BACKGROUND
population density- and growth phase-dependent bacterial cell-to-cell communication involving the production and detection of extracellular signaling molecules  is referred to as quorum sensing .
the detection of a stimulatory concentration of an autoinducer leads to an alteration in gene expression,  enabling bacteria to coordinate their behavior in response to environmental challenges.
a large number of cellular functions are known to be regulated via the qs mechanism in a variety of bacterial species such as biofilm formation,  swarming behavior or the production of antibiotics and virulence factors  <cit> .
n-acyl-homoserine lactones  are the most frequent signaling molecules found in gram-negative bacteria.
this first autoinduction system  was identified in the luminescent marine symbiont vibrio fischeri and is based on the pheromone n-3-oxohexanoyl-l-homoserine lactone  <cit> .
meanwhile several other type i autoinducers ,  whose molecules differ only in the ahl-acyl side chain moiety,  have been discovered in a number of gram-negative bacteria  <cit> .
since every species employs specific receptors for its own ahl molecule,  not every bacterium can automatically cross-talk with all other bacteria,  so that every 'diahlect' can essentially be understood only by those species sharing the same cognate receptor.
however,  due to the fact that different bacteria may simultaneously use several signal molecules and receptors,  and given the chemical similarity of the different ahl molecules,  a limited interaction  is possible even for bacteria carrying non-cognate receptors.
thus,  a limited  communication between different bacterial species is possible through qs- <dig>  <cit> .
in this respect great interest accompanied the discovery,  first in vibrio harveyi  <cit>  then in a variety of other bacteria  <cit> ,  of the luxs protein that catalyzes the production of an autoinducer molecule  for a second quorum sensing system   <cit> .
since the luxs gene was subsequently found to be widespread among both gram-negative and gram-positive bacteria,  it was tempting to presume that qs- <dig> may constitute the basis of an universal language,  a sort of bacterial esperanto  <cit> .
the presence of luxs has been reported in several subgroups of the bacterial kingdom,  being widely found in bacteroidetes,  actinobactetria,  and β- plus γ-proteobacteria,  along with all bacilli,  deinococci and ε-proteobacteria,  but not in archaea or eukarya  <cit>
in vibrio species qs- <dig> is connected with qs- <dig> by partially sharing the same transduction pathway leading through the central signal relay protein luxu to the terminal response regulator luxo  <cit> ,  but displays a separate receptor for ai- <dig>   namely the two component sensor kinase luxpq  <cit> .
although homologs to luxpq have been found in other vibrio species  <cit> ,  other bacterial species were reported to lack similar qs- <dig> related proteins with the exception of luxs,  raising the prospect that this kind of receptor and signal transduction pathway is limited to the vibrionales  <cit> .
in salmonella typhimurium other genes were shown to encode for a different complex that serves as receptor for ai- <dig>  <cit> .
an abc-type transporter named lsr  is responsible for the ai- <dig> uptake into the cell and was reported subsequently also in escherichia coli  <cit> .
ai- <dig> is phosphorylated inside the cell and is anticipated to interact with lsrr,  a protein that contributes to repress the lsr-operon and possibly acts as regulator of gene expression  <cit> .
this mechanism is different from ai- <dig> detection in v. harveyi,  where just the signal but not the ai- <dig> molecule is transduced inside the cell.
thus alternative explanations to qs have been proposed whereby ai- <dig> may be released as a waste product and then reused subsequently as a metabolite  <cit>  or,  as in the case of actinobacillus actinomycetemcomitans,  that the molecule may be used as a borate scavenger by bacteria  <cit> .
there are now several studies that describe the central role of luxs in e. coli as crucial for the transition to pathogenic existence inside the host  <cit>  by controlling virulence determinants such as motility  <cit> ,  biofilm formation  <cit>  or gene expression  <cit> .
ai- <dig> dependent behavior was consequently claimed also in other gram-negative and gram-positive bacteria ,  but many reports are simply based on the presence of the luxs gene,  mutant analysis and/or additional phenotypic tests such as the use of ai- <dig> reporter strain v. harveyi bb <dig>  <cit> .
rarely have investigations examined the existence of potential ai- <dig> receptors or assessed their functionality by proving ai- <dig> depletion of the media by the wildtype or by performing a chemical complementation of luxs mutants using pure ai- <dig> signal or ai- <dig> conditioned supernatants  <cit> .
this is essential since it is now well recognized that luxs displays also a primary role as metabolic enzyme in the activated methyl cycle   <cit> , which is responsible for the generation of the cell major methyl donor s-adenosyl-l-methionine  and the recycling of methionine by detoxification of s-adenosyl-l-homocysteine .
luxs takes part in this cycle by salvaging the homocysteine moiety from the cycle intermediate s-ribosyl-homocysteine .
as a by-product of this reaction, the direct ai- <dig> precursor - <dig> -dihydroxy- <dig> -pentanedione  is formed.
in v. harveyi dpd cyclizes and is spontaneously converted to furanosyl borate diester by the addition of borate  <cit> ,  while s. typhimurium produces and recognizes a chemically distinct form of ai- <dig>  <cit>  .
given this dual nature of luxs the sole use of the commonly exploited mutational approach is inappropriate,  since a simple genetic complementation using the intact gene on a plasmid will also repair the induced metabolic unbalance as well and yield in any case a restored phenotype.
a more detailed description of the findings of the papers cited herein is available as additional file  <dig> .
a +: positive ; : positive ; -: negative ;
b h: hypothesized; nd: not determined; q: questioned;
c +: successful complementation; -: unsuccessful complementation; nd: not determined; ai-2: complementation with pure autoinducer; dpd: complementation with precursor; sn: complementation with co-culture or conditioned supernatants of luxs-positive strain.
a more detailed description of the findings of the papers cited herein is available as additional file  <dig> .
a +: positive ; : positive ; -: negative ;
b c: claimed; h: hypothesized;
c +: successful complementation; -: unsuccessful complementation; nd: not determined; ai-2: complementation with pure autoinducer; dpd: complementation with precursor; sn: complementation with co-culture or conditioned supernatants of luxs-positive strain;
d hypothesized on the basis of the genomes of  <dig> fully sequenced lactobacillales .
a more detailed description of the findings of the papers cited herein is available as additional file  <dig> .
a +: positive ; : positive ; -: negative ; nd: not determined;
b nd: not determined; c: claimed;
c +: successful complementation; -: unsuccessful complementation; nd: not determined; ai-2: complementation with pure autoinducer; dpd: complementation with precursor; sn: complementation with co-culture or conditioned supernatants of luxs-positive strain;
d hypothesized based on serratia proteomaculans sequence;
e hypothesized on the basis of more than  <dig> known streptococcus complete genomes belonging to the species s. pneumoniae, s. mutans, s. pyogenes, s. agalactiae, s. suis, s. equis and s. zooepidemicus.
in order to assess whether the role of luxs in well-described bacterial species is effectively related to ai- <dig> mediated autoinduction ,  we performed a database analysis for established ai- <dig> receptors in fully-sequenced genomes and sequence databases,  and compared the outcome with published results about qs- <dig> in these bacteria.
in this way we were able to corroborate many reports where the role of luxs in qs- <dig> was proven experimentally  or to confirm results of other works where no effect related to autoinduction could be demonstrated .
on the other hand,  in those cases when an ai- <dig> mediated mechanism was claimed,  but no known receptor was found in the corresponding bacterial sequence,  novel or alternative receptors must be postulated or,  without a successful chemical complementation of the luxs mutant,  a sheer metabolic role must be acknowledged.
with the aim of understanding the evolutionary relationships among luxs,  the presence of ai- <dig> receptors and the functionality of the qs- <dig> system,  a phylogenetic analysis was performed to compare the evolution of the luxs gene with the presence and evolution of the genes coding for known ai- <dig> receptors,  transporters and regulators.
since known ai- <dig> receptors genes are  restricted to luxs-positive bacteria and have only partially coevolved with the ai- <dig> synthase,  it can be hypothesized that the qs- <dig> function was acquired and developed as a new trait by some bacteria by building upon the preexistent metabolic function of luxs.
methods
search for known ai- <dig> receptors in complete genomes
complete genome sequences of bacteria known to have a luxs gene were analyzed for the presence of ai- <dig> receptor sequences using the blastx and the tblastx programs with a cut-off e-value of 1e- <dig> by applying a reciprocal best hit strategy,  which allows to distinguish between orthologous and paralogous genes  <cit> .
as starting queries the luxp gene of v. harveyi strain bb <dig>  and the lsrb gene of e. coli strain k <dig>  were used.
the hits were manually checked to confirm that they had the same functional annotation as the query and re-submitted as novel queries until no new relevant positive match was found.
in this second phase also luxs-negative strains were included in the analysis by performing a general blast search across the entire ncbi database .
in bacteria found to carry a gene coding for an ai- <dig> receptor the presence of a complete qs- <dig> system was corroborated by verifying the existence of other genes associated to the respective ai- <dig> transduction pathway.
bacterial species were scored as positive for qs- <dig> receptors only when in addition to luxp,  homologs of the luxq ,  luxu  and luxo  genes which encode the full transduction pathway  <cit>  were also found.
similarly,  positive results obtained with lsrb were confirmed by verifying the presence of genes constituting a complete lsr-operon .
these include lsra ,  lsrc and lsrd ,  lsrr ,  and lsrk   <cit> .
our investigation was extended to incomplete sequenced bacteria known to carry a luxs gene or for which a function of qs- <dig> was documented in the literature through an ncbi database search with relevant terms such as "luxs",  "lsrb",  "luxp",  "ai-2" or "quorum sensing".
discovered pertinent sequences were used in the analysis.
a literature search for published studies on the effect of type- <dig> quorum sensing in the organisms carrying the luxs gene was performed using the ncbi database and the papers were critically read,  with particular attention to those key experiments which are essential to ascertain the presence of a qs- <dig>   such as the identification of ai- <dig> receptors,  chemical complementation of the luxs mutant or detection of ai- <dig> depletion in the culture medium  <cit> .
in cases where these tests were omitted the possibility that a metabolic dysfunction in the amc may have been misinterpreted as disruption of type- <dig> quorum sensing was proposed.
phylogenetic analysis
phylogenetic analysis was performed in order to understand how ai- <dig> receptors may have evolved in different bacteria with respect to the presence of luxs.
phylogenetic trees were generated on the basis of complete luxs- and lrsb-sequences without choosing any outgroup.
housekeeping gene rpob,  encoding the rna polymerase β-subunit,  was used as reference.
retrieved dna sequences were aligned with clustalw  <cit> .
sites presenting alignment gaps were excluded from analysis.
the molecular evolutionary genetics analysis  program version  <dig>   <cit>  was used to calculate evolutionary distances and to infer trees based on the neighbor-joining  method using the jc formula.
nodal robustness of the inferred trees was assessed by  <dig> bootstrap replicates.
genome resources and ncbi accession numbers
complete genomes and key luxs and lsrb sequences were used.
complete genomes
actinobacillus actinomycetemcomitans ,  actinomyces naeslundii ,  bacillus anthracis str.
sterne ,  bacillus cereus atcc  <dig> ,  bacillus cereus subsp.
cytotoxis nvh 391- <dig> ,  bacillus subtilis subsp.
subtilis str.
<dig> ,  bacillus thuringensis serovar israelensis atcc  <dig> ,  bacillus thuringiensis serovar konkukian str.
97- <dig> ,  bacillus weihenstephanensis kbab <dig> ,  bifidobacterium adolescentis ,  bifidobacterium longum , borrelia burgdorferi ,  campylobacter jejuni , clostridium acetobutylicum , clostridium difficile , clostridium perfringens ,  desulfovibrio desulfuricans ,  desulfuromonas acetoxidans ,  erwinia amylovora , escherichia coli ,  haemophilus influenzae ,  haemophilus somnus ,  helicobacter pylori ,  klebsiella pneumoniae , mannheimia succiniciproducens ,  neisseria meningiditis ,  pasteurella multocida ,  photobacterium profundum ,  photorhabdus luminescens ,  rhodobacter sphaeroides  <dig> .
<dig> ,  rhodobacter sphaeroides atcc  <dig> ,  rhodobacter sphaeroides atcc  <dig> ,  salmonella bongori ,  salmonella typhimurium , serratia marcescens ,  shewanella oneidensis mc_ <dig> ,  shighella boydii ,  shighella dysenteriae ,  shighella flexneri ,  shighella sonnei ,  sinorrhizobium meliloti ,  sodalis glossinidius ,  staphylococcus aureus ,  streptococcus pyogenes ,  vibrio angustum ,  vibrio cholerae ,  vibrio fischeri , vibrio harveyi ,  vibrio vulnificus , xenorhabdus sp.
,  yersinia enterocolitica , yersinia frederiksenii ,  yersinia intermedia ,  yersinia mollaretii , yersinia pestis , yersinia pseudotubercolosis .
luxs gene sequences
erwinia billingiae ,  erwinia tasmaniensis ,  vibrio anguillarum .
lsrb gene sequences
rhodobacter capsulatus .
RESULTS
presence of luxp-type receptor genes
the luxp gene of v. harveyi strain bb <dig> was used as a starting query for a search of luxpq-type receptors in bacterial genomes that was successful only for members of the orders of the vibrionales and,  to a much lesser extent,  of the oceanospirillales.
this kind of receptor,  along with the effector kinase luxq,  was already known in several species of the genus vibrio,  i.e., v. anguillarum,  v. cholerae,  v. fischeri,  v. harveyi and v. parahaemolyticus .
in vibrio species for which a response to ai- <dig> system is documented,  but in which no receptor was yet described,  i.e.,  v. vulnificus  <cit>  and v. alginolyticus  <cit> ,  we were able to retrieve the luxpq genes by searching the relevant gene sequences in the corresponding published genomes.
conversely,  the luxpq-receptor was absent from the genome of v. angustum,  which does not respond to the ai- <dig> signal  <cit> ,  even though the luxs gene coding for s-ribosylhomocysteinase is present.
besides that,  we managed to identify the luxpq receptor complex also in other representatives of the genus for which no study about qs- <dig> related phenotype has been performed to date,  i.e.,  v. splendidus,  vibrio sp.
ex <dig> and vibrio sp.
med <dig>   but not in closely related photobacterium profundum ss <dig> .
a luxp homolog was found also in the genomes of some oceanospirillales,  albeit among the bacteria of this order no luxs gene has been sequenced or found to date.
the similarity of these proteins to the luxp-receptor present in v. harveyi was only moderate:  <dig> % identity and  <dig> % similarity for the neptuniibacter caesariensis med <dig> periplasmic protein  which carries a region similar to the rbsb-like abc-type sugar transport system receptor locus and  <dig> % identity and  <dig> % similarity for the marinomonas sp.
med <dig> luxp protein precursor .
however,  both results withstood the reciprocal best hit strategy test and yielded a luxp protein of vibrio as best match in a blastx search.
furthermore,  in neptuniibacter caesariensis med <dig> and marinomonas sp.
med <dig> the genes encoding these putative luxp homologs are arranged in an operon with presumed transcriptional regulators that likewise contain pas domains ,  but share otherwise little homology with each other.
in marinomonas sp.
med <dig>   the transcriptional regulator showed moderate homology with luxq of v. cholerae and contains also aaa-type atpase and dna-binding domains and is followed by the pfs gene coding for a 5'-methylthioadenosine nucleosidase .
a similar protein  in desulfuromonas acetoxidans  showed an equivalent identity to luxp of v. harveyi  and was analogously arranged in an operon with a putative transcriptional regulator containing pas domains , while in desulfovibrio desulfuricans  the matching protein  also carries a putative receptor locus but the corresponding gene was not associated with either a luxq homolog  or the other usual genes of an abc-transporter, indicating the probable absence of a signal transduction pathway toward the inner of the cell.
however, also dace_ <dig> and dde_ <dig> pointed toward luxp in the reciprocal best hit test.
as for oceanospirillales, both d. acetoxidans and d. desulfuricans  were found to lack the luxs gene.
all vibrionales carrying the luxpq genes also carried open reading frames which encoded proteins homologous to the two component, σ54-specific, transcriptional regulator luxuo found in v. harveyi .
in oceanospirillales a similar situation was discovered, but both luxp and luxo were flanked by other transcriptional regulator genes which displayed a less important resemblance with luxq and luxu respectively.
in all strains investigated the luxo protein displayed the characteristic 8-aa glycine-rich consensus  which is needed for interaction with alternative σ <dig>   <cit> .
interestingly, in none of the strains were the genes encoding the single components of the qs- <dig> mechanism  found in proximal positions on the genome , thus they were probably acquired and/or developed independently.
notably the tblastx search using luxp gene of v. harveyi strain bb <dig> resulted in local hits with the lsrb gene of enterobacteriales .
similarity is limited to a small rbsb-like portion on the c-terminus of the luxp protein, but is most probably indicative of the substrate  binding domain of both receptors.
generally, the use of luxq gene of v. harveyi strain bb <dig> as starting query yielded multiple partial positive matches in almost every strain tested, but these were limited to the cytoplasmic transduction domain of other sensor kinases and were not directly related to any known quorum sensing related gene .
presence of lsrb-type receptor genes
our screening of completed genome projects for lsrb, the gene belonging to the lsr-operon encoding the periplasmic ai- <dig> binding component of the abc-type sugar transport apparatus, revealed that this type of receptor is generally more widely spread than the luxpq-system.
we found that lsrb is present both in gram-negative and in gram-positive bacteria, albeit its distribution was not homogeneous in and among the bacterial orders considered .
an open reading frame coding for lsrb was found, for gram-negative γ-proteobacteria, essentially only in part of enterobacteriales  and of pasteurellales .
no evidence for lsrb was found in vibrionales and in the luxs-negative families of pseudomonadaceae and xanthomonadaceae.
no lsrb homolog was located either in the luxs-positive class of ε-proteobacteria  or in spirochaetales, in which the presence of luxs was reported to date only in borrelia spp.
<cit> .
for gram-negative α-proteobacteria, lsrb was found only on the psymb-plasmid of sinorhizobium meliloti and in three rhodobacter species, i.e.
r. capsulatus  and on the high variable chromosome- <dig> of r. sphaeroides  <dig> .
<dig> and r. sphaeroides atcc  <dig>  but was absent from r. sphaeroides atcc  <dig>  these finding are of special interest since α-proteobacteria are lacking the luxs gene coding for the s-ribosylhomocysteinase/ai- <dig> synthase.
in gram-positive bacteria, lsrb was located solely in bacteria belonging to the bacillus cereus group.
b. anthracis, b. thuringensis, b. weihenstephanensis and b. cereus were found to carry the lsr-operon .
however, in b. weihenstephanensis kbab <dig> both lsrb and lsrd show frameshifts and are listed as pseudogenes, in a similar way to lsrb of b. thuringensis serovar konkukian 97- <dig> and to the associated atpase-gene lsra of non-pathogenic b. cereus atcc  <dig>  which is apparently truncated.
in b. thuringensis serovar israeliensis atcc  <dig> a pseudogene with very high homology to part of the lsrb-receptor gene is found well separated from the rest of the operon  suggesting that in the latter strain the abc-transporter was inactivated by a rearrangement of the genome.
the lsrb gene could not be identified in closely related bacillus cereus ssp.
cytotoxis nhv391- <dig> confirming a significant degree of divergence from typical b. cereus  <cit> , which is corroborated also by our luxs-based phylogeny.
more distant bacillus species, including b. subtilis, were all found to be lsrb-negative  suggesting that in gram-positive bacteria the lsr-transporter was acquired or developed by an ancestor of the bacillus cereus group.
no lsrb homolog was found in the actinobacteria bifidobacterium longum  <cit>  and bifidobacterium adolescentis which are so far the only bacteria besides escherichia blattae  <cit> , to possess both known sah-detoxification pathways .
all strains which yielded a positive match for lsrb were further verified by demonstrating the presence of other genes  encoding the lsr-complex.
however, in none of the positive matches the luxs gene and the lsr operon were found to lie in proximal regions of the genome, suggesting that  they were probably acquired and/or developed independently.
on the other hand, in enterobacteriaceae luxs is frequently found in association with another gene involved in sulfur metabolism such as gsha, coding for the γ-glutamate cysteine ligase .
an ambiguous positive match to lsrb  was obtained for substrate binding protein atu <dig>  on the linear chromosome of agrobacterium tumefaciens c <dig>  however, both the sequence of the receptor and the organization of the flanking genes in the operon displayed high similarity with the rhamnose catabolism gene locus of rhizobium leguminosarum bv.
trifolii plasmid prlew14-2c  showing that these genes are probably not involved in quorum sensing.
similarly, a positive match  in actinobacillus pleuropneumoniae to an uncharacterized conserved protein  was discarded after additional analysis yielded that the retrieved sequences showed more similarity to e. coli xylf gene, coding for the membrane receptor protein of the abc-type xylose transport system.
phylogenetic analysis
in order to understand how qs- <dig> developed in the different bacterial taxa during evolution, a phylogenetic analysis was performed by comparing, for those species for which a qs- <dig> has been previously described, the presence of the established ai- <dig> receptor genes luxp and lsrb to the tree based on the luxs-gene sequences .
our results confirmed the conclusions of a previous work  <cit>  in which a few species appeared be misplaced on the luxs tree if compared to species phylogeny and thus to have acquired the latter gene by an horizontal gene transfer  event: these bacteria are β-proteobacterium neisseria meningiditis, ε-proteobacterium helicobacter pylori, the spirochete borrelia burgdorferi, and actinobacteria bifidobacterium longum and bifidobacterium adolescentis.
interestingly, however, in none of these bacteria the gene sequences encoding the luxp- or lsrb-receptors have been found, indicating that most probably only the metabolic function of luxs, and not a complete qs- <dig> system, was transferred.
more generally, as detailed above, the occurrence of luxp was restricted to vibrionales, but the gene was not universally present in every species of this order, missing for instance from vibrio angustum or photobacterium profundum.
similarly, the presence of the lsrb-gene was generally limited to certain species belonging to the gram-negative bacterial orders of enterobacteriales.
pasteurellales, and to the gram-negative positive classes of bacilli and clostridia and was not a collective feature of all the members of each group.
most importantly, several species for which an ai- <dig> dependent behavior was claimed  were found to be devoid of either receptor genes.
analysis of lsrb tree  shows that this gene is well distinct from related abc-receptor genes such as rbsb or xylf.
this result is supported by similar analyses performed on the other genes coding for the abc-receptor complexes  and indicates that the lsr receptor is ortholog to those abc-transporters and did not arose by gene duplication from related sugar transporters in each single species or genus.
the lsrb-phylogeny of luxs-negative species r. sphaeroides,r.
capsulatus and s. meliloti reveals that the lsr-receptor complex of these α-proteobacteria was probably horizontally acquired from unrelated enterobacterial species.
the hgt hypothesis is further supported by the fact that in these species the genes coding for the ai- <dig> receptor are not on the main chromosome, but are found on dynamic genetic elements such as a smaller chromosome as in the case of r. sphaeroides  <dig> .
<dig> or on a plasmid as in s. meliloti  <dig>
taking in consideration only lsrb-positive bacteria, we finally compared the topology of the phylogenetic trees based on luxs, lsrb and the housekeeping gene rpob, encoding a subunit of the rna polymerase and which is commonly used as a molecular marker for microbial evolution studies .
the luxs- and rpob-trees displayed a branching pattern which was more similar to each other than to that of the lsrb-tree, however in the latter most of the differences were found at local level within single clades and  they were not supported by high bootstrap values even when using more rigorous building methods such as minimum evolution or maximum parsimony , indicating the lsrb may have undergone an hgt only in a limited number of cases.
this result, together with the discrete distribution of lsrb among luxs-positive bacteria suggests that for luxs- and lsrb-positive bacteria the ai- <dig> receptor coevolved with the sah-lyase/ai- <dig> synthase, but that for luxs-negative strains it was probably acquired by certain species  as adaptation to a specific niche.
discussion
distribution of luxpq sensor kinase
our search across fully published genomes clearly shows that the presence of this kind of ai- <dig> receptors is primarily limited to a narrow group of bacteria.
corroborating the results of sun and coworkers  <cit>  no complete qs- <dig> receptor complex based on the luxpq sensor kinase and the related signal cascade was found in bacteria not belonging to the order of vibrionales.
within the latter order the presence of luxpq genes was not ubiquitous, but was found to be largely in accordance with the responsiveness to ai- <dig> stimulation and the functionality of the qs- <dig> system that can be deduced from the literature already published on this topic .
no alternative receptor for ai- <dig> has been described so far in bacteria belonging to the vibrionales.
even if an experimental verification is still pending, we predict therefore that other sequenced vibrio species  that carry both luxs, luxpq and luxuo will most probably have a functional qs- <dig> system, while photobacterium profundum ss <dig> will not.
luxp homologs were also found in luxs-negative neptuniibacter caesariensis med <dig>  marinomonas sp.
med <dig> and d. acetoxidans dsm <dig>  since previous evidence suggests that ai- <dig> may also be involved in interspecies gene regulation  <cit>  and mixed-species biofilm formation  <cit> , it is therefore conceivable that some bacteria may be able to recognize the ai- <dig> signal even though they are unable to produce it.
the evolutionary benefit would be the opportunity to sense the presence and abundance of ai- <dig> producers sharing the same niche.
both neptuniibacter and marinomonas are marine bacteria that share the same environment  <cit>  or are associated with the same host as ai- <dig> producer vibrio strains  <cit> .
however, given the less important similarity to the receptor found in vibrionales and the different sensor kinase associated this hypothesis has in any case to be proven experimentally.
in the case of d. desulfuricans, in which the luxp homolog is not physically associated to any gene indicating a signal transduction toward the inner of the cell, the prospect of an ai- <dig> related qs seems less realistic.
distribution of lsr-receptor complex
although in recent years the activity of qs- <dig> was repeatedly hypothesized on the basis of the presence and functionality of the luxs gene in several bacterial species not belonging to the order of vibrionales, few efforts have been made to verify the presence of a cognate ai- <dig> receptor, either experimentally by demonstrating successful chemical complementation of luxs mutant  or genetically by showing the presence of the associated genes in the corresponding bacterial genomes.
the only other clearly established ai- <dig> receptor, the lsr abc-transporter was identified so far only in s. typhimurium  <cit>  and e. coli  <cit> .
for this reason we examined the genomes of completely sequenced bacteria with a reported quorum sensing activity ascribed to ai- <dig> and searched for the presence of dna-sequences coding for receptor protein lsrb, the extracellular substrate-binding component of the lsr-complex.
the outcome of our investigation was at first glance surprising, since a positive match was found only in part of enterobacteriales, pasteurellales and bacillales .
however, a more detailed analysis of the results showed that these were largely congruent with the ability to complement the luxs mutation by the means of chemically pure ai- <dig> signal or ai- <dig> containing supernatants.
the lsrb gene was retrieved exclusively in the genome of bacteria for which, provided that it was performed in the corresponding study, a chemical complementation yielded a successful result .
conversely, our search for lsrb did not yield positive results in any of the species in which exogenous ai- <dig> was unable to restore the original wild-type phenotype in luxs mutants .
these results strongly corroborate the hypothesis that lsrb functions as receptor for ai- <dig> and the fact that in many bacteria the role of luxs is restricted to the amc.
in a few cases we were unable to identify either the lsr-receptor complex or the luxpq sensor kinase in the genome of the corresponding bacteria even if a successful chemical complementation of the luxs mutant using ai- <dig> containing fluids was claimed in the literature , although it must be stressed that for some bacterial species at least one work came to contrasting conclusions and asserted a mere metabolic role for luxs .
strain-specific distribution or functionality of lsr-transporters are in principle possible explanations for this result.
although the lsr-operon seems to be ubiquitous in strains belonging to the bacillus cereus group, in some non-pathogenic strains  or insect-associated species like b. thuringiensis the sequences of the lsr-genes revealed frameshifts or rearrangements that will most likely lead to the production curtailed proteins and non-functional ai- <dig> receptors.
since in none of these strains the lsr-operon was completely absent from the genome, this may be the result of gene deterioration as a consequence of reduced selective pressure in those strains not tightly associated with warm-blooded animals.
alternative receptors
another option to explain successful chemical complementation of luxs-mutants in bacteria lacking both luxpq or the lsr-receptor complex is to postulate the presence of alternative receptors for ai- <dig>  yoshida and coworkers  <cit>  were able to complement the defect in biofilm formation of a streptococcus mutans luxs mutant by culture media of other streptococci or other luxs-positive bacteria which are normally associated with s. mutans in the human oral cavity such as porphyromonas gingivalis and a. actinomycetemcomitans, while in a more recent study  <dig> genes responsive to ai- <dig> were identified in the same strain by global transcriptome analysis  <cit> .
in the latter study, the product of one of the strong ai- <dig> upregulated genes  was hypothesized to act as an uptake mechanism for the autoinducer molecule.
the corresponding protein is a xanthine/uracil/vitamin c permease  and belongs to the major facilitator superfamily  transporters.
homologs to this putative receptor are present in all streptococcus species for which a qs- <dig> related phenotype was described, but also in many other species, including many for which a conventional receptor for ai- <dig> is already known, such as v. vulnificus, b. anthracis or p. multocida .
a role as an alternative ai- <dig> receptor was also suggested for the ribose abc-transporter encoded by the rbs-operon.
stevenson and coworkers speculated that rbsb may serve as the ai- <dig> receptor in borrelia burgdorferi  <cit> , while in actinobacillus actionmycetemcomitans both the lsrb and rbsb proteins were shown to interact with ai- <dig> molecule  <cit> .
the ai- <dig> signal showed differential affinity to two receptors depending if found as thmf  or as borate diester   leading to the hypothesis that the two receptors may be used together by the bacteria to scavange borate from the environment  <cit> .
the distribution of the ribose abc-transporter is almost ubiquitous among most bacterial species regardless if the luxs- and lsrb-genes are present , so that it is conceivable, although far to be proved, that rbsb may account for the internalization of ai- <dig> in a number of species lacking lsrb.
host specificity of lsrb-positive bacteria
interestingly, bacteria that possess a lsr-receptor complex are mostly associated with warm-blooded animals.
in fact, this abc-transporter could not be located in the luxs-positive bacteria s. bongori, which unlike other salmonella spp.
is usually associated with cold-blooded organisms  <cit> , or in b. subtilis, which is a soil organism not considered to be an animal pathogen  <cit> .
exceptions are represented by the human pathogens s. boydii and s. sonnei in which the lsr-genes are absent  <cit>  and insect pathogens b. thuringensis and p. luminescens which conversely feature the lsr-complex.
the case of p. luminescens is of particular significance since in this bacterium the presence of the lsr-locus seems restricted to those strains  forming a mutualistic consortium with entomopathogenic nematode heterorhabditis bacteriophora, but shows ample gene deletions in strains associated with other nematode hosts or in related xenorhabdus species suggesting that the lsr locus is strongly involved in the bacterial association with h. bacteriophora  <cit> .
altogether this data strongly suggest that ai- <dig> may be a relevant signal for many invasive bacteria and that the lsr-receptor complex may generally play an important role in host colonization.
orphan receptors
an unexpected result of our study was the discovery of a region with high homology to the lsr operon   on chromosome ii of two of the three sequenced strains of the free-living photosynthetic bacterium r. sphaeroides and on plasmid psymb of nitrogen-fixing plant symbiont s. meliloti  <dig>  both bacteria belong to the α-subdivision of proteobacteria that regenerates homocysteine not by the means of the amc, but directly from sah using a one-step reaction catalyzed by sah hydrolase.
since they are devoid of the luxs enzyme they can not produce the ai- <dig> signal by themselves and are thus in principle dumb in the qs- <dig> language.
in both bacteria the genes coding for the lsr receptor complex are situated not on the main chromosome, but either on a megaplasmid mainly devoted at the codification of solute uptake systems in s. meliloti  <cit>  or on a smaller chromosome with much lower information content density than the main chromosome, which is thought to provide highly adaptable organisms such as r. sphaeroides with genetic "safe zones" that allow dna to change without affecting the organism itself  <cit> .
the phylogeny of the lsrb gene  suggest that the ai- <dig> receptor complex in r. sphaeroides and s. meliloti was eventually acquired horizontally from unrelated enterobacterial species, such as e.g.
k. pneumoniae which occurs as free-living diazotrophs in soil, plant endophytes, and as opportunistic human pathogens  <cit> .
it is conceivable that k. pneumoniae acquired the lsr receptor from other endotherm associated enterobacteria and passed it to cohabitant nitrogen fixing bacteria in soil.
whether the lsr-complex in r. sphaeroides and s. meliloti really enables them to react to the ai- <dig> signal still has to be proven experimentally, but is tempting to speculate that these α-proteobacteria may use this receptor to eavesdrop on qs- <dig> communication employed by k. pneumoniae in soil.
dual pathways
contrasting with other actinobacteria that bear only the sah hydrolase for the production of homocysteine using sah as substrate, both b. longum and b. adolescentis also possess luxs in their genome.
these species are key commensals of the human gastrointestinal tract and appear to have acquired luxs by horizontal gene transfer in the gut from other gram-positive endotherm-associated bacteria .
the presence of the other genes coding for the complete amc and absence of lsrb from the genome of bifidobacterium suggest that the additional occurrence of luxs in these strains is not aimed to ai- <dig> production , but represents only a redundant metabolic pathway for the recycling of homocysteine as proposed previously  <cit> .
however, the possibility that an eventual ai- <dig> signal may be used for one-way interspecies communication can not be discounted.
another non-pathogenic species, e. blattae, was also identified to have both pathways  <cit> , but given its taxonomical classification among enterobacteriaceae, here the horizontal gene transfer most probably involved sahh instead of luxs.
as for bifidobacterium, also in e. blattae the physiological significance of the double sah detoxification pathway still awaits clarification.
phylogenetic analysis
previous studies have assessed that at the level of bacterial phyla the phylogenetic tree based on luxs largely corresponds to the one constructed using 16s ribosomal rna, demonstrating that the former gene is ancestral in groups like proteobacteria and firmicutes.
ε-proteobacterium h. pylori, spirochete b. burgdorferi and actinobacterium b. longum, which clustered with firmicutes in the luxs tree, represented the exception to this criterion indicating that horizontal gene transfer has occurred in these species  <cit> .
our result confirm these findings and suggest that also b. adolescentis, or more probably a common bifidobacterium ancestor, may have undergone an hgt .
in order to understand if and how the ai- <dig> receptors coevolved with the ai- <dig> synthesis gene, we first analyzed the distribution of the recognized receptor genes on the entire luxs phylogenetic tree , then compared the tree based on the lsrb receptor with the luxs and rpob trees of the matching strains .
as previously reported the presence of the luxp receptor is fundamentally limited to vibrionaceae  <cit>  and its evolution corroborates the fact that this kind of receptor has been constantly present during the evolution of this family of marine bacteria .
on the other hand the lsr complex is widespread  in enterobacteriaceae and bacillaceae, as well as present in two species belonging to the family pasteurellaceae .
despite the fact that these bacterial groups are fairly separate on an evolutionary level, the branching pattern of the lsrb tree is mostly concordant with the corresponding tree based on luxs and housekeeping gene rpob , implicating that this kind of ai- <dig> receptor genes are probably orthologs that have co-evolved with the ai- <dig> producer gene.
an alternative hypothesis postulating the lsr receptor complex being a paralog of closely related abc transporter, such as ribose transporter rbs, is not supported by the available data which demonstrate that rbs genes of distant related species are more similar to each other, than equivalent rbs and lsr genes in the same species .
as expected from previous studies on abc systems  <cit> , also shuffling of genes between different transporters is not supported by phylogenetic analysis .
the fact that in no bacteria the components of the ai- <dig> receptor and signal transduction pathway are found clustered together with luxs in the genome further suggests that the qs- <dig> system was built upon a pre-existent general metabolic function by some bacteria only, which may help to explain why it is not an universal feature of all species.
CONCLUSIONS
although in order to be able to detect the ai- <dig> signal and to react in a qs- <dig> dependent manner bacteria must carry an appropriate receptor for the autoinducer, in the light of our analysis it is apparent that there is still a common misconception in the literature leading to the conclusion that the mere presence of the luxs gene is a sufficient condition to deduce the existence of type- <dig> quorum sensing.
in these works the functionality of luxs is usually demonstrated by the ability of the wildtype strain supernatants to stimulate luminescence in ai- <dig> reporter strain v. harveyi bb <dig>  by the competence of the species-specific luxs gene to induce ai- <dig> production when introduced in a luxs-negative strain such as e. coli dh5α or by in trans complementation of luxs-mutants to restore the original phenotype.
it is critical to understand that these tests are in any case insufficient to demonstrate the existence of a qs- <dig> based communication.
even for bacteria which are devoid of a functional qs- <dig>  the v. harveyi reporter assay will result in a simple test of the activity of the amc, as ai- <dig> is produced spontaneously from dpd  <cit> , which is a by-product as of this metabolic cycle .
the complementation of ai- <dig> production in e. coli dh5α, which has a defective luxs due to a frameshift-mutation  <cit> , using a foreign luxs-gene merely proves its functionality, but does not give any indication about the production and most importantly the detection of ai- <dig> in the original organism.
finally, any defect induced by the luxs mutation in the methionine recycling pathway  will be in most cases fully restored by the straightforward genetic complementation  <cit> .
it is thus imperative for qs- <dig> studies to provide evidence for ai- <dig> receptor also include a chemical complementation of the luxs mutant  <cit> , either by using ai- <dig> conditioned supernatants or by using the chemically defined ai- <dig> molecule  <cit> .
new criteria for establishing the functionality of qs- <dig> in a given species should include also identification and mutational analysis of putative ai- <dig> receptors, rna/protein expression patterns in presence/absence of the ai- <dig> signal and, if the latter is transported inside the cell as in the case of the lsr receptor complex, ai- <dig> consumption profiles.
in bacteria lacking the lsr-operon this would enable assessment of the role as alternative ai- <dig> receptors for related abc-transporters, e.g., ribose rbs receptor complex  <cit>  or xylose xyl receptor complex  <cit> .
an alternative approach may consist in the genetic complementation of the sole metabolic function of the defective luxs gene by replacing it with an exogenous sahh gene encoding the sah hydrolase, which is part of the other known s-adenosylhomocysteine detoxification pathway.
in this way it is possible to repair the function of the amc in e. coli without restoring a potential ai- <dig> production  <cit> .
in any case the possibility that some bacteria lacking an ai- <dig> receptor either produce ai- <dig> for interference with signaling of other bacteria or that inadvertently produced ai- <dig> is detected by other bacteria can not be discarded and may have important ecological implications.
yet, a disease control strategy aimed to interfere with intra- or interspecific qs- <dig> based crosstalk between bacteria , or as a means to enhance qs- <dig> in beneficial bacteria, would have hope of success only for those species which carry a functional ai- <dig> receptor.
as suggested by xavier and bassler  <cit> , this may be true for many human pathogenic bacteria  for which the presence of the lsr-receptor complex was confirmed herein.
on the other hand, the prospective of infection control using ai- <dig> based quorum quenching is less concrete for plant diseases, since no ai- <dig> receptor was unambiguously confirmed in specialized plant pathogens, meaning that in these bacteria the role of luxs is most probably limited to metabolism.
authors' contributions
fr initiated the study, conducted the data mining work and contributed to writing the manuscript.
bd supervised the study and contributed to writing the manuscript.
both authors read and approved the final manuscript.
supplementary material
additional file 1
click here for file
additional file 2
click here for file
acknowledgements
we would like to thank dr. pascal zaffarano  for fruitful discussions on phylogenetic analysis while cycling up the pfannenstiel.
this work was financially supported by the swiss federal department of home affairs, state secretariat for education and research , the swiss federal department of agriculture  and the safecrop centre , funded by fondo per la ricerca, autonomous province of trento.
it was conducted within the european research networks cost <dig> and cost <dig>

 population density- and growth phase-dependent bacterial cell-to-cell communication involving the production and detection of extracellular signaling molecules is referred to as quorum sensing. the detection of a stimulatory concentration of an autoinducer leads to an alteration in gene expression, enabling bacteria to coordinate their behavior in response to environmental challenges. a large number of cellular functions are known to be regulated via the qs mechanism in a variety of bacterial species such as biofilm formation, swarming behavior or the production of antibiotics and virulence factors. n-acyl-homoserine lactones are the most frequent signaling molecules found in gram-negative bacteria. this first autoinduction system was identified in the luminescent marine symbiont vibrio fischeri and is based on the pheromone n-3-oxohexanoyl-l-homoserine lactone. meanwhile several other type i autoinducers, whose molecules differ only in the ahl-acyl side chain moiety, have been discovered in a number of gram-negative bacteria. since every species employs specific receptors for its own ahl molecule, not every bacterium can automatically cross-talk with all other bacteria, so that every'diahlect' can essentially be understood only by those species sharing the same cognate receptor. however, due to the fact that different bacteria may simultaneously use several signal molecules and receptors, and given the chemical similarity of the different ahl molecules, a limited interaction is possible even for bacteria carrying non-cognate receptors. thus, a limited communication between different bacterial species is possible through qs-. in this respect great interest accompanied the discovery, first in vibrio harveyi then in a variety of other bacteria, of the luxs protein that catalyzes the production of an autoinducer molecule for a second quorum sensing system. since the luxs gene was subsequently found to be widespread among both gram-negative and gram-positive bacteria, it was tempting to presume that qs- may constitute the basis of an universal language, a sort of bacterial esperanto. the presence of luxs has been reported in several subgroups of the bacterial kingdom, being widely found in bacteroidetes, actinobactetria, and β- plus γ-proteobacteria, along with all bacilli, deinococci and ε-proteobacteria, but not in archaea or eukarya in vibrio species qs- is connected with qs- by partially sharing the same transduction pathway leading through the central signal relay protein luxu to the terminal response regulator luxo, but displays a separate receptor for ai- namely the two component sensor kinase luxpq. although homologs to luxpq have been found in other vibrio species, other bacterial species were reported to lack similar qs- related proteins with the exception of luxs, raising the prospect that this kind of receptor and signal transduction pathway is limited to the vibrionales. in salmonella typhimurium other genes were shown to encode for a different complex that serves as receptor for ai-. an abc-type transporter named lsr is responsible for the ai- uptake into the cell and was reported subsequently also in escherichia coli. ai- is phosphorylated inside the cell and is anticipated to interact with lsrr, a protein that contributes to repress the lsr-operon and possibly acts as regulator of gene expression. this mechanism is different from ai- detection in v. harveyi, where just the signal but not the ai- molecule is transduced inside the cell. thus alternative explanations to qs have been proposed whereby ai- may be released as a waste product and then reused subsequently as a metabolite or, as in the case of actinobacillus actinomycetemcomitans, that the molecule may be used as a borate scavenger by bacteria. there are now several studies that describe the central role of luxs in e. coli as crucial for the transition to pathogenic existence inside the host by controlling virulence determinants such as motility, biofilm formation or gene expression. ai- dependent behavior was consequently claimed also in other gram-negative and gram-positive bacteria, but many reports are simply based on the presence of the luxs gene, mutant analysis and/or additional phenotypic tests such as the use of ai- reporter strain v. harveyi bb. rarely have investigations examined the existence of potential ai- receptors or assessed their functionality by proving ai- depletion of the media by the wildtype or by performing a chemical complementation of luxs mutants using pure ai- signal or ai- conditioned supernatants. this is essential since it is now well recognized that luxs displays also a primary role as metabolic enzyme in the activated methyl cycle, which is responsible for the generation of the cell major methyl donor s-adenosyl-l-methionine and the recycling of methionine by detoxification of s-adenosyl-l-homocysteine. luxs takes part in this cycle by salvaging the homocysteine moiety from the cycle intermediate s-ribosyl-homocysteine. as a by-product of this reaction, the direct ai- precursor - -dihydroxy- -pentanedione is formed. in v. harveyi dpd cyclizes and is spontaneously converted to furanosyl borate diester by the addition of borate, while s. typhimurium produces and recognizes a chemically distinct form of ai-. given this dual nature of luxs the sole use of the commonly exploited mutational approach is inappropriate, since a simple genetic complementation using the intact gene on a plasmid will also repair the induced metabolic unbalance as well and yield in any case a restored phenotype. a more detailed description of the findings of the papers cited herein is available as additional file. a +: positive;: positive; -: negative; b h: hypothesized; nd: not determined; q: questioned; c +: successful complementation; -: unsuccessful complementation; nd: not determined; ai-2: complementation with pure autoinducer; dpd: complementation with precursor; sn: complementation with co-culture or conditioned supernatants of luxs-positive strain. a more detailed description of the findings of the papers cited herein is available as additional file. a +: positive;: positive; -: negative; b c: claimed; h: hypothesized; c +: successful complementation; -: unsuccessful complementation; nd: not determined; ai-2: complementation with pure autoinducer; dpd: complementation with precursor; sn: complementation with co-culture or conditioned supernatants of luxs-positive strain; d hypothesized on the basis of the genomes of fully sequenced lactobacillales. a more detailed description of the findings of the papers cited herein is available as additional file. a +: positive;: positive; -: negative; nd: not determined; b nd: not determined; c: claimed; c +: successful complementation; -: unsuccessful complementation; nd: not determined; ai-2: complementation with pure autoinducer; dpd: complementation with precursor; sn: complementation with co-culture or conditioned supernatants of luxs-positive strain; d hypothesized based on serratia proteomaculans sequence; e hypothesized on the basis of more than known streptococcus complete genomes belonging to the species s. pneumoniae, s. mutans, s. pyogenes, s. agalactiae, s. suis, s. equis and s. zooepidemicus. in order to assess whether the role of luxs in well-described bacterial species is effectively related to ai- mediated autoinduction, we performed a database analysis for established ai- receptors in fully-sequenced genomes and sequence databases, and compared the outcome with published results about qs- in these bacteria. in this way we were able to corroborate many reports where the role of luxs in qs- was proven experimentally or to confirm results of other works where no effect related to autoinduction could be demonstrated. on the other hand, in those cases when an ai- mediated mechanism was claimed, but no known receptor was found in the corresponding bacterial sequence, novel or alternative receptors must be postulated or, without a successful chemical complementation of the luxs mutant, a sheer metabolic role must be acknowledged. with the aim of understanding the evolutionary relationships among luxs, the presence of ai- receptors and the functionality of the qs- system, a phylogenetic analysis was performed to compare the evolution of the luxs gene with the presence and evolution of the genes coding for known ai- receptors, transporters and regulators. since known ai- receptors genes are restricted to luxs-positive bacteria and have only partially coevolved with the ai- synthase, it can be hypothesized that the qs- function was acquired and developed as a new trait by some bacteria by building upon the preexistent metabolic function of luxs. methods search for known ai- receptors in complete genomes complete genome sequences of bacteria known to have a luxs gene were analyzed for the presence of ai- receptor sequences using the blastx and the tblastx programs with a cut-off e-value of 1e- by applying a reciprocal best hit strategy, which allows to distinguish between orthologous and paralogous genes. as starting queries the luxp gene of v. harveyi strain bb and the lsrb gene of e. coli strain k were used. the hits were manually checked to confirm that they had the same functional annotation as the query and re-submitted as novel queries until no new relevant positive match was found. in this second phase also luxs-negative strains were included in the analysis by performing a general blast search across the entire ncbi database. in bacteria found to carry a gene coding for an ai- receptor the presence of a complete qs- system was corroborated by verifying the existence of other genes associated to the respective ai- transduction pathway. bacterial species were scored as positive for qs- receptors only when in addition to luxp, homologs of the luxq, luxu and luxo genes which encode the full transduction pathway were also found. similarly, positive results obtained with lsrb were confirmed by verifying the presence of genes constituting a complete lsr-operon. these include lsra, lsrc and lsrd, lsrr, and lsrk. our investigation was extended to incomplete sequenced bacteria known to carry a luxs gene or for which a function of qs- was documented in the literature through an ncbi database search with relevant terms such as "luxs", "lsrb", "luxp", "ai-2" or "quorum sensing". discovered pertinent sequences were used in the analysis. a literature search for published studies on the effect of type- quorum sensing in the organisms carrying the luxs gene was performed using the ncbi database and the papers were critically read, with particular attention to those key experiments which are essential to ascertain the presence of a qs- such as the identification of ai- receptors, chemical complementation of the luxs mutant or detection of ai- depletion in the culture medium. in cases where these tests were omitted the possibility that a metabolic dysfunction in the amc may have been misinterpreted as disruption of type- quorum sensing was proposed. phylogenetic analysis phylogenetic analysis was performed in order to understand how ai- receptors may have evolved in different bacteria with respect to the presence of luxs. phylogenetic trees were generated on the basis of complete luxs- and lrsb-sequences without choosing any outgroup. housekeeping gene rpob, encoding the rna polymerase β-subunit, was used as reference. retrieved dna sequences were aligned with clustalw. sites presenting alignment gaps were excluded from analysis. the molecular evolutionary genetics analysis program version was used to calculate evolutionary distances and to infer trees based on the neighbor-joining method using the jc formula. nodal robustness of the inferred trees was assessed by bootstrap replicates. genome resources and ncbi accession numbers complete genomes and key luxs and lsrb sequences were used. complete genomes actinobacillus actinomycetemcomitans, actinomyces naeslundii, bacillus anthracis str. sterne, bacillus cereus atcc, bacillus cereus subsp. cytotoxis nvh 391-, bacillus subtilis subsp. subtilis str., bacillus thuringensis serovar israelensis atcc, bacillus thuringiensis serovar konkukian str. 97-, bacillus weihenstephanensis kbab, bifidobacterium adolescentis, bifidobacterium longum, borrelia burgdorferi, campylobacter jejuni, clostridium acetobutylicum, clostridium difficile, clostridium perfringens, desulfovibrio desulfuricans, desulfuromonas acetoxidans, erwinia amylovora, escherichia coli, haemophilus influenzae, haemophilus somnus, helicobacter pylori, klebsiella pneumoniae, mannheimia succiniciproducens, neisseria meningiditis, pasteurella multocida, photobacterium profundum, photorhabdus luminescens, rhodobacter sphaeroides., rhodobacter sphaeroides atcc, rhodobacter sphaeroides atcc, salmonella bongori, salmonella typhimurium, serratia marcescens, shewanella oneidensis mc_, shighella boydii, shighella dysenteriae, shighella flexneri, shighella sonnei, sinorrhizobium meliloti, sodalis glossinidius, staphylococcus aureus, streptococcus pyogenes, vibrio angustum, vibrio cholerae, vibrio fischeri, vibrio harveyi, vibrio vulnificus, xenorhabdus sp., yersinia enterocolitica, yersinia frederiksenii, yersinia intermedia, yersinia mollaretii, yersinia pestis, yersinia pseudotubercolosis. luxs gene sequences erwinia billingiae, erwinia tasmaniensis, vibrio anguillarum. lsrb gene sequences rhodobacter capsulatus.  presence of luxp-type receptor genes the luxp gene of v. harveyi strain bb was used as a starting query for a search of luxpq-type receptors in bacterial genomes that was successful only for members of the orders of the vibrionales and, to a much lesser extent, of the oceanospirillales. this kind of receptor, along with the effector kinase luxq, was already known in several species of the genus vibrio, i.e., v. anguillarum, v. cholerae, v. fischeri, v. harveyi and v. parahaemolyticus. in vibrio species for which a response to ai- system is documented, but in which no receptor was yet described, i.e., v. vulnificus and v. alginolyticus, we were able to retrieve the luxpq genes by searching the relevant gene sequences in the corresponding published genomes. conversely, the luxpq-receptor was absent from the genome of v. angustum, which does not respond to the ai- signal, even though the luxs gene coding for s-ribosylhomocysteinase is present. besides that, we managed to identify the luxpq receptor complex also in other representatives of the genus for which no study about qs- related phenotype has been performed to date, i.e., v. splendidus, vibrio sp. ex and vibrio sp. med but not in closely related photobacterium profundum ss. a luxp homolog was found also in the genomes of some oceanospirillales, albeit among the bacteria of this order no luxs gene has been sequenced or found to date. the similarity of these proteins to the luxp-receptor present in v. harveyi was only moderate: % identity and % similarity for the neptuniibacter caesariensis med periplasmic protein which carries a region similar to the rbsb-like abc-type sugar transport system receptor locus and % identity and % similarity for the marinomonas sp. med luxp protein precursor. however, both results withstood the reciprocal best hit strategy test and yielded a luxp protein of vibrio as best match in a blastx search. furthermore, in neptuniibacter caesariensis med and marinomonas sp. med the genes encoding these putative luxp homologs are arranged in an operon with presumed transcriptional regulators that likewise contain pas domains, but share otherwise little homology with each other. in marinomonas sp. med the transcriptional regulator showed moderate homology with luxq of v. cholerae and contains also aaa-type atpase and dna-binding domains and is followed by the pfs gene coding for a 5'-methylthioadenosine nucleosidase. a similar protein in desulfuromonas acetoxidans showed an equivalent identity to luxp of v. harveyi and was analogously arranged in an operon with a putative transcriptional regulator containing pas domains, while in desulfovibrio desulfuricans the matching protein also carries a putative receptor locus but the corresponding gene was not associated with either a luxq homolog or the other usual genes of an abc-transporter, indicating the probable absence of a signal transduction pathway toward the inner of the cell. however, also dace_ and dde_ pointed toward luxp in the reciprocal best hit test. as for oceanospirillales, both d. acetoxidans and d. desulfuricans were found to lack the luxs gene. all vibrionales carrying the luxpq genes also carried open reading frames which encoded proteins homologous to the two component, σ54-specific, transcriptional regulator luxuo found in v. harveyi. in oceanospirillales a similar situation was discovered, but both luxp and luxo were flanked by other transcriptional regulator genes which displayed a less important resemblance with luxq and luxu respectively. in all strains investigated the luxo protein displayed the characteristic 8-aa glycine-rich consensus which is needed for interaction with alternative σ. interestingly, in none of the strains were the genes encoding the single components of the qs- mechanism found in proximal positions on the genome, thus they were probably acquired and/or developed independently. notably the tblastx search using luxp gene of v. harveyi strain bb resulted in local hits with the lsrb gene of enterobacteriales. similarity is limited to a small rbsb-like portion on the c-terminus of the luxp protein, but is most probably indicative of the substrate binding domain of both receptors. generally, the use of luxq gene of v. harveyi strain bb as starting query yielded multiple partial positive matches in almost every strain tested, but these were limited to the cytoplasmic transduction domain of other sensor kinases and were not directly related to any known quorum sensing related gene. presence of lsrb-type receptor genes our screening of completed genome projects for lsrb, the gene belonging to the lsr-operon encoding the periplasmic ai- binding component of the abc-type sugar transport apparatus, revealed that this type of receptor is generally more widely spread than the luxpq-system. we found that lsrb is present both in gram-negative and in gram-positive bacteria, albeit its distribution was not homogeneous in and among the bacterial orders considered. an open reading frame coding for lsrb was found, for gram-negative γ-proteobacteria, essentially only in part of enterobacteriales and of pasteurellales. no evidence for lsrb was found in vibrionales and in the luxs-negative families of pseudomonadaceae and xanthomonadaceae. no lsrb homolog was located either in the luxs-positive class of ε-proteobacteria or in spirochaetales, in which the presence of luxs was reported to date only in borrelia spp.. for gram-negative α-proteobacteria, lsrb was found only on the psymb-plasmid of sinorhizobium meliloti and in three rhodobacter species, i.e. r. capsulatus and on the high variable chromosome- of r. sphaeroides. and r. sphaeroides atcc but was absent from r. sphaeroides atcc these finding are of special interest since α-proteobacteria are lacking the luxs gene coding for the s-ribosylhomocysteinase/ai- synthase. in gram-positive bacteria, lsrb was located solely in bacteria belonging to the bacillus cereus group. b. anthracis, b. thuringensis, b. weihenstephanensis and b. cereus were found to carry the lsr-operon. however, in b. weihenstephanensis kbab both lsrb and lsrd show frameshifts and are listed as pseudogenes, in a similar way to lsrb of b. thuringensis serovar konkukian 97- and to the associated atpase-gene lsra of non-pathogenic b. cereus atcc which is apparently truncated. in b. thuringensis serovar israeliensis atcc a pseudogene with very high homology to part of the lsrb-receptor gene is found well separated from the rest of the operon suggesting that in the latter strain the abc-transporter was inactivated by a rearrangement of the genome. the lsrb gene could not be identified in closely related bacillus cereus ssp. cytotoxis nhv391- confirming a significant degree of divergence from typical b. cereus, which is corroborated also by our luxs-based phylogeny. more distant bacillus species, including b. subtilis, were all found to be lsrb-negative suggesting that in gram-positive bacteria the lsr-transporter was acquired or developed by an ancestor of the bacillus cereus group. no lsrb homolog was found in the actinobacteria bifidobacterium longum and bifidobacterium adolescentis which are so far the only bacteria besides escherichia blattae, to possess both known sah-detoxification pathways. all strains which yielded a positive match for lsrb were further verified by demonstrating the presence of other genes encoding the lsr-complex. however, in none of the positive matches the luxs gene and the lsr operon were found to lie in proximal regions of the genome, suggesting that they were probably acquired and/or developed independently. on the other hand, in enterobacteriaceae luxs is frequently found in association with another gene involved in sulfur metabolism such as gsha, coding for the γ-glutamate cysteine ligase. an ambiguous positive match to lsrb was obtained for substrate binding protein atu on the linear chromosome of agrobacterium tumefaciens c however, both the sequence of the receptor and the organization of the flanking genes in the operon displayed high similarity with the rhamnose catabolism gene locus of rhizobium leguminosarum bv. trifolii plasmid prlew14-2c showing that these genes are probably not involved in quorum sensing. similarly, a positive match in actinobacillus pleuropneumoniae to an uncharacterized conserved protein was discarded after additional analysis yielded that the retrieved sequences showed more similarity to e. coli xylf gene, coding for the membrane receptor protein of the abc-type xylose transport system. phylogenetic analysis in order to understand how qs- developed in the different bacterial taxa during evolution, a phylogenetic analysis was performed by comparing, for those species for which a qs- has been previously described, the presence of the established ai- receptor genes luxp and lsrb to the tree based on the luxs-gene sequences. our results confirmed the conclusions of a previous work in which a few species appeared be misplaced on the luxs tree if compared to species phylogeny and thus to have acquired the latter gene by an horizontal gene transfer event: these bacteria are β-proteobacterium neisseria meningiditis, ε-proteobacterium helicobacter pylori, the spirochete borrelia burgdorferi, and actinobacteria bifidobacterium longum and bifidobacterium adolescentis. interestingly, however, in none of these bacteria the gene sequences encoding the luxp- or lsrb-receptors have been found, indicating that most probably only the metabolic function of luxs, and not a complete qs- system, was transferred. more generally, as detailed above, the occurrence of luxp was restricted to vibrionales, but the gene was not universally present in every species of this order, missing for instance from vibrio angustum or photobacterium profundum. similarly, the presence of the lsrb-gene was generally limited to certain species belonging to the gram-negative bacterial orders of enterobacteriales. pasteurellales, and to the gram-negative positive classes of bacilli and clostridia and was not a collective feature of all the members of each group. most importantly, several species for which an ai- dependent behavior was claimed were found to be devoid of either receptor genes. analysis of lsrb tree shows that this gene is well distinct from related abc-receptor genes such as rbsb or xylf. this result is supported by similar analyses performed on the other genes coding for the abc-receptor complexes and indicates that the lsr receptor is ortholog to those abc-transporters and did not arose by gene duplication from related sugar transporters in each single species or genus. the lsrb-phylogeny of luxs-negative species r. sphaeroides,r. capsulatus and s. meliloti reveals that the lsr-receptor complex of these α-proteobacteria was probably horizontally acquired from unrelated enterobacterial species. the hgt hypothesis is further supported by the fact that in these species the genes coding for the ai- receptor are not on the main chromosome, but are found on dynamic genetic elements such as a smaller chromosome as in the case of r. sphaeroides. or on a plasmid as in s. meliloti taking in consideration only lsrb-positive bacteria, we finally compared the topology of the phylogenetic trees based on luxs, lsrb and the housekeeping gene rpob, encoding a subunit of the rna polymerase and which is commonly used as a molecular marker for microbial evolution studies. the luxs- and rpob-trees displayed a branching pattern which was more similar to each other than to that of the lsrb-tree, however in the latter most of the differences were found at local level within single clades and they were not supported by high bootstrap values even when using more rigorous building methods such as minimum evolution or maximum parsimony, indicating the lsrb may have undergone an hgt only in a limited number of cases. this result, together with the discrete distribution of lsrb among luxs-positive bacteria suggests that for luxs- and lsrb-positive bacteria the ai- receptor coevolved with the sah-lyase/ai- synthase, but that for luxs-negative strains it was probably acquired by certain species as adaptation to a specific niche. discussion distribution of luxpq sensor kinase our search across fully published genomes clearly shows that the presence of this kind of ai- receptors is primarily limited to a narrow group of bacteria. corroborating the results of sun and coworkers no complete qs- receptor complex based on the luxpq sensor kinase and the related signal cascade was found in bacteria not belonging to the order of vibrionales. within the latter order the presence of luxpq genes was not ubiquitous, but was found to be largely in accordance with the responsiveness to ai- stimulation and the functionality of the qs- system that can be deduced from the literature already published on this topic. no alternative receptor for ai- has been described so far in bacteria belonging to the vibrionales. even if an experimental verification is still pending, we predict therefore that other sequenced vibrio species that carry both luxs, luxpq and luxuo will most probably have a functional qs- system, while photobacterium profundum ss will not. luxp homologs were also found in luxs-negative neptuniibacter caesariensis med marinomonas sp. med and d. acetoxidans dsm since previous evidence suggests that ai- may also be involved in interspecies gene regulation and mixed-species biofilm formation, it is therefore conceivable that some bacteria may be able to recognize the ai- signal even though they are unable to produce it. the evolutionary benefit would be the opportunity to sense the presence and abundance of ai- producers sharing the same niche. both neptuniibacter and marinomonas are marine bacteria that share the same environment or are associated with the same host as ai- producer vibrio strains. however, given the less important similarity to the receptor found in vibrionales and the different sensor kinase associated this hypothesis has in any case to be proven experimentally. in the case of d. desulfuricans, in which the luxp homolog is not physically associated to any gene indicating a signal transduction toward the inner of the cell, the prospect of an ai- related qs seems less realistic. distribution of lsr-receptor complex although in recent years the activity of qs- was repeatedly hypothesized on the basis of the presence and functionality of the luxs gene in several bacterial species not belonging to the order of vibrionales, few efforts have been made to verify the presence of a cognate ai- receptor, either experimentally by demonstrating successful chemical complementation of luxs mutant or genetically by showing the presence of the associated genes in the corresponding bacterial genomes. the only other clearly established ai- receptor, the lsr abc-transporter was identified so far only in s. typhimurium and e. coli. for this reason we examined the genomes of completely sequenced bacteria with a reported quorum sensing activity ascribed to ai- and searched for the presence of dna-sequences coding for receptor protein lsrb, the extracellular substrate-binding component of the lsr-complex. the outcome of our investigation was at first glance surprising, since a positive match was found only in part of enterobacteriales, pasteurellales and bacillales. however, a more detailed analysis of the results showed that these were largely congruent with the ability to complement the luxs mutation by the means of chemically pure ai- signal or ai- containing supernatants. the lsrb gene was retrieved exclusively in the genome of bacteria for which, provided that it was performed in the corresponding study, a chemical complementation yielded a successful result. conversely, our search for lsrb did not yield positive results in any of the species in which exogenous ai- was unable to restore the original wild-type phenotype in luxs mutants. these results strongly corroborate the hypothesis that lsrb functions as receptor for ai- and the fact that in many bacteria the role of luxs is restricted to the amc. in a few cases we were unable to identify either the lsr-receptor complex or the luxpq sensor kinase in the genome of the corresponding bacteria even if a successful chemical complementation of the luxs mutant using ai- containing fluids was claimed in the literature, although it must be stressed that for some bacterial species at least one work came to contrasting conclusions and asserted a mere metabolic role for luxs. strain-specific distribution or functionality of lsr-transporters are in principle possible explanations for this result. although the lsr-operon seems to be ubiquitous in strains belonging to the bacillus cereus group, in some non-pathogenic strains or insect-associated species like b. thuringiensis the sequences of the lsr-genes revealed frameshifts or rearrangements that will most likely lead to the production curtailed proteins and non-functional ai- receptors. since in none of these strains the lsr-operon was completely absent from the genome, this may be the result of gene deterioration as a consequence of reduced selective pressure in those strains not tightly associated with warm-blooded animals. alternative receptors another option to explain successful chemical complementation of luxs-mutants in bacteria lacking both luxpq or the lsr-receptor complex is to postulate the presence of alternative receptors for ai- yoshida and coworkers were able to complement the defect in biofilm formation of a streptococcus mutans luxs mutant by culture media of other streptococci or other luxs-positive bacteria which are normally associated with s. mutans in the human oral cavity such as porphyromonas gingivalis and a. actinomycetemcomitans, while in a more recent study genes responsive to ai- were identified in the same strain by global transcriptome analysis. in the latter study, the product of one of the strong ai- upregulated genes was hypothesized to act as an uptake mechanism for the autoinducer molecule. the corresponding protein is a xanthine/uracil/vitamin c permease and belongs to the major facilitator superfamily transporters. homologs to this putative receptor are present in all streptococcus species for which a qs- related phenotype was described, but also in many other species, including many for which a conventional receptor for ai- is already known, such as v. vulnificus, b. anthracis or p. multocida. a role as an alternative ai- receptor was also suggested for the ribose abc-transporter encoded by the rbs-operon. stevenson and coworkers speculated that rbsb may serve as the ai- receptor in borrelia burgdorferi, while in actinobacillus actionmycetemcomitans both the lsrb and rbsb proteins were shown to interact with ai- molecule. the ai- signal showed differential affinity to two receptors depending if found as thmf or as borate diester leading to the hypothesis that the two receptors may be used together by the bacteria to scavange borate from the environment. the distribution of the ribose abc-transporter is almost ubiquitous among most bacterial species regardless if the luxs- and lsrb-genes are present, so that it is conceivable, although far to be proved, that rbsb may account for the internalization of ai- in a number of species lacking lsrb. host specificity of lsrb-positive bacteria interestingly, bacteria that possess a lsr-receptor complex are mostly associated with warm-blooded animals. in fact, this abc-transporter could not be located in the luxs-positive bacteria s. bongori, which unlike other salmonella spp. is usually associated with cold-blooded organisms, or in b. subtilis, which is a soil organism not considered to be an animal pathogen. exceptions are represented by the human pathogens s. boydii and s. sonnei in which the lsr-genes are absent and insect pathogens b. thuringensis and p. luminescens which conversely feature the lsr-complex. the case of p. luminescens is of particular significance since in this bacterium the presence of the lsr-locus seems restricted to those strains forming a mutualistic consortium with entomopathogenic nematode heterorhabditis bacteriophora, but shows ample gene deletions in strains associated with other nematode hosts or in related xenorhabdus species suggesting that the lsr locus is strongly involved in the bacterial association with h. bacteriophora. altogether this data strongly suggest that ai- may be a relevant signal for many invasive bacteria and that the lsr-receptor complex may generally play an important role in host colonization. orphan receptors an unexpected result of our study was the discovery of a region with high homology to the lsr operon on chromosome ii of two of the three sequenced strains of the free-living photosynthetic bacterium r. sphaeroides and on plasmid psymb of nitrogen-fixing plant symbiont s. meliloti both bacteria belong to the α-subdivision of proteobacteria that regenerates homocysteine not by the means of the amc, but directly from sah using a one-step reaction catalyzed by sah hydrolase. since they are devoid of the luxs enzyme they can not produce the ai- signal by themselves and are thus in principle dumb in the qs- language. in both bacteria the genes coding for the lsr receptor complex are situated not on the main chromosome, but either on a megaplasmid mainly devoted at the codification of solute uptake systems in s. meliloti or on a smaller chromosome with much lower information content density than the main chromosome, which is thought to provide highly adaptable organisms such as r. sphaeroides with genetic "safe zones" that allow dna to change without affecting the organism itself. the phylogeny of the lsrb gene suggest that the ai- receptor complex in r. sphaeroides and s. meliloti was eventually acquired horizontally from unrelated enterobacterial species, such as e.g. k. pneumoniae which occurs as free-living diazotrophs in soil, plant endophytes, and as opportunistic human pathogens. it is conceivable that k. pneumoniae acquired the lsr receptor from other endotherm associated enterobacteria and passed it to cohabitant nitrogen fixing bacteria in soil. whether the lsr-complex in r. sphaeroides and s. meliloti really enables them to react to the ai- signal still has to be proven experimentally, but is tempting to speculate that these α-proteobacteria may use this receptor to eavesdrop on qs- communication employed by k. pneumoniae in soil. dual pathways contrasting with other actinobacteria that bear only the sah hydrolase for the production of homocysteine using sah as substrate, both b. longum and b. adolescentis also possess luxs in their genome. these species are key commensals of the human gastrointestinal tract and appear to have acquired luxs by horizontal gene transfer in the gut from other gram-positive endotherm-associated bacteria. the presence of the other genes coding for the complete amc and absence of lsrb from the genome of bifidobacterium suggest that the additional occurrence of luxs in these strains is not aimed to ai- production, but represents only a redundant metabolic pathway for the recycling of homocysteine as proposed previously. however, the possibility that an eventual ai- signal may be used for one-way interspecies communication can not be discounted. another non-pathogenic species, e. blattae, was also identified to have both pathways, but given its taxonomical classification among enterobacteriaceae, here the horizontal gene transfer most probably involved sahh instead of luxs. as for bifidobacterium, also in e. blattae the physiological significance of the double sah detoxification pathway still awaits clarification. phylogenetic analysis previous studies have assessed that at the level of bacterial phyla the phylogenetic tree based on luxs largely corresponds to the one constructed using 16s ribosomal rna, demonstrating that the former gene is ancestral in groups like proteobacteria and firmicutes. ε-proteobacterium h. pylori, spirochete b. burgdorferi and actinobacterium b. longum, which clustered with firmicutes in the luxs tree, represented the exception to this criterion indicating that horizontal gene transfer has occurred in these species. our result confirm these findings and suggest that also b. adolescentis, or more probably a common bifidobacterium ancestor, may have undergone an hgt. in order to understand if and how the ai- receptors coevolved with the ai- synthesis gene, we first analyzed the distribution of the recognized receptor genes on the entire luxs phylogenetic tree, then compared the tree based on the lsrb receptor with the luxs and rpob trees of the matching strains. as previously reported the presence of the luxp receptor is fundamentally limited to vibrionaceae and its evolution corroborates the fact that this kind of receptor has been constantly present during the evolution of this family of marine bacteria. on the other hand the lsr complex is widespread in enterobacteriaceae and bacillaceae, as well as present in two species belonging to the family pasteurellaceae. despite the fact that these bacterial groups are fairly separate on an evolutionary level, the branching pattern of the lsrb tree is mostly concordant with the corresponding tree based on luxs and housekeeping gene rpob, implicating that this kind of ai- receptor genes are probably orthologs that have co-evolved with the ai- producer gene. an alternative hypothesis postulating the lsr receptor complex being a paralog of closely related abc transporter, such as ribose transporter rbs, is not supported by the available data which demonstrate that rbs genes of distant related species are more similar to each other, than equivalent rbs and lsr genes in the same species. as expected from previous studies on abc systems, also shuffling of genes between different transporters is not supported by phylogenetic analysis. the fact that in no bacteria the components of the ai- receptor and signal transduction pathway are found clustered together with luxs in the genome further suggests that the qs- system was built upon a pre-existent general metabolic function by some bacteria only, which may help to explain why it is not an universal feature of all species.  although in order to be able to detect the ai- signal and to react in a qs- dependent manner bacteria must carry an appropriate receptor for the autoinducer, in the light of our analysis it is apparent that there is still a common misconception in the literature leading to the conclusion that the mere presence of the luxs gene is a sufficient condition to deduce the existence of type- quorum sensing. in these works the functionality of luxs is usually demonstrated by the ability of the wildtype strain supernatants to stimulate luminescence in ai- reporter strain v. harveyi bb by the competence of the species-specific luxs gene to induce ai- production when introduced in a luxs-negative strain such as e. coli dh5α or by in trans complementation of luxs-mutants to restore the original phenotype. it is critical to understand that these tests are in any case insufficient to demonstrate the existence of a qs- based communication. even for bacteria which are devoid of a functional qs- the v. harveyi reporter assay will result in a simple test of the activity of the amc, as ai- is produced spontaneously from dpd, which is a by-product as of this metabolic cycle. the complementation of ai- production in e. coli dh5α, which has a defective luxs due to a frameshift-mutation, using a foreign luxs-gene merely proves its functionality, but does not give any indication about the production and most importantly the detection of ai- in the original organism. finally, any defect induced by the luxs mutation in the methionine recycling pathway will be in most cases fully restored by the straightforward genetic complementation. it is thus imperative for qs- studies to provide evidence for ai- receptor also include a chemical complementation of the luxs mutant, either by using ai- conditioned supernatants or by using the chemically defined ai- molecule. new criteria for establishing the functionality of qs- in a given species should include also identification and mutational analysis of putative ai- receptors, rna/protein expression patterns in presence/absence of the ai- signal and, if the latter is transported inside the cell as in the case of the lsr receptor complex, ai- consumption profiles. in bacteria lacking the lsr-operon this would enable assessment of the role as alternative ai- receptors for related abc-transporters, e.g., ribose rbs receptor complex or xylose xyl receptor complex. an alternative approach may consist in the genetic complementation of the sole metabolic function of the defective luxs gene by replacing it with an exogenous sahh gene encoding the sah hydrolase, which is part of the other known s-adenosylhomocysteine detoxification pathway. in this way it is possible to repair the function of the amc in e. coli without restoring a potential ai- production. in any case the possibility that some bacteria lacking an ai- receptor either produce ai- for interference with signaling of other bacteria or that inadvertently produced ai- is detected by other bacteria can not be discarded and may have important ecological implications. yet, a disease control strategy aimed to interfere with intra- or interspecific qs- based crosstalk between bacteria, or as a means to enhance qs- in beneficial bacteria, would have hope of success only for those species which carry a functional ai- receptor. as suggested by xavier and bassler, this may be true for many human pathogenic bacteria for which the presence of the lsr-receptor complex was confirmed herein. on the other hand, the prospective of infection control using ai- based quorum quenching is less concrete for plant diseases, since no ai- receptor was unambiguously confirmed in specialized plant pathogens, meaning that in these bacteria the role of luxs is most probably limited to metabolism. authors' contributions fr initiated the study, conducted the data mining work and contributed to writing the manuscript. bd supervised the study and contributed to writing the manuscript. both authors read and approved the final manuscript. supplementary material additional file 1 click here for file additional file 2 click here for file acknowledgements we would like to thank dr. pascal zaffarano for fruitful discussions on phylogenetic analysis while cycling up the pfannenstiel. this work was financially supported by the swiss federal department of home affairs, state secretariat for education and research, the swiss federal department of agriculture and the safecrop centre, funded by fondo per la ricerca, autonomous province of trento. it was conducted within the european research networks cost and cost 
this suggests that qs- may play an important role in interactions with animal hosts. an unequivocal ai- related behavior was restricted primarily to organisms bearing known ai- receptor genes, while phenotypes of luxs mutant bacteria lacking these genes could often be explained simply by assuming deficiencies in sulfur metabolism. in order to assess whether the role of luxs in these bacteria is indeed related to ai- mediated quorum sensing we analyzed genomic databases searching for established ai- receptors and other presumed qs-related proteins and compared the outcome with published results about the role of qs- in these organisms. 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. since the luxs gene was found to be widespread among the most diverse bacterial taxa, it was hypothesized that ai- 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- therein. 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- autoinducer molecule for a second quorum sensing system.
 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- autoinducer molecule for a second quorum sensing system. since the luxs gene was found to be widespread among the most diverse bacterial taxa, it was hypothesized that ai- 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- therein. however, rarely hathe existence of potential ai- 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.  in order to assess whether the role of luxs in these bacteria is indeed related to ai- mediated quorum sensing we analyzed genomic databases searching for established ai- receptors and other presumed qs-related proteins and compared the outcome with published results about the role of qs- in these organisms. an unequivocal ai- related behavior was restricted primarily to organisms bearing known ai- receptor genes, while phenotypes of luxs mutant bacteria lacking these genes could often be explained simply by assuming deficiencies in sulfur metabolism.  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- 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- must be postulated. 