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

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AM1 is composed of a single chromosome of 6.8 Mb and three plasmids of 44, 38, and 25 kb (M. E. Lidstrom et al., unpublished data).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).

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.

To study the potential role of tslI in acyl-HSL production, we constructed a ΔtslI mutant using pCM184 (9). Culture extracts of M. extorquens AM1 ΔtslI::Km^r 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 ΔtslI mutant was indistinguishable from the ΔmsaI mutant (15), i.e., production of C₈- and C₆-HSL was abolished and C_14:1- and C_14:2-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 ΔtslI and ΔmsaI mutants by introducing the respective genes cloned in a broad-host-range vector (10) (Table 1).

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 ΔmsaI mutant did not alter the production of short-chain acyl-HSLs. However, when msaI was cloned into pCM62 (10) and introduced into the ΔtslI 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 ΔmsaI mutant (Fig. 3). While msaI transcripts were clearly detectable in the parental strain, the msaI messengers were not amplified in the ΔtslI strain. In the wild-type strain, mlaI transcripts could clearly be detected, whereas transcription was reduced about seven- to eightfold in ΔmsaI and ΔtslI single mutants, as well as in the ΔmsaI/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.

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, ΔmsaI and ΔtslI, 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 ΔmsaI strain produced about 50% less EPS than the wild-type strain, secretion of EPS was reduced to 25% in the ΔtslI strain (Fig. 4). The ΔmlaI 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.

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 α-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.

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.