Negative Regulation of Ectoine Uptake and Catabolism in Sinorhizobium meliloti: Characterization of the EhuR Gene.

RESEARCH ARTICLE

crossm Negative Regulation of Ectoine Uptake and Catabolism in Sinorhizobium meliloti: Characterization of the EhuR Gene Qinli Yu,a,b Hanlin Cai,a,b Yanfeng Zhang,a,b Yongzhi He,a Lincai Chen,a Justin Merritt,c Shan Zhang,a Zhiyang Donga State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing, Chinaa; University of Chinese Academy of Sciences, Beijing, Chinab; Restorative Dentistry, Oregon Health & Science University, Portland, Oregon, USAc

ABSTRACT Ectoine has osmoprotective effects on Sinorhizobium meliloti that differ

from its effects in other bacteria. Ectoine does not accumulate in S. meliloti cells; instead, it is degraded. The products of the ehuABCD-eutABCDE operon were previously discovered to be responsible for the uptake and catabolism of ectoine in S. meliloti. However, the mechanism by which ectoine is involved in the regulation of the ehuABCD-eutABCDE operon remains unclear. The ehuR gene, which is upstream of and oriented in the same direction as the ehuABCD-eutABCDE operon, encodes a member of the MocR/GntR family of transcriptional regulators. Quantitative reverse transcription-PCR and promoter-lacZ reporter fusion experiments revealed that EhuR represses transcription of the ehuABCD-eutABCDE operon, but this repression is inhibited in the presence of ectoine. Electrophoretic mobility shift assays and DNase I footprinting assays revealed that EhuR bound specifically to the DNA regions overlapping the ⫺35 region of the ehuA promoter and the ⫹1 region of the ehuR promoter. Surface plasmon resonance assays further demonstrated direct interactions between EhuR and the two promoters, although EhuR was found to have higher affinity for the ehuA promoter than for the ehuR promoter. In vitro, DNA binding by EhuR could be directly inhibited by a degradation product of ectoine. Our work demonstrates that EhuR is an important negative transcriptional regulator involved in the regulation of ectoine uptake and catabolism and is likely regulated by one or more end products of ectoine catabolism.

Received 12 February 2016 Accepted 20 September 2016 Accepted manuscript posted online 17 October 2016 Citation Yu Q, Cai H, Zhang Y, He Y, Chen L, Merritt J, Zhang S, Dong Z. 2017. Negative regulation of ectoine uptake and catabolism in Sinorhizobium meliloti: characterization of the EhuR gene. J Bacteriol 199:e00119-16. https:// doi.org/10.1128/JB.00119-16. Editor Anke Becker, Philipps-Universität Marburg Copyright © 2016 American Society for Microbiology. All Rights Reserved. Address correspondence to Shan Zhang, [email protected], or Zhiyang Dong, [email protected].

IMPORTANCE Sinorhizobium meliloti is an important soil bacterium that displays

symbiotic interactions with legume hosts. Ectoine serves as a key osmoprotectant for S. meliloti. However, ectoine does not accumulate in the cells; rather, it is degraded. In this study, we characterized the transcriptional regulation of the operon responsible for ectoine uptake and catabolism in S. meliloti. We identified and characterized the transcription repressor EhuR, which is the first MocR/GntR family member found to be involved in the regulation of compatible solute uptake and catabolism. More importantly, we demonstrated for the first time that an ectoine catabolic end product could modulate EhuR DNA-binding activity. Therefore, this work provides new insights into the unique mechanism of ectoine-induced osmoprotection in S. meliloti. KEYWORDS Sinorhizobium meliloti, EhuR, MocR, ectoine, transcriptional regulator

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inorhizobium meliloti is a soil bacterium that can be found either free-living or on the roots of leguminous plants. The ability to adapt to changes in the osmolality of the external soil environment is of vital importance for the growth and survival of soil bacteria, and ectoine serves as a key osmoprotectant for S. meliloti (1). Unlike many other bacteria, however, S. meliloti does not produce ectoine. Therefore, other soil Volume 199 Issue 1 e00119-16

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bacteria presumably serve as potential sources of ectoine for S. meliloti (1). In addition, ectoine does not accumulate within S. meliloti cells; instead, it is degraded. It has been demonstrated that the ehuABCD-eutABCDE operon is involved in ectoine uptake and catabolism in S. meliloti, and an ABC transporter encoded by four genes (ehuA, ehuB, ehuC, and ehuD) has been reported to be responsible for ectoine uptake in this species (1). The ehuABCD genes form an operon with another cluster of putative ectoinecatabolic genes, previously named eutA, eutB, eutC, eutD, and eutE. Although the function of eutABCDE has not been assigned, a homologous operon in Halomonas elongata has been demonstrated, via mutational experiments and comparative analyses, to function in ectoine degradation (2). The ectoine degradation pathway in H. elongata has been reported to be as follows: ectoine is hydrolyzed to N-␣-acetyl-L-2,4diaminobutyric acid via DoeA (EutD); N-␣-acetyl-L-2,4-diaminobutyric acid is deacetylated to L-2,4-diaminobutyric acid (DABA) via DoeB (EutE); and DABA is converted to aspartate semialdehyde in a transaminase reaction via DoeD (SMB_20423) and finally is oxidized to aspartate via DoeC (SMB_20424) (2). A previous study revealed that the expression of the ehuABCD-eutABCDE operon is induced by ectoine and hydroxyectoine, rather than by other osmoprotectants or high osmolality (1). EhuR was suggested to be associated with the ehuABCD and eutABCDE genes (1). In this report, we describe the function of the regulator EhuR, which is encoded by the ehuR gene, upstream of and in the same orientation as the ehuABCDeutABCDE operon. The deduced product of ehuR belongs to the MocR/GntR family. The MocR subfamily belongs to the large GntR superfamily, members of which are characterized by a helix-turn-helix (HTH) DNA-binding domain and an effector-binding domain (3). The C-terminal domain of MocR proteins shows homology to class I aminotransferase proteins (3–6). Here, the role of ehuR in ectoine uptake and catabolism was determined. Both in vitro and in vivo experiments indicated that EhuR is a transcriptional regulator that negatively regulates the expression of genes involved in ectoine uptake and catabolism in S. meliloti. RESULTS ehuR encodes a putative MocR family transcriptional regulator. The ehuR gene (SMB_20426) is located upstream of the ehuABCD-eutABCDE operon and is oriented in the same direction as the other genes in the operon (Fig. 1A). A search of the Conserved Domain Database (CDD) (http://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml) revealed that the predicted gene product, EhuR, belongs to the MocR/GntR subfamily, members of which have been reported to be transcriptional regulators (3, 4, 7, 8). Similar to other MocR proteins described previously, EhuR is predicted to consist of an N-terminal helix-turn-helix DNA-binding domain and a C-terminal domain with homology to members of the aspartate aminotransferase superfamily. A multiple-sequence alignment of EhuR and various MocR-like regulators identified a homologous region in EhuR that appeared truncated relative to the other MocR-like regulators (Fig. 1C). Because of this apparent discrepancy, we decided to scrutinize the sequence features of ehuR. We scanned the ehuR gene sequence to identify potential alternative start codons and noticed that the open reading frame (ORF) continued 105 bp further upstream to an alternative start codon (Fig. 1B). Our 5= rapid amplification of cDNA ends (5=-RACE) analysis revealed that the transcription start site (TSS) of ehuR was identical to the adenine residue of our manually annotated upstream start codon, indicating a leaderless transcript. Leaderless transcripts are mRNAs that are completely devoid of 5= untranslated regions (UTRs), and such transcripts are known to occur in S. meliloti (9) as well as in other organisms, including Escherichia coli, Rhodobacter sphaeroides, and Synechocystis sp. strain PCC6803 (10–12). The 5=-terminal AUG triplet of leaderless mRNAs in E. coli was shown to be competent for ribosome binding (12). Although there was an apparent lack of a potential ribosome-binding site, the upstream start codon could serve as a translation start site. In addition, a recent TSS sequencing study with S. meliloti 1021 identified the same ⫹1 site for ehuR (9), which confirmed our 5=-RACE Volume 199 Issue 1 e00119-16

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EhuR, Ectoine-Responsive Regulator


FIG 1 Amendment of the EhuR translation start site. (A) Genetic organization of the ehuR and ehuABCD-eutABCDE loci in S. meliloti. (B) Region surrounding the annotated ehuR translation start site. Bent arrow, TSS determined by 5=-RACE. Potential start codons are shown in bold and underlined. Black star, amended start codon; gray star, predicted start codon. Putative ⫺35 and ⫺10 regions are underlined. (C) N-terminal ClustalW alignment, including the amended sequence of EhuR and the sequences of several MocR-like regulators. Solid arrow and solid box, amended first amino acid of EhuR; dashed arrow and dashed box, predicted first amino acid of EhuR. The second, third, and fourth MocR-like regulators have been functionally characterized, while the others are predicted. Large box and two thick arrows, DNA-binding domains identified by the Conserved Domain Database.

result. Close to this ⫹1 site, we identified a consensus ⫺35 motif, ideally spaced upstream of the putative ⫺10 sequence. Next, we performed a ClustalW alignment of the first 100 amino acids of the amended EhuR sequence with those of several other MocR-like regulators. As shown in Fig. 1C, the helix-turn-helix DNA-binding domains of EhuR predicted by the Conserved Domain Database are homologous to several MocR-like regulators. The predicted DNA-binding domain of EhuR is located at amino acids 14 to 78, including 22 amino acids from the amended portion (as shown in Fig. 1C). Moreover, we expressed a recombinant EhuR protein in E. coli using the annotated start site and found the protein to be totally insoluble, whereas this was not a problem when the amended ehuR sequence was used (see Fig. S1 in the supplemental material), which suggests that the N terminus is important for the proper folding of EhuR. These results support the hypothesis that the upstream start codon serves as the true translation start site. Therefore, for our subsequent studies, we used the amended EhuR protein of S. meliloti 1021, which is 461 amino acids in length, with a theoretical molecular mass of 50.2 kDa. ehuR and the ehuABCD-eutABCDE operon are cotranscribed. The ehuABCD and eutABCDE genes are organized as an operon in the genome of S. meliloti (Fig. 1A) (1). Volume 199 Issue 1 e00119-16

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FIG 2 Effects of ehuR disruption on gene expression. S. meliloti cells were cultured in LB/MC medium, with or without ectoine, until they reached the mid-exponential phase. The strains used were the WT strain and the ΔehuR strain transformed with a control vector plasmid or a plasmid encoding ehuR. The transcription levels of ehuA and eutA in cells with or without ectoine were determined by quantitative RT-PCR. The transcript levels of the genes are presented relative to those of the WT strain without ectoine. Data are presented as the averages from three independent experiments conducted in triplicate. The transcription of rpsF was monitored and used as the internal control. Error bars, standard deviations.

Since ehuR is in the same orientation as the ehuABCD-eutABCDE operon, we were curious whether ehuR and the ehuABCD-eutABCDE operon could be cotranscribed. To test this, total RNA was isolated from S. meliloti cells grown to the mid-log phase and was used as the template for cDNA synthesis. Primers flanking ehuA and ehuR were used for PCR amplification. If transcription was occurring across the intergenic region between ehuA and ehuR due to cotranscription, it could be verified based on the generation of a corresponding PCR-amplified product. Reverse transcription (RT) reaction mixtures lacking reverse transcriptase, containing genomic DNA from wild-type (WT) S. meliloti, and containing deionized water were used as negative, positive, and blank controls, respectively. It was demonstrated that ehuR and ehuABCD-eutABCDE indeed formed a polycistronic transcriptional unit (Fig. S2). However, it should be mentioned that ehuABCD-eutABCDE has its own promoter, as demonstrated by the promoter-lacZ fusion assays and 5=-RACE analysis. The expression of ehuA and eutA is negatively regulated by S. meliloti EhuR. To examine the role of EhuR in gene expression of the ectoine utilization gene cluster, a S. meliloti ehuR in-frame deletion mutant (ΔehuR) was constructed via double-crossover recombination. In the ΔehuR strain, a 1,275-bp fragment (from bp 109 to 1383 relative to the ehuR translation start site) was deleted by markerless exchange. The 5=-terminal 108-bp fragment of ehuR was preserved for examination of the transcription level of the ehuR promoter in the ΔehuR strain. Moreover, a plasmid carrying the ehuR gene under the control of the lac promoter was introduced into the ΔehuR strain to complement the loss of EhuR. The ehuA and eutA transcript levels in the WT pSRK strain were low in the absence of added ectoine, compared with the levels in the ΔehuR pSRK strain (Fig. 2). In the ehuR-complemented strain, in which ehuR was overexpressed, the ehuA and eutA transcript levels were decreased drastically without added ectoine (Fig. 2). When ectoine was supplemented in the culture medium, the ehuA and eutA transcript levels were drastically increased (about 45-fold) in the WT pSRK and ΔehuR pSRK-ehuR strains. These results suggest that EhuR regulates the expression of ehuA and eutA and that ectoine is also involved in this process, as a direct or indirect antirepressor. To confirm these results, the promoter of ehuA was fused to the lacZ reporter gene and introduced into the WT and ΔehuR strains. For complementation, ehuR with its own promoter was cloned into the lacZ fusion plasmid and introduced into the ΔehuR strain. The activity of the ehuA promoter was increased 9-fold in the ΔehuR strain, compared with the WT strain (Fig. 3). The ␤-galactosidase activity was decreased significantly in the ehuRcomplemented strain, compared with the WT and ΔehuR strains (Fig. 3). With the Volume 199 Issue 1 e00119-16

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FIG 3 ␤-Galactosidase activities of the S. meliloti PehuA-lacZ reporter gene fusions. S. meliloti strains were grown on LB/MC medium with or without ectoine. The levels of expression of the ehuABCD-eutABCDE operon promoter and the ehuR promoter in a WT background (left) and an ehuR-defective background (middle) and of the ehuABCD-eutABCDE operon promoter in an ehuR-complemented strain (right) are shown. All data were derived from three independent experiments conducted in triplicate. Error bars, standard deviations.

addition of ectoine, the activity of the ehuA promoter was increased in the WT strain and the complemented strain but remained unchanged in the ΔehuR strain (Fig. 3). These results indicate that EhuR negatively regulates the expression of the ehuABCDeutABCDE operon. EhuR binds specifically to the upstream regions of ehuA and ehuR. To determine whether EhuR directly regulates the transcription of the ehuABCD-eutABCDE operon, recombinant EhuR protein was expressed in E. coli and purified as an N-terminal His6-tagged protein by using Ni-nitrilotriacetic acid (NTA)-agarose chromatography (Fig. 4A). Because ehuR is cotranscribed with ehuABCD-eutABCDE, probes covering the promoter regions of both ehuR and ehuA were used. In electrophoretic mobility shift assays (EMSA) experiments, EhuR bound to the ehuA promoter, forming two stable protein-DNA complexes, while EhuR bound to the ehuR promoter and formed only one stable complex (Fig. 4B and C). These results indicate that the negative regulation of ehuA by EhuR likely occurs through its direct interaction with the two promoters. To identify the specific binding sites of EhuR, the TSSs of ehuA and ehuR were first determined by 5=-RACE analysis. For ehuA, transcription was found to begin 77 bp

FIG 4 EMSAs for detecting the binding of EhuR to the promoter regions of ehuA and ehuR. (A) SDS-PAGE analysis of purified His6-EhuR. (B) Binding of His6-EhuR to the promoter region of ehuA. The DNA probe (0.7 ng/␮l) was incubated with increasing concentrations of purified EhuR. Lanes 1 to 9, 0, 0.05, 0.1, 0.25, 0.4, 0.55, 0.7, 0.85, and 1 ␮g purified EhuR, respectively. (C) Binding of His6-EhuR to the promoter region of ehuR. The DNA probe (0.7 ng/␮l) was incubated with increasing concentrations of purified EhuR. Lanes 1 to 8, 0, 0.1, 0.25, 0.4, 0.65, 0.8, 1, and 1.3 ␮g purified EhuR, respectively. As a control, 0.7 ng/␮l of the rpsF promoter DNA probe was incubated with 1.3 ␮g purified EhuR. Volume 199 Issue 1 e00119-16

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FIG 5 Identification of EhuR-binding sites in the promoter regions of ehuA (A and B) and ehuR (C and D) in DNase I footprinting assays. (A and C) Fluorograms corresponding to the control DNA (10 ␮M BSA) and DNase protection (400 and 800 nM EhuR) reactions. (B and D) Nucleotide sequences of the promoter regions of ehuA and ehuR. The numbers on the left indicate the length of the sequence. The transcription start sites are indicated by bent arrows. The ehuA and ehuR translation start codons are marked by underlining and asterisks. Sequences protected from DNase I digestion are indicated with shaded boxes. The dashed arrows indicate the conserved motifs.

upstream of the translation start site (Fig. 5B), whereas the 5= end of ehuR was identical to the adenine residue of the amended start codon (Fig. 5D), confirming the presence of a leaderless transcript for ehuR (9). Next, DNase I footprinting analysis was performed to determine the EhuR-binding site within the ehuABCD-eutABCDE promoter. EhuR protected two regions, ranging from bp ⫺23 to ⫺38 and from bp ⫺44 to ⫺61 relative to the ehuA TSS (Fig. 5A), and these regions were included in the promoter fragment used in the EMSAs described above. Analysis of these two DNA regions revealed a highly conserved sequence (ATTGWCA TGATWT [W indicates A or T]) that may serve as a consensus binding site (Fig. 5B). DNase I footprinting analysis with the ehuR promoter indicated that EhuR protects a region from bp ⫺9 to ⫹10 relative to the ehuR TSS (Fig. 5C). EhuR displays different binding affinities for its target promoters. To compare the affinities of EhuR for the two promoters, surface plasmon resonance (SPR) experiments were performed. Two DNA fragments, covering the binding regions revealed in the DNase I footprinting experiments, were immobilized on a streptavidin-coated SA sensor chip (GE Healthcare). The promoter of rpsF (encoding a ribosomal protein) was used as a negative control. As the concentration of EhuR increased, the amount of EhuR bound to the two ligands increased (Fig. 6). The KD (equilibrium dissociation constant) values for the promoters of the ehuABCD-eutABCDE operon and the ehuR gene could be best fitted as 9.14 nM and 32.9 nM, respectively. This result indicated that EhuR bound more tightly to the promoter of the ehuABCD-eutABCDE operon than to the promoter of the ehuR gene. DABA modulates the DNA-binding activity of EhuR. Previous studies showed that the DNA-binding abilities of MocR family transcriptional regulators were modulated by small molecules (4, 13). Because ehuA and eutA expression was found to be ectoine inducible in vivo (Fig. 2), we questioned whether ectoine and/or its degradation products could influence DNA binding by EhuR in vitro. In addition, members of the MocR family have a highly conserved aspartate (class I) aminotransferase domain at Volume 199 Issue 1 e00119-16

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FIG 6 SPR analysis of EhuR binding to the ehuA and ehuR promoter regions. Biotinylated PehuA, PehuR, and PrpsF were immobilized on the SA sensor chip. RU, relative units.

their C terminus, which can bind pyridoxal 5=-phosphate (PLP) as a cofactor (3, 5, 13, 14). PLP, alone or with other small molecules, proved to modulate the DNA-binding activities of MocR family regulators (5, 13–16). Since the putative PLP-binding site is highly conserved in EhuR (data not shown), we analyzed EhuR-mediated mobility shifts in the presence of PLP as well as ectoine, DABA (a catabolic end product), and aspartate (Fig. 7). Other catabolic end products in the ectoine degradation pathway were not tested because the molecules were not available. As shown in Fig. 7, DABA alone could inhibit the binding of EhuR to both promoters (PehuA and PehuR) in a concentrationdependent manner, although we noted that the DABA concentration required for inhibition in the in vitro assay was rather high. DABA exhibited a slightly stronger inhibitory effect on the binding of EhuR to PehuA than to PehuR. In addition, 24 mM DABA completely inhibited the binding to PehuA, whereas 30 mM DABA was required to achieve total inhibition of the binding to PehuR. In contrast, ectoine, aspartate, and PLP exhibited no effects on the binding of EhuR to DNA probes. These results suggest that not ectoine itself but one or more of the degradation products in the ectoine catabolism pathway modulate the repression activity of EhuR. DISCUSSION Uptake of exogenous compatible solutes such as ectoine is preferable to de novo synthesis because it is more energetically favorable (17, 18). Ectoine is always trans-

FIG 7 Effects of ectoine, catabolic degradation products, and PLP on the binding of EhuR to PehuA and PehuR. A range of concentrations of ectoine, DABA, or aspartate (Asp), either alone or in combination with PLP, were added in 50 ␮l of reaction buffer, and the mixture was incubated at 30°C for 30 min. The final concentrations of the compounds are shown at the top of each image. (A and B) Effects of small molecules on the binding of EhuR to PehuA. The EMSAs were performed with the same concentrations of EhuR (0.02 ␮g/␮l) and PehuA DNA (0.7 ng/␮l). (C and D) Effects of small molecules on the binding of EhuR to PehuR. The EMSAs were performed with the same concentrations of EhuR (0.03 ␮g/␮l) and PehuR DNA (0.7 ng/␮l). Volume 199 Issue 1 e00119-16

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ported by osmoprotectant transporters together with other osmoprotectants. In S. meliloti, however, ectoine uptake is conducted by a specific transporter, the Ehu transporter (1, 19). Additionally, ectoine does not accumulate within S. meliloti cells; instead, it is degraded (1). Previous studies of the ectoine uptake and catabolism operon revealed that the expression of the operon is induced by its substrate but not by elevated osmolality, which indicates that a new regulatory mechanism may exist (1). In the present study, we found that EhuR, a MocR family transcription factor, negatively regulates the transcription of an operon involved in ectoine uptake and catabolism, ehuABCD-eutABCDE, by directly binding to the region from bp ⫺61 to ⫺23 of the operon promoter and the region from bp ⫺9 to ⫹10 of the ehuR promoter. Moreover, DABA, a degradation product of ectoine, appears to release the repression of the ehuABCD-eutABCDE operon through dissociation of EhuR from its targets. The ectoine transporter in S. meliloti, encoded by ehuABCD, belongs to the ATPbinding cassette transporter family and is mainly subject to transcriptional regulation (20, 21). Bioinformatic analysis revealed that ehuR belongs to the MocR family, which has a characteristic structure, i.e., an N-terminal HTH DNA-binding domain and a C-terminal domain with homology to members of the aspartate aminotransferase superfamily (3). These proteins require the cofactor PLP to catalyze the reversible transfer of an amino group from an amino acid to an acceptor (8, 13, 14). Members of the MocR family can be found in both Gram-negative and Gram-positive bacteria. Previous studies demonstrated that a MocR-like protein in S. meliloti regulates the synthesis of the symbiosis-specific compound rhizopine to facilitate competition in the rhizosphere by initiating nodulation of Medicago sativa (22, 23). However, only a few members of the MocR family have been functionally characterized and shown to be bona fide transcriptional regulators, based on direct binding to their target genes (5, 6, 13, 15). One MocR protein (GabR) of Bacillus subtilis was characterized as an activator of genes involved in ␥-aminobutyrate (GABA) utilization (4). GabR activates transcription of the gabTD operon in the presence of PLP and GABA and represses its own transcription in the absence of GABA (13). PdxR, another MocR protein, directly activates the divergently transcribed pdxST operon (which is responsible for the de novo synthesis of PLP) and serves as a negative autoregulator in Corynebacterium glutamicum, Listeria monocytogenes, and Bacillus clausii. PLP decreases the activation activity and increases the repression activity of PdxR (5, 7, 14, 15). Still another MocR family member, TauR, was found to activate taurine-dependent tpa expression (6). Five of 54 putative GntR family transcriptional regulators in S. meliloti can be classified into the MocR subfamily (24), and none of those proteins has been characterized previously. Unlike other MocR family transcriptional regulators that were shown to activate their targets, we discovered that EhuR negatively regulates the transcription of ehuABCDeutABCDE. RT-PCR analysis revealed that ehuR is cotranscribed with the ehuABCD-eutABCDE operon. To determine whether the regulation of ehuA is dependent on the regulation of ehuR, a SPR assay was conducted. The results revealed that EhuR has slightly higher affinity for the ehuA promoter than for the ehuR promoter, indicating that EhuR has a dominant regulatory effect on the transcription of ehuA. Typically, transcriptional regulators from the MocR family bind to short direct repeats (5, 6, 13, 14). Two regions of the ehuA promoter protected by EhuR (ATTGTC ATGATATTAT, bp ⫺59 to ⫺44 relative to the ehuA TSS, and ATTGACATGATTTGGG, bp ⫺36 to ⫺21 relative to the ehuA TSS) were consistent with the motifs predicted by the RegPrecise database (http://regprecise.lbl.gov/RegPrecise/sites.jsp?regulog_id⫽6597) (25). Sequence analyses of the EhuR protected regions from the ehuA promoter and other motifs predicted by RegPrecise for members of Rhizobiales (the nucleotide sequences extracted from the RegPrecise database are shown in Table S2 in the supplemental material) revealed the presence of a conserved motif (Fig. 8A). A further multiple-sequence alignment of the promoter regions of ectoine transporter genes from seven different Rhizobium species showed a high level of sequence conservation (Fig. 8B). A putative MocR/GntR family regulator is typically found near the transporter Volume 199 Issue 1 e00119-16

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FIG 8 Sequence logos of EhuR-binding motifs from ehuA promoters and multiple-sequence alignment of the ehuA promoter regions from different Rhizobium species. (A) The consensus sequence was created by aligning the DNA sequences of 17 motifs. The consensus logo was created using WEBLOGO (32). (B) Nucleotide sequences of the promoter regions from seven Rhizobium strains were aligned using Clustal Omega software (http://www.ebi.ac.uk /Tools/msa/clustalo). Bent arrow, transcription start site determined by 5=-RACE. Dashed arrows, direct repeats within the EhuR-binding site. Asterisks, nucleotides identical in all genomes. M. opportunistum, Mesorhizobium opportunistum; S. medicae, Sinorhizobium medicae; S. fredii, Sinorhizobium fredii; E. adhaerens, Ensifer adhaerens; R. leguminosarum, Rhizhobium leguminosarum.

genes, strongly suggesting that the EhuR regulation mechanism may exist in these Rhizobium strains as well. A motif (TTCAAATCATGACAAA, bp ⫺8 to ⫹8 relative to the ehuR TSS) within its own promoter was also found to be protected by EhuR. Interestingly, this motif is reverse complementary to the binding sequence for the ehuA promoter. Other reverse complementary motifs can be found in the RegPrecise database, and this is apparently not a unique phenomenon. The reverse complementary motif might also account for the lower binding affinity of EhuR for its own promoter, compared to the ehuA promoter. We also assayed the transcript abundance of smb_20425, and no obvious regulation by EhuR was detected (data not shown). Thus, this reverse complementary region is unlikely to be a binding motif for smb_20425. In previous studies, small-molecule ligands were found to trigger dissociation of MocR from its target DNA sequence. In B. subtilis, GABA and PLP are essential for transcription activation of the gabTD operon by GabR but not for gabR autoregulation (13). Similarly, in L. monocytogenes, PLP is the only effector known to reduce the activation activity of PdxR while increasing its repressor activity (15). Regarding EhuR, quantitative RT-PCR and promoter-lacZ reporter assays clearly indicated that the expression of ehuA and eutA was enhanced by ectoine in vivo. Interestingly, ectoine alone or in combination with PLP had no effect on the binding of EhuR to its targets, while its degradation product DABA was found to inhibit the binding of EhuR to both of its DNA targets. We propose a hypothetical model for EhuR-mediated regulation of ehuR and ehuABCD-eutABCDE expression. Based on quantitative RT-PCR and 5=-RACE data, ehuA is expressed from two promoters. The shorter transcript starts 77 bp upstream of the ehuA translation start codon, whereas the longer transcript starts further upstream, at the adenine residue of the amended start codon of ehuR. EhuR represses the transcription of ehuABCD-eutABCDE by directly binding to the region from bp ⫺61 to ⫺23 of its promoter and the region from bp ⫺9 to ⫹10 of the ehuR promoter. The SPR assay revealed that EhuR has higher affinity for the promoter of the ehuABCD-eutABCDE operon than for its own promoter. The repression maintains an extremely low level of Volume 199 Issue 1 e00119-16

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TABLE 1 Strains and plasmids used in this study Strain or plasmid E. coli strains DH5␣ BL21(DE3) Sinorhizobium strains WT ΔehuR mutant Plasmids pK18mobsacB pK18ER pSRK pSRK-ehuR pRK2013 pGD926 PehuA-pGD926 ehuR-PehuA-pGD926 pSMER

Relevant properties

Reference or source

F⫺ ␾80lacZΔM15 Δ(lacZYA-argF)U169 deoR recA1 endA1 hsdR17(rK⫺ mK⫹) phoA supE44 ␭⫺ thi-1 gyrA96 relA1 F⫺ dcm ompT hsdS(rB⫺ mB⫹) gal ␭ (DE3)

Invitrogen Novagen

S. meliloti 1021 S. meliloti 1021 ΔehuR Smr

33 This study

Suicide vector; Kmr pK18mobsacB carrying 1,500-bp joint DNA fragment from S. meliloti smb_20425 and ehuA Expression vector under control of lac promoter; Tcr pSRK carrying S. meliloti ehuR gene; Tcr Kmr ␤-Galactosidase expression vector pGD926 carrying 170-bp DNA fragment upstream of ehuA; Smr Kmr pGDAP derivate with insertion of fragment containing coding region of ehuR and its upstream region pET28a carrying S. meliloti ehuR gene; Kmr

27 This 34 This 35 28 This This

study study

study study

This study

basal ehuABCD-eutABCDE transcription. When ectoine is present outside the cells, the basal production of ehuABCD-eutABCDE allows a small amount of ectoine to be transported into the cells; the ectoine is subsequently degraded, yielding DABA and other degradation products. One or more of the degradation products, such as DABA, inhibit EhuR repression of the ehuABCD-eutABCDE operon. This, in turn, triggers increased production of the Ehu transporter, allowing a large amount of ectoine to be transported into the cells for osmoprotection and catabolism. With the decline in the external concentration of DABA, EhuR binds to the DNA again and recovers its repression activity. At present, DABA is the only molecule found to directly affect EhuR activity, although the possibility that other catabolic end products or other small molecules also serve as ehuABCD-eutABCDE operon inducers cannot be excluded. MATERIALS AND METHODS Bacterial strains and growth conditions. The bacterial strains and plasmids used in this study are listed in Table 1. Escherichia coli strains were grown at 37°C in Luria-Bertani (LB) medium. S. meliloti strains were grown at 28°C in LB medium supplemented with 2.5 mM CaCl2 and 2.5 mM MgCl2 (LB/MC) (26). For the selection of E. coli and S. meliloti strains, antibiotics were added at the following final concentrations: 100 ␮g/ml ampicillin, 50 ␮g/ml kanamycin, 100 ␮g/ml streptomycin, 25 ␮g/ml chloramphenicol, and 10 ␮g/ml tetracycline. Construction of ehuR in-frame deletion mutant. Homologous regions upstream and downstream of the ehuR ORF were amplified using Fast Pfu DNA polymerase (TransGen Biotech) with the P1-P2 and P3-P4 primer pairs, respectively. After purification, both PCR products were assembled using the P1-P4 primer pair. The PCR product was digested with EcoRI and XbaI and cloned into pK18mobsacB (27). The recombinant plasmid pK18ER was mobilized into S. meliloti by triparental mating with pRK2013. Positive selection was performed by plating on LB/MC agar containing streptomycin and kanamycin, to identify S. meliloti individuals that had integrated the narrow-host-range plasmid into the genome. After selection of the transconjugants, individuals that had undergone a second recombination and allelic replacement were selected on LB/MC agar containing 5% sucrose. The resulting mutant ΔehuR was verified by PCR and sequencing. The ehuR ORF was also amplified with the P5-P6 primer pair and cloned into the broad-host-range vector pSRK, to generate pSRK-ehuR (Table 1). The recombinant plasmid pSRK-ehuR was introduced into the ehuR mutant (ΔehuR) to obtain the complemented strain (ΔehuR pSRK-ehuR). Isolation of RNA and analysis of gene transcription. Total RNA was isolated from cells grown to the log phase (optical density at 600 nm [OD600] of ⬃0.8) in LB/MC medium, with or without ectoine (Sigma), using TRIzol reagent (Life Technologies, Gaithersburg, MD). RNA was treated with RNase-free DNase (TaKaRa) to remove the remaining genomic DNA. The purified RNA was reverse transcribed using the Superscript III first-strand synthesis kit (Invitrogen, Carlsbad, CA). Real-time PCR was performed using the TransStart Top Green quantitative PCR (qPCR) SuperMix kit (TransGen Biotech) and the ABI 7500 real-time PCR detection system. Each 20 ␮l of PCR mixture contained the following: 10 ␮l of 2⫻ TransStart Top Green qPCR SuperMix, 0.2 ␮M forward primer, 0.2 ␮M reverse primer, and 0.5 ␮l of the diluted cDNA template. The real-time PCR amplification parameters were as follows: 95°C for 10 s, followed by 40 cycles of 5 s of denaturation at 94°C and 30 s of annealing and extension at 60°C. A melting curve analysis with a temperature gradient from 50°C to 99°C, at 0.1°C/s, was performed. All PCRs Volume 199 Issue 1 e00119-16

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were performed in triplicate. The transcripts of ehuA, eutA, and rpsF were amplified with primers P7 to P12 (listed in Table S1 in the supplemental material). Promoter-lacZ fusion constructs and ␤-galactosidase assays. A DNA fragment of approximately 200 bp containing the ehuA promoter region was amplified with the P13-P14 primer pair (Table S1). The amplicons were digested with HindIII or BamHI and cloned into pGD926 (28), yielding the recombinant plasmid PehuA-pGD926 (Table 1). For the complementation experiment, a fragment containing the 1,386-bp open reading frame of ehuR and its upstream region was introduced into PehuA-pGD926 upstream of the lacZ promoter, yielding the recombinant plasmid ehuR-PehuA-pGD926. To assay ␤-galactosidase activity, the cells were grown to the mid-exponential phase in 50 ml of LB/MC medium and then were collected by centrifugation at 6,000 ⫻ g for 5 min at 4°C. ␤-Galactosidase activity was assessed by measuring the hydrolysis of o-nitrophenyl-␤-D-galactoside, as described by Miller (29). The assays were conducted with at least three technical replicates per strain, and at least three biological replicates were used. 5=-RACE analysis. To determine the TSSs of ehuA and ehuR, 5=-RACE was performed as described previously (10). RNA was extracted with TRIzol reagent (Life Technologies), and 20 ␮g of total RNA was digested with 2.5 ␮l of tobacco acid pyrophosphatase (TAP) (Epicentre) for 2 h at 37°C. An aliquot of RNA that had not been treated with TAP was included as a control. After ethanol precipitation, the RNA was ligated to a 5=-RNA adapter (5=-CAGACUGGAUCCGUCGUC-3=; Integrated DNA Technologies). After a second ethanol precipitation, adapter-ligated RNA was used as the template for reverse transcription with SuperScript III reverse transcriptase (Invitrogen), using adapter primers and gene-specific primers (P15 and P17) (listed in Table S1). The resulting cDNA was used for first-run PCR amplification with adapter primers and gene-specific primers (P15 and P17). In the second-run PCR amplification, the products of the first-run PCR and genespecific primers (P16 and P18) that targeted a region less than 200 nucleotides downstream were used. The PCR products were separated by agarose gel electrophoresis and sequenced. Expression and purification of EhuR-His6 protein. The ehuR coding region was amplified from genomic DNA using primers P5 and P6. The amplified DNA fragment was digested with NdeI and XhoI and ligated into pET28a (Novagen). The resultant plasmid, pSMER, contains the recombinant gene encoding EhuR fused to a His6 tag sequence at the N terminus. The plasmid was confirmed by DNA sequencing and introduced into E. coli strain BL21(DE3). The recombinant gene was expressed in exponentially growing cells (OD600 of 0.6) through the addition of isopropyl-␤-D-thiogalactopyranoside (IPTG) to a final concentration of 1 mM. The cultures were incubated for 20 h at 16°C. The cultures were collected by centrifugation and washed with binding buffer (20 mM phosphate buffer, 500 mM NaCl, 5 mM imidazole [pH 7.0]). The recombinant protein was purified by nickel affinity chromatography as described previously (4). Electrophoretic mobility shift assays. EMSAs were performed as described by Wang et al. (30). DNA probes containing the promoter regions of ehuA and ehuR were generated by PCR from genomic DNA. For the ehuA upstream region, primers P19 and P20 (Table S1) were used to generate a 246-bp fragment containing the ehuA promoter. For the ehuR upstream region, primers P21 and P22 (Table S1) were used to generate a 204-bp fragment containing the ehuR promoter. A probe containing the rpsF (encoding a ribosomal protein) promoter was used as a negative control. The DNA probe (0.7 ng/␮l) was incubated for 30 min at 30°C with various concentrations of purified His6-tagged EhuR in reaction buffer (20 mM phosphate [pH 7.0], 2 mM dithiothreitol [DTT], 5 mM MgCl2, 5 mg/ml bovine serum albumin [BSA], 5% [vol/vol] glycerol); each reaction mixture had a final volume of 20 ␮l. The samples were loaded on a native 6% polyacrylamide gel. After electrophoresis, the gels were stained for 30 min with SYBR Gold nucleic acid gel stain (Invitrogen) and photographed under UV transillumination using a Bio-Rad Gel Doc XR system. DNase I footprinting assays. DNase I footprinting assays were performed using a fluorescent labeling procedure, as described previously (31). Briefly, DNA fragments were prepared by PCR using the fluorescently labeled primers listed in Table S1; primers P19 and P20 were used for the EhuR-binding site in the promoter of the ehuABCD-eutABCDE operon, and primers P21 and P22 were used for the EhuR-binding site in the ehuR promoter. After gel purification, 200 ng of labeled DNA fragments were incubated with various concentrations of proteins in a total volume of 50 ␮l. After incubation at 30°C for 30 min, 0.8 U of DNase I (Promega) and 5.5 ␮l of DNase I reaction buffer were added and the mixture was incubated at 37°C for 1 min. To stop the reaction, 10 ␮l of stop buffer was added. After phenol-chloroform extraction and ethanol precipitation, the samples were sequenced by Sangon Biotech (China). Electropherograms were analyzed with GeneMapper v4.0 (Applied Biosystems). Surface plasmon resonance assays. SPR assays were performed on a Biacore 3000 system (GE Healthcare). The experiments were carried out in running buffer composed of 20 mM Tris-HCl, 150 mM NaCl, and 0.005% Tween 20 (pH 7.0), with a flow rate of 30 ␮l/min. The 5=-biotinylated double-stranded DNA fragments of the ehuA promoter and ehuR promoter were immobilized on a SA sensor chip (GE Healthcare). A DNA fragment of the rpsF promoter was used as a negative control. His6-tagged EhuR, with or without small-molecule ligands, was diluted in running buffer to achieve solutions of different concentrations and was injected with the K-inject command. At the end of each cycle, 0.05% sodium dodecyl sulfate was used to regenerate the surface of the sensor chip. The data were fit to the binding model by using BIAevaluation 4.1 software (GE Healthcare).

SUPPLEMENTAL MATERIAL Supplemental material for this article may be found at https://doi.org/10.1128/ JB.00119-16. TEXT S1, PDF file, 0.4 MB. Volume 199 Issue 1 e00119-16

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Yu et al.


ACKNOWLEDGMENTS We thank Wang Yipping and Tian Zhexian (School of Life Sciences, Peking University) for sharing their expertise and skills and Luo Li (School of Life Sciences, Shanghai University) for providing pSRK. This work was supported by the National Natural Science Foundation of China (grant 31100066) and the National High Technology Research and Development Program (863 Program, grant 2012AA02A703).

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L-Hydroxyproline and d-Proline Catabolism in Sinorhizobium meliloti.

Role and Regulation of ACC Deaminase Gene in Sinorhizobium meliloti: Is It a Symbiotic, Rhizospheric or Endophytic Gene?

Regulation of Polyhydroxybutyrate Accumulation in Sinorhizobium meliloti by the Trans-Encoded Small RNA MmgR.

Genomic characterization of Sinorhizobium meliloti AK21, a wild isolate from the Aral Sea Region.

Biogeography of Sinorhizobium meliloti nodulating alfalfa in different Croatian regions.

Use of the computer-retargeted group II intron RmInt1 of Sinorhizobium meliloti for gene targeting.

Biochemical and functional characterization of SpdA, a 2', 3'cyclic nucleotide phosphodiesterase from Sinorhizobium meliloti.

Global analysis of cell cycle gene expression of the legume symbiont Sinorhizobium meliloti.

Formation of chloroform and tetrachloroethene by Sinorhizobium meliloti strain 1021.

Identification of a dominant gene in Medicago truncatula that restricts nodulation by Sinorhizobium meliloti strain Rm41.

Functional characterization of the CDF transporter SMc02724 (SmYiiP) in Sinorhizobium meliloti: Roles in manganese homeostasis and nodulation.

The underlying process of early ecological and genetic differentiation in a facultative mutualistic Sinorhizobium meliloti population.

Rhizobial galactoglucan determines the predatory pattern of Myxococcus xanthus and protects Sinorhizobium meliloti from predation.

Sinorhizobium meliloti flavin secretion and bacteria-host interaction: role of the bifunctional RibBA protein.

Inactivation of group II intron RmInt1 in the Sinorhizobium meliloti genome.

Osmoprotection of Escherichia coli by ectoine: uptake and accumulation characteristics.

Correction: Quantitative Proteomic Analysis of the Hfq-Regulon in Sinorhizobium meliloti 2011.

Metabolic modelling reveals the specialization of secondary replicons for niche adaptation in Sinorhizobium meliloti.

Mixed Nodule Infection in Sinorhizobium meliloti-Medicago sativa Symbiosis Suggest the Presence of Cheating Behavior.

Local and systemic proteomic changes in medicago truncatula at an early phase of Sinorhizobium meliloti infection.

Complete Genome Sequence of the RmInt1 Group II Intronless Sinorhizobium meliloti Strain RMO17.

The Sinorhizobium meliloti EmrR regulator is required for efficient colonization of Medicago sativa root nodules.

Cloning and characterization of the mouse JDP2 gene promoter reveal negative regulation by p53.

Insights into the history of a bacterial group II intron remnant from the genomes of the nitrogen-fixing symbionts Sinorhizobium meliloti and Sinorhizobium medicae.