Lack of genomic evidence of AI-2 receptors suggests a non-quorum sensing role for luxS in most bacteria

Abstract

Background: Great excitement accompanied discoveries over the last decade in several Gram-negative and Gram-positive bacteria of the LuxS protein, which catalyzes production of the AI-2 autoinducer molecule for a second quorum sensing system (QS-2). Since the luxS gene was found to be widespread among the most diverse bacterial taxa, it was hypothesized that AI-2 may constitute the basis of a universal microbial language, a kind of bacterial Esperanto. Many of the studies published in this field have drawn a direct correlation between the occurrence of the luxS gene in a given organism and the presence and functionality of a QS-2 therein. However, rarely hathe existence of potential AI-2 receptors been examined. This is important, since it is now well recognized that LuxS also holds a central role as a metabolic enzyme in the activated methyl cycle which is responsible for the generation of S-adenosyl-L-methionine, the major methyl donor in the cell.

Results: In order to assess whether the role of LuxS in these bacteria is indeed related to AI-2 mediated quorum sensing we analyzed genomic databases searching for established AI-2 receptors (i.e., LuxPQ-receptor of Vibrio harveyi and Lsr ABC-transporter of Salmonella typhimurium) and other presumed QS-related proteins and compared the outcome with published results about the role of QS-2 in these organisms. An unequivocal AI-2 related behavior was restricted primarily to organisms bearing known AI-2 receptor genes, while phenotypes of luxS mutant bacteria lacking these genes could often be explained simply by assuming deficiencies in sulfur metabolism.

Conclusion: Genomic analysis shows that while LuxPQ is restricted to Vibrionales, the Lsr-receptor complex is mainly present in pathogenic bacteria associated with endotherms. This suggests that QS-2 may play an important role in interactions with animal hosts. In most other species, however, the role of LuxS appears to be limited to metabolism, although in a few cases the presence of yet unknown receptors or the adaptation of pre-existent effectors to QS-2 must be postulated.

Figure 1

Relation between the Activated Methyl Cycle (AMC) and AI-2 production in bacteria. The AMC is responsible for the generation of the major methyl donor in the cell, S-adenosyl-L-methionine (SAM) and the recycling of methionine by detoxification of S-adenosyl-L-homocysteine (SAH). LuxS takes part in this cycle by salvaging the homocysteine moiety from the cycle intermediate S-ribosyl-homocysteine (SRH). As a by-product of this reaction the direct AI-2 precursor 4,5-dihydroxy-2,3-pentadione (DPD) is formed. DPD undergoes further reactions to form distinct biologically active signal molecules generically termed AI-2. (2S,4S)-2-methyl-2,3,3,4-tetrahydroxytetrahydrofuran-borate (S-THMF-borate), the AI-2 signal of Vibrionales, is produced without the help on any known enzyme in the presence of boric acid (lower pathway), while in other bacteria (e.g., S. typhimurium) DPD rearranges spontaneously to form (2R,4S)-2-methyl-2,3,3,4-tetrahydroxytetrahydrofuran (R-THMF) as AI-2 signal (upper pathway). CH₃-THPG: N⁵-methyltetrahydropteroryl glutamate, CH₃-THF: N⁵-methyltetrahydrofolate.

Figure 2

Phylogenetic relationships among Gram-positive and Gram-negative bacteria on the basis of complete luxS sequences and presence of established AI-2 receptors in the respective genomes. The distance tree was generated by the NJ method with the JC formula, without choosing any outgroup. Nodal supports were assessed by 1000 bootstrap replicates. Only bootstrap values greater than 50% are shown. The scale bar represents the number of substitutions per site. The presence of recognized AI-2 receptor genes (luxP, lsrB) is indicated between parentheses. The asterisk shows the truncated Lsr-receptor complex of X. bovienii. With the exception of the E. billingiae, which was produced in this work (accession number DQ977724), all luxS sequences were retrieved at the NCBI database or in published genome projects (see Methods for accession numbers).

Figure 3

Phylogenetic relationships on the basis of complete lsrB. The distance tree was generated by the NJ method with the JC formula, without choosing any outgroup. Nodal supports were assessed by 1000 bootstrap replicates. Only bootstrap values greater than 50% are shown. All lsrB sequences were retrieved at the NCBI database or in published genome projects (see Methods for accession numbers).

Figure 4

Comparison of phylogenetic trees based on complete sequences of luxS (A), lsrB (B) and rpoB (C). All trees are restricted to strains which are both luxS- and lsrB-positve. The distance tree was generated by the NJ method with the JC formula, without choosing any outgroup. Nodal supports were assessed by 1000 bootstrap replicates. Only bootstrap values greater than 50% are shown. The scale bar represents the number of substitutions per site. All sequences were retrieved at the NCBI database or in published genome projects (see Methods for accession numbers).

Figure 5

Comparison of the operons coding for the Lsr-like receptors in the luxS-negative α-proteobacteria R. sphaeroides 2.4.1 and S. meliloti 1021 with the lsr cluster of AI-2 producers S. typhimurium and E. coli K12. The lsr-like operons are located on the highly variable chromosome II of R. sphaeroides 2.4.1 (accession number NC_007494) and on the pSymB megaplasmid of S. meliloti 1021 (NC_003078) respectively. The transporter core is composed of a periplasmic AI-2 binding protein (LsrB), two hydrophobic proteins which for an homodimeric transmembrane channel (LsrC and LsrD) and a hydrophilic ATP-binding protein (LsrA). The expression of the lsr operon is controlled by repressor protein LsrR and AI-2 kinase LsrK which is responsible for the production of phospho-AI-2, the lsr operon inducer. In S. typhimurium (NC_003197), the LsrF and LsrG proteins are involved in modifying phospho-AI-2 [21]. For comparison ribose and xylose ABC transporters of E. coli K12 (NC_000913) are shown below. Proteins with the same function are coded with the same color. As point of reference, locus tags for selected genes are marked inside the arrows.