Global Transcriptional Repression of Diguanylate Cyclases by MucR1 Is Essential for Sinorhizobium-Soybean Symbiosis

ABSTRACT The ubiquitous bacterial second messenger c-di-GMP is intensively studied in pathogens but less so in mutualistic bacteria. Here, we report a genome-wide investigation of functional diguanylate cyclases (DGCs) synthesizing c-di-GMP from two molecules of GTP in Sinorhizobium fredii CCBAU45436, a facultative microsymbiont fixing nitrogen in nodules of diverse legumes, including soybean. Among 25 proteins harboring a putative GGDEF domain catalyzing the biosynthesis of c-di-GMP, eight functional DGCs were identified by heterogenous expression in Escherichia coli in a Congo red binding assay. This screening result was further verified by in vitro enzymatic assay with purified full proteins or the GGDEF domains from representative functional and nonfunctional DGCs. In the same in vitro assay, a functional EAL domain catalyzing the degradation of c-di-GMP into pGpG was identified in a protein that has an inactive GGDEF domain but with an active phosphodiesterase (PDE) function. The identified functional DGCs generally exhibited low transcription levels in soybean nodules compared to free-living cultures, as revealed in transcriptomes. An engineered upregulation of a functional DGC in nodules led to a significant increase of c-di-GMP level and symbiotic defects, which were not observed when a functional EAL domain was upregulated at the same level. Further transcriptional analysis and gel shift assay demonstrated that these functional DGCs were all transcriptionally repressed in nodules by a global pleiotropic regulator, MucR1, that is essential in Sinorhizobium-soybean symbiosis. These findings shed novel insights onto the systematic regulation of c-di-GMP biosynthesis in mutualistic symbiosis.

GTP molecules by diguanylate cyclases (DGCs) containing the GGDEF domain and can be degraded into 59-phosphoguanylyl-(39-59)-guanosine (pGpG) and/or two GMP molecules by phosphodiesterases (PDEs) bearing the EAL or HD-GYP domains (3,4). c-di-GMP homeostasis is modulated by DGCs and PDEs and can be sensed by effectors, including the PilZ domain, GIL domain, MshEN domain, riboswitch, transcriptional factors, and degenerate GGDEF or EAL domain (4,5). The high diversity in DGCs, PDEs, and c-di-GMP effectors and their subsequent regulation accounts for the multiple roles of c-di-GMP in bacterial adaptations to fluctuating abiotic and biotic conditions. c-di-GMP signaling has been mainly and intensively studied in bacterial pathogens but less so in mutualistic bacteria (2,(6)(7)(8)(9)(10)(11)(12). As model mutualistic microsymbionts, rhizobia induce and intracellularly infect root nodules, where they fix atmospheric N 2 into ammonia to support legume growth (13). The energy-consuming process of rhizobial nitrogen fixation is sustained by nutrients provided by host cells (14). Rhizobia can live saprophytically in soils in the absence of a compatible legume host and represent a typical facultative microsymbiont bearing larger genomes than those obligate microsymbionts to cope with fluctuating stimuli (15). Constitutive expression of a DGC (PleD) from Caulobacter crescentus in Rhizobium etli and Rhizobium leguminosarum strains favored exopolysaccharide (EPS) production and adhesion to legume roots but decreased the fresh weight of inoculated plants (9). A similar constitutive expression of PleD from C. crescentus in Sinorhizobium meliloti allowed the discovery of cryptic EPSs such as a linear mixed-linkage beta-glucan and an arabinose-containing polysaccharide (6,7,12), although the plasmid carrying this heterologous PleD was lost rapidly under nonselective conditions, including the rhizosphere of the legume host alfalfa (7). A later study showed that an S. meliloti mutant lacking 16 out of 17 GGDEF-encoding genes had no salient phenotypes under tested free-living and symbiotic conditions except for its decreased tolerance to acid stress (11), although this strain had no detectable c-di-GMP under test conditions. In contrast, a c-di-GMP-free derivative of C. crescentus showed severe defects in its bimodal life cycle, motility, and surface attachment (16), and a Salmonella enterica serovar Typhimurium mutant lacking all GGDEF-encoding genes lost virulence and exhibited various defects in free-living processes, such as motility, biofilm formation, and cellulose biosynthesis (17). In short, despite open pangenomes of rhizobial species and dozens of c-di-GMP signaling components in individual genomes (10,18), c-di-GMP signaling in rhizobia remains largely unexplored.
In this work, we focused on Sinorhizobium fredii CCBAU45436 (SF45436), which can establish effective symbiosis with soybean and many other legumes (19,20). A genome-wide bioinformatic analysis of proteins with putative GGDEF, EAL, and HD-GYP domains was performed. The functional DGCs were screened by heterogenous expression of the full proteins or GGDEF domain alone in Escherichia coli in a canonical Congo red binding assay. Enzymatic activities of DGC or PDE were further tested for purified full proteins, GGDEF or EAL domains from representative functional, and nonfunctional DGCs. Transcriptome sequencing (RNA-seq) was used to determine transcriptional profiles of DGCs at exponential and stationary phases in free-living culture and in soybean nodules. The potential role of c-di-GMP in symbiosis was tested by using deletion mutants of major DGCs transcribed in soybean nodules or engineered strains harboring nodule-specific overexpression of a functional DGC or an active EAL domain. Finally, we studied the direct transcriptional regulation of functional DGCs by a pleotropic regulator, MucR, which modulates various canonical processes responding to c-di-GMP (21) and is essential for symbiotic efficiency of SF45436 on soybean (22). such as S. fredii SF25509 (26 proteins), S. fredii SF83666 (35 proteins), S. sojae SJ05684 (26 proteins), and Sinorhizobium sp. strain SS05631 (28 proteins) (Fig. 1B).
Among those GGDEF-containing proteins in SF45436 ( Fig. 2A), 19 of them have GGDEF domains of a conserved motif [D X(7 aa) N X(8 aa) D X(21 aa) R X G /S/A GD /E EF] reported in functional diguanylate cyclases (1). In E. coli, the c-di-GMP biosynthesis mediated by a functional diguanylate cyclase can be indicated by the biosynthesis of cellulose with strong Congo red binding ability (25). To verify the activity of putative diguanylate cyclases in SF45436, Congo red binding ability of E. coli strains expressing individual GGDEF-containing proteins from SF45436 was tested (Fig. 2B). Western blot analysis showed notable induced expression by isopropyl-b-D-thiogalactopyranoside (IPTG) for individual proteins in E. coli BL21 or Rosetta, although leak expression was observed for those strains harboring ydeH, SFb59510, SFc17580, and SFc05240 (Fig. 2B). Similar to the known diguanylate cyclase YdeH from E. coli (26), overexpressing SFc17580, SFc15850, SFc19200, SFc31640, SFb52570, SFc11920, SFc23720, and SFb47640 from SF45436 was able to enhance the ability of engineered E. coli cells to bind Congo red. All eight of these proteins and YdeH have the conserved motif [D X(7 aa) N X(8 aa) D X(21 aa) R X G /S/A GD /E EF], while overexpression of some other proteins with this motif could not enable E. coli to bind Congo red (Fig. 2B).
Diverse N-terminal domains, such as REC-REC (SFc19200), 5TM-5TMR_LYT-PAS_4-PAS_7 (SFc31640), CHASE4 (SFc11920), and HAMP-PAS (SFb47640 and SFc23720), are associated with GGDEF in these functional DGCs, implying their potential roles in directly sensing fluctuating stimuli or interacting with other proteins in the life cycle of this facultative microsymbiont of various legumes (19,20,27). For example, diverse PAS domains can serve as direct sensors of various ligands, including oxygen, blue light, cellular redox, carboxylatecontaining substrates, divalent metal, and fatty acid (28). DGCs with the REC domain are responsive regulators of two-component signal transduction systems, responding to extracellular or intracellular signals perceived by their cognate sensor His kinases (1). When only the GGDEF domain from 20 proteins of multiple domains was overexpressed in E. coli, none of them was functional (see Fig. S1 in the supplemental material). Since stable single GGDEF-domain-bearing DGCs are not rare (Fig. 2B), this implies potential misfolding of the cloned GGDEF domain from SFc19200, SFc31640, SFb52570, SFc11920, SFc23720, and SFb47640. This view was further supported by high-performance liquid chromatography mass spectrometry (HPLC-MS) analysis of c-di-GMP production using purified His-  YdeH is a known functional DGC from E. coli. For proteins not expressed in BL21(DE3) harboring pET28a(1) derivatives, the Rosetta strains (#) containing the same expressing plasmids or BL21(DE3) carrying pET30a derivatives ($) were tested. (C and D) HPLC-MS determination of c-di-GMP (C) or pGpG (D) content in 1 mM purified protein prepared from E. coli strains harboring corresponding vectors derived from pET30a or pET28a(1). Error bars represent standard deviations. Different letters above error bars indicate significant difference between means based on three independent experiments (ANOVA followed by Bonferroni's multiple-comparison test, a = 0.05). (E) HPLC-MS determination of c-di-GMP content in E. coli strains expressing the indicated proteins relative to that of strains carrying the empty vector (*, P , 0.05; **, P , 0.01; ***, P , 0.001; t test). Error bars represent SD. Protein purification for these five proteins was not successful under test conditions, and in vitro enzymatic assay was not done. SFb52570 GGDEF and His-SFb52570 (Fig. S2) in the presence of GTP, i.e., the full protein of SFb52570 rather than SFb52570 GGDEF alone had diguanylate cyclase activity (Fig. 2C). Moreover, phosphodiesterase activity was not detectable for His-SFb52570 EAL and His-SFb52570, suggesting a degenerated EAL domain in this protein (Fig. 2D). The degenerate EAL domain of SFb52570 may play a structural or regulatory function, as deletion of this enzymatically inactive EAL domain abolished the DGC activity of SFb52570 (Fig. 2C,  Fig. S1). Similarly, DGCs with degenerate EAL domains are also experimentally demonstrated in Gluconacetobacter xylinus (29,30).
Both purified His-SFc33230 and His-SFc33230 EAL (Fig. S2) exhibited phosphodiesterase activity generating 59-pGpG from c-di-GMP, whereas no diguanylate cyclase activity was detected for His-SFc33230 and His-SFc33230 GGDEF ( Fig. 2D and C). The purified His-SUMO-SFc17580 with only the GGDEF domain and His-SUMO-SFc19200 ( Fig. S2; His-SFc17580 and His-SFc19200 were poorly soluble) were able to produce c-di-GMP from GTP (Fig. 2C). Although protein purification for the other five functional DGCs (SFc23720, SFc11920, SFc31640, SFb47640, and SFc15850) was not successful under test conditions, HPLC-MS analysis revealed significantly higher levels of c-di-GMP in E. coli strains expressing these five DGCs than in strains carrying empty vectors ( Fig. 2E; P , 0.05, t test). These results are consistent with those of Congo red binding assay.
Transcriptional profiles of functional diguanylate cyclases and the effect of c-di-GMP elevation in soybean nodules. RNA-seq analysis revealed that eight functional diguanylate cyclases exhibited contrasting transcriptional profiles during exponential (log) and stationary phases and within soybean nodules (Fig. 3). The largest proportion of total variation (34.64%) was explained by different diguanylate cyclases, followed by different conditions (25.34%) and interaction between these two main effects (23.9%). SFb52570 and SFc17580 were major diguanylate cyclases transcribed during the log phase and within symbiotic nodules (Fig. 3), although all functional DGCs were actively transcribed at stationary phase, suggesting that multiple stress signals arise under this nutrient-starving condition. In line with these findings, two DGCs had relatively strong expression during all growth phases of E. coli, while a number of DGCs were induced during stationary phase (25).
In-frame deletion of either SFb52570 (Db52570) or SFc17580 (Dc17580) or both major DGC genes (Dc17580 Db52570) had no significant symbiotic defects on soybean Transcriptional changes of SFc17580 and SFc33230 EAL in test strains (three biological replicates, ANOVA followed by Dunnett's multiple comparisons; ***, P , 0.001; ****, P , 0.0001; error bars represent SD). plants ( Fig. 4A and B). In contrast, a derivative carrying the functional DGC SFc17580 (Fig. 2C) driven by the nifH promoter (P nifH -DGC) induced more but inefficient nodules, leading to a significant decline in chlorophyll content of soybean leaves ( Fig. 4A to C). Since nifH encodes the nitrogenase reductase and is specifically expressed in nodules induced by most rhizobia without free-living nitrogen fixation ability (22,31), the impaired symbiotic performance of the P nifH -DGC strain is strictly nodule specific. This is in contrast to earlier studies of R. etli and R. leguminosarum using a constitutive expression version of heterogenous PleD from C. crescentus, which showed a decline in both nodule number and nitrogen content of corresponding host plants Phaseolus vulgaris and Vicia sativa (9). When the transcription of the functional EAL domain of SFc33230 (Fig. 2D) was driven by P nifH (P nifH -PDE), no symbiotic defects were observed on soybean plants (Fig. 4A and B). This is consistent with the normal phenotype of alfalfa plants inoculated with an S. meliloti mutant lacking 16 out of 17 GGDEF-encoding genes and having no detectable c-di-GMP (11).
The HPLC-MS analysis (Fig. 4D) further showed that c-di-GMP was at the level of pmol per microgram protein in nodules infected by the PnifH-DGC strain but undetectable in the other treatments (the wild-type SF45436, DmucR1, and P nifH -PDE strains). Further quantitative reverse transcription-PCR (qRT-PCR) analysis (Fig. 4E) revealed that the DGC gene SFc17580 and the EAL-encoding fragment SFc33230 EAL were actively transcribed in the P nifH -DGC and P nifH -PDE strains, respectively. This demonstrated the efficiency of the test P nifH promoter in nodules, and the high expression of SFc33230 EAL had no significant effect on symbiosis. In short, elevating rhizobial c-di-GMP in nodules exerted a negative effect on symbiotic performance.
For comparison, the in-frame deletion mutant of the functional PDE gene SFc33230 (Fig. 2D) or the pleotropic regulator gene mucR1 (mucR2, the other mucR copy in SF45436, is not functional due to a frameshift mutation) (21,22) were also tested for their symbiotic performance. As many as 31 EAL domain-containing proteins are present in SF45436 (Fig. 1A); consequently, it is not unexpected that Dc33230 strain was indistinguishable from the wild-type SF45436 in symbiotic performance ( Fig. 4A and  B). The DmucR1 mutant showed more severe symbiotic defects than the P nifH -DGC strain regarding the chlorophyll content of leaves ( Fig. 4A and C), although it induced as many nodules as the P nifH -DGC strain ( Fig. 4B and C). Both SFc17580 and SFc33230 EAL were upregulated in the DmucR1 mutant (Fig. 4E). This may at least partially explain the undetectable c-di-GMP in nodules infected by the DmucR1 mutant, implying an intriguing regulation role of MucR1 on c-di-GMP signaling components.
Moreover, bacteroids of the P nifH -DGC strain and the DmucR1 mutant but not those of the P nifH -PDE strain showed significant upregulation of genes involved in various c-di-GMP-responsive processes (Fig. 5), such as the c-di-GMP receptor McrA, regulating motility (11), and CuxR, activating arabinose-containing polysaccharide production (6), the biosynthesis of mixed-linkage beta-glucan (bgsA) and adhesion polysaccharides (uppE), and a key flagellar component (fliG) (12,32,33). Notably, both polysaccharides and motility machinery have energetic cost in bacterial physiology and are downregulated during nitrogen fixation that consumes as many as 16 ATP to reduce one molecule of N 2 (22,27,34). This is supported by recent evidence that optimum energy metabolism status of bacteroids is required for efficient symbiosis in soybean nodules (24).
Since no c-di-GMP could be detected in bacteroids of the DmucR1 mutant while the P nifH -DGC strain significantly accumulated c-di-GMP in nodules (Fig. 4E), the transcription of the c-di-GMP-responsive processes mentioned above may be subject to a general negative regulation by MucR1, which can be relieved to a certain extent by elevating c-di-GMP levels (Fig. 5). Notably, MucR1 can also act as a positive regulator required for active transcription of high-affinity transporters for phosphate and zinc ions and the iron-responsive regulator RirA, which are all essential for efficient symbiosis of S. fredii within soybean nodules (22,(35)(36)(37)(38). The downregulation of rirA in bacteroids of the mucR1 mutant was confirmed in this work but not observed in bacteroids of the P nifH -DGC strain (Fig. 5). This may partially explain the more severe symbiotic defects associated with the DmucR1 mutant than the P nifH -DGC strain (Fig. 4A to C).
Direct regulation of functional DGCs by MucR1. The previous transcriptomic analyses in alphaproteobacteria have revealed various processes responding to c-di-GMP, and some GGDEF/EAL-encoding genes are differentially transcribed when mucR1 is mutated (21,22). As mentioned above, certain functional DGCs were transcribed at relatively low levels in bacteroids and exponential-phase cells, although a global active transcription of functional DGCs was observed at the stationary phase (reads per kilobase per million mapped reads [RPKM] . 67; Fig. 3). In line with these transcriptional profiles of functional DGCs, the average RPKM value of mucR1 in stationary-phase cells was 12% and 52% of those in exponential-phase cells and bacteroids, respectively (Data Set S1). However, the relatively low transcription level of mucR1 at the stationary phase can still be considered active transcription within the whole transcriptome (RPKM . 156; Data Set S1). qRT-PCR analyses of soybean nodules and the free-living cells in rich medium TY (optical density at 600 nm [OD 600 ] of 1.2) showed that transcriptional levels of most functional DGCs were significantly upregulated in the DmucR1 mutant compared to the wild-type SF45436 (Fig. 6A and B), with more drastic changes in nodules. The transcription of functional PDE SFc33230 was also significantly upregulated in the DmucR1 mutant under both symbiotic and free-living conditions (Fig. 6A  and B). Although these DGC/PDE genes have a scattered distribution pattern on the chromosome and chromid, the observed overall upregulation transcription profiles, particularly in nodules, imply that these genes are regulated by a shared regulation machinery.
Cumulative evidence suggests the zinc-finger bearing MucR can be a global repressor preferring low-GC target sequences of low consensus (21,39,40). Sequence analysis revealed that the average %GC of upstream intergenic region for genes encoding functional or nonfunctional DGCs (Fig. 6C) is significantly lower than the genome average of 63.1% (*, P , 0.05; **, P , 0.01; t test) and the average value of the chromosome (63.45%, P , 0.05; t test) and chromid (63.04%, P , 0.05; t test) while indistinguishable from the average %GC of the symbiosis plasmid pSymA (59.92%, P . 0.097; t test). Further electrophoretic mobility shift assay (EMSA) with the purified Sumo-MucR1 and its derivative MucR1 (with Sumo removed by HRV-3C protease) demonstrated that MucR1 can directly bind the promoter regions of genes encoding eight functional DGCs and the functional PDE SFc33230 (Fig. 6D) but not on three tested FIG 5 Transcriptional changes of c-di-GMP responsive genes of the DmucR mutant and the P nifH -DGC and P nifH -PDE strains in soybean nodules. An iron-responsive regulator gene, rirA, is used as a control. Significant differences compared to the wild-type SF45436 are indicated (three biological replicates, ANOVA followed by Dunnett's multiple comparisons; **, P , 0.01; ***, P , 0.001).
nonfunctional DGCs (Fig. S3). A clear gradient of band shift for test probes was observed when the ratio of MucR1 to probe was increased (Fig. 6D), suggesting oligomeric or multiple MucR1 binding events. This is in line with the hypothesis that MucR1 works in a similar nonspecific way in binding DNA as the well-known H-NS (histonelike nucleoid structuring protein) of E. coli (21,(40)(41)(42)(43). The global repression of functional DGC genes located on different replicons by MucR1, particularly in nodules, represents a largely unexplored scenario in the evolution of the facultative life cycle of rhizobia.
The MucR/Ros family proteins are mainly found in alpha and deltaproteobacteria, particularly conserved in the former class that is enriched with various pathogenic and mutualistic bacteria associated with eukaryote hosts (21). Intensive studies of MucR/Ros homologs from Agrobacterium, Sinorhizobium, Rhizobium, Mesorhizobium, Caulobacter, and Brucella uncovered not only the conserved phenotype of rough colonies of the mucR or ros mutant but also their pleotropic transcriptional regulatory role in various cellular processes, such as the production of exopolysaccharides, motility and chemotaxis, cell cycle, ion uptake, and protein secretion systems (22, 38, 39, 44-53), some of which are involved in symbiosis and virulence. In the model rhizobium S. meliloti, for example, MucR directly activates the Repression of c-di-GMP Synthesis in Legume Nodules ® transcription of exoY, encoding a galactosyltransferase initiating the repeating unit assembly process during the biosynthesis of succinoglycan exopolysaccharide (54) that is essential for S. meliloti host invasion (48,55). MucR can directly repress the transcription of Rem, which is a transcriptional activator of motility genes (56). It has been established that c-di-GMP is a ubiquitous second messenger modulating motility, biofilm formation, virulence, and cell cycle in bacteria (57), and several common cellular process regulated by both c-di-GMP and MucR recently have been reviewed for the free-living lifestyle of S. meliloti (58). This work further demonstrated that, at the nitrogen-fixing stage of mutualistic interaction between rhizobium and legumes, MucR can globally repress functional DGCs to downshift various energetically expensive processes induced by c-di-GMP. This strategic regulation mechanism also can be tested in other bacterium-host interactions.
Conclusions. Rhizobia are characterized by their facultative symbiotic life cycle, in which various stimuli should be sensed and properly responded to. Dozens of c-di-GMP signaling components are present in rhizobia but are largely unexplored. This work made a systematic screening of functional DGCs in S. fredii SF45436 by using both in vitro and in vivo experiments ( Fig. 1 and 2). The condition-dependent transcriptomic profiles suggest a general downregulation of functional DGC genes in soybean nodules (Fig. 3). An engineered nodulespecific accumulation of c-di-GMP led to an increased number of inefficient nodules, while no c-di-GMP could be detected in efficient nodules infected by the wild-type SF45436 and the PDE-overexpressing strain (Fig. 4). The elevated c-di-GMP induced various c-di-GMP-responsive processes, which are energetically costly and negatively regulated by the pleotropic regulator MucR1 in nodules (Fig. 5). It was further revealed that functional DGCs with scattered distributions in the multipartite genome of SF45436 were all directly repressed by MucR1 through oligomeric or multiple binding events on their promoter regions (Fig. 6). Collectively, these findings demonstrate a strategic repression of the c-di-GMP biosynthesis arsenal in legume nodules (Fig. 7), which represents a novel adaptation mechanism potentially explored by many other prokaryotes harboring a rich pool of c-di-GMP signaling components and their distinct global silencers, such as MucR (alphaproteobacteria, G-), H-NS and MvaT (gammaproteobacteria, G-), Lsr2 (actinobacteria, G 1 ), and Rok (bacilli, G 1 ) (21).

MATERIALS AND METHODS
Bacteria and culture conditions. Bacterial strains and plasmids used in this work are shown in Table S1 in the supplemental material. E. coli was grown at 37°C in Luria broth (LB) medium. S. fredii SF45436 and its derivatives were grown at 28°C in TY medium (tryptone at 5 g/liter, yeast extract at 3 g/ liter, and CaCl 2 at 0.6 g/liter). The antibiotic concentrations used were 50 mg/ml kanamycin (Km), 10 mg/ ml trimethoprim (Tmp), 30 mg/ml gentamicin (Gen), and 30 mg/ml carbenicillin (Cb).
Genetic procedures. The primers used are listed in Table S2. All plasmid constructs in this work were verified by Sanger sequencing. Plasmids were transformed into E. coli DH5a (unless indicated) before conjugation into rhizobia with pRK2013 as the helper plasmid.
The DmucR1 mutant was generated by removing the gentamicin resistance cassette within the DmucR1:: Gm mutant constructed previously (22). The pCM157 plasmid carrying Cre recombinase that recognizes the loxP sites flanking the gentamicin cassette was introduced into the DmucR1::Gm mutant, and transconjugants sensitive to gentamicin were selected for subsequent screening of pCM157-cured strains (59). To construct in-frame deletion mutations of SFc33230, SFc17580, and SFb52570, a seamless assembly method was used. Briefly, gene-flanking regions of 500 to 1,000 bp were amplified using primers carrying 59-homologous sequences containing the ends of SmaI restriction sites (CCC) in pJQ200SK (60). Two flanking fragments were mixed with SmaI-linearized pJQ200SK and incubated at 50°C for 15 min using a seamless cloning kit (Taihe Biotechnology), and positive transformants with correct sequences were used for conjugation with SF45436. Single-crossover clones resistant to Gen were selected for sequencing verification and subsequent cultivation in liquid TY medium for 36 h. The resulting liquid culture was subjected to double-crossover screening on a TY plate containing 7% (wt/vol) sucrose and Tmp as previously described (61).
Individual coding sequences for 26 DGCs (including YdeH from E. coli K-12), 20 GGDEF domains, and two EAL domains (SFc33230 EAL and SFb52570 EAL ) were amplified using primers harboring 59-homologous sequences, including the ends of the NdeI restriction site (CAT) of pET28a(1), or primers carrying 59-homologous sequences, including the ends of BamHI site (GGA) of pET30a-SUMO. These amplified fragments were individually subjected to seamless cloning with the plasmids pET28a(1) and pET30a-SUMO linearized by NdeI and BamHI, respectively. The resulting plasmids were then transformed into E. coli BL21(DE3) or Rosetta(DE3) before Congo red binding assay or protein purification experiments.
To construct pJQ-P nifH -c17580 and pJQ-P nifH -c33230 EAL , a promoter region (496 bp of SF45436_a46030) and the upstream and downstream fragments of the coding sequences of SFc17580 or SFc33230 EAL were amplified using primers containing the ends of the SmaI restriction site of pJQ200SK. These fragments were linked with pJQ200SK linearized by SmaI using the seamless assembly method described above.
The EZ-T Simple Zero pTOPO cloning kit (GenStar) was used to clone intergenic regions, which were then used as DNA probes in EMSA.
Protein expression and purification. E. coli BL21(DE3) or Rosetta(DE3) carrying the expression plasmids were grown in LB medium until the OD 600 had reached 0.8. Gene expression was induced by adding 0.2 mM IPTG for 14 h at 18°C. The cells were harvested (4,000 Â g, 5 min, 4°C) and resuspended in a lysis buffer (25 mM Tris-HCl, pH 8.0, 250 mM NaCl, 50 mM imidazole, 5% glycerol), supplemented with EDTA-free protease-inhibitor cocktail (1 tablet/50 ml buffer; Roche) and 200 mg/ml lysozyme. After sonication, the supernatant was loaded onto nickel columns washed with the same lysis buffer as described above and eluted with gradient imidazole elution from 100 mM to 500 mM. For the enzyme assay, the elution fractions were purified by size exclusion chromatography using a Superdex 200 10/30 column (GE Healthcare) and the SEC buffer (20 mM Tris-HCl, pH 8.0, 250 mM NaCl, 5% glycerol).
Congo red binding assays. E. coli BL21(DE3) or Rosetta(DE3) strains containing pET28a(1) or pET30a-SUMO were grown overnight, subcultured in fresh LB medium, and grown to an OD 600 of 0.8. Next, 5 ml culture was dropped onto the LB agar medium containing Congo red (50 mg/ml) and IPTG (0.5 mM). Plates were incubated at 23°C, 28°C, and 37°C for 3 days before recording, and the Congo red binding phenotype at 23°C was more obvious under test conditions and was shown in Fig. 2 and Fig. S1.
Western blotting. To determine relative expression levels of cloned genes in E. coli strains used in Congo red binding assay, bacteria cultured in LB medium at an OD 600 of 0.8 were subject to protein expression induced by 0.5 mM (final concentration) IPTG (23°C for 16 h). Cells from pre-and postinduction cultures (adjusted to OD 600 of 0.8) were harvested by centrifugation (14,000 Â g for 2 min, 4°C). The pellets were resuspended in SDS-loading buffer and lysed by boiling for 5 min. Each lysate was separated on SDS-PAGE gels and then transferred to nitrocellulose membrane. For immunodetection of individual proteins, HRP (horseradish peroxidase)-conjugated anti-His-Tag mouse monoclonal antibody (CWBIO, China) and ECL Western detection reagents (Solarbio, China) were used.