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Journal of Bacteriology, January 2009, p. 434-438, Vol. 191, No. 1
0021-9193/09/$08.00+0     doi:10.1128/JB.01247-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

The LuxR Family Quorum-Sensing Activator MrtR Requires Its Cognate Autoinducer for Dimerization and Activation but Not for Protein Folding ^,

Menghua Yang,¹ Jennifer L. Giel,² Tao Cai,¹ Zengtao Zhong,³ and Jun Zhu^1,2,3*

Department of Microbiology, MOA Key Lab of Microbiological Engineering of Agricultural Environment, Nanjing Agricultural University, Nanjing, China,¹ Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania,² The State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing, China³

Received 6 September 2008/ Accepted 21 October 2008

Top ABSTRACT INTRODUCTION REFERENCES

 
MrtR, a LuxR homolog in Mesorhizobium tianshanense, is important for symbiosis. We found that MrtR requires its cognate N-acylhomoserine lactone for forming dimers, binding to a single DNA site and activating the downstream promoter. However, MrtR is able to fold independently of its ligand.

Top ABSTRACT INTRODUCTION REFERENCES

 
Many bacteria communicate by releasing and detecting diffusible chemical signals to regulate various physiological processes, including virulence, symbiosis, and biofilm formation (10, 30). A family of these quorum-sensing regulatory systems that widely exist in gram-negative bacteria resembles the LuxR and LuxI proteins of Vibrio fischeri, where LuxI-type proteins generate N-acylhomoserine lactones (AHLs; also called autoinducers) that act as pheromones and LuxR type proteins serve as pheromone receptors and transcriptional regulators (8).

LuxR type proteins have been the subject of extensive studies. They are thought to contain an amino-terminal module (comprising about two-thirds of the protein) that binds autoinducers and mediates dimerization as well as a carboxyl-terminal module that binds particular DNA sites near target promoters (9). Of these LuxR family proteins, TraR from Agrobacterium tumefaciens has been well characterized. Biochemical and structural analyses show that 3-oxooctanoyl homoserine lactone (3OC₈-HSL), the cognate autoinducer for TraR, binds to TraR monomers in a 1:1 molar ratio and that these dimerized TraR-3OC₈-HSL complexes bind to TraR target DNA (24, 32, 36). Moreover, TraR requires 3OC₈-HSL for stability. Autoinducers act as scaffolds for the correct folding of the TraR protein, and TraR synthesized in the absence of 3OC₈-HSL is targeted for rapid proteolysis or forms inclusion bodies (36, 37). Other LuxR type proteins, however, have various AHL receptor interaction patterns. Based on the recent biochemical characterization of different LuxR type proteins, Schuster and Greenburg proposed that LuxR type proteins can be divided into three classes (25). Class I proteins, including TraR, LasR of Pseudomonas aeruginosa (26), and CepR of Burkholderia cenocepacia (31), require autoinducer for folding and, once folded, bind AHL tightly. Class II proteins, which include QscR from P. aeruginosa (14) and LuxR from V. fischeri (28), require AHLs for folding, but binding of AHLs is reversible. Class III proteins, including EsaR of Pantoea stewartii (22) and ExpR of Erwinia chrysanthemi (2), do not require AHLs for folding, and the mature proteins bind AHLs reversibly. Interestingly, both EsaR and ExpR function as repressors that bind to their target DNA sequence in the absence of AHLs. Interactions with the signal cause dissociation from the DNA, thereby derepressing target genes. The transcriptional activator RhlR of P. aeruginosa may also belong to this class (20), but AHL binding has yet to be assessed with purified RhlR in vitro.

Previously, we identified the LuxR type protein MrtR in Mesorhizobium tianshanense, which forms nodules and fixes nitrogen on the roots of Glycyrrhiza (licorice) (34). The quorum-sensing regulatory components MrtR and MrtI, the LuxI type protein that is responsible for synthesis of 3OC₁₂-HSL and 3OC₁₄-HSL (33), are indispensable for nodulation. How MrtR regulates nodulation is currently unknown, but genetic studies show that MrtR and its cognate autoinducers are required to activate the expression of mrtI (34). To further investigate how MrtR functions compared to other LuxR type proteins, we PCR amplified the mrtR coding sequence and cloned it into pET-28 (Invitrogen) to create a P_T7-His₆·mrtR fusion. The N-terminal six-histidine tags did not affect MrtR function, as the His-tagged MrtR could restore AHL production in the mrtR mutant (data not shown). To test whether autoinducers are required for MrtR protein folding, we grew Escherichia coli BL21 DE3 containing the P_T7-His₆·mrtR plasmid in the absence or in the presence of cognate AHLs. Total and soluble protein fractions were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and Western blot analyses were performed using an anti-His₆ antibody (Rockland) (Fig. 1A). Even in the absence of autoinducers, most of the MrtR protein was soluble, unlike many other LuxR type proteins such as TraR (36), LasR (26), and LuxR (28) that form inclusion bodies when overexpressed in E. coli in the absence of their cognate autoinducers. These data suggest that MrtR protein can fold independently of its ligand.

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FIG. 1. MrtR requires cognate autoinducer for activity but not for folding. (A) Autoinducer does not affect MrtR solubility. E. coli BL21 DE3 containing a PT7-His6·mrtR plasmid was grown in LB medium without autoinducer or with 1 µM 3OC12-HSL (Cayman Chemical Co.). After induction, cells were harvested, both total and soluble proteins (cleared by ultracentrifugation) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and MrtR proteins were detected with anti-His6 antibodies. (B) MrtR gel retardation assays were performed as described previously (36) with a 32P-end-labeled 300-bp DNA fragment containing the mrtI promoter region. (C) DNase I protection of a predicted MrtR binding site. The top strand of a 284-bp mrtI promoter fragment was 32P end labeled and incubated without MrtR and with 100 nM MrtR in the absence or presence of 1 µM 3OC12-HSL. The +1 transcriptional start was based on the prediction described in reference 19. G+A sequencing ladders were generated as previously described (18). (D) Gel filtration assay results. A total of 0.5 mg of apoMrtRwt (), apoMrtRG114S (an AHL-independent MrtR mutant) (), and MrtRwt –3OC12-HSL () was loaded on a Bio-Gel P-100 column (Bio-Rad) and eluted with PBS with or without 0.1 µM 3OC12-HSL at a rate of 0.25 ml/min. The fractions (0.25 ml) were then assayed for protein concentration using the Bradford assay (Bio-Rad). The positions of the standard proteins (carbonic anhydrase from bovine erythrocytes and albumin from bovine serum) used for column calibration are indicated.

To test whether MrtR requires autoinducers to bind to target DNA, we performed gel retardation assays. When 1 µM 3OC₁₂-HSL was included in the binding reaction, MrtR protein purified in the absence of autoinducers retarded the mobility of a 300-bp DNA fragment containing the mrtI promoter region, forming a single complex (Fig. 1B). The K_d for binding was approximately 10^–7 M, and the Hill coefficient was 3.4, indicating that MrtR cooperatively bound this site. No binding was detected when the gel shift reaction contained no autoinducers (Fig. 1B) or contained noncognate autoinducer 3OC₈-HSL (data not shown). These data suggest that MrtR specifically binds the target mrtI promoter DNA in a cognate autoinducer-dependent manner. DNase I footprinting localized 3OC₁₂-HSL-dependent MrtR binding to an 18-bp inverted repeat from –69 to –52 relative to the predicted mrtI transcriptional start site, based on the closely orthologous gene cinI from Rhizobium leguminosarum biovar viciae (19) (Fig. 1C). Deletion of this 18-bp repeat sequence in mrtI promoter DNA completely abolished the ability of MrtR to bind to the DNA in vitro (see Fig. S1A in the supplemental material) and to induce transcription from the mrtI promoter in M. tianshanense (see Fig. S1B in the supplemental material), confirming the essential role of the 18-bp repeat sequence in MrtR binding.

Extensive studies of LuxR type proteins in vitro and in vivo reveal that an important consequence of autoinducer binding is dimerization (25). The ability to purify abundant soluble apoMrtR enables us to test directly whether addition of cognate autoinducers can induce dimerization in vitro. To confirm that MrtR protein could form a complex with 3OC₁₂-HSL in vitro, we incubated MrtR with 3OC₁₂-HSL for 30 min and precipitated MrtR proteins with trichloroacetic acid. AHL activity was detected in the sample with AHL bioassays (35) (see Fig. S2A in the supplemental material). Binding of 3OC₁₂-HSL to MrtR was reversible, as no AHL activity was observed once MrtR-3OC₁₂-HSL was dialyzed against phosphate-buffered saline (PBS) buffer for 30 min (see Fig. S2A in the supplemental material). Control experiments showed no detectable AHL activity from trichloroacetic acid-precipitated samples that contained 3OC₁₂-HSL alone or MrtR incubated with 3OC₈-HSL (see Fig. S2B in the supplemental material). We then used gel filtration chromatography to estimate the molecular weights of apoMrtR and the MrtR-3OC₁₂-HSL complex by passing 0.5 mg of either apoMrtR or MrtR incubated with 1 µM 3OC₁₂-HSL over a sizing column (BioGel P100; Bio-Rad) and eluting with PBS buffer with or without 0.1 µM 3OC₁₂-HSL (Fig. 1D). apoMrtR eluted as a monomer (31 kDa), but in the presence of 3OC₁₂-HSL, MrtR protein produced a higher peak at the position corresponding to the dimer. These results indicate that binding of autoinducers promotes the shift of MrtR from monomers to dimers.

The above biochemical studies indicate that the transcriptional activator MrtR protein belongs to class III of the LuxR type proteins that do not require autoinducers for folding and that bind ligands reversibly. To date, most class III LuxR family proteins studied are repressors (25). Intriguingly, MrtR is the activator for mrtI in an autoinducer-dependent manner in M. tianshanense. To determine the regions of MrtR necessary for transcriptional activation of mrtI, eight truncated forms of MrtR were generated by PCR amplification and cloning of the mrtR truncation mutations under the control of the P_lac promoter on the vector pBBR1-MCS5 (12) and introduced into an M. tianshanense mrtR mrtI strain (34) containing a P_mrtI-lacZ translational fusion plasmid (1). Western blot analysis with anti-MrtR antibodies indicated that all of the truncated forms of MrtR were stable in M. tianshanense in the absence or presence of 3OC₁₂-HSL (data not shown). Figure 2A shows that, as predicted, full-length MrtR activated the expression of mrtI only in the presence of 3OC₁₂-HSL. Similar to C-terminal truncations of LuxR (5), RhlR (13), and TraR (17), MrtR 233-271, which lacks the conserved HTH DNA binding domain, had no activity. MrtR 2-30 was similar to full-length MrtR. However, there are three different potential translation start sites that are all conserved in MtrR and CinR from R. leguminosarum and Rhizobium etli (shown highlighted in Fig. S3 in the supplemental material) (11, 15). Currently, we do not have experimental evidence to show which one is used for MrtR. Deletion of up to 50 N-terminal amino acids enhanced MrtR activity even in the absence of 3OC₁₂-HSL. Interestingly, MrtR protein is one of largest known LuxR type proteins, and this additional region (from amino acid 30 to 50) is present only in a few LuxR type proteins from other rhizobia, such as CinR (6) and BisR (see Fig. S3 in the supplemental material) (7). It will be intriguing to test whether these 20-amino-acid regions in other proteins also play a similar regulatory role. MrtR 2-80, 2-120, and 2-140 showed no activity; however, MrtR 2-160 displayed a low level of autoinducer-independent activity. Previous studies of LuxR and TraR suggest that in activators of the LuxR family, the N-terminal domain masks the C-terminal DNA binding domain in the absence of autoinducer, thus interfering with DNA binding, and the C-terminal domain alone was shown to function in AHL-independent activation (4, 16).

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FIG. 2. Transcriptional activity of MrtR and its derivatives. M. tianshanense mrtR mrtI mutants containing a plasmid harboring the PmrtI-lacZ translational fusion reporter and a plasmid harboring Plac-mrtR wild-type truncations (A) or point mutations (B) were grown in TY medium (29) at 28°C to mid-log in the absence or in the presence of 1 µM 3OC12-HSL. β-Galactosidase activity was then measured and presented as Miller units (21). The results are the average of three experiments ± the standard deviation.

To further characterize the structure and function of MrtR, we screened for MrtR constitutive mutants. We used the E. coli mutator strain XL-1 Red (Stratagene) to introduce mutations into the P_lac-mrtR plasmid. The mutated plasmids were then transformed into an M. tianshanense mrtR mrtI strain containing a P_mrtI-lacZ plasmid, and the transformants were spread onto X-Gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside) plates without any autoinducer. Approximately 25,000 colonies were screened, of which 8 exhibited a LacZ⁺ phenotype in the absence of autoinducer and had point mutations in the mrtR gene. To ensure that the phenotype was due to the mutation within the mrtR gene and not due to another mutation carried on the plasmid, we recloned mrtR genes from the mutated plasmids to a new vector and examined the phenotypes. Figure 2B shows that compared to wild-type MrtR, all eight MrtR mutants had enhanced ability to activate the expression of mrtI in the absence of 3OC₁₂-HSL. However, a cluster of mutations located in the C-terminal DNA binding domain (P208S, H219R, T221M, and Y227C) produced hypersensitive mutants, as they still responded to the 3OC₁₂-HSL stimulation (Fig. 2B). The constitutive MrtR mutations that activated mrtI independently of and with no further stimulation by 3OC₁₂-HSL were clustered in the N-terminal autoinducer binding domain (G114S, M135I, H142R, and R182C). We further characterized the mutant with the highest activity in the absence of 3OC₁₂-HSL, MrtR^G114S, by purifying it as a His₆-tagged recombinant protein. The MrtR^G114S protein could efficiently retard mrtI promoter DNA in the absence of 3OC₁₂-HSL (Fig. 1B, last lane). Moreover, gel filtration chromatography showed that MrtR^G114S could dimerize in the absence of autoinducers (Fig. 1D), which may explain the constitutive activation of mrtI by MrtR^G114S (Fig. 2B). Point mutations that result in the activation of LuxR in the absence of the cognate autoinducer have been identified throughout the whole protein (23, 27), but no such mutants were identified in TraR (3). However, the mutations we identified in MrtR are not homologous to those in LuxR, so future work will compare those mutants biochemically to further understand the protein structure and autoinducer-induced conformational changes.

 
We thank Anatol Eberhard for the generous gift of 3OC₈-HSL and Jun Dai for assistance in making MrtR antibodies.

This study was supported by the NSFC grants 30570011 and 30770074.

 
* Corresponding author. Mailing address: The State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing, China. Phone and fax: 25-843-96645. E-mail: junzhu{at}nju.edu.cn

Published ahead of print on 31 October 2008.

Supplemental material for this article may be found at http://jb.asm.org/.

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Journal of Bacteriology, January 2009, p. 434-438, Vol. 191, No. 1
0021-9193/09/$08.00+0     doi:10.1128/JB.01247-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

This Article Abstract Full Text (PDF) Supplemental material Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Yang, M. Articles by Zhu, J. Search for Related Content PubMed PubMed Citation Articles by Yang, M. Articles by Zhu, J.