Classical and omic approaches to the analysis of acid-stressed alfalfa nodulating rhizobia.

DRAGHI, W. O.; DEL PAPA, M.F.; PISTORIO, M.; LOZANO, M. J.; GIUSTI, M. A.; TORRES TEJERIZO, G. A.; HELLWEG, CH.; WEIDNER, S.; NIEHAUS, K.; PUHLER, A.; LAGARES, A.

Los Cocos, Córdoba, Argentina

Congreso; XXIII Reunión Latinoamericana de Rizobiología; 2007

Asociación Latinoamericana de Rhizobiología

Alfalfa-nodulating rhizobia present in agricultural soils from Argentina and Uruguay: Diversity, acid tolerance, symbiosis, and genotypic characteristics.Soil acidity is a worldwide distributed stress-factor (von Uexküll y Mutert, 1995) that severely restricts rhizobial survival and symbiosis in agricultural soils (Glenn y Dilworth, 1994). Thus, diverse experimental approaches have been used to increase knowledge on the survival, persistence, and biology of rhizobia in the acid underground.In Argentina, large areas of arable lands have progressively acidified over the last 10 to 20 years, where the continuous cultivation over time without crop rotation are thought to be main factors that favored soil acidification.In central Argentina a gradual acidification of a significant proportion of cultured soils has been observed during the last 20 years. Such observation, together with the known sensitivity of Sinorhizobium mellioti to the hydrogen ions moved our laboratories to investigate the kind and characteristics of the alfalfa-nodulating rhizobia present in the acidic soils from Argentina and Uruguay. As a result, Del Papa et al. (1999) identified in the local soils the presence of two kinds of rhizobia: a major group represented by S. meliloti, and a second group geno/phenotypically related to the previously described Or191 isolate recovered from a soil of Oregon, USA. The Or191-like rhizobia recovered from our soils showed to be promiscuous, markedly acid tolerant, inefficient to fix atmospheric nitrogen, and highly competitive to nodulate alfalfa (Del Papa et al., 1999; Segundo et al., 1999; Wegener et al., 2001). Such evidences prompt us to investigate more deeply the symbiosis and ecology of such rhizobia in order to estimate both their evolution in local soils, and the impact on the population of efficient S. meliloti. Thus, we analyzed the behavior of S. meliloti and Or191-like rhizobia in sterile microcosms containing acidic and neutralized soil samples, both, with and without alfalfa plants (Del Papa et al., 2003). Although the acid-tolerant rhizobia showed a slightly better survival during the first months in acid soil (pH=5.6), none of the strains could be detected 2 months after inoculation (bacterial counts were below 10 colony forming units (cfu)/30 g of soil). The inclusion of two alfalfa plants/glass tube with soil, however, supported the persistence of both types of rhizobia at pH 5.6 for over 2 months with counts higher than 9 x 106 cfu/30 g of soil. Remarkably, in the presence of alfalfa the cell densities reached by S. meliloti were higher than those reached by the Or191-like rhizobia, which started to decline 1 week after inoculation. Although more acid-sensitive in the culture medium than the Or191-like rhizobia, in the presence of the host plant the S. meliloti strains showed to be better adapted to the free-living condition, irrespective of the pH of the soil (Del Papa et al., 2003). The collected evidence provide direct proof that the critical pH for growth in culture media is not the only factor influencing rhizobial survival under acid stress in the soil. The differential behavior between S. meliloti and the acid-tolerant and inefficient Or191-like rhizobia is consistent with a report by Barber (1980) on the relevance of the host plant to keep a field population of S. melilotiagainst inefficient and acid-tolerant isolates which grew up in the laboratory at pH 5.0. It is thus possible that the promotion of acid-sensitive and efficient S. melilotiby alfalfa in the field might have been partially due to a differential ability of these rhizobia to benefit from the presence of the host root. It will of interest the evaluation of non-host plants in similar experiment to elucidate if the positive effect on S. meliloti it is or not a general phenomenon extended to other plants. In any case, the presence of alfalfa (in the laboratory, and in the field ) appears to be an important biotic factor to favor the establishment of S. meliloti over the Or191-like rhizobia. Unfortunately, it is not ease to establish the presence real number of Or191-like rhizobia in agricultural soils since: a) the topology of the infections by these rhizobia results in their poor detection (late infections), and b) there is a no knowledge on the distribution of these rhizobia trough the different soil horizons. In a field assay we observed that 90% of isolates recovered from nodules located between 30-120 cm deepness were Or191-like rhizobia. Such results account for the need of a reliable assay to monitor the presence and number of Or191-like bacteria in agricultural fields, in order to trace the distribution and dynamics of these bacteria in productive soils. Though we developed a PCR assay to specifically detect the Or191-like rhizobia, the achieved sensitivity (104 genomes/PCR reaction) makes the protocol unsuitable to be used with DNA samples extracted from soils.The interest of our group to characterize the Or191-like bacteria relies on several peculiar properties of such bacteria: a) they are present in diverse geographic locations, b) they are inefficient to fix atmospheric nitrogen, c) they are markedly acid tolerant, and d) they are highly competitive to nodulate alfalfa against S. meliloti. Interestingly, while the 16S rDNA of these rhizobia are related to the homologs from current bean/peas/clover-nodulating rhizobia, their nifH and nodC genes are phylogenetically related to the S. meliloti homologs (Laguerre et al., 2001). Such evidences are of particular interest considering the known capacity of Or191-like rhizobia to nodulate common bean, alfalfa and Leucaena leucocephala. The remarkable specificity of the alfalfa-S. meliloti interaction together with its strict requirement for a C6 sulfation at the reducing end of the Nod factors, keep yet open the following questions: a) are Nod factor produced by these rhizobia sulfated?, or b) is there a chemically different modification/structure in the signal molecules that allow for the symbiosis to occur? We have recently shown that the Or191-like rhizobia carry a single copy of a nodH ortholog in, which encodes for an active sulfotransferase with ability to complement both Nod factor sulfation and symbiosis in S. melilotinodH mutants (Del Papa et al., 2007). Interestingly, the nodH sequence as well as the immediate synteny support a close relationship with the homolog region in S. meliloti. In agreement with evidence from other authors, the nodH sequence analysis strongly supports the idea that the Or191-like rhizobia most likely represent a genetic mosaic resulting from the horizontal transfer of symbiotic genes from a sinorhizobial megaplasmid to a not yet clearly identified ancestor. Thus, current observations taken together suggest that either i) the Or191-like rhizobia have not yet acquired the complete set of functions to became a full legume symbiont (this also is supported by structural data of deficiently infected nodules) or ii) they are a residual form of formerly active symbionts, which is unlikely in terms of the coevolution of rhizobia with their host plants. Whatever the case, the biology and the interaction of these bacteria with the environment severely conditioned the genetic diversity of the Or191-like germ plasm irrespective of their distant genetic location. The absence of a known true host plant for these bacteria, their capacity to induce inefficient root nodules in different legumes (temperate and tropical), and the unusual absence of detectable genetic polymorphisms among independent isolates (Wegener et al. 2001) make them an interesting target of analysis to improve our knowledge on the rhizobial evolution strategies. With a genetic structure that preserves clear evidences of horizontal gene transfer events (likely via a sinorhizobial symbiotic megaplasmid), the Or191-like isolates represent a valuable system to better understand the underlying mechanisms of rhizobial diversification and evolution. How do the Or191-like rhizobia preserve their extremely low genetic variation in such a genomic context? (Wegener et al. 2001). The molecular (and functional) characterization of new genetic markers from the Or191-like rhizobia, together with upcoming information from new, fully sequenced rhizobial genomes, will be key elements toward a deeper tracing and reconstruction of the Or191 genomic structure and genealogy.Omic approaches to the analysis of rhizobial responses to the acid medium.A most used approach to the analysis of rhizobial genes involved in acid tolerance has been the transposon mutagenesis and search for those clones displaying acid sensitive phenotypes (Glenn et al., 1999). In such a way, several act (for acid tolerance) genes have been identified in S. medicae. However, it has not yet been elucidated the precise biochemical function/s of any of the act translation products. It is well known that the sinorhizobial profile of acid tolerance results from the individual contribution of a number of different genes (multigenic phenotype), where none of them play a dominant role in the expression of the strain acid tolerance. In addition, there is no currently any evidence of act allelic variants that could account for the more tolerant phenotypes. Such a situation presents unfortunately a modest basic knowledge, with poor practical potential towards the rational improvement of acid tolerance in rhizobia. Thus, in the last years omic approaches have been considered a valuable new tool to try getting a deeper insight into the biochemical responses to acidity (S. medicae, Reeve et al., 2004; S. meliloti, our laboratory unpublished results). With that aim we analyzed the differential proteomes of S. meliloti 2011 grown in chemostat at pH 7.0, and 6.1 under continuous cultivation. The 2D-SDS-PAGE peptide fingerprint UV-MALTI-TOF analysis of cytoplasmic proteins of S. meliloti 2011 allowed us for the identification of several markers differentially expressed at each pH condition. In parallel, we ran the transcriptome analysis of the same bacteria: a) to evaluate the degree of correlation between expression of markers at the proteome and transcriptome level, and b) to have a gene expression screening at the complete genome level. The analysis using glass microarrays showed a very high correspondence between the analyzed transcriptomes (from rhizobia grown at pH 7.0; and 6.1) and the differentially expressed proteome markers. The omic analysis showed that: a) while cells growing at pH 7.0 cover a diverse set of metabolic functions, cell growing under acidity concentrate cell activities in the translation machinery, preservation and biosynthesis of the cell envelope, and energy production; b) while aminoacid metabolism appeared intense at pH 7.0, markers associated to carbohydrate metabolism are over-expressed under acidity, c) as expected from the subcellular location ofthe protein fraction analyzed, markers associated to the cell envelope were under represented (motility, lipid and cell envelope metabolism, membrane-associated respiratory components). d) transcription factors were under represented (inappropriate pH range during the IEF?, few molecules/cell?,). Currently, a collection of S. meliloti mutants generated by transposon STM (signature tagged mutagenesis) (Pobigaylo et al., 2006) and affected in individual protein markers of interest are being evaluated in their growth and symbiosis at different pH. The omic approximation to changes in the metabolism as a consequence of the external pH provided a reach view on how metabolic functions are adjusted by rhizobia against a severe acid challenge. The maintenance/renewal of cell structures, and the preservation of the machinery of energy production appeared as the central priorities for the stressed rhizobial cells. We have now initiated the careful evaluation of each differential marker to know which of them (if any) affect growth and symbiosis in acid media. The availability of such information is expected to provide new and useful targets to attempt the yet difficult manipulation of acid tolerance in S. meliloti.