A variety of bacterial species have been shown to be capable of responding to AI-2 by the regulation of a range of niche-specific functions, but the mechanisms for AI-2 detection have been characterized in only a few cases (17
). This constitutes a major obstacle in work toward an understanding of the function of AI-2. While sequence analysis of bacterial genomes reveals the presence of orthologs of LsrB-like AI-2 receptors in gram-negative as well as gram-positive bacteria (41
; this study), establishing which orthologs are, in fact, functional as AI-2 receptors is important for determining if and how these species use AI-2 as a chemical signal. Thus, after analyzing the sequences and predicted structures of LsrB orthologs, we identified criteria for predicting which LsrB orthologs are functional AI-2 receptors and assayed the AI-2 binding ability of selected candidates to test our criteria. Our results not only support our predictions but also provide the first biochemical confirmation of the presence of functional AI-2 receptors in gram-positive bacteria, specifically in B. anthracis
and B. cereus
Our sequence and structural analyses allowed us to categorize the organisms with LsrB orthologs into two different groups. Members of group I have (i) LsrB orthologs with greater than 60% sequence identity with S.
Typhimurium LsrB, (ii) orthologs to the other key transport proteins encoded by the lsr
operon, and (iii) complete conservation of all six residues that hydrogen bond with AI-2 in S.
Typhimurium LsrB (based on structure predictions). On the other hand, in organisms belonging to group II, the LsrB orthologs have a sequence identity below 36%, are missing orthologs to key proteins encoded by the lsr
operon, and lack at least two of the six residues in the AI-2 binding pocket. These characteristics led us to hypothesize that the organisms from group I had functional AI-2 binding proteins, whereas the LsrB orthologs in group II were likely to have a different function. In all organisms where the function of either the LsrB protein or its gene has been studied, LsrB has been shown, along with other proteins that form the Lsr transport system, to participate in the uptake of AI-2 (37
); thus, we further predicted that organisms with a functional LsrB and orthologs to all the proteins from the Lsr system would take up AI-2. Accordingly, all the organisms from group I tested for the binding of AI-2 by LsrB or for in vivo AI-2 removal (S.
Typhimurium, S. meliloti
], E. coli
K-12 [MG1655], B. cereus
, and B. anthracis
) were capable of both of these functions. None of the proteins from the organisms that we tested from group II (UPEC UT189, R. etli
, R. leguminosarum
, and A. tumefaciens
) were capable of binding AI-2, nor were these organisms able to take up AI-2. In addition, our analysis of predicted structures of the LsrB orthologs identified key binding-site residues that are not conserved in group II organisms. Mutagenesis of the B. anthracis
LsrB ortholog (classified as group I and demonstrated to bind AI-2) with the two most common group II substitutions (D166N and A222T) confirmed that these residues are critical for AI-2 binding. This result strongly supports our use of binding-site conservation as a key criterion in identifying group I orthologs.
These results offer experimental evidence that functional LsrB-AI-2 receptors are present in particular members of the Enterobacteriaceae (S. Typhimurium and E. coli), Rhizobiaceae (S. meliloti), and Bacillaceae (B. cereus and B. anthracis), and given the correlation of our experimental results with our classification scheme, we predict that all the other LsrB orthologs from group I are functional AI-2 receptors and that these organisms are competent for AI-2 uptake. Accordingly, we expect that the members of the families Pasteurellaceae and Rhodobacteraceae in group I (Table ) also have functional AI-2 transporters. On the other hand, we believe that it is likely that all group II members have orthologs that are not involved in AI-2 transport and thus that these organisms do not take up AI-2 via an LsrB-type mechanism. The criteria described here can be used to predict the presence (or absence) of functional LsrB-like AI-2 receptors in newly sequenced species, and as new species are sequenced, we expect the number of organisms in group I to increase.
The large majority of the organisms from group I belong to the Enterobacteriales
and the Pasteurellales
. This, coupled with the fact that the diversification pattern of the lsrB
gene largely mimics the bacterial phylogenetic relationships within this group, is consistent with a single origin for the LsrB-AI-2 receptor that likely occurred in an ancestor of these organisms after the diversification of the Enterobacteriales
and the Pasteurellales
from the Vibrionales
. Thus, the occurrence of LsrB receptors in one species of the Rhizobiales
) and the Rhodobacterales
) and in three species of the Bacillales
was very surprising and immediately raised the possibility of LGT. The hypothesis of LGT between organisms from the Enterobacteriales
or the Pasteurellales
and these three orders was supported by the comparison of the lsrB
gene tree and the rpoB
organismal tree. Specifically, in the lsrB
gene tree, Bacillus
species are clustered with the Pasteurellales
, and S. meliloti
and R. sphaeroides
are nested within the Enterobacteriales
. These are nested patterns where species appeared to be “misplaced” in the gene phylogeny and can be interpreted as an indication of events of LGT. Often, genes that have been acquired by LGT have an atypical nucleotide distribution (reflected in GC content or codon usage) compared with the rest of the genome (25
). However, in this case, analysis of GC usage and codon bias provided no information to argue for or against the hypothesis of LGT (data not shown). Certainly, other occurrences such as convergent evolution by natural selection or the ancient origin of lsrB
at the base of the Bacteria
tree with a large number of events of gene loss could also explain the observed patterns, but since we do not have specific data to support a particular explanation over the others, we favor LGT as the most parsimonious explanation, as it requires the minimum number of assumptions. LGT events are now well accepted as a major force in the evolution of bacterial genomes (8
) leading to an increment in the number of genes (35
) and pathways (19
) and often enabling bacteria to acquire new functions, such as traits associated with pathogenicity, that allow adaptation to novel environments. In the specific cases of S. meliloti
and R. sphaeroides
, it is intriguing that that these organisms have acquired the AI-2 receptor but not its synthase (LuxS); thus, these organisms have potentially gained the ability to eavesdrop on their neighbor's signal, as previously suggested (37
). It will also be interesting to determine the adaptive value of this new function and explore its impact on the physiology of these organisms. LGT has been proposed for other autoinducer receptors and regulators from the LuxI/LuxR family of species-specific quorum-sensing proteins, where it was previously proposed that the acquisition of this family of proteins has benefited certain bacterial species by allowing them to gain an efficient mechanism for regulating virulence genes (8
Interestingly, the LsrB ortholog in R. leguminosarum
bv. trifolii, which we identified as belonging to group II, has been shown to be essential for rhamnose (a methyl-pentose sugar) uptake and growth in this sugar and is thus likely to be a rhamnose binding protein (42
). Motivated by this finding, we used the protein sequence of R. leguminosarum
bv. trifolii (KEGG identification number pRL110413) to carry out a reciprocal best-hit analysis against all the genome sequences used in the previous analysis. We found that there are 12 orthologs of the R. leguminosarum
binding protein (along with the proteins from the rhamnose transport operon) present in group II (shown in Table ). Thus, these 12 binding proteins are orthologs of both LsrB of S.
Typhimurium and the rhamnose binding protein of R. leguminosarum
. These proteins have more than 65% sequence identity with the R. leguminosarum
protein but less than 36% identity with S.
Typhimurium LsrB. We interpret this as strong evidence that these 12 proteins in group II are functioning as rhamnose binding proteins, in agreement with our prediction that they are not AI-2 receptors (these proteins are highlighted in the Table S1 in the supplemental material). These 12 organisms correspond to species belonging to Alphaproteobacteria
that cluster together in the organismal rpoB
tree (Fig. ). Interestingly, S. meliloti
is the only organism that has an LsrB ortholog belonging to group I and also a different set of proteins that are orthologs to the R. leguminosarum
proteins from the rhamnose transport operon, further corroborating our hypothesis that the acquisition of LsrB occurred by LGT in S. meliloti.
While the presence of a functional LsrB ortholog does not prove that AI-2 import is involved in the control of AI-2-mediated behavior, it is suggestive. Accordingly, the function of the Lsr system in AI-2 signaling has already been shown for a member of the Pasteurellaceae
, A. actinomycetemcomitans
(an organism not present in Table because, to date, its genome is not present in the KEGG database). Shao and coworkers previously showed that this oral pathogen is capable of internalizing AI-2 via the Lsr system and, importantly, that LsrB is required to mediate the complete AI-2-dependent activation of biofilm formation in this organism (48
). In other cases such as that of Photorhabdus luminescens
, an insect pathogen belonging to the Enterobacteriaceae
, the transcription of the lsr
operon was shown to be induced by AI-2, and AI-2 was also implicated in the regulation of biofilm formation and motility (24
). However, it remains to be demonstrated whether or not the Lsr system is involved in mediating these AI-2-regulated behaviors. Likewise, it will be interesting to determine whether the Lsr system is involved in mediating AI-2 signal transduction in B. cereus
and B. anthracis
, where AI-2 has been implicated in regulating biofilm formation (2
) and growth rate (21
). Certainly, the results presented here give support to that possibility.
This study, along with the two previous studies based on sequence analysis (41
), also reveals that certain bacteria such as Helicobacter pylori
), Streptococcus mutans
), Staphylococcus epidermidis
), Porphyromonas gingivalis
), Pseudomonas aeruginosa
), and Bacillus subtilis
), which have been shown to respond to AI-2, do not have either of the known types of AI-2 receptors (neither LuxP nor LsrB), and thus we expect that other receptors for AI-2 remain to be discovered. These receptors may be of entirely new classes or may be promiscuous receptors for other small molecules. Novel receptor classes are likely to be identified by approaches that rely on genetic screens to isolate mutants involved in modulating AI-2-regulated phenotypes, and as shown here, integration with approaches that use sequence analysis coupled with biochemical assays may prove very useful. Clearly, an elucidation of the proteins involved in AI-2 recognition and signal relay is essential for studying the potential functions of this class of signal molecule in intra- and interspecies cell-to-cell communication and/or intra- and intercellular signal transduction. The identification and experimental confirmation of functional LsrB receptors in this study open the door to the understanding of the molecular basis of AI-2-mediated behavioral regulation in a variety of new species.