Characterization of the Twin-Arginine Transport Secretome in Sinorhizobium meliloti and Evidence for Host-Dependent Phenotypes

Abstract

The twin-arginine transport (Tat) system is essential for cell viability in Sinorhizobium meliloti and may play a role during the development of root nodules. Utilizing an in vivo recombination strategy, we have constructed 28 strains that contain deletions in predicted Tat substrates. Testing of these mutations for symbiotic proficiency on the plant hosts alfalfa and sweet clover shows that some of these mutations affect associations with these hosts differentially.

TEXT

Sinorhizobium meliloti is an alphaproteobacterium capable of forming nitrogen-fixing nodules on the roots of leguminous plants, including alfalfa, sweet clover, and fenugreek. The interaction of S. meliloti with its host has been used as a model system to study plant-microbe interactions. Completion of the S. meliloti genome sequence has provided an invaluable tool to help study plant-microbe interactions and to gain insight into how it functions as an organism (2, 6, 15, 16). As a result, a large number of previously uncharacterized genes have been examined using genome-based analysis (3, 10, 23–25, 27, 44).

The ability to transport and target proteins to the inner membrane is an important process involved in many cellular events. The twin-arginine transport (Tat) system is a highly conserved translocation pathway that transports proteins across the cytoplasmic membrane. These protein substrates are typically inserted into the inner membrane or shuttled into the periplasmic space; many of these substrates are redox proteins containing Fe-S or molybdopterin cofactors. Unlike the general secretory (Sec) system, Tat substrates are prefolded and often require cofactor insertion for proper enzymatic function. An important characteristic of Tat substrates is a leader signal motif at the N terminus (S/TRRxFLK), which directs transport through the Tat pathway. The number of substrates transported through the Tat pathway varies, depending upon the organism. This can range from very few proteins to the majority of proteins, such as in the haloarchaeal bacteria (38).

The Tat system has been implicated in pathogenesis of bacteria, including Agrobacterium tumefaciens, Pseudomonas aeruginosa, and many others (9, 13, 31). It has been previously reported that in Rhizobium leguminosarum, the twin-arginine transport pathway was required for correct association with its host plant (29). A mutation in the tatC operon resulted in Fix⁻ plants producing empty nodules. The authors proposed that a developmental breakdown occurred during the infection process due to the loss of the twin-arginine transport system and, therefore, protein substrates exported via the twin-arginine transport mechanism may be required for development.

Bioinformatic data predict that the S. meliloti genome contains at least 100 putative Tat substrates (1, 29). Since the Tat transport system is necessary for cell viability in S. meliloti (35), little is known about the precise role some of these proteins may play during the symbiotic process. To address this, we initiated a systematic approach to delete putative genes which are predicted to encode Tat substrates, utilizing the ORFeome platform that was developed for the S. meliloti genome (22, 40).

Targeting of putative Tat substrates in S. meliloti.

When this work was initiated, TATFIND analyses (38) had been carried out on the S. meliloti genome, identifying 94 putative proteins with Tat-like motifs (29). With the release of TatP (4), the number of predicted proteins encoded in the genome of S. meliloti with Tat-like motifs was 128. Fifty-four of these proteins scored above the suggested trusted cutoff. A comparison of the outputs showed that in addition to proteins that were common, each program had identified proteins that were not recognized by the other program; 26 of the proteins identified by TATFIND were not found by TatP. In addition, by manual scanning of the annotated genome, other candidates that were not identified by either program were also identified. Taken altogether, a list of 154 proteins with Tat-like motifs was generated. Other than fbcF (SMc00187), which encodes Rieske protein involved in electron transport and known to affect nitrogen fixation (43, 45), no other obvious candidates that might affect symbiotic development were found.

To generate a list of genes for deletion, the following criteria were used. First, genes encoding proteins that were identified by TatP and TATFIND below the trusted cutoff value were eliminated. Second, genes at the beginning or at the end of operons were also eliminated because the in vivo deletion strategy employs the recombination of frt sequences into sites adjacent to genes flanking the target; final products of these could potentially delete intergenic promoter regions, yielding pleiotropic mutations. The remaining genes, listed in Table S1 in the supplemental material, were targeted for deletion.

More recently a new program, PRED-TAT (1), predicted 142 putative Tat substrates with cleavage sites. Using the list of GI numbers, the predicted proteins were retrieved from GenBank, and each sequence was reanalyzed. It was found that 13 of the 142 proteins did not contain Tat-like leaders (see Table S1 in the supplemental material). As well, of the 10 proteins manually predicted to be Tat substrates, 5 were corroborated by PRED-TAT, whereas 4 were predicted to have transmembrane helices in the N-terminal region, and the remaining protein contained a leader that was below the trusted cutoff (see Table S1).

To determine if S. meliloti leaders carried any characteristic signatures, the remaining 129 leaders were aligned using ClustalX (42). The final alignments were manually inspected and optimized by removing 8 sequences. The remaining 121 were used to generate an S. meliloti Tat leader consensus using WebLogo (11) (Fig. 1). The logo is similar to the previously published logos; however, it may be of note that our analysis shows that a number of the putative leaders seem to contain a methionine 6 amino acids prior to the canonical twin-arginine motif. The significance of this observation at this time is not clear.

Fig 1.

Consensus sequence of twin-arginine transport motif. The N-terminal amino acids of predicted S. meliloti Tat substrates were aligned around the twin arginines using ClustalX. Conservation of sequence is shown in bits as previously described (39).

Construction of in vivo deletions using the S. meliloti ORFeome.

To generate targeted in vivo deletions, we employed the ORFeome system with some modifications as previously described (17, 22, 40). Following the final recombination between the frt sites that flanked the targeted gene, each junction was PCR amplified and sequenced to ensure that the proper mutation was constructed. The strains, plasmids, and primers that were used or generated are listed in Tables S2 and S3 in the supplemental material.

Evidence for host-dependent phenotypes.

To address the hypothesis that Tat-dependent substrates may play a role in symbiosis, mutants were screened for symbiotic capabilities by inoculation onto two host plants—alfafa and sweet clover. The inoculated plants were assessed qualitatively by their appearance, as well as the number and positioning of nodules. Plant dry matter accumulation was used to assay symbiotic efficiency, as previously described, and independent trials were normalized on the basis of plant dry matter accumulation from plants inoculated with the wild type (32, 36, 37).

Except for SRmD232 (fbcF), which was obviously not providing fixed nitrogen, most plants did not appear to be drastically different with respect to the appearance or the number, shape, or color of nodules on each root system compared with the wild type. However, based on dry matter accumulation, the mutants fell into 4 distinct classes (Fig. 2). The majority of the mutants did not have different dry matter accumulation on either host (Fig. 2). SRmD232 (fbcF), did not show any dry matter accumulation greater than that of the uninoculated plant controls. Two strains—SRmD230 (SMb21380) and SRmD214 (SMb21067)—showed reduced dry matter accumulation on both hosts (Fig. 2). Five strains—SRmA691, SRmD224, SRmD236, SRmD228, and SRmD242—showed significantly reduced dry matter accumulation on alfalfa but not on sweet clover.

Fig 2.

Plant phenotypes exhibited by putative Tat substrate deletions. Shown are plant dry weights of alfalfa (black bars) or sweet clover (white bars) inoculated with the strain indicated. The data represent between 4 and 6 independent experiments, each consisting of 30 plants, and are presented as percentages of the wild type. Wild-type plant weights (100%) were 57 ± 6 mg/plant (alfalfa, n = 6) and 85 ± 11 mg/plant (sweet clover, n = 6). Differences between the plants inoculated with mutant and the wild type are based on Student's t test at the 95% confidence level (P < 0.0001).

The mutants that displayed phenotypes were generally those whose genes encode proteins that putatively affect either metabolism or metabolite transport. Two of these mutant genes, SMa02021 and SMb20671, encode sugar-binding proteins that are associated with ABC-type transporters. Whereas a deletion of SMb20671 displays an effect on both alfalfa and sweet clover, a deletion of SMa02021 appears to only show an affect on alfalfa (Fig. 2). Both of these genes have been previously screened for expression against a bank of 117 carbon substrates (27). Neither of these was strongly induced by any of the conditions assayed; however, it may be of note that each did show a modest 3-fold induction with lyxose. The significance of this observation is unclear at this time.

Based on the annotation, as well as rudimentary sequence analysis, three of the remaining mutants that displayed a plant-dependent phenotype appear to affect carbon catabolism. Both SRmD224, carrying a deletion in SMc00817, and SRmD214, carrying a deletion of SMb21380, putatively affect the degradation of aromatic compounds. Whereas some plant-derived aromatic compounds have been identified as being important to the physiology of S. meliloti, our knowledge is limited by the characterization of relatively few compounds (19, 26, 30, 34, 41). In addition, the mutation in SRmD242 (SMb20342) also falls into the category of an enzyme that is likely a redox enzyme that can potentially oxidize compounds within the periplasmic space and contribute to the overall energetics of the organism.

Two of the mutants tested, SRmA691, carrying a deletion of napA, and SRmD236, carrying a deletion of nosZ, affect denitrification (Fig. 2). The existence of enzymes involved in denitrification has long been known in S. meliloti (7, 8, 20). The existence of a complete denitrification pathway became clear following the complete sequencing of the genome (16).

The involvement of nitric oxide (NO) in symbiotic and pathogenic associations is well known (5, 14). The production of NO has clearly been shown in nodule tissue and infection threads (12). Microarray analyses have shown that both nos and nap are upregulated in response to nitric oxide in a FixL-independent manner and that NO within nodules can be synthesized by both the bacteria and the plant and used as a source of electrons (and thus an energy source) in Medicago truncatula nodules (21, 28). More recently it has been shown that an hmp mutant in S. meliloti both is affected in competition for nodule occupancy (12) and has lower levels of nitrogenase activity (28). The finding that nos and nap mutations have differential symbiotic phenotypes adds to this body of evidence.

This work was initiated to identify putative Tat-dependent substrates that have a role in symbiosis. Based on the previous findings in R. leguminosarum (29), we had anticipated finding mutants with severe developmental phenotypes. Based on simple BLAST searches, only the genes associated with denitrification had E values of zero in R. leguminosarum. As the work proceeded, it became clear that the strategy we had employed was not yielding the expected results; however, additional screening on a second host, sweet clover, revealed a number of host-dependent effects (Fig. 2). Taken together, the results presented here identify a number of genes that have not been previously implicated in having a role in symbiosis, as well as genes that appear to have roles that are host dependent.

At present, our data do not allow us to distinguish whether we have caused a subtle developmental delay or affected nitrogen fixation or whether the phenotypes are due to a combination of events. It is important to remember that although our results are reproducible, these mutants have not been complemented. Many of these mutations are in complex operons, and their phenotypes do not lend themselves to simple isolation of complementing cosmids, as has been previously described (18, 32, 33, 36). In this aspect, it is noteworthy that both mutations affecting denitrification yielded the same plant phenotype (Fig. 2).

To our knowledge, the systematic deletion of genes encoding putative twin-arginine transport substrates to find physiological function is unique. The data show that Tat substrates may play a more complex role in symbiosis than previously envisaged. To properly define the role that these proteins play within the plant-microbe interaction is going to be dependent on careful physiological, biochemical, and microscopic approaches.