BACKGROUND
cell to cell communication – also called quorum sensing – by self-produced small signalling molecules  is a mechanism by which bacteria coordinate gene expression within their population and in such a way obtain optimum timing and efficiency, for example when infecting a eukaryotic host, degrading polymers, or colonizing surfaces.
while most quorum sensing mechanisms studied to date are species specific, bassler et al.
<cit>  discovered an autoinducer in the marine pathogen vibrio harveyi which is the byproduct of a reaction carried out by a metabolic enzyme and thus has the potential to act as a universal bacterial signal.
autoinducer- <dig>  refers to a group of compounds resulting from spontaneous cyclisation and hydration of dihydroxypentanedione , which yields -2-methyl- <dig> , <dig> -tetrahydroxytetrahydrofuran  and -2-methyl- <dig> , <dig> -tetrahydroxytetrahydrofuran  in chemical equilibrium.
s-thmf spontaneously reacts with borate ions to form the active compound detected by v. harveyi  <cit> , while r-thmf, the other stereoisomere, is unable to complex borate and is sensed by salmonella typhimurium  <cit>  and probably by escherichia coli  <cit> , but not by v. harveyi.
the receptor proteins for s-thmf and r-thmf are not homologous, and their three dimensional structures determine the specificity of their interaction with the respective signalling molecules  <cit> .
dpd is produced only in the domain bacteria as a by-product of the activated methyl cycle.
the pathway leading to dpd involves a two-step detoxification of s-adenosylhomocysteine , namely by a mta/sah nucleosidase  and a s-ribosylhomocysteinase.
the latter enzyme is also known as luxs enzyme or ai- <dig> synthase.
the products of the reaction carried out by the luxs enzyme are homocysteine and dpd, which spontaneously forms the ai- <dig> group of compounds.
however, all eukarya, archaea, and some phyla within the domain bacteria employ an alternative one-step detoxification of sah using only a sah hydrolase  enzyme.
these bacteria are therefore unable to produce ai- <dig>  <cit> .
the presence of the luxs gene  versus the sahh gene  was previously surveyed in the genomes of fully sequenced bacteria  <cit> .
thus, the luxs gene was found in some gamma- and betaproteobacteria, all epsilonproteobacteria, none of the alphaproteobacteria, none of the actinobacteria, all firmicutes and some spirochaetes.
since the majority of the fully sequenced bacteria at the time were pathogens, and the number of genomes within the various phylogenetic groups was sometimes very small, it had to remain open if the presence of the luxs gene is phylogenetically conserved or if it has been acquired through lateral gene transfer by pathogens.
in the sea, the three most abundant groups of heterotrophic bacteria are members of the alphaproteobacteria, gammaproteobacteria, and bacteroidetes  <cit> .
therefore, here we studied a collection of  <dig> marine isolates from these three phylogenetic groups  <cit>  for the presence of the luxs gene using pcr amplification with conserved degenerated primers and sequencing of selected bands.
since the absence of the luxs gene could theoretically be due to lack of primer specificity, we confirmed our results by determining the presence of the sahh gene, representing the alternative pathway for sah detoxification.
most bacteria have either the luxs or the sahh gene in monocopy.
there are several exceptions, e.g.
escherichia blattae carries both genes, and some bacteria have neither luxs nor sahh  <cit> .
within the gammaproteobacteria, the luxs gene was detected in all shewanella strains consistently.
the genus shewanella was first described in  <dig>  <cit>  and includes organisms that have been associated with spoilage of proteinacous food  as well as opportunistic human pathogens   <cit> .
during the past two decades, members of the genus shewanella have mostly been studied for their involvement in anaerobic transformations important for biogeochemical cycling of elements.
the coupling of growth and energy generation to the dissimilatory reduction of iron and manganese oxide have been demonstrated in s. oneidensis mr- <dig> for the first time in  <dig>  <cit> .
although the luxs gene has been found in s. oneidensis and in several draft genomes of shewanella strains which are currently sequenced, ai- <dig> production has not yet been investigated in this genus, which comprises both pathogenic and free living species.
therefore, here we determined the ability of nine shewanella type strains and one new marine shewanella isolate to stimulate luminescence of the ai- <dig> biosensor strain v. harveyi bb <dig> and indeed confirmed the production of ai- <dig> in all of them for the first time.
RESULTS
in this study,  <dig> strains were investigated which have been isolated from various habitats of the north sea near the island of helgoland, e.g.
the surface of microoalgae  and macroalgae , suspended particles, in situ grown biofilms, and the picoplankton  <cit> .
in addition,  <dig> strains from the hypersaline antarctic ekho lake were also included  <cit> .
the marine isolates were identified on the basis of near complete 16s rrna gene sequences and belonged to  <dig> different phylogenetic groups: alphaproteobacteria , bacteroidetes , and gammaproteobacteria .
in order to confirm the results which were obtained on the marine shewanella isolates, eight type strains of the genus shewanella and one alishewanella fetalis type strain were also investigated .
identification of the luxs gene
all marine isolates were screened with primers luxs_degfor <dig> and luxs_degrev4; for the gammaproteobacteria, the primer set luxs_gamma_for and luxs_gamma_rev  was also used.
the luxs gene was not found in any of the alphaproteobacteria and bacteroidetes strains tested , consistent with the survey of fully sequenced microbial genomes published previously  <cit> .
in the gammaproteobacteria, the fully sequenced genomes available through ncbi in january  <dig> revealed that some representatives use the two step detoxification of sah, hence contain the pfs gene and the luxs gene, while others use the one-step detoxification of sah, thus harbour the sahh gene.
there are several parasitic and also some free living species which have neither luxs nor sahh, but only the pfs gene.
we screened  <dig> marine gammaproteobacteria for the presence of the luxs gene .
it was found in all strains of alteromonas, consistent with the full genome sequence of alteromonas hydrophila.
it was also found in seven of  <dig> strains of halomonas tested; no complete genome sequence is available in this genus yet.
in the genus pseudoalteromonas, two genome sequences have been completed, i.e.
p. haloplanktis and p. atlantica.
both species have neither a luxs nor a sahh homologue, but contain only the pfs gene.
we tested  <dig> pseudoalteromonas isolates, and found a pcr band of the expected size in  <dig> of them.
in one strain the band could be confirmed by sequencing to represent a luxs gene fragment.
finally, the luxs gene was identified in the marine shewanella  and vibrio  isolates, as expected from the completed genomes in these genera.
in order to determine if the luxs gene was consistently present in the shewanellaceae family, nine type strains were investigated .
in all of them the luxs gene could be amplified and verified by sequencing.
identification of the sahh gene
the sahh gene is responsible for the s-adenosylhomocysteinase enzyme, which is able to detoxify sah in one step without production of ai- <dig>  in bacteria, usually either sahh or luxs is present as a single copy.
all strains were tested with both sahh primers .
the sahh gene was identified in  <dig> out of  <dig> alphaproteobacteria and in all  <dig> bacteroidetes strains , confirming that the strains from these phyla do not have the ability to produce ai- <dig> through the luxs enzyme.
interestingly, the sahh gene was also found in some gammaproteobacteria, namely in three strains of psychrobacter sp.
.
two psychrobacter species have been fully sequenced and also have the sahh gene .
full genome sequences of representatives of two additional species of gammaproteobacteria  also revealed the presence of sahh .
lack of both the sahh and the luxs gene
in five of the  <dig> alphaproteobacteria investigated, and in  <dig> of the  <dig> gammaproteobacteria investigated, neither the luxs nor the sahh gene was detected.
available whole genome sequences of the phylogenetically closest organisms in the ncbi database were checked for comparison, since it can never be excluded that a negative result in pcr is caused by a primer mismatch.
within the alphaproteobacteria, lack of both luxs and sahh has previously only been seen in the parasitic ricksettia species  <cit> , as well as in the genera neoricksettia, wolbachia, anaplasma, and ehrlichia  <cit> .
in the gammaproteobacteria, the lack of both genes has previously been observed, and sah recycling presumably occurs through the mta/sah nucleoidase alone  <cit> .
ten of the  <dig> strains where neither luxs nor sahh could be identified belonged to the genera glaciecola, halomonas or marinobacter, where genome sequences are not yet available.
however,  <dig> strains belonged into the genus pseudoalteromonas.
the fully sequenced genomes of p. haloplanktis and p. atlantica also have neither luxs nor sahh, suggesting that this result might not be caused by primer mismatch but is actually correct and indicates sah detoxification by the pfs enzyme in these strains.
however, confirmation of band identity proved difficult due to the simultaneous amplification of another gene of similar size.
it can therefore not be excluded that more luxs genes might have been detected using different primers.
ai- <dig> production in shewanella and alishewanella species
several of the gammaproteobacteria species which did not have the luxs gene were tested for ai- <dig> activity using the v. harveyi bioassay.
ai- <dig> could not be detected, confirming the pcr result .
all shewanella and alishewanella strains contained the luxs gene and were tested for ai- <dig> activity, which was detected in all of them.
table  <dig> shows the summary of all  <dig> bioassays conducted; for each species, the maximum a1- <dig> activity is averaged for the indicated number of bioassays.
average maximum fold induction values were between 4%  and 46% .
figure  <dig> shows a subset of these data; for each species, two different cultures are shown, each of which was tested in two different bioassays independently.
variability between cultures and between replicate bioassays was sometimes very high.
independent of the level of ai- <dig> activity detected, the same growth phase dependent pattern of production was observed in all investigated strains.
fig.
2a shows this for two representative species, a. fetalis with low, and s. japonica with high maximum ai- <dig> activity.
in all shewanella strains ai- <dig> activity reached its maximum towards the end of the exponential growth phase and decreased during stationary phase.
in species with high ai- <dig> activity, this decrease occurred gradually.
by contrast, if the ai- <dig> activity was low, it disappeared rapidly at the beginning of the stationary phase within  <dig> –  <dig> hours.
at the late stationary phase , no ai- <dig> activity was detected in any of the strains tested.
the same pattern was observed in all cultivation media used.
as an example, fig.
2b shows ai- <dig> production of s. hafniensis dt- <dig> in two different media.
the strain produced similar ai- <dig> activities at the same growth phase in both of them.
s. oneidensis was also cultivated anaerobically using iron as an electron acceptor .
under these conditions, growth was very slow and ai- <dig> activity was not found .
discussion
phylogenetic distribution of the sahh and luxs genes
in all bacteroidetes isolates tested here, and in all fully sequenced bacteroidetes strains, the sahh pathway was present, with the exception of porphyromonas gingivalis, which uses the pfs/luxs pathway  <cit> .
this may indicate that horizontal gene transfer occurred in this species.
the main habitat of this bacterium is the oral cavity, where many of the cultivated species use the pfs/luxs pathway  <cit>  and gene transfer might have a high probability.
several events of gene transfer have been demonstrated for the luxs enzyme previously  <cit> .
a recent report demonstrated ai- <dig> activity in some bacteroides isolates, although the luxs gene could not be identified  <cit> .
isolates belonging into the alphaproteobacteria had the sahh gene consistently and did not contain the luxs gene.
therefore, they use the one-step detoxification of sah and do not have the potential for communication by ai- <dig>  these results confirm previous work based on fully sequenced genomes  <cit> .
within the gammaproteobacteria, the luxs gene was found in all strains of the genera alteromonas, shewanella and vibrio consistent with results from fully sequenced genomes in these genera.
it was for the first time found in some strains of the genus pseudoalteromonas.
in this genus, whole genome sequencing and pcr amplification indicate that many representatives have neither the luxs nor the sahh gene and thus use only the pfs enzyme for removal of sah.
the same is true for the genus halomonas, where the luxs gene was found in some but not all representatives.
species from several other genera of fully sequenced gammaproteobacteria  also contained neither the luxs nor the sahh gene.
in addition, in the genus psychrobacter  the sahh gene was found consistently, in accordance with fully sequenced genomes in this genus.
the data strongly support the conclusion that the distribution of the luxs gene is dependent on the phylogenetic position of an organism rather than on the ecological niche which it has occupied, with rare exceptions which may have been caused by horizontal gene transfer.
the complex pattern of presence/absence of luxs versus sahh in the gammaproteobacteria requires a thorough analysis of the organismal phylogeny and possible gene transfer events in this group, which may not be monophyletic  <cit> .
the genus shewanella and the production of autoinducer-2
the shewanella type strains investigated here were isolated from the sediment of lake oneida   <cit> , the intestines of marine animals  <cit> , antarctic sea ice   <cit> , a human fetus   <cit> , and marine seawater and mussel samples   <cit> .
s. algae was isolated from red algae  <cit>  and was differentiated from the first described member of the genus, s. putrefaciens, only later <cit> .
s. hafniensis  <cit>  was isolated from fish in the baltic sea, and is 98% similar to the marine isolate dt- <dig> obtained from the surface of a diatom  <cit> .
all shewanella/alishewanella strains were able to induce luminescence in the v. harveyi bb <dig> reporter strain, indicating that they produced the dpd derivative s-thmf which can be sensed by v. harveyi and which is usually referred to as ai- <dig>  the large differences in the levels of ai- <dig> seen in the various species of shewanella are partly due to the variability of the bioassay; however compounds inhibitory to v. harveyi might also be produced by some strains.
for example, stationary phase culture supernatants of s. algae and s. oneidensis were able to inhibit v. harveyi luminescence .
alternatively, the highly unstable dpd might be converted to compounds other than ai- <dig> in shewanella, or the chemical equilibrium between s-thmf and r-thmf might be shifted towards the r-thmf molecule which cannot be detected by vibrio.
production of ai- <dig> during growth
in all shewanella strains, the pattern of ai- <dig> production during growth was the same.
the amount of ai- <dig> detected in the culture supernatant had a maximum towards the end of the logarithmic growth phase.
subsequently, it decreased and disappeared completely in the late stationary phase.
a similar pattern is observed in salmonella typhimurium and escherichia coli  <cit> .
in these bacteria, ai- <dig> triggers its own uptake through induction of an abc transporter in the late logarithmic growth phase, followed by intracellular degradation.
ai- <dig>  or rather, r-thmf, is specifically sensed by salmonella through the lsrr protein which bears remote resemblance to ribose transporters.
in salmonella and e. coli, there is little evidence of cell-cell communication through ai- <dig>  however, nothing is known at the moment about the mechanism responsible for the disappearance of ai- <dig> from the culture supernatant in shewanella species.
a protein with weak homology  to the lsrb protein is present in s. oneidensis, but most of the other genes in the lsr operon are missing.
CONCLUSIONS
in this study, a phylogenetically and ecologically well characterized collection of  <dig> marine isolates was examined for their potential for communication by ai- <dig>  for this aim, the presence of the luxs and sahh genes – representing the two pathways for sah recycling – was determined by pcr with degenerate primers and sequencing.
the majority of the isolates tested here belonged to phyla where whole genome sequence analysis had indicated the presence of the sahh gene rather than the luxs gene , and this was clearly confirmed in our work.
within the marine gammaproteobacteria, the more complex picture which was already seen in earlier investigations  <cit>  was confirmed in this study, with some strains harbouring the luxs gene, while the sahh gene was present in others, and many strains containing neither of those genes, raising questions regarding the evolution of the gammaproteobacteria and of the pfs/luxs and sahh pathways, respectively.
in the second part of our work, we focused on the genus shewanella.
the luxs gene was consistently found in all marine shewanella isolates and all shewanella type strains tested.
the ability to induce luminescence in the v. harveyi reporter strain was demonstrated in  <dig> species of the genus shewanella, both opportunistic pathogens and free living species.
the kinetics of ai- <dig> production was very similar in all shewanella species, showing a peak in the late logarithmic growth phase, and the disappearance of ai- <dig> from the culture medium during stationary phase.
it will be the aim of future studies to elucidate the role of ai- <dig> in shewanella.

 cell to cell communication – also called quorum sensing – by self-produced small signalling molecules is a mechanism by which bacteria coordinate gene expression within their population and in such a way obtain optimum timing and efficiency, for example when infecting a eukaryotic host, degrading polymers, or colonizing surfaces. while most quorum sensing mechanisms studied to date are species specific, bassler et al. discovered an autoinducer in the marine pathogen vibrio harveyi which is the byproduct of a reaction carried out by a metabolic enzyme and thus has the potential to act as a universal bacterial signal. autoinducer- refers to a group of compounds resulting from spontaneous cyclisation and hydration of dihydroxypentanedione, which yields -2-methyl-, -tetrahydroxytetrahydrofuran and -2-methyl-, -tetrahydroxytetrahydrofuran in chemical equilibrium. s-thmf spontaneously reacts with borate ions to form the active compound detected by v. harveyi, while r-thmf, the other stereoisomere, is unable to complex borate and is sensed by salmonella typhimurium and probably by escherichia coli, but not by v. harveyi. the receptor proteins for s-thmf and r-thmf are not homologous, and their three dimensional structures determine the specificity of their interaction with the respective signalling molecules. dpd is produced only in the domain bacteria as a by-product of the activated methyl cycle. the pathway leading to dpd involves a two-step detoxification of s-adenosylhomocysteine, namely by a mta/sah nucleosidase and a s-ribosylhomocysteinase. the latter enzyme is also known as luxs enzyme or ai- synthase. the products of the reaction carried out by the luxs enzyme are homocysteine and dpd, which spontaneously forms the ai- group of compounds. however, all eukarya, archaea, and some phyla within the domain bacteria employ an alternative one-step detoxification of sah using only a sah hydrolase enzyme. these bacteria are therefore unable to produce ai-. the presence of the luxs gene versus the sahh gene was previously surveyed in the genomes of fully sequenced bacteria. thus, the luxs gene was found in some gamma- and betaproteobacteria, all epsilonproteobacteria, none of the alphaproteobacteria, none of the actinobacteria, all firmicutes and some spirochaetes. since the majority of the fully sequenced bacteria at the time were pathogens, and the number of genomes within the various phylogenetic groups was sometimes very small, it had to remain open if the presence of the luxs gene is phylogenetically conserved or if it has been acquired through lateral gene transfer by pathogens. in the sea, the three most abundant groups of heterotrophic bacteria are members of the alphaproteobacteria, gammaproteobacteria, and bacteroidetes. therefore, here we studied a collection of marine isolates from these three phylogenetic groups for the presence of the luxs gene using pcr amplification with conserved degenerated primers and sequencing of selected bands. since the absence of the luxs gene could theoretically be due to lack of primer specificity, we confirmed our results by determining the presence of the sahh gene, representing the alternative pathway for sah detoxification. most bacteria have either the luxs or the sahh gene in monocopy. there are several exceptions, e.g. escherichia blattae carries both genes, and some bacteria have neither luxs nor sahh. within the gammaproteobacteria, the luxs gene was detected in all shewanella strains consistently. the genus shewanella was first described in and includes organisms that have been associated with spoilage of proteinacous food as well as opportunistic human pathogens. during the past two decades, members of the genus shewanella have mostly been studied for their involvement in anaerobic transformations important for biogeochemical cycling of elements. the coupling of growth and energy generation to the dissimilatory reduction of iron and manganese oxide have been demonstrated in s. oneidensis mr- for the first time in. although the luxs gene has been found in s. oneidensis and in several draft genomes of shewanella strains which are currently sequenced, ai- production has not yet been investigated in this genus, which comprises both pathogenic and free living species. therefore, here we determined the ability of nine shewanella type strains and one new marine shewanella isolate to stimulate luminescence of the ai- biosensor strain v. harveyi bb and indeed confirmed the production of ai- in all of them for the first time.  in this study, strains were investigated which have been isolated from various habitats of the north sea near the island of helgoland, e.g. the surface of microoalgae and macroalgae, suspended particles, in situ grown biofilms, and the picoplankton. in addition, strains from the hypersaline antarctic ekho lake were also included. the marine isolates were identified on the basis of near complete 16s rrna gene sequences and belonged to different phylogenetic groups: alphaproteobacteria, bacteroidetes, and gammaproteobacteria. in order to confirm the results which were obtained on the marine shewanella isolates, eight type strains of the genus shewanella and one alishewanella fetalis type strain were also investigated. identification of the luxs gene all marine isolates were screened with primers luxs_degfor and luxs_degrev4; for the gammaproteobacteria, the primer set luxs_gamma_for and luxs_gamma_rev was also used. the luxs gene was not found in any of the alphaproteobacteria and bacteroidetes strains tested, consistent with the survey of fully sequenced microbial genomes published previously. in the gammaproteobacteria, the fully sequenced genomes available through ncbi in january revealed that some representatives use the two step detoxification of sah, hence contain the pfs gene and the luxs gene, while others use the one-step detoxification of sah, thus harbour the sahh gene. there are several parasitic and also some free living species which have neither luxs nor sahh, but only the pfs gene. we screened marine gammaproteobacteria for the presence of the luxs gene. it was found in all strains of alteromonas, consistent with the full genome sequence of alteromonas hydrophila. it was also found in seven of strains of halomonas tested; no complete genome sequence is available in this genus yet. in the genus pseudoalteromonas, two genome sequences have been completed, i.e. p. haloplanktis and p. atlantica. both species have neither a luxs nor a sahh homologue, but contain only the pfs gene. we tested pseudoalteromonas isolates, and found a pcr band of the expected size in of them. in one strain the band could be confirmed by sequencing to represent a luxs gene fragment. finally, the luxs gene was identified in the marine shewanella and vibrio isolates, as expected from the completed genomes in these genera. in order to determine if the luxs gene was consistently present in the shewanellaceae family, nine type strains were investigated. in all of them the luxs gene could be amplified and verified by sequencing. identification of the sahh gene the sahh gene is responsible for the s-adenosylhomocysteinase enzyme, which is able to detoxify sah in one step without production of ai- in bacteria, usually either sahh or luxs is present as a single copy. all strains were tested with both sahh primers. the sahh gene was identified in out of alphaproteobacteria and in all bacteroidetes strains, confirming that the strains from these phyla do not have the ability to produce ai- through the luxs enzyme. interestingly, the sahh gene was also found in some gammaproteobacteria, namely in three strains of psychrobacter sp.. two psychrobacter species have been fully sequenced and also have the sahh gene. full genome sequences of representatives of two additional species of gammaproteobacteria also revealed the presence of sahh. lack of both the sahh and the luxs gene in five of the alphaproteobacteria investigated, and in of the gammaproteobacteria investigated, neither the luxs nor the sahh gene was detected. available whole genome sequences of the phylogenetically closest organisms in the ncbi database were checked for comparison, since it can never be excluded that a negative result in pcr is caused by a primer mismatch. within the alphaproteobacteria, lack of both luxs and sahh has previously only been seen in the parasitic ricksettia species, as well as in the genera neoricksettia, wolbachia, anaplasma, and ehrlichia. in the gammaproteobacteria, the lack of both genes has previously been observed, and sah recycling presumably occurs through the mta/sah nucleoidase alone. ten of the strains where neither luxs nor sahh could be identified belonged to the genera glaciecola, halomonas or marinobacter, where genome sequences are not yet available. however, strains belonged into the genus pseudoalteromonas. the fully sequenced genomes of p. haloplanktis and p. atlantica also have neither luxs nor sahh, suggesting that this result might not be caused by primer mismatch but is actually correct and indicates sah detoxification by the pfs enzyme in these strains. however, confirmation of band identity proved difficult due to the simultaneous amplification of another gene of similar size. it can therefore not be excluded that more luxs genes might have been detected using different primers. ai- production in shewanella and alishewanella species several of the gammaproteobacteria species which did not have the luxs gene were tested for ai- activity using the v. harveyi bioassay. ai- could not be detected, confirming the pcr result. all shewanella and alishewanella strains contained the luxs gene and were tested for ai- activity, which was detected in all of them. table shows the summary of all bioassays conducted; for each species, the maximum a1- activity is averaged for the indicated number of bioassays. average maximum fold induction values were between 4% and 46%. figure shows a subset of these data; for each species, two different cultures are shown, each of which was tested in two different bioassays independently. variability between cultures and between replicate bioassays was sometimes very high. independent of the level of ai- activity detected, the same growth phase dependent pattern of production was observed in all investigated strains. fig. 2a shows this for two representative species, a. fetalis with low, and s. japonica with high maximum ai- activity. in all shewanella strains ai- activity reached its maximum towards the end of the exponential growth phase and decreased during stationary phase. in species with high ai- activity, this decrease occurred gradually. by contrast, if the ai- activity was low, it disappeared rapidly at the beginning of the stationary phase within – hours. at the late stationary phase, no ai- activity was detected in any of the strains tested. the same pattern was observed in all cultivation media used. as an example, fig. 2b shows ai- production of s. hafniensis dt- in two different media. the strain produced similar ai- activities at the same growth phase in both of them. s. oneidensis was also cultivated anaerobically using iron as an electron acceptor. under these conditions, growth was very slow and ai- activity was not found. discussion phylogenetic distribution of the sahh and luxs genes in all bacteroidetes isolates tested here, and in all fully sequenced bacteroidetes strains, the sahh pathway was present, with the exception of porphyromonas gingivalis, which uses the pfs/luxs pathway. this may indicate that horizontal gene transfer occurred in this species. the main habitat of this bacterium is the oral cavity, where many of the cultivated species use the pfs/luxs pathway and gene transfer might have a high probability. several events of gene transfer have been demonstrated for the luxs enzyme previously. a recent report demonstrated ai- activity in some bacteroides isolates, although the luxs gene could not be identified. isolates belonging into the alphaproteobacteria had the sahh gene consistently and did not contain the luxs gene. therefore, they use the one-step detoxification of sah and do not have the potential for communication by ai- these results confirm previous work based on fully sequenced genomes. within the gammaproteobacteria, the luxs gene was found in all strains of the genera alteromonas, shewanella and vibrio consistent with results from fully sequenced genomes in these genera. it was for the first time found in some strains of the genus pseudoalteromonas. in this genus, whole genome sequencing and pcr amplification indicate that many representatives have neither the luxs nor the sahh gene and thus use only the pfs enzyme for removal of sah. the same is true for the genus halomonas, where the luxs gene was found in some but not all representatives. species from several other genera of fully sequenced gammaproteobacteria also contained neither the luxs nor the sahh gene. in addition, in the genus psychrobacter the sahh gene was found consistently, in accordance with fully sequenced genomes in this genus. the data strongly support the conclusion that the distribution of the luxs gene is dependent on the phylogenetic position of an organism rather than on the ecological niche which it has occupied, with rare exceptions which may have been caused by horizontal gene transfer. the complex pattern of presence/absence of luxs versus sahh in the gammaproteobacteria requires a thorough analysis of the organismal phylogeny and possible gene transfer events in this group, which may not be monophyletic. the genus shewanella and the production of autoinducer-2 the shewanella type strains investigated here were isolated from the sediment of lake oneida, the intestines of marine animals, antarctic sea ice, a human fetus, and marine seawater and mussel samples. s. algae was isolated from red algae and was differentiated from the first described member of the genus, s. putrefaciens, only later. s. hafniensis was isolated from fish in the baltic sea, and is 98% similar to the marine isolate dt- obtained from the surface of a diatom. all shewanella/alishewanella strains were able to induce luminescence in the v. harveyi bb reporter strain, indicating that they produced the dpd derivative s-thmf which can be sensed by v. harveyi and which is usually referred to as ai- the large differences in the levels of ai- seen in the various species of shewanella are partly due to the variability of the bioassay; however compounds inhibitory to v. harveyi might also be produced by some strains. for example, stationary phase culture supernatants of s. algae and s. oneidensis were able to inhibit v. harveyi luminescence. alternatively, the highly unstable dpd might be converted to compounds other than ai- in shewanella, or the chemical equilibrium between s-thmf and r-thmf might be shifted towards the r-thmf molecule which cannot be detected by vibrio. production of ai- during growth in all shewanella strains, the pattern of ai- production during growth was the same. the amount of ai- detected in the culture supernatant had a maximum towards the end of the logarithmic growth phase. subsequently, it decreased and disappeared completely in the late stationary phase. a similar pattern is observed in salmonella typhimurium and escherichia coli. in these bacteria, ai- triggers its own uptake through induction of an abc transporter in the late logarithmic growth phase, followed by intracellular degradation. ai- or rather, r-thmf, is specifically sensed by salmonella through the lsrr protein which bears remote resemblance to ribose transporters. in salmonella and e. coli, there is little evidence of cell-cell communication through ai- however, nothing is known at the moment about the mechanism responsible for the disappearance of ai- from the culture supernatant in shewanella species. a protein with weak homology to the lsrb protein is present in s. oneidensis, but most of the other genes in the lsr operon are missing.  in this study, a phylogenetically and ecologically well characterized collection of marine isolates was examined for their potential for communication by ai- for this aim, the presence of the luxs and sahh genes – representing the two pathways for sah recycling – was determined by pcr with degenerate primers and sequencing. the majority of the isolates tested here belonged to phyla where whole genome sequence analysis had indicated the presence of the sahh gene rather than the luxs gene, and this was clearly confirmed in our work. within the marine gammaproteobacteria, the more complex picture which was already seen in earlier investigations was confirmed in this study, with some strains harbouring the luxs gene, while the sahh gene was present in others, and many strains containing neither of those genes, raising questions regarding the evolution of the gammaproteobacteria and of the pfs/luxs and sahh pathways, respectively. in the second part of our work, we focused on the genus shewanella. the luxs gene was consistently found in all marine shewanella isolates and all shewanella type strains tested. the ability to induce luminescence in the v. harveyi reporter strain was demonstrated in species of the genus shewanella, both opportunistic pathogens and free living species. the kinetics of ai- production was very similar in all shewanella species, showing a peak in the late logarithmic growth phase, and the disappearance of ai- from the culture medium during stationary phase. it will be the aim of future studies to elucidate the role of ai- in shewanella. 
the autoinducer- group of signalling molecules are produced by both gram positive and gram negative bacteria as the by-product of a metabolic transformation carried out by the luxs enzyme. all ten species of shewanella tested produced a pronounced peak of ai- towards the end of the exponential growth phase in several media investigated. the luxs gene was not present in any of the alphaproteobacteria and bacteroidetes strains tested; by contrast, these bacteria harboured the sahh gene, coding for an alternative enzyme for the detoxification of s-adenosylhomocysteine in the activated methyl cycle. a number of gammaproteobacteria appeared to have neither the luxs nor the sahh gene. we then studied the production of ai- in the genus shewanella using the vibrio harveyi bioassay. the maximum of ai- activity was different in each shewanella species, ranging from 4% to 46% of the positive control. this is the first report on ai- production in shewanella species; its signalling role in these organisms remains to be elucidated. the luxs gene coding for the ai- synthase enzyme was found in many important pathogens. here, we surveyed its occurrence in a collection of marine isolates belonging to abundant marine phyla using conserved degenerated pcr primers and sequencing of selected positive bands to determine if the presence of the luxs gene is phylogenetically conserved or dependent on the habitat. the data are consistent with those of fully sequenced bacterial genomes and show that the potential for luxs related signalling is dependent on phylogenetic affiliation rather than ecological niche and is largest in certain groups of gammaproteobacteria in the marine environment.
 the autoinducer- group of signalling molecules are produced by both gram positive and gram negative bacteria as the by-product of a metabolic transformation carried out by the luxs enzyme. they are the only non species-specific quorum sensing compounds presently known in bacteria. the luxs gene coding for the ai- synthase enzyme was found in many important pathogens. here, we surveyed its occurrence in a collection of marine isolates belonging to abundant marine phyla using conserved degenerated pcr primers and sequencing of selected positive bands to determine if the presence of the luxs gene is phylogenetically conserved or dependent on the habitat.  the luxs gene was not present in any of the alphaproteobacteria and bacteroidetes strains tested; by contrast, these bacteria harboured the sahh gene, coding for an alternative enzyme for the detoxification of s-adenosylhomocysteine in the activated methyl cycle. within the gammaproteobacteria, luxs was found in all shewanella, vibrio and alteromonas isolates and some pseudoalteromonas and halomonas species, while sahh was detected in psychrobacter strains. a number of gammaproteobacteria appeared to have neither the luxs nor the sahh gene. we then studied the production of ai- in the genus shewanella using the vibrio harveyi bioassay. all ten species of shewanella tested produced a pronounced peak of ai- towards the end of the exponential growth phase in several media investigated. the maximum of ai- activity was different in each shewanella species, ranging from 4% to 46% of the positive control.  the data are consistent with those of fully sequenced bacterial genomes and show that the potential for luxs related signalling is dependent on phylogenetic affiliation rather than ecological niche and is largest in certain groups of gammaproteobacteria in the marine environment. this is the first report on ai- production in shewanella species; its signalling role in these organisms remains to be elucidated. 