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Journal of Bacteriology, October 2006, p. 7321-7324, Vol. 188, No. 20
0021-9193/06/$08.00+0     doi:10.1128/JB.00649-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

A Plasmid-Borne Truncated luxI Homolog Controls Quorum-Sensing Systems and Extracellular Carbohydrate Production in Methylobacterium extorquens AM1

Carlos G. Nieto Penalver,1 Franck Cantet,1 Danièle Morin,2 Dominique Haras,2 and Julia A. Vorholt1*

Laboratoire des Interaction Plantes Micro-organismes, INRA/CNRS, BP52627, 31326 Castanet-Tolosan, France,1 Laboratoire de Biotechnologie et Chimie Marines, 56321 Lorient, France2

Received 6 May 2006/ Accepted 3 August 2006


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ABSTRACT
 
A cryptic plasmid of Methylobacterium extorquens AM1 was found to encode tslI, a truncated luxI homolog. tslI was shown to be expressed and to control transcription of the acyl-homoserine lactone (HSL) synthase gene msaI and thus, indirectly, acyl-HSL production. In addition, tslI was found to positively regulate extracellular polysaccharide production.


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TEXT
 
Many bacteria coordinate physiological processes, including biofilm differentiation, conjugation, and motility, by intercellular signaling. This type of regulation, termed quorum sensing (QS), allows the coordination of gene expression through the perception of signal molecules in a concentration-dependent manner. Produced by homoserine lactone synthases, many gram-negative bacteria secrete N-acyl-homoserine lactones (acyl-HSLs) as the QS signal molecules (6). Acyl-HSLs vary in the acyl group length, the substitution on the third carbon, and the degree of saturation of the acyl chain. These differences confer signal specificity through the affinity of receptor proteins of the LuxR family (6).

Methylobacterium spp. are systematically found in association with plants and potentially dominate the epiphytic population (4, 8). These {alpha}-Proteobacteria are capable of utilizing substrates lacking carbon-carbon bonds (3, 21) and take advantage of methanol produced by plants (19). We have recently shown that the model methylotroph Methylobacterium extorquens AM1 possesses two functional LuxI homologs: MsaI, responsible for the synthesis of C8-HSL and C6-HSL, and MlaI, responsible for C14:1-HSL and C14:2-HSL (15), which are organized in hierarchical fashion, with MsaI activity required for full expression of mlaI.

Identification of a truncated LuxI homolog. The genome of M. extorquens AM1 is composed of a single chromosome of 6.8 Mb and three plasmids of 44, 38, and 25 kb (M. E. Lidstrom et al., unpublished data). To date, these plasmids are cryptic and no functions could be attributed to them. A BLAST search in the genome sequence of M. extorquens AM1 (http://www.integratedgenomics.com/genomereleases.html#6) permitted us to identify an open reading frame (ORF) (RMQ03963) which is preceded by a ribosome binding site and which encodes a putative protein of 123 amino acids that is located on the 44-kb plasmid. The predicted product of RMQ03963 exhibits 24% sequence identity to Msi039, a predicted LuxI homolog in Mesorhizobium loti M7A (accession no. CAD31444) (18). However, the predicted product of RMQ03963 shows 48% identity in local pair-wise alignments with Msi039 and 29% and 26% identity, respectively, with MlaI and MsaI (15). RMQ03963 is probably not part of an operon. In its upstream region, RMQ03963 is flanked by an ORF predicted to encode a transposase and in the downstream region by an ORF predicted to encode MobC, involved in conjugation (5) (Fig. 1A). The RMQ03963 gene product is remarkably short, since described LuxI homologs are 180 to 230 amino acids long (6). Protein sequence alignment showed that eight conserved residues in LuxI-type enzymes were not present in RMQ03963: Arg-25, Phe-29, Trp-35, Glu-44, Asp-46, Asp-49, Arg-70, and Phe-84 (according to Vibrio fischeri) (Fig. 1B). Indeed, the N-terminal region that possesses all these residues involved in binding of the substrate S-adenosyl-L-methionine and essential for acyl-HSL synthase activity (7, 23) is absent in RMQ03963, which shows similarity with the central and C-terminal sequences of LuxI-like enzymes (Fig. 1B). Sequencing errors resulting in a potential frameshift were ruled out through resequencing of the genomic region and evaluation of alternative reading frames through BLAST analysis. Since the putative translated product of RMQ03963 possesses a truncated N-terminal region compared to all characterized acyl-HSL synthases, we named RMQ03963 tslI (truncated synthase-like I).


Figure 1
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  		FIG. 1. A. Genome region of tslI on the 44-kb plasmid of M. extorquens AM1. B. Alignment of the translated product of tslI and the sequence of other representative members of the LuxI enzyme family. The alignment was constructed using the ClustalW algorithm followed by manual editing. Black highlighting indicates completely conserved residues in all LuxI homologs described to date, and gray shading indicates conserved amino acids between TslI and the closest LuxI homolog, Msi039. Abbreviations of bacterial species (and accession numbers) are as follows: LuxI, V. fischeri (AAW87994); EsaI, Pantoea stewartii subsp. stewartii (AAA82096); TraI, Rhizobium sp. strain NGR234 (AAB92427); Msi039, M. loti (CAD31444).

Several altered luxI homologs are apparent from whole-genome sequencing projects, none of which have been analyzed in detail yet. A Sinorhizobium meliloti strain harbors on one plasmid an ORF for a putative protein of only 74 residues showing similarity with known LuxI-like enzymes (GenBank accession no. AAX19272) (22). This predicted protein also aligns with the central region of these enzymes. Agrobacterium tumefaciens C58 possesses on its pAT plasmid, which is involved in virulence and control of quorum sensing (2, 14), an ORF that encodes a hypothetical protein of 136 residues (accession no. AAL45719) (24) that, similar to tslI, possesses similarity with the central and C-terminal regions of LuxI-like enzymes.

tslI is involved in the regulation of acyl-HSL production and required for msaI expression. To study the potential role of tslI in acyl-HSL production, we constructed a {Delta}tslI mutant using pCM184 (9). Culture extracts of M. extorquens AM1 {Delta}tslI::Kmr were analyzed in bioassays using Chromobacterium violaceum CV026, Agrobacterium tumefaciens NTL4, and Pseudomonas putida F117 as biosensor strains (11, 17, 25) and using liquid chromatography-tandem mass spectrometry (LC-MS/MS) (12, 15). Surprisingly, the acyl-HSL profile of the {Delta}tslI mutant was indistinguishable from the {Delta}msaI mutant (15), i.e., production of C8- and C6-HSL was abolished and C14:1- and C14:2-HSL synthesis was reduced (Fig. 2; Table 1). To show that these phenotypes were indeed due to alterations in the mutated genes, we complemented the {Delta}tslI and {Delta}msaI mutants by introducing the respective genes cloned in a broad-host-range vector (10) (Table 1).


Figure 2
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  		FIG. 2. Reverse-phase thin-layer chromatography of extracts from methanol-grown M. extorquens AM1 and derived mutants (1 µl of 250-fold concentrate) at early (a), mid (b), and late stationary (c) growth phases. Reverse-phase thin-layer chromatography was developed with C. violaceum CV026 as the indicator strain (11) for the detection of C6-HSL and C8-HSL. Lanes: 1, M. extorquens AM1; 2, M. extorquens {Delta}mlaI; 3, M. extorquens {Delta}msaI; 4, M. extorquens {Delta}tslI; 5, 5 x 10–9 mol C8-HSL; 6, 0.25 x 10–9 mol C6-HSL. Samples were also analyzed with A. tumefaciens NTL4 (25) and P. putida F117 (17) (not shown) and, in addition, by LC-MS (12) (Table 1). No acyl-HSL could be attributed to tslI (see text for details).


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  		TABLE 1. Detection of acyl-HSLs in concentrated extracts prepared from whole cultures of wild-type M. extorquens AM1 and mutant derivatives

Since it is unlikely that tslI encodes a functional acyl-HSL synthase, due to the predicted N-terminal deletion, it might be indirectly involved in expression of msaI, as evidenced via two experiments. tslI introduced in trans into a {Delta}msaI mutant did not alter the production of short-chain acyl-HSLs. However, when msaI was cloned into pCM62 (10) and introduced into the {Delta}tslI mutant, we detected short-chain acyl-HSLs, albeit at lower levels than the wild type (Table 1). These results indicate that msaI is sufficient to ensure synthesis of C6- and C8-HSL and encodes a true acyl-HSL synthase. In addition, we performed transcriptional analysis of the luxI homologs of M. extorquens at the late exponential growth phase using reverse transcription-PCR (1) and of the mxaF gene as an internal standard. Transcripts of tslI could be detected in wild-type M. extorquens and in the {Delta}msaI mutant (Fig. 3). While msaI transcripts were clearly detectable in the parental strain, the msaI messengers were not amplified in the {Delta}tslI strain. In the wild-type strain, mlaI transcripts could clearly be detected, whereas transcription was reduced about seven- to eightfold in {Delta}msaI and {Delta}tslI single mutants, as well as in the {Delta}msaI/tslI double mutant. These findings confirm that tslI is expressed and required for the transcription of msaI. These results are also consistent with our observations of lower levels of long-chain acyl-HSLs detectable in the msaI and tslI deletion strains.


Figure 3
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  		FIG. 3. Quantitative reverse transcription-PCR analysis of mlaI, msaI, and tslI transcripts in the late exponential growth phase. Total RNA was isolated from wild-type M. extorquens AM1 (WT), {Delta}msaI, and {Delta}tslI and also {Delta}msaI/tslI as a negative control. The level of transcript is expressed relative to the wild-type level. Standard deviations from three experiments were less than 10% (not shown). <, transcript not detectable.

tslI is involved in the regulation of EPS. Exopolysaccharide (EPS) carbohydrate production is a physiological trait important for bacteria in their natural environment and has been shown to be controlled through QS (16, 20). To test whether this is also the case for M. extorquens AM1, we cultivated the wild type and the different mutants in methanol minimal medium. At regular intervals, we took samples and quantified EPS in cell-free supernatants (13). The two strains defective in short-chain acyl-HSL production, {Delta}msaI and {Delta}tslI, were severely impaired in EPS production (Fig. 4). When the msaI mutant was complemented with 1 µM C8-HSL or a concentrated extract of spent medium from a parent strain culture, the wild-type level was restored (Fig. 4), showing the role of C8-HSL as a positive regulatory molecule for this trait. EPS levels could not be recovered in the tslI mutant by the addition of either 1 µM C8-HSL or wild-type extracts (Fig. 4), suggesting that tslI not only influences the extracellular carbohydrate level through the production of C8-HSL but also through a different, as-yet-unidentified regulatory mechanism. The regulation of EPS concentration through tslI independently of MsaI is also indicated by the differences in the level of EPS when comparing the two deletion strains: while the {Delta}msaI strain produced about 50% less EPS than the wild-type strain, secretion of EPS was reduced to 25% in the {Delta}tslI strain (Fig. 4). The {Delta}mlaI mutant showed an increase of ~15% in EPS level compared to the wild-type strain, which was restored when the mutant was complemented with extracts from a parent strain culture (Fig. 4), suggesting that long-chain acyl-HSLs play an inhibitory role in the regulation of EPS in M. extorquens AM1.


Figure 4
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  		FIG. 4. Total extracellular carbohydrate production by M. extorquens AM1 wild type (WT) and derived mutants. Total carbohydrate content was determined in cell-free supernatants from late exponential cultures using the anthrone reagent (13). Extracellular complementation of single mutants was performed with crude extracts from wild-type strain (Ext) and 1 µM C8-HSL (C8) (note that C14:1-HSL and C14:2-HSL are not commercially available). The bars indicate means, and the error bars indicate one standard error of the mean of five (wild type and single mutants) and three (complemented mutants) independent experiments, respectively.

Conclusion. We demonstrated that a cryptic plasmid of 44 kb encodes a truncated luxI homolog, tslI, which represents a higher level of control of the msaI and mlaI quorum-sensing systems in M. extorquens AM1. To the best of our knowledge, this is the first report that shows a function for a plasmid that M. extorquens AM1 harbors. In line with the loss of the N-terminal region of the predicted product, there was no evidence that tslI encodes a bona fide acyl-HSL synthase. However, tslI is expressed and involved in acyl-HSL synthesis by MsaI and MlaI (15). We hypothesize that tslI encodes an enzyme that might be responsible for the biosynthesis of a regulatory molecule whose biosynthesis does not require S-adenosyl-L-methionine as substrate. Beyond its indirect role in acyl-HSL production, tslI is involved in at least one other physiological process, i.e., the regulation of the extracellular polysaccharide concentration. Further studies are now required to understand how tslI controls msaI expression and extracellular carbohydrate production. Truncated luxI homologs are also found in the {alpha}-Proteobacteria Sinorhizobium meliloti and Agrobacterium tumefaciens and thus might represent a novel subgroup of proteins influencing the regulatory cascades of quorum systems in other bacteria.


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ACKNOWLEDGMENTS
 
This work was supported by the Centre National de la Recherche Scientifique and the Max-Planck-Gesellschaft.

We thank Mary E. Lidstrom and Stéphane Vuilleumier for access to unpublished genome data. We are grateful to Linda Dombrowsky for collaboration in LC-MS/MS analysis and identification of acyl-HSLs.


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FOOTNOTES
 
* Corresponding author. Present address: Institute of Microbiology, Swiss Federal Institute of Technology Zurich, ETH Hönggerberg, Zurich, Switzerland. Phone: 41-44-632-5524. Fax: 41-44-663-1307. E-mail: vorholt{at}micro.biol.ethz.ch. Back


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Journal of Bacteriology, October 2006, p. 7321-7324, Vol. 188, No. 20
0021-9193/06/$08.00+0     doi:10.1128/JB.00649-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.




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