The Symbiosis Interactome: a computational approach reveals novel components, functional interactions and modules in Sinorhizobium meliloti
- Ignacio Rodriguez-Llorente1,
- Miguel A Caviedes1,
- Mohammed Dary1,
- Antonio J Palomares^1,
- Francisco M Cánovas2 and
- José M Peregrín-Alvarez2, 3Email author
© Rodriguez-Llorente et al; licensee BioMed Central Ltd. 2009
Received: 29 January 2009
Accepted: 16 June 2009
Published: 16 June 2009
Rhizobium-Legume symbiosis is an attractive biological process that has been studied for decades because of its importance in agriculture. However, this system has undergone extensive study and although many of the major factors underpinning the process have been discovered using traditional methods, much remains to be discovered.
Here we present an analysis of the 'Symbiosis Interactome' using novel computational methods in order to address the complex dynamic interactions between proteins involved in the symbiosis of the model bacteria Sinorhizobium meliloti with its plant hosts. Our study constitutes the first large-scale analysis attempting to reconstruct this complex biological process, and to identify novel proteins involved in establishing symbiosis. We identified 263 novel proteins potentially associated with the Symbiosis Interactome. The topology of the Symbiosis Interactome was used to guide experimental techniques attempting to validate novel proteins involved in different stages of symbiosis. The contribution of a set of novel proteins was tested analyzing the symbiotic properties of several S. meliloti mutants. We found mutants with altered symbiotic phenotypes suggesting novel proteins that provide key complementary roles for symbiosis.
Our 'systems-based model' represents a novel framework for studying host-microbe interactions, provides a theoretical basis for further experimental validations, and can also be applied to the study of other complex processes such as diseases.
Plant-microbe interactions play an important role in agriculture and a lot of effort has been dedicated to analyse these interactions in detail. One of these interactions is the Rhizobium-Legume symbiosis, a process that allows the growth of the plant in the absence of externally supplied nitrogen. This is a well studied agronomically important process that is also used as a model to study general genetic aspects of plant-microbe interactions [1, 2]. Rhizobial bacteria and legumes have evolved complex signal exchange mechanisms in which a lot of genes are involved . To probe this complexity further we chose to study the model rhizobial symbiont genome Sinorhizobium meliloti . S. meliloti is a model bacterium that can engage in a symbiotic interaction by infecting the roots of members of the genera Medicago and Melilotus, being the S. meliloti-Medicago truncatula interaction the model system for indeterminate type nodules .
The sequencing of hundreds of complete genomes from diverse species is having a tremendous impact on our understanding of biology by enabling the identification of all proteins and the analysis of their function. Despite the vast body of literature about the Rhizobium-legume interaction there have been no systematic large-scale attempts to identify its components and function using a systems biology perspective, and most studies have been restricted to the analysis of individual proteins. However, biological functions results from the interactions of proteins so that understanding the network of biological linkages utilizing functional genomics information is becoming a hot topic in current research projects [6–11]. The main advantage of creating these networks lies in the ability to understand biological processes from a system level perspective. This would ideally require the application of computational and experimental techniques to combine experimental observations of protein-protein interactions (PPIs) and computational predictions derived from different data sources. To date a variety of methods have been developed to derive large scale networks of PPIs for a variety of organisms. These range from experimental methods such as yeast two-hybrid screens, or tandem affinity purification coupled with mass spectrometry [6, 8, 9, 12], to computational methods such as genome context methods [13, 14]. The integration of these types of data helps to provide a complete overview of gene networks of high value for characterizing many biological processes, and ultimately, for understanding the basis of host-microbe interactions including diseases [15–17]. However, experimental information is sometimes missing and deriving gene networks from different computational approaches is not an easy task. Computational predictions such as those obtained by applying genome context methods usually measure functional interactions between proteins. The assumption is that proteins are most likely to interact if: a) their proteins are either present or absent together across multiple genomes (the Phylogenetic Profile method) ; b) a gene fusion event occurred in other species (the Gene Fusion or Rosetta Stone method) [19, 20]; c) the genes are in physical proximity (the Gene Cluster method) ; or d) the genes are conserved in physical proximity and in phylogenetically distant genomes (the Gene Neighbor method) . These methods have the advantage over experimental methods and other computational methods based on protein conservation such as Interologs  or literature mining , that they are not biased towards well studied or conserved proteins or interactions . Therefore, genome context methods are able to highlight organism-specific features since they just rely on genome structure. The outputs derived by these methods can be computationally integrated in order to reconstruct network models of the relations between genes [13, 14]. Data integration for inferring protein associations is advantageous for two main reasons. First, combining data from diverse studies and methods generates data sets of higher quality, and second, integration effectively captures different aspects of organism's biology [25–27]. Further exploiting the topological properties of these networks, clustering algorithms have subsequently allowed proteins to be organized into discrete interconnected units known as functional modules representing either protein complexes or biochemical pathways [28, 29]. In addition, integration of additional functional and comparative genomics data sets are further providing insights into how these modules and their components are co-ordinated and how they may have evolved [9, 30].
Due to the scarcity of large-scale experimental assays aiming to study this important microorganism-host interaction, we chose to apply a systems-based computational approach to evaluate and organize our current knowledge about this complex biological process further. Here, we first reconstruct an extensive and accurate functional network in S. meliloti by integrating the functional associations present in the two well known databases PROLINKS  and STRING  (see methods). These databases host functional linkage predictions obtained mainly by the four different computational genome context approaches described above. Second, we present an analysis of the 'Symbiosis Interactome' (a detailed functional interaction network of the proteins involved in the S. meliloti-Legume symbiosis) by first mapping proteins known to be involved in symbiosis on top of the S. meliloti network, and secondly, by extending this resulting network by means of a novel method, referred here as 'phenotypic profiling', which is further extended by incorporating data from the computational prediction of functional modules. This computational approach potentially revealed the complex interplay of functional interactions between proteins involved in S. meliloti-Medicago symbiosis providing a way to expand the current understanding of symbiosis by enabling hypothesis generation based on our predicted network. Finally, since one of the major advantages of constructing PPI networks is the ability to predict functions for proteins based on their association with well known proteins, we identified and tested the functions of candidate proteins and demonstrate that novel Symbiosis Interactome proteins can still be discovered despite the many decades of effort dedicated to study this important and complex biological process.
The S. meliloti network
The S. meliloti network demonstrated to have properties of scale-free network [see Additional file 1] like other biological networks, the Internet and social networks . Most of the proteins had few interacting partners, where a subset of 'hubs' form a far greater number of connections. Scale-free networks are predicted to be robust against random node removal but vulnerable to hub removal, a property that might be preserved across evolution . Furthermore, the average clustering coefficient (ACC) of the intersection network and its diameter or average shortest path length (L) (see methods) suggests properties of a small-word network (L ~ Lrandom, ACC >> ACCrandom) typical of intracellular network in which the nodes are connected when they are involved in the same biological processes .
Prediction of functional modules
While defining accurate PPI networks is important, the ultimate goal of interactome analyses is to identify the functional modules in these networks, that is, proteins with related functions that tend to be clustered into highly interconnected subnetworks [10, 33, 34], and to validate them. To assess if our network could also be clustered into such subnetworks, we first tested the capacity of the S. meliloti network to form groups of highly interconnected proteins, as indicated by its Average Clustering Coefficient (ACC) (see methods). Indeed, the ACC of the S. meliloti network is much higher (ACC = 0.41) than other large-scale E. coli (ACC = 0.15  and ACC = 0.08 ) and H. pylori  (ACC = 0.02) experimental, and random networks (ACC = 0.0002) suggesting the organization of the S. meliloti network in functional modules.
The Symbiosis Interactome network
We first undertook an exhaustive literature-search analysis to identify and compile a list of bacterial proteins whose role in the symbiosis Rhizobium-Legume has been widely studied (Additional file 2 and methods). These proteins were classified as "classical-known" proteins in different categories according to the stage of symbiosis they are involved in.
Prediction of functional annotation and stage of symbiosis
A major goal for many functional genomics and proteomics projects is the generation of accurate functional information for every gene and its product. Although tremendous progress has been made through the application of such systematic studies, we found that within the S. meliloti proteome 3,376 (54%) proteins were not assigned to a functional category according to COGs, 290 (5%) have been assigned category S (function unknown), and a further 307 (5%) proteins have only been assigned into category 'R' ('general function prediction'). There has been recent progress in the development of novel methods of functional inference based on network connectivity . The availability of our S. meliloti functional network thus provides a valuable resource for future studies aimed at predicting the functions of these high number of functionally 'orphan' proteins. In order to test the ability of our functional network to accurately infer reliable functional annotations and the stage of symbiosis where components of the Symbiosis Interactome may participate, we investigated a basic network-based approach based on functional category membership within predicted functional modules. To provide estimates of the accuracy of functional modules on inferring reliable functional annotations, we applied a cross-validation procedure to predict functional annotations (see methods). We were able to identify correct annotations for 87%–100% of the proteins contained in modules depending on the stringency of COGs category assignments (see methods and Fig. 3c). The accuracy of this type of functional module predictions has been found to be superior to other methods based merely on direct interacting partners [Peregrín-Alvarez JM, Xiong X, Su C, Parkinson J: The modular organization of protein interactions in Escherichia coli, submitted]. These findings highlight both the quality of the network and the predicted functional modules for hypothesis generation and future experimental validation.
Based on these results, module 266, for example, includes three proteins Q92QS6 (Smc01792), Q92QS4 (SMc01794) and Q92VP9 (Smb21071) [see Additional file 2]. The first two proteins are involved in M (cell wall/membrane/envelope biogenesis) while the third one has no COGs category assignment. We therefore predict the latter is potentially involved in this biological process. Furthermore, interestingly, we correctly identify the stage of symbiosis for 92%–100% of the proteins contained in modules depending on stringency (see methods and Fig. 3c). Again based on these promising results, module 208, for example, includes two nodulation proteins: nodP2 (Smb21223) and nodQ2 (Smb21224); and the novel protein Q92VH5 (SMb21225) [see Additional file 2], therefore, being tempting to speculate the participation of the latter in nodulation.
The conservation and evolution of the Symbiosis Interactome network
To investigate the conserved nature and evolution of our predicted Symbiosis Interactome network, the classical-known and novel Symbiosis Interactome components were classified into different node ages according to their phylogenetic distribution (see methods). A total of 313 (~ 68%) proteins were classified as old nodes (with broad phenotypic profiles (i.e with homologs in 7 or 8 phenotypic categories) suggesting an old evolutionary origin for symbiosis [8, 46]. Furthermore, of the 92 classical-known proteins previously identified as components of the Symbiosis Interactome 62 (~ 67%) had homologs with distantly related genomes, indicating that these highly conserved proteins were a valid system from which to derive a model of symbiosis. In addition, highly conserved genes tend to involve essential genes [8, 9]. Since most of the genes known to be involved in symbiosis are highly conserved [see Additional file 2] this suggests that these genes could be essential for organism's survival or at least determinant for symbiosis. Indeed, many of the novel genes predicted by our approach are missing from a S. meliloti mutant collection recently published  (data not shown) suggesting an essential role for many of these novel genes. It has also been shown that nodes with high network connectivity tend to be essential nodes [8, 9, 15]. Since most of the 'classical-known' and other novel Symbiosis Interactome proteins have multiple interacting partners (315 (~ 68%) and 341 (~ 74%) proteins using the Symbiosis Interactome and the complete intersection S. meliloti network, respectively, interact with more than one protein in the network) (see methods), this suggests that these proteins may indeed have a key role in this important biological process. It follows from these findings that the number of interactions of the Symbiosis Interactome proteins are positively correlated with its conservation [see Additional file 1] supporting a model of evolution of the Symbiosis Interactome from core components by adding additional ones over time .
M. sativa plants were inoculated with S. meliloti strains mutated at these genes, using S. meliloti 1021 as control wild-type strain (see methods). We could not observe any difference in nodulation phenotypes between plants inoculated with the strain mutated in Q92TC2 and the 1021 control strain (Fig. 5b). On the other hand, differences in nodulation were observed when plants were inoculated with the other mutants. A 20–30% decrease in nodule number (depending on the experiment these are maximum and minimum values) was observed in plants inoculated with the strain mutated in etfB1, and a 20–25% decrease in nodule number in plants inoculated with the mutant in Q92P53. These differences have been shown as biologically significant in other symbiosis studies [48–50]. In addition, it is important to notice that a high percentage of small nodules (white and probably non-fixing nodules) was also observed in plants inoculated with etfB1 mutant. Surprisingly, plants inoculated with the strain mutated in msbA1 showed a 20–25% increase in nodule number when compared with control strain (Fig. 5b). In summary, these results clearly suggest that still there could be a number of non-described proteins involved in the Rhizobium-Legume interaction.
Further functional predictions
Based on our experimental results and the interactions of the novel targeted proteins, etfB1 acts in a module involved in energy production and conversion, and we predict it to be potentially involved in nitrogen fixation [see Additional file 2]; in fact, the high percentage of small non-fixing nodules induced by the strain mutated in this gene is consistent with this role. msbA1 functions in a module together with ndvA and exsA genes and is potentially involved in glucan synthesis; and Q92P53 is functioning within a module involved in lipid transport and metabolism in coordination with nod genes, and may be potentially involved in the regulation of the first stages of nodule formation. These novel findings only represents hypothesis and still have to be analysed in more detail to shed more light on their precise biological role and mechanistic details but, nonetheless, the predictions highlighted here represent a tempting guide for further experimental validation.
The building of our final 'Symbiosis Interactome network' complemented our initial classical-known list in many different ways. First, we extended the initial set from 92 to 163 known components (92 from the intersection and 71 from the union network). Second, we identified 263 potential novel Symbiosis Interactome components, representing ideal targets for further experimental validation [see Additional file 2]. Third, the incorporation of functional modules in the network provides additional information concerning the structure and functional organization of the Symbiosis Interactome. Interestingly, functional modules tend to be formed by proteins involved in the same stage of symbiosis [see Additional file 2] suggesting that distinct symbiosis-stages are organized and coordinated as distinct functional modules. Therefore, the incorporation of modules apart from providing another structural dimension to the Symbiosis Interactome also allows the prediction of both protein function and the symbiosis-stage a novel component may participate (Fig. 3c). This highlight both the quality of the network and the functional modules we predicted as guide for direct experimental validation. The final 'Symbiosis Interactome network', therefore, hosts the organization of the Symbiosis Interactome into functional interactions and modules, and constitutes the first attempt toward the representation of this complex biological process (Fig. 4).
Novel predicted components include many conserved proteins of unknown functions and others participating in a variety of cellular processes (Fig. 4). Novel proteins may represent false negatives components not identified by current experimental techniques perhaps because they are highly specialized components or maybe recruited to the Symbiosis Interactome under specific conditions that have escaped from detection and are therefore absent from our 'classical-known' preliminary data. Our experimental results yielded a preliminary notable success (3 positive cases out of 4 proteins tested experimentally) for predicting novel S. meliloti-M. sativa symbiotic components by using our computational approach. The results also provide tempting clues in regard to the predictive potential of our approach for hypothesis generation and guiding future experimental validation. For example, the two module-network scenarios presented here suggest high accuracy at predicting novel components and functional modules. Furthermore, high scored interactions based on our probability scores are experimentally validated as opposed to low quality interactions for which we could not find any direct experimental evidence, at least not for the gene Q92TC2 tested here. For this particular protein and the remaining 259 non-tested novel proteins, it is difficult to determine how many of them could be really involved in this complex biological process. It has been described that mutations in some bacterial nodulation genes do not have any influence in the symbiotic properties of the bacteria. For example, S. meliloti cells mutated in fixT gene are not affected in nodulation with M. sativa host plants . The expression of this fixation gene is regulated by FixH protein, which is essential for nodulation (mutations in fixH gives a Fix- phenotype, that is, non-fixing nodules). It has been suggested that some nodulation proteins could have a role in symbiosis when the expression of essential proteins is blocked. In the same manner, there are proteins that could be essential for nodulation in special situations, such as biotic and abiotic stress. In addition, there are proteins that could be involved in the symbiotic competitiveness of the rhizobial strain. Finally, another alternative explanation is that the potential involvement of the gene Q92TC2 in symbiosis might be compensated by other genes performing similar functions. Indeed, a gene family analysis by using sequence similarity clustering through the MCL algorithm  (see methods) revealed an intriguing gene family expansion in this particular case (31 genes in this family), whereas in the other 3 mutated genes we do not observe such drastic family expansions (with 1 (singleton family), 3, and 14 gene family members, for the genes Q92P53, etfB1, and msbA1, respectively). This interesting result suggests that other members of this large gene family might rescue its potential role in symbiosis through the establishment of backup circuits, such as occurs in other well studied model organisms . There is evidence of direct backup compensation between gene duplicates with overlapping functions where one gene can cover for the loss of its paralogue, and sometimes these compensations occur only for certain functions under given conditions . In all these situations, the single mutation of these genes in conventional laboratory conditions would not be the best experiment to assess their role in symbiosis. We believe this novel finding supports the model of network robustness through gene duplication , and it also has very interesting implications regarding the selection of the right candidate genes and experimental method in future validation studies.
While the functional network presented here provides valuable clues about the components of the bacterial Symbiosis Interactome, the main limitation of our study is the lack of experimental information on PPIs which made us to consider as input only computationally derived functional genomics data. Integration of computational approaches with recently published  and future experimental interaction data would likely improve the quality of our network and the prediction of novel components. This can be done by using Bayesian or probabilistic models shown to result in accurate confidence scoring systems [[26, 27, 55], Peregrín-Alvarez JM, Xiong X, Su C, Parkinson J: The modular organization of protein interactions in Escherichia coli, submitted]. Furthermore, although we believe we have been very flexible by allowing interactions between proteins with potential phenotypic profiles and not directly interacting with the giant-central network component, our Symbiosis Interactome network can still serve as a platform to add other interactions and components potentially involved in symbiosis. For example, we can choose other proteins with other interesting phenotypic profiles to extend our network such as those profiles showing homologs in other symbionts and/or pathogenic species since these bacteria often use the same core molecular mechanism to maintain their associations with hosts . Future analyses will also include further network extensions based on recently characterized symbiosis components [57–59], inclusion of other interesting phenotypic profiles (see above), a larger-scale experimental validation of the novel components predicted to be involved in symbiosis, and further analyses of the components and pathways involved in host-microbe, and host (i.e. plant) interactions. Finally, through an iterative process, novel Symbiosis Interactome components once experimentally confirmed, can be then added to the known set, potentially increasing the list of novel components and finally revealing the complete picture of the Symbiosis Interactome network.
The essential contribution of symbiosis to understand host-microbe interactions underscores the importance of further studying the structure and organization of the Symbiosis Interactome. Here we presented a novel 'systems-based model' that provided for the very first time new insights into the functional organization of the S. meliloti Symbiosis Interactome and the necessary framework on which to build, in an iterative manner, to further our understanding of symbiosis. We have identified 263 potential novel symbiosis components, and have demonstrated experimentally the participation of novel proteins involved in this important process. These novel proteins might not be essential for symbiosis but still determinant for the microbe-plant interaction since most of the essential components for this process have been described through decades of effort. Understanding the biology of this important model organism is essential not only for having a network view of how this biological process functions at a molecular level but also for the development of anti-microbial drugs since many of the proteins and modules involved in bacterial-symbiosis may be conserved, and thus, performing similar functions, in other microbial pathogens . Furthermore, we can use our network as a template to derive other Symbiosis Interactome networks for other bacteria-related species which is particularly important given the difficulty and cost of obtaining high throughput screens. Those maps should provide an useful starting point for predicting functional interactions and modules, and the function of unknown proteins. It remain to be seen which of these interactions and components do indeed occur and what is the specific role they play in each of these organisms. We believe that this model adds a new view and dimension to our understanding of host-microbe interactions, and can be extended to study other complex biological processes such as those involved in diseases.
An initial list of proteins known to be involved in the Rhizobium-Legume symbiosis was obtained and manually curated using PubMed, Google, journal-specific searches, and literature reviews and citations. We have called this list the 'classical-known' set.
We used S. meliloti genome context data from the PROLINKS  and STRING  databases. While both databases use the same genome context methods to derive functional linkages they both differ in the statistical procedures and scoring systems they use to provide high quality interactions. We reasoned that the overlap between both databases (intersection) represents interactions more likely to be true positives, and that the union of both databases represents a dataset with higher coverage (see below). We used all medium-to-high confidence functional linkages provided by the STRING database. From PROLINKS database we used those functional linkages in S. meliloti over 0.6 confidence. This cut-off provided a true positive rate similar to the one obtained by using the medium-to-high confidence data from the STRING database. The genome context data obtained from these two databases were combined into two single non-redundant datasets: one based on the overlapping between these two databases (the intersection dataset), and another one based on the union of the databases (the union dataset).
The confidence scores associated to each functional linkage provided by the original STRING and PROLINKS databases were re-scored according to the following criteria: STRING provides unified scores representing the confidence of a given functional linkage. The bigger the score, the more reliable the interaction. We reasoned that those interactions present in both databases are the most reliable ones, and we tested it by calculating ROC curves (see below). STRING scores were transformed into a scoring scale 0 – 0.5, the closer to 0.5, the bigger the confidence of the interaction. PROLINKS provides independent confidence scores for each applied independent genome context method. The scores were combined into an unified score by summing all confidence scores for a particular functional linkage and transforming the resulting number to a 0 – 0.5 scale. This procedure resulted in a 0 – 1 confidence score for those functional linkages present in both databases (the intersection data set) and a 0 – 0.5 confidence score for those interactions present in only one of the databases.
The validity of our re-scoring approach and the integrated networks was tested by calculating Receiving Operating Curves (ROC) and the Area Under the Curve (AUC) of the intersection, union, PROLINKS and STRING data sets as a measure of accuracy.
To be able to calculate accurate ROC curves and AUCs it is crucial to complement a positive gold standard set with a negative one. Because a reference set of known interactions is not available for S. meliloti, here we consider as positive set those functional linkages belonging to the same COGs functional category [37, 60]. The construction of a negative set is rather problematic because it is impossible to be sure that two proteins do not interact. However, by using those pairs of proteins that are present in different COGs functional categories and do not colocalize in the same cell compartment it is possible to make a list of protein pairs that are unlikely to interact, thus representing a good approximation to a negative set. COGs annotations were mapped to functional linkages and the periplasmic location of all the proteins was predicted (see below).
The periplasmic location of all S. meliloti proteins was predicted using the SIGCLEAVAGE software . The proteins were considered as periplasmic if they contained at least one predicted signal sequence within 50 residues from the N terminus. The proteins that did not contain any signal sequence throughout the entire sequence were considered cytoplasmic, and the remaining proteins were not classified.
The protein IDs of the functional linkage data from the intersection and union networks were converted to gene names using UNIPROT database  and these were used to map the list of classical-known proteins in S. meliloti.
For each S. meliloti sequence, a BLASTP  search was performed against 200 complete genome datasets. Both S. meliloti and other complete genomes were downloaded from the COGENT database  [see Additional file 2]. Homologs for each S. meliloti protein were determined based on a raw bit score threshold of 50, and were used to generate phenotypic profiles as follow: the complete genomes were manually curated and assigned to the following 8 phenotype categories [see Additional file 2] using PubMed, Google, and other web-specific searches: C, root colonizing bacteria; Fn, nitrogen-fixing bacteria in symbiosis with plants; Fl, free living nitrogen-fixing bacteria; P, pathogen; Pp, plant pathogen; S, soil cohabitant; Sy, symbiont/commensal; and O, other organisms. The only restriction for categorizing was that the genome in question have to be classified into one category only, the one with the most relevant phenotype for the study of symbiosis [see Additional file 2]. For example, if a genome could be classified as C and S, we considered only the category C because all C are also category S; or if a genome could be classified as P and S, P was considered more important for our analysis and thus classified as P only.
We then built phylogenetic profiles  for each S. meliloti protein and mapped the phenotypic data on top of the phylogenetic profiles yielding what we term 'phenotypic profiles'. For example, a protein with a phenotypic profile "FnFl" stands for a protein with homologs in plant nitrogen fixing bacteria and free-living nitrogen fixing bacteria only, thus representing a protein that may be potentially involved in symbiosis.
Unless otherwise noted network analyses were performed using Perl scripts developed in house. The degree (k) of a node (protein) in an interaction network is defined by the number of interactions of the node with other nodes in the network. For a node of degree k, its clustering coefficient (CC) is defined as 2N/k(k-1), where N is the number of interactions between the node's k neighbors and k(k-1)/2 is the number of possible interactions between its neighbors. A CC of 1 means that all the neighbors of a node are fully interconnected. The shortest path length between two nodes in the network is the number of edges in a shortest path connecting them. The shortest path length is infinity if there are no paths between two nodes. Network diameters were obtained using Pajek , and cluster coefficients and shortest path lengths were obtained using tYNA .
To act as controls, random networks were created by randomly selecting equal numbers of proteins (compared with the comparator network) from the S. meliloti network and randomly connecting them with equal numbers of interactions.
where A and B represents different networks, SAB the similarity (i.e. the frequency of common interactions) of A versus B, and SBA the similarity of B versus A.
Detection of functional modules
We identified highly connected functional modules operating within the intersection S. meliloti network by using the Markov Cluster (MCL) algorithm . MCL was applied to our S. meliloti network by testing several inflation operators, and settling on values that provided the highest clusters size, and the best overlap (semantic similarity)  of the computed clusters with the functional categories of the highly curated database COGs .
To compute the significant of finding specific COGs modules, a p-value for each module was calculated based on the distribution of 10,000 random module sets of the same size (assuming a normal distribution) and our module predictions, therefore, representing the probability of seeing such modules at chance. COGs categories with general function prediction, unknown or unassigned were not considered in this analysis. Only modules with at least three components with COGs assignments were statistically computed.
Prediction of functional annotation and stage of symbiosis
Predictions of functional annotation and the stage of symbiosis were performed using enrichment of COGs terms in functional modules (see above). Module prediction for a protein employed the predicted functional modules and derived COGs/symbiosis-stage annotations for the target proteins based on the highest percentage of common COGs/symbiosis-stage terms among the different components of the functional module. Correct COGs/symbiosis-stage assignments additionally required at least 20% of the interaction module components to have the same COGs/symbiosis-stage category. Two measures of stringency were employed: high stringency predictions required the majority of interaction module components to be assigned to the same COGs/symbiosis-stage category; low stringency predictions only required any of the interaction module components to possess the same COGs/symbiosis-stage category (albeit with the additional proviso that at least 20% of the module partners were so annotated). To measure the accuracy of module predictions we used a leave-one-out (LOO) cross-validation procedure, i.e. only proteins which itself and one of its module components possessed an annotation were used in cross-validation. The LOO method randomly selects a protein and compares its known annotation with that predicted by the functional module method.
Gene family analyses
S. meliloti mutants
S. meliloti mutants were obtained from a Mini-Tn5 transposon library constructed in the Lehrstuhl für Genetik (Bielefeld University, Germany) . Based on four network scenarios (see results) we selected the following S. meliloti mutants for experimental validation of our approach: 2011mTn5 STM.3.02.D12_transposon(etfB1), 2011mTn5 STM.4.10.F09_transposon(Q92P53), 2011mTn5 STM.3.08.C10_transposon(Q92TC2), and 2011mTn5 STM.1.06.E11_transposon (msbA1).
Seeds of alfalfa (Medicago sativa L. ecotype. Aragon) were surface sterilised on 70% ethanol for 10 minutes, exhaustively washed on distilled water and placed in water-agar plates for 36 hours at 22°C in the dark. 0.5–1 cm root pre-germinated seedlings were carefully transferred to squared plates containing a slope of BNM-agar medium . Seedlings were inoculated with 100 μl of an overnight culture of S. meliloti mutants or the strain 1021 as control. The lower part of the plate was covered with black paper in order to avoid the roots getting exposed to light. Plates were placed on an Ibercex G-28 plant growth cabinet at 22°C with 16 hours photoperiod. Plants were taken out of the plates at 28 days post-inoculation (dpi) for nodule analyses (counting, size, color, etc). Three independent experiments with 50 plants per experiment were done (150 plants in total). General aspects of plants were also analysed.
This work was supported by operating funds from the Canadian Institutes of Health Research (CIHR). P-A.JM also acknowledges the Ramon y Cajal Program from the Ministry of Science and Technology, Spain. R-L.I, C.MA and D.M also acknowledge Dr. Eloisa Pajuelo and the Spanish Ministry of Education (Plant Biotechnology Program) for financial support. We would like to thank Dr. Anke Becker (Lehrstuhl für Genetik, Bielefeld University, Germany) for providing S. meliloti mutants.
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