions are processed and mobilized by members of either clade. We conclude that Group I and Group II plasmids diverged based upon where the cargo DNA is located and moreover that this divergence in organization extends to function

Molecular markers to study competition and diversity of Rhizobium

The research described in this thesis was directed to the development of molecular identification and detection techniques for studying the ecology of Rhizobium, a nitrogen- fixing bacterium of agricultural importance. Competition of inoculant strains with indigenous microbes is a serious problem in agricultural practice and was therefore addressed in this work using the developed tools. Furthermore, various molecular techniques have been applied to analyse rhizobial populations nodulating common bean and a new species was characterized.In this chapter the results obtained are summarized and potential future applications are discussed.Development of gusA - and celB -minitransposons and their use in rhizobial competition studiesThe use of marker genes in rhizobial competition studies is reviewed in Chapter 1. Specific attention is given to the gusA gene, encoding β-glucuronidase (GUS). This gene is a highly suitable marker for studying plant-microbe interactions due to the absence of GUS activity in plants and in most bacteria that are of relevance in agriculture. In Chapter 2 the construction of several GUS transposons containing the marker gene in combination with different regulation systems to be used for ecological and genetic studies is described. The minitransposon mTn5SS gusA 20 contains the aph promoter which was demonstrated to be expressed in a wide variety of Gram-negative bacteria (de Bruijn and Lupski, 1984) and its use in Rhizobium resulted in high-level constitutive expression. Transposon mTn5SS gusA 21 is similar to mTn5SS gusA 20, except that it contains a unique site for Spe I, a rare-cutting enzyme in bacteria with high G+C contents such as rhizobia (Sobral et al., 1991). The use of mTn5SS gusA 2I therefore may be instrumental for the genetic mapping of insertions. The tac promoter was used to drive the expression of the gusA gene in the transposon mTn5SS gusA 11 resulting in high GUS activity in the free-living state. The transposons with constitutive gusA expression are optimal for studying rhizosphere colonization and for studying nodule occupancy in young plants. In order to reduce any metabolic load due to GUS production, the mTn5SS gusA 10 transposon with regulated gusA expression was constructed. It contains the tac promoter in combination with the lacl q repressor gene and gusA gene expression is repressed until an inducer, such as IPTG, is added. This regulation should avoid possible effects on the ecological fitness, Two transposons carrying symbiotically activated gusA genes, mTn5SS gusA 30 and mTn5SS gusA 31, were made by using the nifH promoter of a Rhizobium and a Bradyrhizobium strain, respectively. The nifH gene encodes the Fecomponent of nitrogenase and is only expressed in symbiotic or microaerobic conditions (Fischer, 1994). These constructs are recommended for longer-term nodule occupancy experiments. Furthermore, a promoter-less GUS transposon,mTn5SS gusA 40, is described that should be of use for molecular genetic studies as well as for screening bacteria for their response to specific environmental conditions or signals. The developed transposons carry a gene conferring resistance tospectinomycin and streptomycin that proved to be an appropriate marker for many strains. Nevertheless, few strains with an endogenous resistance exist. Therefore, the development of additional transposons conferring an alternative resistance would be advantageous. This may be realized by inserting the developed GUS expression cassettes into minitransposons: containing other antibiotic (de Lorenzo et al., 1990) or natural resistance markers (Herrero et al., 1990). Chapter 2 also addresses also theapplication of the different transposons in studies on root colonization and nodule occupancy, while various GUS assays are described in detail.Potential effects on the fitness of a strain due to insertion of a GUS transposon were evaluated in Chapter 3. Only few data exist on the impact of foreign genes on the fitness of an organism (Doyle et al., 1995) and before using any marker system for ecological studies its ecological effects have to be studied rigorously. In the case of Rhizobium, it is essential that the nodulation behaviour and competitive ability are maintained. The competitive abilities, nodulation characteristics, and growth rates of five independent derivatives of R. tropici strain CIAT899 marked with the gusA gene on minitransposon mTn5SS gusA 10 were determined relative to the parent strain. Insertion of mTn5SS gusA 10 did not affect the nodulation or nitrogen fixation efficiency of the wild-type strain. Nevertheless, the competitiveness index of the different gusA derivatives relative to the parental strain CIAT899 varied between isolates. One isolate was less competitive than the wild-type strain in three independent experiments, while the other isolates proved to be either equally competitive or more competitive. The utilization of this methodology to assess competitivity resulted in highly significant calculations as all the nodules on each plant were analysed for nodule occupancy. The results showed that the insertion of mTn5SS gusA 10 may have an impact on the ecological behaviour of a strain, but derivatives indistinguishable from the parent strain can be obtained. A primary selection of marked strains is recommended, which may be achieved by coinoculating the parent and the marked derivative in a one to one ratio and ensuring that the proportion of blue nodules does not differ significantly from the expected 50%.Furthermore, in Chapter 3, the detection of dual nodule occupancy is discussed. The appearance of partially stained nodules in mixed inoculum treatments but not in single strain treatments led to the conclusion that these were due to mixed infections. This was confirmed by nodule isolation and plating experiments. Partially stained nodules were also observed by Krishnan and Pueppke (1992) who reported that nodules were occupied by either a lacZ- marked or a non-marked R. fredii strain and X-gal was used for detection. However, the lacZ marker system has several disadvantages due to high background activity in plant and rhizobia whereas the gusA marker gene can be used to readily detect dual nodule occupancy on plant.A new marker gene system based on the celB gene is presented in Chapter 4. The celB gene has been isolated from the hyperthermophilic archaeon Pyrococcus juriosus and it encodes a thermostable and thermoactive β-glucosidase with a high β-galactosidase activity (Voorhorst et al., 1995). The latter enzyme activity can be used for the detection of rhizobia as endogenous background activity in plants as well as in bacteria can be easily eliminated by a heat treatment. Moreover, cheap histochemical substrates are available to determine β-galactosidase activity. The E. coli β-galactosidase gene, lacZ , has been used to monitor engineered soil bacteria (Drahos et al., 1986; Hartel et al., 1994) but was only found to be appropiate when used with Lac- bacteria. Transposons containing the ce1B gene were constructed in E. coli , based on the existing gusA transposons. The first transposon, mTn5SS celB 10 , contains the tac promoter which is regulated by the lacI q gene product and should reduce any metabolic stress to the marked strain due to marker gene activity. Transposon mTn5SS celB 31 carries celB expressed from a Bradyrhizobium nifH promoter and is active in nitrogen-fixing legume nodules. A third celB minitransposon, which contains the marker gene constitutively expressed is described elsewhere (Sessitsch et al., submitted). The celB marker gene system has several advantages in rhizobial competition studies over conventional techniques as the assay is simple to perform and the histochemical substrates are cheap. However, the greatest advantage is that gusA and celB marked strains can be localized simultaneously on a plant and a combined gusA/celB assay will enable studies of multi-strain rhizobial inocula competing with indigenous rhizobial populations. Although simultaneous detection of differently marked strains has been reported (Thompson et al., 1995; Bauchrowitz et al., 1996), the celB gene encoding the thermostable marker is better suited for double staining. In addition, a procedure has been described allowing detection of gusA and celB marked strains on plates (Sessitsch et al., submitted). In Chapter 4 the application of the celB marker gene is demonstrated for Rhizobium. However, because of the wide host range of the Tn5 based transposons and the portable expression signals this marking system is suitable for use in a variety of Gram-negative bacteria.The advantages of the different gusA and celB marker gene cassettes are discussed in Chapter 1, but also other reporter genes and their applications in studies on microbial ecology are presented. Moreover, the development of a GUS Gene Marking Kit is reported. This kit was made particularly for agronomists and microbiologists in developing countries who are not familiar with molecular techniques and who do not have the resources to establish this methodology in their laboratories. Meanwhile, a CelB Gene Marking Kit is also available that can be used either in combination with or instead of the GUS Gene Marking Kit (FAO/IAEA, 1992-1997).Ecology of rhizobia nodulating common beanIn the fields around the Seibersdorf laboratory common bean has not been cultivated during the last decades but is well nodulated. In earlier studies, common bean rhizobia populations in this soil have been found to be very competitive in nodulation (see Chapter 1). They were shown to outcompete R. tropici strain CIAT899 when inoculating Phaseolus vulgaris at an inoculation level of 10 5cells per seed. When increasing inoculation to 10 8cells per seed, 65% of the nodules were still occupied by the native strains. In Chapter 5, rhizobial populations isolated from common bean nodules grown in the Seibersdorf soil were characterized. Molecular methods targeting the whole genome such as PCR with repetitive primers were used, and specific chromosomal loci such as the 16S rRNA gene or the 16S- 23S rDNA intergenic spacer were analyzed. Plasmid profiles and Southern hybridization with a nifH probe gave information on symbiotic regions. In addition, the nodulation host range was determined. Two distinct groups were found, one of them was classified as R. etli according to the RFLP analysis of the 16S rRNA gene and because of the presence of three copies of the nifH gene. The members of the second group could not be assigned to any recognized common bean nodulating Rhizobium species, i.e. R. leguminosarum bv. phaseoli, R. etli and R. tropici, but showed high similarity to Rhizobium sp. (Phaseolus) strain R602sp isolated in France (Laguerre et al., 1993). Isolates of this group also formed nodules on cowpea, Leucaena and Gliricidia. For a long time, R. leguminosarum bv. phaseoli was believed to be the only microsymbiont in Europe, but recently R. tropici and two new species have been found in French soils (Laguerre et al., 1993; Amarger, 1994). These studies and the results obtained in Chapter 5 indicate that strains originating in Mesoamerica could establish well in European soils. However, diversity was not high among the Austrian isolates due to the long absence of the host plant in this soil.The focus of Chapter 6 is on the taxonomy and phylogeny of the Austrian isolates showing high similarity to Rhizobium sp. R602sp. A Mexican common bean isolate, FL27, was included in this study since Laguerre et al. (1993) found the partial 16S rRNA gene sequence of R602sp to be identical to FL27. Sequence analysis of the 16S rRNA gene, determination of the copy number and heterogeneity of ribosomal genes, plasmid profiles and DNA-DNA hybridization resulted in valuable taxonomic information on these strains. Based on these results it was proposed that these strains belong to a new species that was named R. pueblae sp. nov., referring to the state Puebla, Mexico, where FL27 was isolated. The Mexican, French and Austrian isolates showed very similar 16S rDNA sequences with a maximum of two nucleotide substitutions. Comparison of the 16S rDNA sequences with those of other bacteria revealed highest similarity to R. leguminosarum strain IAM 12609, R. sp. OK50 and to R. etli. Although phylogenetic dendrograms always positioned R. pueblae sp. nov. strains in the vicinity of the above-mentioned species, the new species was found to belong to a lineage different from those of described Rhizobium species. The whole DNA relatedness among the European isolates was very high but showed lower levels with FL27, probably due to the presence of different plasmids. The DNA homology to other bean-nodulating species was very low. R. pueblae sp. nov. strains possess at least three copies of the 16S rRNA gene and the ribosomal gene organization is different to other species. Despite the high competitive ability of some strains in the Seibersdorf soil, little is known on the agronomic value of this species.Concluding remarksTo improve biological nitrogen fixation, adapted efficient nitrogen-fixing plant genotypes, effective rhizobial inoculants and appropiate agricultural management practices are needed. Consequently, plant breeders, microbiologists, soil scientists, agronomists as well as farmers have to cooperate in order to achieve this goal.The selection of superior sources of natural plant genetic variability and plant breeding in the presence of rhizobia instead of applying nitrogen fertilizers may lead to the identification of high-fixing lines. Other desirable traits such as disease resistance or stress tolerance could be transferred by appropiate breeding methods or by genetic engineering. Soils, in which legumes are cultivated, vary greatly and can be opposed to various environmental stresses such as low pH or high temperature. Efforts have been undertaken to develop appropiate plant genotypes whereas for a long time the stress tolerance of inoculant strains has not been considered. Inoculant strains have been recommended based on good symbiotic performance in a particular environmentbeen recommended based on good symbiotic performance in a particular environment while the soil status or the agroecological zone of the final application has not been taken into account. The vast genetic pool of natural soils containing not yet identified strains and species can provide a variety of inoculant strains that may show better performance in the field. A strong correlation between the indigenous population size and the nodule occupancy of the inoculant strain has been established (Thies et al., 1991). However, the effect of the diversity of indigenous rhizobia on competition has not been determined. Probably, different strategies are needed in order to outcompete highly dominant field isolates or to achieve successful competition of an inoculant strain with a variety of different indigenous strains that are present in low numbers. In addition, inoculation practices have to be developed that are convenient for the farmer and that allow distribution of the introduced strain into the entire rooting zone.It seems that the various aspects important for efficient nodulation and nitrogen fixation are presently not linked sufficiently. A more rational selection of efficient and competitive strains could be realized when a database existed containing data on soil properties, environmental conditions, rhizobial diversity and population size, as well as on the competitive ability and effectiveness of rhizobial strains in combination with particular plant genotypes. Furthermore, these data could be of use for the development of new strains or plants by genetic engineering.In this thesis, the development of new methods to assess rhizobial competition is presented. These marker gene-based techniques are appropiate for the large-scale screening of inoculant strains but can be also used for genetic analysis of a variety of Gram-negative bacteria. Molecular methods have facilitated the analysis of strains nodulating common bean and resulted in the description of a new Rhizobium species that includes strains with possible beneficial properties

Determinação do papel das proteínas NodD1 e NodD2 na ativação dos genes nod em Bradyrhizobium elkanii

Na simbiose entre soja e bactérias diazotróficas ocorre o processo de Fixação Biológica de Nitrogênio (FBN) no nódulo. Nessa associação há uma comunicação constante, pois a liberação de exsudatos pela planta é percebida pela bactéria, que, então, produz lipo-quito-oligossacarídeos, chamados fatores Nod (FNs), moléculas de sinalização na nodulação. Esses FNs são produtos da atividade dos genes nod bacterianos. Os genes nod regulatórios codificam fatores de transcrição (FTs) responsáveis pela regulação dos genes nod estruturais, que codificam enzimas para a biossíntese dos FNs. Na região promotora dos genes nod atua o FT NodD, que se liga em sequências específicas conservadas, chamadas nod boxes. Essa comunicação abrange a regulação de vários genes, como os genes nod regulatórios nodD1 e nodD2 de Bradyrhizobium elkanii SEMIA 587. Bradyrhizobium elkanii são bactérias Gram-negativas, fixadoras de nitrogênio, usadas comercialmente para a produção de inoculantes na agricultura, devido à eficiência do processo de FBN na simbiose, o que justifica o interesse em torno dessa interação. No micro-organismo modelo Bradyrhizobium diazoefficiens USDA110, as proteínas NodD1 e NodD2, sintetizadas a partir dos genes nodD1 e nodD2, respectivamente, têm ações contrárias. Enquanto NodD1 age como um regulador transcricional positivo dos operons nod e regula a sua própria transcrição, NodD2 atua como um regulador negativo desses operons. Isso despertou o interesse de investigar o papel dessas proteínas em B. elkanii SEMIA 587, com a finalidade de testar se elas apresentam funções semelhantes àquelas demonstradas na estirpe padrão USDA110. Sendo assim, o objetivo do trabalho foi contribuir, através da aplicação de diferentes metodologias, para um melhor entendimento em relação à regulação dos genes nod em B. elkanii SEMIA 587. Para tanto, fragmentos de DNA contendo os genes nodD1 e nodD2 dessa bactéria foram clonados nos vetores pGEM e pGEX-4T2, para produção das proteínas recombinantes em Escherichia coli BL21, a fim de realizar experimentos de retardamento em gel para comprovar a ligação das proteínas NodD1 e NodD2 nos nod boxes identificados nas regiões reguladoras dos respectivos genes, bem como determinar a eficiência de cada ligação. A expressão de ambas as proteínas em E. coli foi visualizada em gel SDS-PAGE e as proteínas estão em fase de purificação. Clonagens posteriores realizadas usando o sistema Gateway® para inserção dos genes nodD1 e nodD2 no vetor de clonagem pENTR foram confirmadas por sequenciamento e recombinadas a dois vetores de levedura (pDEST-22 e pDEST-32). Esses procedimentos visam à execução de ensaios de duplo híbrido para verificar se as proteínas NodD1 e NodD2 são capazes de formar heterodímeros funcionais.The process of Biological Nitrogen Fixation (BNF) in the symbiosis between soybean and diazotrophic bacteria occurs in the nodule. There is a constant communication in this association. Plant exudates are perceived by the bacteria that produce lipo-chito-oligosaccharides (LCOs), called Nod Factors (NF), signaling molecules in nodulation. These NFs are product of bacterial nod genes activity. Transcription Factors (TFs) are encoded by regulatory nod genes, responsible for structural nod genes regulation. These structural nod genes encode enzymes for NFs biosynthesis. TF NodD acts in the promoter region of nod genes and binds to specific conserved sequences, called nod boxes. The Bradyrhizobium elkanii SEMIA 587 regulatory nod genes nodD1 and nodD2 are involved in this communication. B. elkanii are diazotroph gram-negative bacteria used commercially as inoculants source in agriculture, due to the efficiency of the BNF process in the symbiosis, which justifies the interest regarding this interaction. NodD1 and NodD2 proteins are synthesized by nodD1 and nodD2 and display contrary actions in the model strain B. diazoefficiens USDA 110. While NodD1 acts as a positive transcriptional regulator of nod operons and regulates its own transcription, NodD2 acts as a negative regulator of these operons. These proteins roles in B. elkanii SEMIA 587 aroused the interest of investigating, in order to test whether they have similar functions to those demonstrated in the model strain. Therefore, the goal of this work was to contribute, through the application of different methodologies, to a better understanding regarding nod genes regulation in B. elkanii SEMIA 587. B. elkanii SEMIA 587 nodD1 and nodD2 coding sequences were cloned into pGEM and pGEX-4T-2 vectors. The recombinant proteins were expressed in Escherichia coli BL21 in the order to carry out gel retardation experiments to verify purified proteins binding to the nod boxes identified in nod promoters, as well as the efficiency of each binding. The expressed proteins in E. coli were visualized by SDS-PAGE and are undergoing purification. Subsequent cloning was realized with the Gateway® system for nodD1 and nodD2 insertion into pENTR vector. The constructs were confirmed by sequencing and recombined to yeast vectors (pDEST-22 and pDEST-32).These procedures aim to perform two-hybrid assays to verify if NodD1 and NodD2 are capable of forming functional heterodimers

Integrated functional anlaysis of quorum-sensing in the rice pathogenic bacterium Burkholderia glumae

Quorum sensing (QS) is a cell-to-cell communication mechanism that allows bacterial cells to collectively behave like a multicellular organism. It regulates the expression of toxoflavin, one of the major virulence factors of the rice pathogen, Burkholderia glumae. The QS system of B. glumae is mediated by the core genes, tofI and tofR. N-octanoyl-L-homoserine lactone, the primary QS signal molecule of B. glumae, is synthesized by tofI and binds to the cognate receptor tofR at the quorum point. However, tofI and tofR null mutants produce toxoflavin in certain growth conditions, indicating the presence of tofI- and tofR-independent pathways for toxoflavin production. The present study identified regulators required for the tofI- and tofR-independent pathways, including flagella transcriptional activator, diguanylate cyclase, O-antigen polymerase family protein, QsmR QS-dependent master regulator and one hypothetical protein with its encoding gene located upstream of toxJ (encoding toxoflavin production activator). A novel QS regulatory element, tofM, was identified as a positive regulator of pathogenicity and a putative modulator of tofR in B. glumae. RNA-sequencing was also performed to investigate the QS regulon and medium condition-dependent gene expression in B. glumae. A large collection of target genes and noncoding RNAs was detected by comparative transcriptome analysis. From a comparison of the transcriptional profile of the wild type (336gr-1) and quorum sensing mutants grown on solid and liquid media, it is postulated that an alternative global regulator is activated to compensate for the dysfunction of AHL QS on solid medium