BACKGROUND
members of the vibrionaceae family are found in a wide range of aquatic environments, as free-living planktonic cells or attached to surfaces as bacterial aggregates or biofilms.
some members within this family have evolved to become pathogens whereas others have developed symbiotic relationships with their hosts  <cit> .
aliivibrio salmonicida, previously designated vibrio salmonicida  <cit> , is the causative agent of cold water vibriosis in atlantic salmon , rainbow trout  and captive atlantic cod   <cit> , a disease which is kept under control due to successful vaccination  <cit> .
the mechanisms of a. salmonicida virulence and pathogenicity are not known in detail, however, temperature-sensitive iron sequestration, motility and flagella activity, as well as quorum sensing  have been suggested to be involved in virulence  <cit> .
moreover, the genome of a. salmonicida lfi <dig> encodes putative hemolysins, proteases and several protein secretion systems that may be found to be important in pathogenesis  <cit> .
bacteria use qs to regulate and coordinate gene expression by secreting and responding to signal molecules in a cell density dependent manner  <cit> .
the most common qs autoinducer signal molecules in gram-negative bacteria are n-acyl homoserines lactones , which consist of a homoserine lactone  linked to an acyl side chain with 4– <dig> carbons  <cit> .
several qs systems have been identified in vibrios and aliivibrios, and each species seems to utilize a unique combination of qs systems that can act in parallel, or in a hierarchal manner to regulate species-specific activities including biofilm formation, virulence and colonization factors  <cit> .
the well-studied squid symbiont aliivibrio  fischeri, a close relative of a. salmonicida, regulates bioluminescence and colonization by the qs systems luxs/luxpq, ains/ainr and luxi/luxr, where luxs, ains and luxi are the autoinducer synthases  <cit> .
ains in a. fischeri produces n-octanoyl-hsl  which is recognized by the membrane-bound two-component hybrid sensor kinase ainr  <cit> .
luxs in a. fischeri does not produce ahls, but instead makes a signal molecule referred to as autoinducer  <dig>   <cit>  that likely binds the periplasmic receptor luxp and modulates the activity of the sensor kinase luxq, similar to the homologous system in vibrio harveyi  <cit> .
the luxs/luxpq and ains/ainr systems in a. fischeri work in parallel, and in the absence of signal molecules luxpq and ainr are believed to function as kinases and phosphorylate the response regulator luxo via luxu  <cit> .
phosphorylated luxo then activates expression of qrr which encodes a small regulatory rna.
qrr destabilizes the litr mrna that encodes the master regulator of qs.
in the presence of signal molecules at high cell density, luxo becomes dephosphorylated leading to suppression of qrr allowing litr to be expressed  <cit> .
the third system in a. fischeri, luxi/luxr, is activated by the ains/ainr system, and at medium cell densities luxr binds ains-produced c8-hsl and activates transcription of the lux operon .
at higher cell densities, luxi-produced n-3-oxo-hexanoyl-l-hsl  binds to luxr and increases transcription of the luxicdabeg and luxr genes  <cit> .
moreover, the master regulator litr is a positive regulator of ains and is indirectly involved in expression of luxi by controlling expression of luxr.
hence, litr links the ains/ainr and luxs/luxpq systems to the luxi/luxr system in a. fischeri  <cit> .
the number and types of ahls vary between different species of vibrio and aliivibrio, as well as between strains of the same species  <cit> .
however, the specific autoinducer synthases responsible for the different ahls are only known for a few bacteria, such as the above mentioned in a. fischeri.
in v. anguillarum, another fish pathogen, the ains homolog vanm is reported to produce n-3-hydroxy-hexanoyl-hsl  and n-hexanoyl-hsl   <cit> , whereas the luxi homolog vani produces n-3-oxo-decanoyl-hsl   <cit> .
in the bioluminescent bacteria v. harveyi the luxm synthase is reported to produce n-3-hydroxy-butyryl-l-hsl   <cit> .
thus, while some autoinducer synthases produce only one ahl others are able to produce several.
the genome of a. salmonicida lfl <dig> encodes five qs systems: the ains/ainr, luxi/luxr, vars/vara, luxm/luxn and luxs/luxpq systems.
the latter two systems are probably inactive since luxm is absent, and luxn and luxp contain frame-shift mutations  <cit> .
the lux operon of a. salmonicida has a novel organization compared to a. fischeri, with two flanking luxr genes  and the luxi gene located outside the luxcdabeg locus  <cit> , and a. salmonicida is only able to produce bioluminescence after addition of decyl aldehyde  <cit> .
deletion of luxa in the lux operon of a. salmonicida has been associated with decreased virulence  <cit> .
the master regulator of qs, litr, is also involved in regulation of virulence in a. salmonicida lfl <dig> as well as biofilm formation, motility and cryptic bioluminescence  <cit> .
in recent work we identified and quantified ahls in  <dig> members of the vibrionaceae family using high-performance liquid chromatography combined with mass spectrometry   <cit> .
mapping of the resulting ahl profiles onto a host 16s rdna phylogenetic tree revealed that closely related strains produce similar ahl profiles.
one of the  <dig> isolates included was a. salmonicida strain lfi <dig>  from which we were able to identify a total of eight different ahls.
the ahls were analyzed at only one cell density from cultures grown at one temperature in this study  <cit> .
since in a. salmonicida the regulatory role of litr on some phenotypes is temperature dependent  <cit>  we wanted to analyze the impact of temperature on ahl production.
hence, in the work presented here we have analyzed the ahl profiles of a. salmonicida wild type lfi <dig> and a Δlitr mutant at different cell densities during growth at three different temperatures.
we have also inactivated the ahl synthetases ains and luxi in order to identify which synthetase makes which ahl.
finally, we studied ahl production in luxr mutants, and analyzed the involvement of the ains/ainr and luxi/luxr systems in biofilm formation.
methods
chemicals and ahl standards
the following ahl standards were purchased from university of nottingham, uk: n-3-oxo-butyryl-l-homoserine lactone , n-3-hydroxy-butyryl-l-homoserine lactone , n-3-hydroxy-hexanoyl-l-homoserine lactone , n-3-hydroxy-octanoyl-l-homoserine lactone , n-3-hydroxy-decanoyl-l-homoserine lactone .
standards purchased from sigma-aldrich were: n-butyryl-dl-homoserine lactone , n-hexanoyl-l-homoserine lactone , n-3-oxo-hexanoyl-l- homoserine lactone , n-octanoyl-l-homoserine lactone , n-3-oxo-octanoyl-l-homoserine lactone , n-decanoyl-dl-homoserine lactone , n-3-oxo-decanoyl-l-homoserine lactone , n-dodecanoyl-dl-homoserine lactone , n-3-oxo-dodecanoyl-l-homoserine lactone , and n-3-hydroxy-dodecanoyl-dl-homoserine lactone .
hplc grade acetonitrile and formic acid were purchased from merck.
strains and culture conditions
bacterial strains and plasmids used in this study are listed in table  <dig>  the a. salmonicida strains were grown at 12°c in either lysogeny broth  or tryptic soy broth   supplemented with  <dig> % or  <dig> % nacl, respectively.
the escherichia coli strains s <dig>  and dh5α were cultured in lb medium, whereas dh5αλpir was cultivated in brain heart infusion   medium at 37°c.
the plasmids pdm <dig> and pnq <dig> were propagated in s <dig>  cells  <cit>  and pevs <dig> and pevs <dig> were propagated in dh5αλpir and dh5α, respectively  <cit> .table  <dig>
bacterial strains and plasmids used in this study
bacterial strain or plasmid
description
a
source or reference
a. salmonicida
luxi ˉ
luxr1ˉ
luxr2ˉ
e. coli
plasmids
rp <dig>  laczα,cosn, loxp, incd
acmr, chloremphenicol resistance gene.
ermr, erythromycin resistance gene.
e. coli carrying pnq <dig> and pdm <dig> constructs were grown on media containing 25 μg/ml chloramphenicol, whereas 100 μg/ml kanamycin or 250 μg/ml erythromycin were used in the media for propagating e. coli carrying pevs <dig> and pevs <dig>  a. salmonicida transconjugants were selected on lb agar plates containing  <dig> % nacl and 2 μg/ml chloramphenicol, or on tsb agar plates containing  <dig> % nacl and 25 μg/ml erythromycin.
dna extraction, pcr and sequencing
extraction of dna, recombinant dna techniques and transformations were performed according to standard protocols  <cit> .
restriction enzyme digestion, ligation and plasmid purification were performed as recommended by the manufacturers .
pcr using phusion  or taq polymerase  as well as big dye sequencing  were performed with custom made primers .
the primers used for pcr and sequencing are listed in additional file 1: table s <dig>
construction of a. salmonicida lfi <dig> mutants
construction of the Δlitr in-frame mutant has been described elsewhere  <cit> .
similarly, the ains gene was deleted in a. salmonicida by allelic exchange.
in brief, the sequences flanking ains were amplified by pcr from genomic dna of lfi <dig>  the a and b primers were used for amplification of the region upstream ains, and primers c and d for amplification of the downstream region .
primers b and c contain complementary sequences that enable fusion of the upstream and the downstream pcr products by an overlap pcr using the outermost primers a and d. this results in a removal of  <dig> codons  from the ains open reading frame, and hence, only  <dig> codons remain.
the primers a and d contain spei and xhoi restriction enzyme sites, respectively, in their 5’end.
the overlapping pcr product was digested with spei and xhoi, ligated into the corresponding sites of the suicide vector pdm <dig> and transformed directly into e. coli s17- <dig> cells.
the luxi gene was inactivated by plasmid insertion in the wild type a. salmonicida lfi <dig> as well as in the Δains mutant.
the plasmid used for insertional inactivation was made by amplifying an internal part of luxi  by pcr.
adenine overhangs were added to the pcr products using a taq polymerase before being ligated into the pgem t-easy vector  and transformed into e. coli dh5α.
the resulting plasmid was purified, digested with spei and xhoi and finally the re-purified pcr products were cloned into the corresponding sites of the suicide vector pnq <dig>  similarly, internal fragments of luxr <dig>  and luxr <dig>  were pcr amplified and cloned into a pcr™4-topo® ta cloning vector according to the manufacturer’s instructions .
the luxr <dig> and luxr <dig> fragments were cut from the resulting plasmids using bamhi and cloned into the corresponding sites of pevs <dig>
the pdm <dig> and pnq <dig> constructs were transformed into e. coli s17- <dig> and used as donors in mating experiments with their respective parental a. salmonicida strain as described previously  <cit> .
for inactivation of luxr <dig> and luxr <dig> tri-parental mating was performed  <cit> .
to this end, pevsluxr <dig> or pevsluxr <dig> was transferred from e. coli dh5αλpir to a. salmonicida lfi <dig> using pevs <dig> contained in e. coli dh5α as helper plasmid.
briefly, dh5αλpir, dh5α and lfi <dig> were grown to their stationary phases.
in this experiment the lfi <dig> was grown at 15°c in tsb containing  <dig> % nacl.
the volume of each culture was adjusted to account for a 1:1: <dig> ratio of cells.
the cells were pelleted by centrifugation, washed twice and combined by re-suspension in chilled tsb before being spotted onto chilled blood agar plates with  <dig> % nacl.
the plates were incubated at 21°c for 5– <dig> hours followed by incubation at 15°c for 16 hours.
the resulting confluent growth of cells was re-suspended in chilled tsb and cultivated for 24 hours at 12°c with 200 rpm.
finally, the suspension was plated onto tsb agar plates with  <dig> % nacl and erythromycin.
after 5– <dig> days of growth, erythromycin resistant luxr <dig> and luxr <dig> mutants were isolated.
preparation of bacterial supernatants for ahl measurements
two or more biological replicates were used for all a. salmonicida strains, with three technical replicates of each sample collected at different time points.
the primary cultures  were grown from individual colonies in lb medium with  <dig> % nacl at 12°c with 220 rpm.
after 48 hours, a secondary culture was made by diluting the primary culture 1: <dig>  the secondary cultures were grown 24 hours before being diluted to od600 =  <dig>  or  <dig>   in a total volume of 60 ml lb with  <dig> % nacl.
the cultures were grown further in 250 ml baffled flask at 220 rpm.
samples  were harvested at regular intervals and centrifuged at 17000 g for 1 minute .
the supernatants were acidified before ethyl acetate extraction as previously described  <cit> .
the ethyl acetate phase was dried using a rotary vacuum centrifuge  and re-dissolved in 150 μl of 20% acetonitrile containing  <dig> % formic acid and  <dig> nm of the internal standard 3-oxo-c12-hsl.
samples from three separate experiments were harvested and prepared as described above.
in the first experiment, the secondary cultures of lfi <dig> and Δlitr were diluted to an od <dig> of  <dig>  and grown at  <dig>   <dig> and 16°c.
here, the samples were harvested at eight different time points starting from an od <dig> of ~  <dig>  through to the stationary phase.
in the second experiment, cultures of lfi <dig>  Δains, luxiˉ and Δains/luxiˉ were started at an od <dig> of  <dig> , grown at 12°c and harvested after 50 hours.
in the third experiment the different cultures  were started at an od <dig> of  <dig>  and grown at 12°c before samples were collected at an od <dig> of  <dig>  .
the ahl molecules were separated by hplc before being identified using ms/ms with selective reaction monitoring   or using a full scan high resolution  ms method .
detection of ahl profiles using hplc-ms/ms analysis
the hplc-ms/ms analysis was performed as previously described  <cit> .
in brief, hplc was performed using a hypersil gold c <dig> reverse phase column  and eluted with a 162 second gradient of 5-95% acetonitrile in  <dig> % formic acid at a flow rate of 500 μl/min.
the ltq  part of the ltq orbitrap xl  was used in srm mode for detection of fragment m/z  <dig> from parent ions.
the retention time was divided into  <dig> segments of varying lengths with two to three scan events for each segment.
the data was collected from  <dig> to 103 m/z.
detection of ahl profiles using hplc with full scan hr-ms analysis
hplc with full scan hr-ms was performed using the ltq orbitrap xl and accela autosampler .
the samples  were injected onto the same reverse phase column as described above.
the elution procedure was performed with an acetonitrile gradient in  <dig> % formic acid, and consisted of 5% acetonitrile for 18 seconds, followed by a linear gradient up to 90% acetonitrile over 402 seconds, and finally 90% acetonitrile for 150 seconds.
the column was re-equilibrated for 150 seconds with 5% acetonitrile in  <dig> % formic acid before the next sample was injected.
the flow rate was 200 μl/min for all steps.
the separated compounds were detected under positive ion conditions by electrospray ionization using the following settings: sheath gas flow rate  <dig>  auxiliary gas flow rate  <dig>  sweep gas flow rate  <dig>  spray voltage + <dig>  kv, capillary temperature 300°c, capillary voltage 47 v, and tube lens 80–90 v. the orbitrap was operated in the full scan mode from m/z 165– <dig> at a resolution of  <dig>  with target setting of 5 ×  <dig> ions per scan and collection of data in the profile mode.
the maximum ion injection time was 500 ms. lock mass was enabled for correction of background ions from caffeine  and diisooctyl phthalate .
the system was calibrated with a mixture of  <dig> ahls including the internal standard 3-oxo-c12-hsl, and the ion chromatograms were analyzed using the xcalibur v.  <dig> .
<dig> software package.
the mass window was set to  <dig> parts per million.
the limit of detection  and the limit of quantification  for the different ahls were calculated as previously described  <cit>  and are shown in additional file 2: table s <dig>  this method does not allow quantification of c <dig> chained ahls.
biofilm assay
the biofilm assay was performed mainly as described elsewhere  <cit> .
in brief, the different strains were grown in lb with  <dig> % nacl at 12°c before being diluted to od600 =  <dig>  in swt medium , 3 g/l yeast extract , 28 g/l marine sea salt ).
a total volume of 300 μl of each dilution was added to wells in a flat bottom non tissue culture treated falcon 24-well tray .
the plates were incubated statically at 4°c for 3 days before being stained with  <dig> % crystal violet.
the air-dried biofilms were dissolved in 96% ethanol  before the absorbance was read at 590 nm .
three biological replicates were used for each strain, and the experiments were repeated several times.
statistical analysis
student’s t test was performed to calculate statistical significance  using the microsoft excel  <dig> software.
RESULTS
ahl signal production in a. salmonicida is cell density and temperature dependent
we recently established a method for detection of ahls using hplc-ms/ms and reported that a. salmonicida lfi <dig> produces  <dig> different ahls: 3-oxo-c4-hsl, c4-hsl, c6-hsl, 3-oxo-c6-hsl, c8-hsl, 3-oxo-c8-hsl, 3-oxo-c10-hsl and 3-oh-c10-hsl.
in this previous study the bacterium was grown at 12°c and the supernatants were harvested and analyzed after the bacterium had reached the stationary phase  <cit> .
under laboratory conditions, a. salmonicida has a growth optimum between 12-16°c in liquid cultures  <cit> .
however, a seawater temperature below 12°c is normally a prerequisite for a. salmonicida to cause cold water vibrosis in atlantic salmon  <cit> , as well as for siderophore production and iron-regulated outer membrane protein expression  <cit> .
moreover, growth temperatures below 14-16°c are required for the bacteria to express a number of host-bacterium related phenotypes such as adhesion, colony morphology and biofilm formation  <cit> .
therefore, to investigate whether ahl production is also affected by growth temperature, supernatants from the wild type lfi <dig> were harvested at different cell densities during growth at  <dig>   <dig> and 16°c.
as shown in figure  <dig>  the different ahls had reached detectable levels when the measurements started , and increased thereafter in a cell density dependent manner.
for most ahls, the highest concentrations were observed near the stationary phase, after which they decreased.
of the dominating ahls, 3-oxo-c6-hsl reached its maximum concentration  after 38 hours growth at 12°c, whereas c6-hsl and 3-oxo-c8-hsl, which were both detected in significantly lower concentrations, reached maxima  after  <dig> and 38 hours growth respectively, at 12°c.
the production of ahls was highly dependent on temperature, being dramatically lower during growth at 16°c.
at this temperature, the maximum concentrations of 3-oxo-c6-hsl, c6-hsl, 3-oxo-c8-hsl and 3-oh-c10-hsl were less than 5% of those produced after growth at 6°c.
only small amounts of 3-oxo-c4-hsl, c4-hsl, c8-hsl and 3-oxo-c10-hsl could be detected at 16°c, and these were at concentrations below the loq  <cit> .
aside from c6-hsl and 3-oh-c10-hsl, the different ahls were produced at approximately similar levels during growth at  <dig> and 12°c.
the concentration of c6-hsl produced at 12°c was approximately twice that compared to at 6°c.
interestingly, the opposite was observed for 3-oh-c10-hsl where the highest concentration was produced at 6°c, with the concentration rising steadily throughout the experiment reaching a final value of  <dig> nm compared to only  <dig> nm after growth at 12°c.
thus, the decrease in growth temperature to 6°c resulted in a four-fold increase of the 3-oh-c10-hsl concentration.figure  <dig>
ahls produced by
a. salmonicida
lfi <dig> at different temperatures and cell densities.
supernatants were harvested at different time points during growth at  <dig>   <dig> and 16°c.
the ahl concentrations were determined by hplc-ms/ms and are shown with respect to time  and cell density .
symbols indicate the different ahls: 3-oxo-c6-hsl , c6-hsl , 3-oxo-c8-hsl , 3-oh-c10-hsl , 3-oxo-c10-hsl , c4-hsl , 3-oxo-c4-hsl  and c8-hsl .
each value represents the mean of triplicates from three biological replicates.
the error bars represent the standard deviations.
the dotted lines in the top panels display the growth curves of lfi <dig> at different temperatures.
identification of ahls produced by luxi and ains
comparison of the genomes of a. salmonicida and a. fischeri, which are ~65% identical, allowed identification of the genes encoding the ahl synthases luxi and ains in a. salmonicida lfi <dig>  <cit> .
to determine which ahls are produced by which synthases in lfi <dig>  the ains and luxi genes were interrupted by allelic exchange and plasmid insertion respectively, giving rise to the ∆ains and the luxiˉ mutants.
a double mutant  was also made by interrupting both genes.
the wild type lfi <dig> and all three mutants were grown at 12°c for 50 hours  before samples were collected and analyzed.
as shown in figure  <dig>  lfi <dig> produced  <dig> different ahls which were identified by their retention times  and mass-to-charge ratio .
the ∆ains mutant was unable to produce 3-oh-c10-hsl but the remaining seven ahls were still detected; whereas the luxiˉ mutant produced only 3-oh-c10-hsl, and the double mutant ∆ains/luxiˉ showed no ahl production.
thus, the ains synthase is responsible for the 3-oh-c10-hsl production whereas luxi is responsible for production of 3-oxo-c4-hsl, c4-hsl, 3-oxo-c6-hsl, c6-hsl, c8-hsl, 3-oxo-c8-hsl and 3-oxo-c10-hsl.figure  <dig>
hplc-ms/ms ion chromatograms of ahls produced by
a. salmonicida
lfi <dig> and qs mutants.
the wild type lfi <dig> produces eight ahls those being the c4-hsl, 3-oxo-c4-hsl, c6-hsl, 3-oxo-c6-hsl, c8-hsl, 3-oxo-c8-hsl, and 3-oxo-c10-hsl and 3-oh-c10-hsl.
the Δains mutant lacks 3-oh-c10-hsl production, and the chromatogram shows the seven remaining ahls.
the luxiˉ mutant produces only 3-oh-c10-hsl and  no ahls were detected in the supernatants harvested from the double mutant Δains/ luxiˉ.
the peaks of the different ahls were scaled so that the different ahls could be shown in the same chromatogram.
the scaling factors were as follows:  <dig>  ×  <dig> for 3-oxo-c4-hsl,  <dig>  ×  <dig> for c4-hsl,  <dig>  ×  <dig> for 3-oxo-c6-hsl,  <dig>  ×  <dig> for c6-hsl,  <dig>  ×  <dig> for 3-oxo-c8-hsl,  <dig>  ×  <dig> for c8-hsl,  <dig>  ×  <dig> for 3-oh-c10-hsl and  <dig>  x  <dig> for 3-oxo-c10-hsl.
the same scaling was used for all four chromatograms .
the numbers above the peaks are mass-to-charge ratios .
litr is a positive regulator of ains ahl production
in a. fischeri the master regulator litr is a positive regulator of the luxi/luxr and the ains/ainr systems  <cit> .
we therefore wanted to determine if litr has a similar role in a. salmonicida.
the ∆litr mutant was grown at different temperatures as described above and samples were collected throughout the growth curve.
in contrast to a previous report  <cit>  the Δlitr mutant did not reach to the same cell densities as the wild type lfi <dig> in this experiment , which may be due to different culturing conditions or media; however ahl production was still affected by deletion of litr.
compared to the wild type lfi <dig>  the maximum concentrations of 3-oh-c10-hsl  produced by Δlitr were only 11% at 6°c and 14% at 12°c  , while the amounts of 3-oh-c10-hsl detected after growth at 16°c were below the quantification limit  <cit> .
similarly, inactivation of litr resulted in decreased production of 3-oxo-c6-hsl, c6-hsl and 3-oxo-c8-hsl , and at 12°c the maximum concentrations reached by the ∆litr mutant were only 50-60% of those reached by lfi <dig> .
the opposite situation was observed when the bacteria were grown at 16°c, where in the absence of litr the concentrations of 3-oxo-c6-hsl, c6-hsl and 3-oxo-c8-hsl were approximately two-three times higher than in the wild type.
still, these levels were much lower than those of lfi <dig> at 6°c .
no significant differences in the production of 3-oxo-c4-hsl, c4-hsl, c8-hsl and 3-oxo-c10-hsl were detected between lfi <dig> and Δlitr at any of the conditions.figure  <dig>
ahls produced by the
a. salmonicida Δlitr
mutant at different temperatures and cell densities.
supernatants were harvested at different time points during growth at  <dig>   <dig> and 16°c.
the ahl concentrations were measured by hplc-ms/ms and are shown with respect to time  and cell density .
symbols indicate the different ahls; 3-oxo-c6-hsl , c6-hsl , 3-oxo-c8-hsl , 3-oh-c10-hsl , 3-oxo-c10-hsl , c4-hsl , 3-oxo-c4-hsl  and c8-hsl .
each value represents the mean of triplicates from three biological replicates.
the error bars represent the standard deviations.
the dotted lines in the top panels display the growth curves of the Δlitr mutant at the different temperatures.
luxr <dig> and luxr <dig> are required for production of luxi ahls
in a. fischeri, luxr and autoinducer activate transcription the lux operon encoding the ahl synthetase luxi and proteins required for bioluminescence  <cit> .
we therefore inactivated both copies of luxr  in a. salmonicida lfi <dig> by plasmid insertion and analyzed the ahl profiles of the resulting mutants  using hplc with full scan hr-ms. the wild type and mutants were grown at 12°c and samples collected in the stationary phase.
as shown in table  <dig>  inactivation of either copy caused a significant decrease in the production of luxi-dependent ahls, with mutants producing only  <dig> % c6-hsl,  <dig> % 3-oxo-c6-hsl and  <dig> % 3-oxo-c8-hsl relative to wild type lfi <dig> concentrations.
small amounts of c8-hsl and 3-oxo-c10-hsl were detected but in concentrations close to or below the quantification limit .
finally, production of the ains signal  was not affected by mutations in luxr <dig> or luxr <dig>  it should be noted that the ahl concentrations produced by the wild type were much lower here  than detected in the experiment above .
a reason for this could be that the cultures were started at od600 =  <dig>  and not at od600 =  <dig>  as above.
the concentrations of 3-oxo-c4-hsl and c4-hsl were not determined in this experiment.table  <dig>
ahl production in
a. salmonicida
lfi <dig> and
luxrˉ
mutants
a
bacterial strain
c <dig>
3-oxo-c <dig>
c <dig>
3-oxo-c <dig>
3-oxo-c <dig>
3-oh-c <dig>
lfi1238
luxr1ˉ
luxr2ˉ
aeach value represents the mean of triplicates from two biological replicates harvested at od600 ~  <dig> .
the samples were analyzed by the hplc full scan hr-ms method.
the concentrations of c4-hsl or 3-oxo-c4-hsl were not determined.
*below the limit of quantification  given in additional file 2: table s <dig>
the ains/ainr and luxi/luxr pathways regulate biofilm formation
litr is a negative regulator of biofilm in a. salmonicida lfi <dig>  hence deletion of litr results in increased biofilm formation.
the biofilm formation is both temperature- and medium-dependent  <cit> .
to establish the impact of ahls in this process, we analyzed the biofilm formation capabilities of the different mutants.
as shown in figure  <dig>  disruption of either ains or luxi alone did not alter biofilm formation, however interruption of both genes simultaneously resulted in a phenotype similar to Δlitr.
although luxr <dig> and luxr <dig> are necessary for manufacture of luxi-produced ahls, interruption of these genes did not affect biofilm formation in the assays performed here.figure  <dig>
biofilm formation of
a. salmonicida
lfi <dig> and qs mutants.
the biofilms were grown statically for 72 hours at 4°c before being stained with crystal violet.
the absorbance was read at 590 nm.
the error bars represent the standard deviation of three biological replicates.
discussion
although a. salmonicida is no longer an immediate threat to the aquaculture industry it would be beneficial to understand factors involved in its virulence.
disruption of bacterial communication or qs may present a way to reduce virulence, with the usual strategies being destruction of signals by enzymes, or inactivation by chemicals or natural compounds with antagonistic activities  <cit> .
the relationship between ahl production and cell density is thoroughly documented for a variety of gram-negative bacteria  <cit> .
similarly, in the study presented here we show that the eight ahls identified in a. salmonicida lfi <dig> are also produced in a cell density-dependent manner.
a. salmonicida was initially reported to produce only two ahls: c6-hsl and 3-oxo-c6-hsl  <cit> , and reasons for this discrepancy are likely due to different detection methods, strains, growth medium and cultivation temperatures between the different studies.
however, by inactivation of two putative ahl synthases in a. salmonicida lfi <dig> we found that luxi produces seven ahls  whereas ains only produces one ahl .
luxi homologues in other bacteria are known to catalyze the acylation and lactonization reactions between the substrates s-adenosylmethionine and the acylated acp   <cit> , thus it is reasonable to assume that luxi perform the same reaction in a. salmonicida.
the ability of luxi to produce an array of different ahls is intriguing and suggests that this enzyme in a. salmonicida lfi <dig> does not discriminate well between different substrates, and can accept acp carrying acyl chains of different lengths as well as acyl chains without or with a keto substitution in the third position .
the ability of luxi homologues to produce a broad spectrum of ahls is not restricted to a. salmonicida.
indeed, the human pathogenic bacteria yersinia pseudotuberculosis has been reported to produce a far greater range of ahls.
this bacterium possesses two luxi homologous, ypsi amd ytbi, which are responsible for producing over  <dig> different ahls containing acyl chains of both odd and even numbers of carbons  <cit> .
the majority of vibrios and aliivibrios produce multiple types of ahls suggesting that it provides some biological advantages  <cit> .
this was shown for a. fischeri where c8-hsl binds and activates both ainr and luxr at intermediate cell densities, and at higher cell densities luxr binds 3-oxo-c6-hsl  <cit> .
this sequential activation allows specific regulation of early colonization factors by ains/ainr, whereas late colonization factors and luminescence are preferentially regulated by luxi/luxr.
both systems however are required for persistent colonization of the squid host  <cit> .
thus, it is possible that a. salmonicida exploits its ahl diversity to regulate specific activities in response to temperature or other environmental changes, or during transmission from the seawater into the host and vice versa.
cold-water vibriosis in atlantic salmon occurs mainly in late autumn, winter and early spring when the water temperature is below 12°c  <cit> .
we have recently shown that litr regulates a number of phenotypes, including virulence, in a. salmonicida lfi <dig> and that this regulation was stronger at lower temperatures  <cit> .
similarly, as shown here, the growth temperature of wild type lfi <dig> was found to have a significant impact on ahl production, and increasing the temperature from  <dig> to 16°c resulted in a drastic decrease of all eight ahls.
this decline cannot be explained by differences in growth rate, since the lfi <dig> cultures all reached stationary phase at similar cell densities  irrespective of their growth temperatures.
the luxi ahls were produced at comparable or higher levels after growth at 12°c compared to 6°c.
on the other hand, the ains signal concentration produced at 6°c was approximately four times higher than at 12°c in lfi <dig>  this is interesting considering that litr was found to regulate production of the ains signal 3-oh-c10-hsl, and the Δlitr mutant produced 3-oh-c10-hsl corresponding to ≤ 14% of the wild type concentrations at  <dig> and 12°c.
this finding strengthens the suggestion that litr exerts its regulatory function more strongly at low temperatures.
similar to litr in a. fischeri  <cit> , our results suggest that litr also connects the luxi/luxr systems to the ains/ainr and luxs/luxpq systems in a. salmonicida .
this conclusion is based on the finding that;  deletion of litr influenced production of luxi ahls,  inactivation of ains and luxi simultaneously was needed to produce a biofilm similar to the Δlitr mutant, as well as our previous results showing that  the ∆litr mutant produces up to 20-fold less cryptic bioluminescence compared to the wild type lfi <dig>  <cit> .
although litr is involved in regulation of the lux operon, deletion of litr does not completely prevent ahl production through luxi, and the Δlitr mutant still produced the luxi-dependent ahls 3-oxo-c6-hsl, c6-hsl and 3-oxo-c8-hsl at 50-60% of wild type levels at  <dig> and 12°c.
this suggests that litr is not essential for luxr transcription and that other regulators may be involved in the transcriptional regulation of the two luxr genes and hence luxi.
autoregulation of luxr  <cit> , and litr independent activation of luxr have been demonstrated in a. fischeri.
one of those litr independent mechanisms involves camp and camp receptor protein   <cit> , and more recently the regulatory rna-binding protein carbon storage regulator a  has been shown to increase transcription of luxr independently of litr  <cit> .
a. salmonicida lfi <dig> encodes the genes for csra and crp  <cit>  and similar mechanisms may therefore explain why ahl production of luxi is only reduced when litr is deleted.
on the other hand, our study shows that both luxrs in a. salmonicida lfi <dig> are necessary for producing luxi-ahls, and inactivation of either luxr <dig> or luxr <dig> reduced luxi-produced ahls significantly without affecting the ains signal production.
a possible explanation for this is that luxr <dig> and luxr <dig> function as a heterodimer in order to activate transcription of luxi, however further studies are needed to elucidate this.figure  <dig>
illustration of the proposed model of the qs system in
a. salmonicida
lfi <dig>  the autoinducer synthases luxs, luxi and ains, produce the different ahls and ai- <dig> which are transported across the inner membrane  and the outer membrane .
their respective receivers are believed to be luxpq, a luxr1-luxr <dig> heterodimer, and ainr.
the luxs/luxpq pathway may be inactive due to a frame shift mutation within luxp.
it is unknown which ahls bind the luxrs .
at low cell density, a phosphorylation cascade is believed to start from the receivers luxpq and ainr, and proceed downstream to luxo via luxu .
luxo probably regulates expression of qrr, which in turn controls the expression of the master regulator litr.
when the autoinducer concentrations are high, litr is expressed and regulates the production of the ains ahl, as well as activities such as motility, biofilm, adhesion, virulence and bioluminescence  <cit> .
both litr and the luxrs are probably involved in regulation of the lux operon as illustrated.
in our work we have studied the role of different qs genes using gene inactivation.
it should be pointed out that although our observations with the different mutants are coherent complementation studies for the different mutants would be required to unambiguously prove the function of the inactivated genes.
CONCLUSIONS
in this study we have shown that the ahl autoinducer synthases luxi and ains in a. salmonicida lfi <dig> produced the eight different ahls.
their production is dependent on both the cell density and cultivation temperature of the bacteria.
production of numerous ahls suggests that the qs signaling cascade is complex and probably important to fine-tune activities such as virulence, biofilm and other adaption processes to respond to changes in the environment.
the ains/ainr and luxr/luxi systems are connected to, and needed for down-regulation of biofilm formation.
however, further investigations are needed to understand the regulation and complexity of qs in a. salmonicida.
ethics statement
the work presented in this paper does not involve human subjects, and we see no ethical issues.
additional files
additional file 1: table s <dig>  the table lists primers used in this study.
additional file 2: table s <dig>  the table lists ahl detection limits and quantification parameters for the hplc full scan hr-ms method.
abbreviations
ahlsn-acyl-homoserine lactones
3-oh-c4-hsln-3-hydroxy-butyryl hsl
3-oh-c6-hsln-3-hydroxy-hexanoyl hsl
3-oh-c8-hsln-3-hydroxy-octanoyl hsl
3-oh-c10-hsln-3-hydroxy-decanoyl hsl
3-oh-c12-hsln-3-hydroxy-dodecanoyl hsl
3-oxo-c4-hsln-3-oxo-butyryl hsl
3-oxo-c6-hsln-3-oxo-hexanoyl hsl
3-oxo-c8-hsln-3-oxo-octanoyl hsl
3-oxo-c10-hsln-3-oxo-decanoyl hsl
3-oxo-c12-hsln-3-oxo-dodecanoyl hsl
c4-hsln-butyryl hsl
c6-hsln-hexanoyl hsl
c8-hsln-octanoyl hsl
c10-hsln-decanoyl hsl
c12-hsln-dodecanoyl hsl
hplc-ms/mshigh-performance liquid chromatography tandem mass spectrometry
hrhigh resolution
m/zmass-to-charge ratio
hslhomoserine lactone
lblysogeny broth
minminutes
od600optical density measured at 600 nm
pcrpolymerase chain reaction
qsquorum sensing
rpmrounds per minute
srmselective reaction
tsbtryptic soy broth
hilde hansen and amit anand purohit contributed equally to this work.
competing interests
the authors declare that they have no competing interests.
authors’ contributions
hh, aap and npw conceived and designed the experiments.
hh, aap and amb constructed the mutants.
aap, sjk and jaj performed the ahl measurements.
hh performed the biofilm assay.
all authors were involved in analyzing the data.
hh, aap, hksl and npw wrote the paper.
all authors read and approved the final manuscript.

 members of the vibrionaceae family are found in a wide range of aquatic environments, as free-living planktonic cells or attached to surfaces as bacterial aggregates or biofilms. some members within this family have evolved to become pathogens whereas others have developed symbiotic relationships with their hosts. aliivibrio salmonicida, previously designated vibrio salmonicida, is the causative agent of cold water vibriosis in atlantic salmon, rainbow trout and captive atlantic cod, a disease which is kept under control due to successful vaccination. the mechanisms of a. salmonicida virulence and pathogenicity are not known in detail, however, temperature-sensitive iron sequestration, motility and flagella activity, as well as quorum sensing have been suggested to be involved in virulence. moreover, the genome of a. salmonicida lfi encodes putative hemolysins, proteases and several protein secretion systems that may be found to be important in pathogenesis. bacteria use qs to regulate and coordinate gene expression by secreting and responding to signal molecules in a cell density dependent manner. the most common qs autoinducer signal molecules in gram-negative bacteria are n-acyl homoserines lactones, which consist of a homoserine lactone linked to an acyl side chain with 4– carbons. several qs systems have been identified in vibrios and aliivibrios, and each species seems to utilize a unique combination of qs systems that can act in parallel, or in a hierarchal manner to regulate species-specific activities including biofilm formation, virulence and colonization factors. the well-studied squid symbiont aliivibrio fischeri, a close relative of a. salmonicida, regulates bioluminescence and colonization by the qs systems luxs/luxpq, ains/ainr and luxi/luxr, where luxs, ains and luxi are the autoinducer synthases. ains in a. fischeri produces n-octanoyl-hsl which is recognized by the membrane-bound two-component hybrid sensor kinase ainr. luxs in a. fischeri does not produce ahls, but instead makes a signal molecule referred to as autoinducer that likely binds the periplasmic receptor luxp and modulates the activity of the sensor kinase luxq, similar to the homologous system in vibrio harveyi. the luxs/luxpq and ains/ainr systems in a. fischeri work in parallel, and in the absence of signal molecules luxpq and ainr are believed to function as kinases and phosphorylate the response regulator luxo via luxu. phosphorylated luxo then activates expression of qrr which encodes a small regulatory rna. qrr destabilizes the litr mrna that encodes the master regulator of qs. in the presence of signal molecules at high cell density, luxo becomes dephosphorylated leading to suppression of qrr allowing litr to be expressed. the third system in a. fischeri, luxi/luxr, is activated by the ains/ainr system, and at medium cell densities luxr binds ains-produced c8-hsl and activates transcription of the lux operon. at higher cell densities, luxi-produced n-3-oxo-hexanoyl-l-hsl binds to luxr and increases transcription of the luxicdabeg and luxr genes. moreover, the master regulator litr is a positive regulator of ains and is indirectly involved in expression of luxi by controlling expression of luxr. hence, litr links the ains/ainr and luxs/luxpq systems to the luxi/luxr system in a. fischeri. the number and types of ahls vary between different species of vibrio and aliivibrio, as well as between strains of the same species. however, the specific autoinducer synthases responsible for the different ahls are only known for a few bacteria, such as the above mentioned in a. fischeri. in v. anguillarum, another fish pathogen, the ains homolog vanm is reported to produce n-3-hydroxy-hexanoyl-hsl and n-hexanoyl-hsl, whereas the luxi homolog vani produces n-3-oxo-decanoyl-hsl. in the bioluminescent bacteria v. harveyi the luxm synthase is reported to produce n-3-hydroxy-butyryl-l-hsl. thus, while some autoinducer synthases produce only one ahl others are able to produce several. the genome of a. salmonicida lfl encodes five qs systems: the ains/ainr, luxi/luxr, vars/vara, luxm/luxn and luxs/luxpq systems. the latter two systems are probably inactive since luxm is absent, and luxn and luxp contain frame-shift mutations. the lux operon of a. salmonicida has a novel organization compared to a. fischeri, with two flanking luxr genes and the luxi gene located outside the luxcdabeg locus, and a. salmonicida is only able to produce bioluminescence after addition of decyl aldehyde. deletion of luxa in the lux operon of a. salmonicida has been associated with decreased virulence. the master regulator of qs, litr, is also involved in regulation of virulence in a. salmonicida lfl as well as biofilm formation, motility and cryptic bioluminescence. in recent work we identified and quantified ahls in members of the vibrionaceae family using high-performance liquid chromatography combined with mass spectrometry. mapping of the resulting ahl profiles onto a host 16s rdna phylogenetic tree revealed that closely related strains produce similar ahl profiles. one of the isolates included was a. salmonicida strain lfi from which we were able to identify a total of eight different ahls. the ahls were analyzed at only one cell density from cultures grown at one temperature in this study. since in a. salmonicida the regulatory role of litr on some phenotypes is temperature dependent we wanted to analyze the impact of temperature on ahl production. hence, in the work presented here we have analyzed the ahl profiles of a. salmonicida wild type lfi and a Δlitr mutant at different cell densities during growth at three different temperatures. we have also inactivated the ahl synthetases ains and luxi in order to identify which synthetase makes which ahl. finally, we studied ahl production in luxr mutants, and analyzed the involvement of the ains/ainr and luxi/luxr systems in biofilm formation. methods chemicals and ahl standards the following ahl standards were purchased from university of nottingham, uk: n-3-oxo-butyryl-l-homoserine lactone, n-3-hydroxy-butyryl-l-homoserine lactone, n-3-hydroxy-hexanoyl-l-homoserine lactone, n-3-hydroxy-octanoyl-l-homoserine lactone, n-3-hydroxy-decanoyl-l-homoserine lactone. standards purchased from sigma-aldrich were: n-butyryl-dl-homoserine lactone, n-hexanoyl-l-homoserine lactone, n-3-oxo-hexanoyl-l- homoserine lactone, n-octanoyl-l-homoserine lactone, n-3-oxo-octanoyl-l-homoserine lactone, n-decanoyl-dl-homoserine lactone, n-3-oxo-decanoyl-l-homoserine lactone, n-dodecanoyl-dl-homoserine lactone, n-3-oxo-dodecanoyl-l-homoserine lactone, and n-3-hydroxy-dodecanoyl-dl-homoserine lactone. hplc grade acetonitrile and formic acid were purchased from merck. strains and culture conditions bacterial strains and plasmids used in this study are listed in table  the a. salmonicida strains were grown at 12°c in either lysogeny broth or tryptic soy broth supplemented with % or % nacl, respectively. the escherichia coli strains s and dh5α were cultured in lb medium, whereas dh5αλpir was cultivated in brain heart infusion medium at 37°c. the plasmids pdm and pnq were propagated in s cells and pevs and pevs were propagated in dh5αλpir and dh5α, respectively.table bacterial strains and plasmids used in this study bacterial strain or plasmid description a source or reference a. salmonicida luxi ˉ luxr1ˉ luxr2ˉ e. coli plasmids rp laczα,cosn, loxp, incd acmr, chloremphenicol resistance gene. ermr, erythromycin resistance gene. e. coli carrying pnq and pdm constructs were grown on media containing 25 μg/ml chloramphenicol, whereas 100 μg/ml kanamycin or 250 μg/ml erythromycin were used in the media for propagating e. coli carrying pevs and pevs a. salmonicida transconjugants were selected on lb agar plates containing % nacl and 2 μg/ml chloramphenicol, or on tsb agar plates containing % nacl and 25 μg/ml erythromycin. dna extraction, pcr and sequencing extraction of dna, recombinant dna techniques and transformations were performed according to standard protocols. restriction enzyme digestion, ligation and plasmid purification were performed as recommended by the manufacturers. pcr using phusion or taq polymerase as well as big dye sequencing were performed with custom made primers. the primers used for pcr and sequencing are listed in additional file 1: table s construction of a. salmonicida lfi mutants construction of the Δlitr in-frame mutant has been described elsewhere. similarly, the ains gene was deleted in a. salmonicida by allelic exchange. in brief, the sequences flanking ains were amplified by pcr from genomic dna of lfi the a and b primers were used for amplification of the region upstream ains, and primers c and d for amplification of the downstream region. primers b and c contain complementary sequences that enable fusion of the upstream and the downstream pcr products by an overlap pcr using the outermost primers a and d. this results in a removal of codons from the ains open reading frame, and hence, only codons remain. the primers a and d contain spei and xhoi restriction enzyme sites, respectively, in their 5’end. the overlapping pcr product was digested with spei and xhoi, ligated into the corresponding sites of the suicide vector pdm and transformed directly into e. coli s17- cells. the luxi gene was inactivated by plasmid insertion in the wild type a. salmonicida lfi as well as in the Δains mutant. the plasmid used for insertional inactivation was made by amplifying an internal part of luxi by pcr. adenine overhangs were added to the pcr products using a taq polymerase before being ligated into the pgem t-easy vector and transformed into e. coli dh5α. the resulting plasmid was purified, digested with spei and xhoi and finally the re-purified pcr products were cloned into the corresponding sites of the suicide vector pnq similarly, internal fragments of luxr and luxr were pcr amplified and cloned into a pcr™4-topo® ta cloning vector according to the manufacturer’s instructions. the luxr and luxr fragments were cut from the resulting plasmids using bamhi and cloned into the corresponding sites of pevs the pdm and pnq constructs were transformed into e. coli s17- and used as donors in mating experiments with their respective parental a. salmonicida strain as described previously. for inactivation of luxr and luxr tri-parental mating was performed. to this end, pevsluxr or pevsluxr was transferred from e. coli dh5αλpir to a. salmonicida lfi using pevs contained in e. coli dh5α as helper plasmid. briefly, dh5αλpir, dh5α and lfi were grown to their stationary phases. in this experiment the lfi was grown at 15°c in tsb containing % nacl. the volume of each culture was adjusted to account for a 1:1: ratio of cells. the cells were pelleted by centrifugation, washed twice and combined by re-suspension in chilled tsb before being spotted onto chilled blood agar plates with % nacl. the plates were incubated at 21°c for 5– hours followed by incubation at 15°c for 16 hours. the resulting confluent growth of cells was re-suspended in chilled tsb and cultivated for 24 hours at 12°c with 200 rpm. finally, the suspension was plated onto tsb agar plates with % nacl and erythromycin. after 5– days of growth, erythromycin resistant luxr and luxr mutants were isolated. preparation of bacterial supernatants for ahl measurements two or more biological replicates were used for all a. salmonicida strains, with three technical replicates of each sample collected at different time points. the primary cultures were grown from individual colonies in lb medium with % nacl at 12°c with 220 rpm. after 48 hours, a secondary culture was made by diluting the primary culture 1: the secondary cultures were grown 24 hours before being diluted to od600 =  or in a total volume of 60 ml lb with % nacl. the cultures were grown further in 250 ml baffled flask at 220 rpm. samples were harvested at regular intervals and centrifuged at 17000 g for 1 minute. the supernatants were acidified before ethyl acetate extraction as previously described. the ethyl acetate phase was dried using a rotary vacuum centrifuge and re-dissolved in 150 μl of 20% acetonitrile containing % formic acid and nm of the internal standard 3-oxo-c12-hsl. samples from three separate experiments were harvested and prepared as described above. in the first experiment, the secondary cultures of lfi and Δlitr were diluted to an od of and grown at and 16°c. here, the samples were harvested at eight different time points starting from an od of ~  through to the stationary phase. in the second experiment, cultures of lfi Δains, luxiˉ and Δains/luxiˉ were started at an od of, grown at 12°c and harvested after 50 hours. in the third experiment the different cultures were started at an od of and grown at 12°c before samples were collected at an od of. the ahl molecules were separated by hplc before being identified using ms/ms with selective reaction monitoring or using a full scan high resolution ms method. detection of ahl profiles using hplc-ms/ms analysis the hplc-ms/ms analysis was performed as previously described. in brief, hplc was performed using a hypersil gold c reverse phase column and eluted with a 162 second gradient of 5-95% acetonitrile in % formic acid at a flow rate of 500 μl/min. the ltq part of the ltq orbitrap xl was used in srm mode for detection of fragment m/z from parent ions. the retention time was divided into segments of varying lengths with two to three scan events for each segment. the data was collected from to 103 m/z. detection of ahl profiles using hplc with full scan hr-ms analysis hplc with full scan hr-ms was performed using the ltq orbitrap xl and accela autosampler. the samples were injected onto the same reverse phase column as described above. the elution procedure was performed with an acetonitrile gradient in % formic acid, and consisted of 5% acetonitrile for 18 seconds, followed by a linear gradient up to 90% acetonitrile over 402 seconds, and finally 90% acetonitrile for 150 seconds. the column was re-equilibrated for 150 seconds with 5% acetonitrile in % formic acid before the next sample was injected. the flow rate was 200 μl/min for all steps. the separated compounds were detected under positive ion conditions by electrospray ionization using the following settings: sheath gas flow rate auxiliary gas flow rate sweep gas flow rate spray voltage +  kv, capillary temperature 300°c, capillary voltage 47 v, and tube lens 80–90 v. the orbitrap was operated in the full scan mode from m/z 165– at a resolution of with target setting of 5 ×  ions per scan and collection of data in the profile mode. the maximum ion injection time was 500 ms. lock mass was enabled for correction of background ions from caffeine and diisooctyl phthalate. the system was calibrated with a mixture of ahls including the internal standard 3-oxo-c12-hsl, and the ion chromatograms were analyzed using the xcalibur v.. software package. the mass window was set to parts per million. the limit of detection and the limit of quantification for the different ahls were calculated as previously described and are shown in additional file 2: table s this method does not allow quantification of c chained ahls. biofilm assay the biofilm assay was performed mainly as described elsewhere. in brief, the different strains were grown in lb with % nacl at 12°c before being diluted to od600 =  in swt medium, 3 g/l yeast extract, 28 g/l marine sea salt ). a total volume of 300 μl of each dilution was added to wells in a flat bottom non tissue culture treated falcon 24-well tray. the plates were incubated statically at 4°c for 3 days before being stained with % crystal violet. the air-dried biofilms were dissolved in 96% ethanol before the absorbance was read at 590 nm. three biological replicates were used for each strain, and the experiments were repeated several times. statistical analysis student’s t test was performed to calculate statistical significance using the microsoft excel software.  ahl signal production in a. salmonicida is cell density and temperature dependent we recently established a method for detection of ahls using hplc-ms/ms and reported that a. salmonicida lfi produces different ahls: 3-oxo-c4-hsl, c4-hsl, c6-hsl, 3-oxo-c6-hsl, c8-hsl, 3-oxo-c8-hsl, 3-oxo-c10-hsl and 3-oh-c10-hsl. in this previous study the bacterium was grown at 12°c and the supernatants were harvested and analyzed after the bacterium had reached the stationary phase. under laboratory conditions, a. salmonicida has a growth optimum between 12-16°c in liquid cultures. however, a seawater temperature below 12°c is normally a prerequisite for a. salmonicida to cause cold water vibrosis in atlantic salmon, as well as for siderophore production and iron-regulated outer membrane protein expression. moreover, growth temperatures below 14-16°c are required for the bacteria to express a number of host-bacterium related phenotypes such as adhesion, colony morphology and biofilm formation. therefore, to investigate whether ahl production is also affected by growth temperature, supernatants from the wild type lfi were harvested at different cell densities during growth at and 16°c. as shown in figure  the different ahls had reached detectable levels when the measurements started, and increased thereafter in a cell density dependent manner. for most ahls, the highest concentrations were observed near the stationary phase, after which they decreased. of the dominating ahls, 3-oxo-c6-hsl reached its maximum concentration after 38 hours growth at 12°c, whereas c6-hsl and 3-oxo-c8-hsl, which were both detected in significantly lower concentrations, reached maxima after and 38 hours growth respectively, at 12°c. the production of ahls was highly dependent on temperature, being dramatically lower during growth at 16°c. at this temperature, the maximum concentrations of 3-oxo-c6-hsl, c6-hsl, 3-oxo-c8-hsl and 3-oh-c10-hsl were less than 5% of those produced after growth at 6°c. only small amounts of 3-oxo-c4-hsl, c4-hsl, c8-hsl and 3-oxo-c10-hsl could be detected at 16°c, and these were at concentrations below the loq. aside from c6-hsl and 3-oh-c10-hsl, the different ahls were produced at approximately similar levels during growth at and 12°c. the concentration of c6-hsl produced at 12°c was approximately twice that compared to at 6°c. interestingly, the opposite was observed for 3-oh-c10-hsl where the highest concentration was produced at 6°c, with the concentration rising steadily throughout the experiment reaching a final value of nm compared to only nm after growth at 12°c. thus, the decrease in growth temperature to 6°c resulted in a four-fold increase of the 3-oh-c10-hsl concentration.figure ahls produced by a. salmonicida lfi at different temperatures and cell densities. supernatants were harvested at different time points during growth at   and 16°c. the ahl concentrations were determined by hplc-ms/ms and are shown with respect to time and cell density. symbols indicate the different ahls: 3-oxo-c6-hsl, c6-hsl, 3-oxo-c8-hsl, 3-oh-c10-hsl, 3-oxo-c10-hsl, c4-hsl, 3-oxo-c4-hsl and c8-hsl. each value represents the mean of triplicates from three biological replicates. the error bars represent the standard deviations. the dotted lines in the top panels display the growth curves of lfi at different temperatures. identification of ahls produced by luxi and ains comparison of the genomes of a. salmonicida and a. fischeri, which are ~65% identical, allowed identification of the genes encoding the ahl synthases luxi and ains in a. salmonicida lfi. to determine which ahls are produced by which synthases in lfi the ains and luxi genes were interrupted by allelic exchange and plasmid insertion respectively, giving rise to the ∆ains and the luxiˉ mutants. a double mutant was also made by interrupting both genes. the wild type lfi and all three mutants were grown at 12°c for 50 hours before samples were collected and analyzed. as shown in figure  lfi produced different ahls which were identified by their retention times and mass-to-charge ratio. the ∆ains mutant was unable to produce 3-oh-c10-hsl but the remaining seven ahls were still detected; whereas the luxiˉ mutant produced only 3-oh-c10-hsl, and the double mutant ∆ains/luxiˉ showed no ahl production. thus, the ains synthase is responsible for the 3-oh-c10-hsl production whereas luxi is responsible for production of 3-oxo-c4-hsl, c4-hsl, 3-oxo-c6-hsl, c6-hsl, c8-hsl, 3-oxo-c8-hsl and 3-oxo-c10-hsl.figure hplc-ms/ms ion chromatograms of ahls produced by a. salmonicida lfi and qs mutants. the wild type lfi produces eight ahls those being the c4-hsl, 3-oxo-c4-hsl, c6-hsl, 3-oxo-c6-hsl, c8-hsl, 3-oxo-c8-hsl, and 3-oxo-c10-hsl and 3-oh-c10-hsl. the Δains mutant lacks 3-oh-c10-hsl production, and the chromatogram shows the seven remaining ahls. the luxiˉ mutant produces only 3-oh-c10-hsl and no ahls were detected in the supernatants harvested from the double mutant Δains/ luxiˉ. the peaks of the different ahls were scaled so that the different ahls could be shown in the same chromatogram. the scaling factors were as follows:  ×  for 3-oxo-c4-hsl,  ×  for c4-hsl,  ×  for 3-oxo-c6-hsl,  ×  for c6-hsl,  ×  for 3-oxo-c8-hsl,  ×  for c8-hsl,  ×  for 3-oh-c10-hsl and x for 3-oxo-c10-hsl. the same scaling was used for all four chromatograms. the numbers above the peaks are mass-to-charge ratios. litr is a positive regulator of ains ahl production in a. fischeri the master regulator litr is a positive regulator of the luxi/luxr and the ains/ainr systems. we therefore wanted to determine if litr has a similar role in a. salmonicida. the ∆litr mutant was grown at different temperatures as described above and samples were collected throughout the growth curve. in contrast to a previous report the Δlitr mutant did not reach to the same cell densities as the wild type lfi in this experiment, which may be due to different culturing conditions or media; however ahl production was still affected by deletion of litr. compared to the wild type lfi the maximum concentrations of 3-oh-c10-hsl produced by Δlitr were only 11% at 6°c and 14% at 12°c, while the amounts of 3-oh-c10-hsl detected after growth at 16°c were below the quantification limit. similarly, inactivation of litr resulted in decreased production of 3-oxo-c6-hsl, c6-hsl and 3-oxo-c8-hsl, and at 12°c the maximum concentrations reached by the ∆litr mutant were only 50-60% of those reached by lfi. the opposite situation was observed when the bacteria were grown at 16°c, where in the absence of litr the concentrations of 3-oxo-c6-hsl, c6-hsl and 3-oxo-c8-hsl were approximately two-three times higher than in the wild type. still, these levels were much lower than those of lfi at 6°c. no significant differences in the production of 3-oxo-c4-hsl, c4-hsl, c8-hsl and 3-oxo-c10-hsl were detected between lfi and Δlitr at any of the conditions.figure ahls produced by the a. salmonicida Δlitr mutant at different temperatures and cell densities. supernatants were harvested at different time points during growth at   and 16°c. the ahl concentrations were measured by hplc-ms/ms and are shown with respect to time and cell density. symbols indicate the different ahls; 3-oxo-c6-hsl, c6-hsl, 3-oxo-c8-hsl, 3-oh-c10-hsl, 3-oxo-c10-hsl, c4-hsl, 3-oxo-c4-hsl and c8-hsl. each value represents the mean of triplicates from three biological replicates. the error bars represent the standard deviations. the dotted lines in the top panels display the growth curves of the Δlitr mutant at the different temperatures. luxr and luxr are required for production of luxi ahls in a. fischeri, luxr and autoinducer activate transcription the lux operon encoding the ahl synthetase luxi and proteins required for bioluminescence. we therefore inactivated both copies of luxr in a. salmonicida lfi by plasmid insertion and analyzed the ahl profiles of the resulting mutants using hplc with full scan hr-ms. the wild type and mutants were grown at 12°c and samples collected in the stationary phase. as shown in table  inactivation of either copy caused a significant decrease in the production of luxi-dependent ahls, with mutants producing only % c6-hsl, % 3-oxo-c6-hsl and % 3-oxo-c8-hsl relative to wild type lfi concentrations. small amounts of c8-hsl and 3-oxo-c10-hsl were detected but in concentrations close to or below the quantification limit. finally, production of the ains signal was not affected by mutations in luxr or luxr it should be noted that the ahl concentrations produced by the wild type were much lower here than detected in the experiment above. a reason for this could be that the cultures were started at od600 =  and not at od600 =  as above. the concentrations of 3-oxo-c4-hsl and c4-hsl were not determined in this experiment.table ahl production in a. salmonicida lfi and luxrˉ mutants a bacterial strain c 3-oxo-c c 3-oxo-c 3-oxo-c 3-oh-c lfi1238 luxr1ˉ luxr2ˉ aeach value represents the mean of triplicates from two biological replicates harvested at od600 ~ . the samples were analyzed by the hplc full scan hr-ms method. the concentrations of c4-hsl or 3-oxo-c4-hsl were not determined. *below the limit of quantification given in additional file 2: table s the ains/ainr and luxi/luxr pathways regulate biofilm formation litr is a negative regulator of biofilm in a. salmonicida lfi hence deletion of litr results in increased biofilm formation. the biofilm formation is both temperature- and medium-dependent. to establish the impact of ahls in this process, we analyzed the biofilm formation capabilities of the different mutants. as shown in figure  disruption of either ains or luxi alone did not alter biofilm formation, however interruption of both genes simultaneously resulted in a phenotype similar to Δlitr. although luxr and luxr are necessary for manufacture of luxi-produced ahls, interruption of these genes did not affect biofilm formation in the assays performed here.figure biofilm formation of a. salmonicida lfi and qs mutants. the biofilms were grown statically for 72 hours at 4°c before being stained with crystal violet. the absorbance was read at 590 nm. the error bars represent the standard deviation of three biological replicates. discussion although a. salmonicida is no longer an immediate threat to the aquaculture industry it would be beneficial to understand factors involved in its virulence. disruption of bacterial communication or qs may present a way to reduce virulence, with the usual strategies being destruction of signals by enzymes, or inactivation by chemicals or natural compounds with antagonistic activities. the relationship between ahl production and cell density is thoroughly documented for a variety of gram-negative bacteria. similarly, in the study presented here we show that the eight ahls identified in a. salmonicida lfi are also produced in a cell density-dependent manner. a. salmonicida was initially reported to produce only two ahls: c6-hsl and 3-oxo-c6-hsl, and reasons for this discrepancy are likely due to different detection methods, strains, growth medium and cultivation temperatures between the different studies. however, by inactivation of two putative ahl synthases in a. salmonicida lfi we found that luxi produces seven ahls whereas ains only produces one ahl. luxi homologues in other bacteria are known to catalyze the acylation and lactonization reactions between the substrates s-adenosylmethionine and the acylated acp, thus it is reasonable to assume that luxi perform the same reaction in a. salmonicida. the ability of luxi to produce an array of different ahls is intriguing and suggests that this enzyme in a. salmonicida lfi does not discriminate well between different substrates, and can accept acp carrying acyl chains of different lengths as well as acyl chains without or with a keto substitution in the third position. the ability of luxi homologues to produce a broad spectrum of ahls is not restricted to a. salmonicida. indeed, the human pathogenic bacteria yersinia pseudotuberculosis has been reported to produce a far greater range of ahls. this bacterium possesses two luxi homologous, ypsi amd ytbi, which are responsible for producing over different ahls containing acyl chains of both odd and even numbers of carbons. the majority of vibrios and aliivibrios produce multiple types of ahls suggesting that it provides some biological advantages. this was shown for a. fischeri where c8-hsl binds and activates both ainr and luxr at intermediate cell densities, and at higher cell densities luxr binds 3-oxo-c6-hsl. this sequential activation allows specific regulation of early colonization factors by ains/ainr, whereas late colonization factors and luminescence are preferentially regulated by luxi/luxr. both systems however are required for persistent colonization of the squid host. thus, it is possible that a. salmonicida exploits its ahl diversity to regulate specific activities in response to temperature or other environmental changes, or during transmission from the seawater into the host and vice versa. cold-water vibriosis in atlantic salmon occurs mainly in late autumn, winter and early spring when the water temperature is below 12°c. we have recently shown that litr regulates a number of phenotypes, including virulence, in a. salmonicida lfi and that this regulation was stronger at lower temperatures. similarly, as shown here, the growth temperature of wild type lfi was found to have a significant impact on ahl production, and increasing the temperature from to 16°c resulted in a drastic decrease of all eight ahls. this decline cannot be explained by differences in growth rate, since the lfi cultures all reached stationary phase at similar cell densities irrespective of their growth temperatures. the luxi ahls were produced at comparable or higher levels after growth at 12°c compared to 6°c. on the other hand, the ains signal concentration produced at 6°c was approximately four times higher than at 12°c in lfi this is interesting considering that litr was found to regulate production of the ains signal 3-oh-c10-hsl, and the Δlitr mutant produced 3-oh-c10-hsl corresponding to ≤ 14% of the wild type concentrations at and 12°c. this finding strengthens the suggestion that litr exerts its regulatory function more strongly at low temperatures. similar to litr in a. fischeri, our results suggest that litr also connects the luxi/luxr systems to the ains/ainr and luxs/luxpq systems in a. salmonicida. this conclusion is based on the finding that; deletion of litr influenced production of luxi ahls, inactivation of ains and luxi simultaneously was needed to produce a biofilm similar to the Δlitr mutant, as well as our previous results showing that the ∆litr mutant produces up to 20-fold less cryptic bioluminescence compared to the wild type lfi. although litr is involved in regulation of the lux operon, deletion of litr does not completely prevent ahl production through luxi, and the Δlitr mutant still produced the luxi-dependent ahls 3-oxo-c6-hsl, c6-hsl and 3-oxo-c8-hsl at 50-60% of wild type levels at and 12°c. this suggests that litr is not essential for luxr transcription and that other regulators may be involved in the transcriptional regulation of the two luxr genes and hence luxi. autoregulation of luxr, and litr independent activation of luxr have been demonstrated in a. fischeri. one of those litr independent mechanisms involves camp and camp receptor protein, and more recently the regulatory rna-binding protein carbon storage regulator a has been shown to increase transcription of luxr independently of litr. a. salmonicida lfi encodes the genes for csra and crp and similar mechanisms may therefore explain why ahl production of luxi is only reduced when litr is deleted. on the other hand, our study shows that both luxrs in a. salmonicida lfi are necessary for producing luxi-ahls, and inactivation of either luxr or luxr reduced luxi-produced ahls significantly without affecting the ains signal production. a possible explanation for this is that luxr and luxr function as a heterodimer in order to activate transcription of luxi, however further studies are needed to elucidate this.figure illustration of the proposed model of the qs system in a. salmonicida lfi the autoinducer synthases luxs, luxi and ains, produce the different ahls and ai- which are transported across the inner membrane and the outer membrane. their respective receivers are believed to be luxpq, a luxr1-luxr heterodimer, and ainr. the luxs/luxpq pathway may be inactive due to a frame shift mutation within luxp. it is unknown which ahls bind the luxrs. at low cell density, a phosphorylation cascade is believed to start from the receivers luxpq and ainr, and proceed downstream to luxo via luxu. luxo probably regulates expression of qrr, which in turn controls the expression of the master regulator litr. when the autoinducer concentrations are high, litr is expressed and regulates the production of the ains ahl, as well as activities such as motility, biofilm, adhesion, virulence and bioluminescence. both litr and the luxrs are probably involved in regulation of the lux operon as illustrated. in our work we have studied the role of different qs genes using gene inactivation. it should be pointed out that although our observations with the different mutants are coherent complementation studies for the different mutants would be required to unambiguously prove the function of the inactivated genes.  in this study we have shown that the ahl autoinducer synthases luxi and ains in a. salmonicida lfi produced the eight different ahls. their production is dependent on both the cell density and cultivation temperature of the bacteria. production of numerous ahls suggests that the qs signaling cascade is complex and probably important to fine-tune activities such as virulence, biofilm and other adaption processes to respond to changes in the environment. the ains/ainr and luxr/luxi systems are connected to, and needed for down-regulation of biofilm formation. however, further investigations are needed to understand the regulation and complexity of qs in a. salmonicida. ethics statement the work presented in this paper does not involve human subjects, and we see no ethical issues. additional files additional file 1: table s the table lists primers used in this study. additional file 2: table s the table lists ahl detection limits and quantification parameters for the hplc full scan hr-ms method. abbreviations ahlsn-acyl-homoserine lactones 3-oh-c4-hsln-3-hydroxy-butyryl hsl 3-oh-c6-hsln-3-hydroxy-hexanoyl hsl 3-oh-c8-hsln-3-hydroxy-octanoyl hsl 3-oh-c10-hsln-3-hydroxy-decanoyl hsl 3-oh-c12-hsln-3-hydroxy-dodecanoyl hsl 3-oxo-c4-hsln-3-oxo-butyryl hsl 3-oxo-c6-hsln-3-oxo-hexanoyl hsl 3-oxo-c8-hsln-3-oxo-octanoyl hsl 3-oxo-c10-hsln-3-oxo-decanoyl hsl 3-oxo-c12-hsln-3-oxo-dodecanoyl hsl c4-hsln-butyryl hsl c6-hsln-hexanoyl hsl c8-hsln-octanoyl hsl c10-hsln-decanoyl hsl c12-hsln-dodecanoyl hsl hplc-ms/mshigh-performance liquid chromatography tandem mass spectrometry hrhigh resolution m/zmass-to-charge ratio hslhomoserine lactone lblysogeny broth minminutes od600optical density measured at 600 nm pcrpolymerase chain reaction qsquorum sensing rpmrounds per minute srmselective reaction tsbtryptic soy broth hilde hansen and amit anand purohit contributed equally to this work. competing interests the authors declare that they have no competing interests. authors’ contributions hh, aap and npw conceived and designed the experiments. hh, aap and amb constructed the mutants. aap, sjk and jaj performed the ahl measurements. hh performed the biofilm assay. all authors were involved in analyzing the data. hh, aap, hksl and npw wrote the paper. all authors read and approved the final manuscript. 
quorum sensing is a cell-to-cell communication system used by bacteria to regulate activities such as virulence, bioluminescence and biofilm formation. in this work we have constructed several mutants of a. salmonicida lfi in order to study the luxi/luxr and ains/ainr qs systems with respect to ahl production and biofilm formation. this implies a connection between the two systems, and both systems were found to be involved in regulation of biofilm formation. using high-performance liquid chromatography-tandem mass spectrometry we found that luxi in a. salmonicida lfi is responsible for producing seven of the different ahls, whereas ains is responsible for producing only one. the production of these various ahls is dependent on both cell density and growth temperature. ahl production is highly dependent on growth temperature, and a significant decrease was observed when the bacterium was grown at a temperature above its limit for disease outbreak. the most common qs signals in gram-negative bacteria are n-acyl-homoserine lactones. finally, inactivation of either luxr or luxr in the lux operon significantly reduced production of luxi-produced ahls. litr, the master regulator of qs, was found to be a positive regulator of ains-dependent ahl production, and to a lesser extent luxi-dependent ahl production. the ahls were efficiently produced when wild type lfi was grown at or 12°c, however at 16°c ahl production decreased dramatically, and lfi produced less than 5% of the maximum concentrations observed at 6°c. luxi and ains are the autoinducer synthases responsible for the eight ahls in a. salmonicida. aliivibrio salmonicida is the etiological agent of cold water vibriosis in atlantic salmon, a disease which occurs mainly during seasons when the seawater is below 12°c. aliivibrio salmonicidaquorum sensingbiofilmacyl homoserine lactonetemperatureissue-copyright-statement© the author 2015
 quorum sensing is a cell-to-cell communication system used by bacteria to regulate activities such as virulence, bioluminescence and biofilm formation. the most common qs signals in gram-negative bacteria are n-acyl-homoserine lactones. aliivibrio salmonicida is the etiological agent of cold water vibriosis in atlantic salmon, a disease which occurs mainly during seasons when the seawater is below 12°c. in this work we have constructed several mutants of a. salmonicida lfi in order to study the luxi/luxr and ains/ainr qs systems with respect to ahl production and biofilm formation.  using high-performance liquid chromatography-tandem mass spectrometry we found that luxi in a. salmonicida lfi is responsible for producing seven of the different ahls, whereas ains is responsible for producing only one. the production of these various ahls is dependent on both cell density and growth temperature. the ahls were efficiently produced when wild type lfi was grown at or 12°c, however at 16°c ahl production decreased dramatically, and lfi produced less than 5% of the maximum concentrations observed at 6°c. litr, the master regulator of qs, was found to be a positive regulator of ains-dependent ahl production, and to a lesser extent luxi-dependent ahl production. this implies a connection between the two systems, and both systems were found to be involved in regulation of biofilm formation. finally, inactivation of either luxr or luxr in the lux operon significantly reduced production of luxi-produced ahls.  luxi and ains are the autoinducer synthases responsible for the eight ahls in a. salmonicida. ahl production is highly dependent on growth temperature, and a significant decrease was observed when the bacterium was grown at a temperature above its limit for disease outbreak. numerous ahls could offer the opportunity for fine-tuning responses to changes in the environment. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords aliivibrio salmonicidaquorum sensingbiofilmacyl homoserine lactonetemperatureissue-copyright-statement© the author 2015 