A solo luxI-type gene directs acylhomoserine lactone synthesis and contributes to motility control in the marine sponge symbiont Ruegeria sp. KLH11

Abstract

Marine sponges harbour abundant and diverse bacterial communities, providing an ideal environment for bacterial cell-density-dependent cell–cell signalling, termed quorum sensing. The marine sponge symbiont Ruegeria sp. KLH11 produces mainly long chain acylhomoserine lactones (AHLs) and has been developed as a quorum sensing model for roseobacterial sponge symbionts. Two pairs of luxR/I homologues were identified by genetic screening and were designated ssaRI and ssbRI (sponge-associated symbiont locus A or B, luxR/luxI homologue). In this study, we identified a third luxI-type gene, named sscI. The sscI gene does not have a cognate luxR homologue present at an adjacent locus and thus sscI is an AHL synthase solo. The sscI gene is required for production of long-chain hydroxylated AHLs, contributes to AHL pools and modestly influences flagellar motility in KLH11. A triple mutant for all luxI-type genes cannot produce AHLs, but still synthesizes para-coumaroyl-homoserine lactone.

Introduction

Marine sponges harbour highly diverse and dense microbial communities and in some cases up to 30–40 % of the sponge biomass is from the associated bacteria (Taylor et al., 2007). These sponges provide a highly conducive environment for bacterial quorum sensing (QS), a process by which bacteria use chemical signals to communicate with each other and coordinate group behaviours, such as bioluminescence, antibiotic production and virulence at high cell density (Ahlgren et al., 2011; Fuqua & Greenberg, 2002). Acylhomoserine lactone (AHL)-based QS was first discovered in the marine squid symbiont Vibrio fisheri about 40 years ago and since then it has been reported in over 100 different bacterial species, although mainly restricted to the phylum Proteobacteria (Ahlgren et al., 2011; Fuqua & Greenberg, 2002).

Typically, each QS circuit has a luxI-type gene, responsible for AHL synthesis, and a luxR-type gene, encoding a protein that binds and provides a response to AHL(s). These two genes are often genetically linked, arranged in tandem or convergently expressed (Fuqua & Greenberg, 2002). However, in many different bacteria, luxR-type genes without a linked luxI-type gene have been discovered and these luxR-type genes are termed luxR solos. These solos occur both in bacteria that have complete LuxR-LuxI-type QS systems and bacteria that do not (Subramoni & Venturi, 2009). LuxR solos can regulate gene expression by binding to AHLs produced by other luxI genes encoded elsewhere in the same bacterial genome, such as in the cases of QscR in Pseudomonas aeruginosa (Lequette et al., 2006) and ExpR in Sinorhizobium meliloti (McIntosh et al., 2008), or by binding to AHLs produced by other bacteria, such as in the case of SdiA in Escherichia coli and Salmonella enterica (Ahmer, 2004; Yao et al., 2006). Furthermore, some LuxR-type proteins can regulate gene expression in response to non-AHL signals or independently of ligand binding (Subramoni & Venturi, 2009). Although progress has been made in understanding these luxR solos, there is little information about the less common scenario in which functional luxI genes are not linked to luxR-type genes, which we designate as luxI solos.

We have found that members of the Silicibacter-Ruegeria (SR) subgroup of the ecologically important Roseobacter clade are the primary AHL producers among cultivatable bacterial isolates from the marine sponges Mycale laxissima and Ircinia strobilina (Mohamed et al., 2008) and over 80 % of available roseobacterial genomes encode at least one luxI homologue (Zan et al., 2014). We have developed Ruegeria sp. KLH11 (hereafter referred to as KLH11) as a model to study QS in marine sponge symbionts. We previously reported detailed analyses of two luxRI systems: ssaRI and ssbRI in KLH11 (Zan et al., 2012). SsaI and SsbI direct the synthesis of long chain AHLs ranging from C₁₂- to C₁₆-homoserine lactone (HSL), dominated by 3-oxo and 3-hydroxy moieties at the beta-position in the acyl chain, respectively. The SsaRI system provides QS-dependent control of flagellar motility in KLH11, functioning through the CtrA master regulator (Zan et al., 2013). Analysis of whole sponge tissues revealed the presence of ssaI transcripts and AHLs (Zan et al., 2012). The function of the SsbRI system remains unclear, but it is indirectly regulated by SsaRI. We have also presented preliminary evidence for a solo luxI-type gene, sscI, in KLH11, which is not genetically linked to a luxR homologue (Zan et al., 2012). In the current study, we have analysed the functional characteristics of the sscI solo in KLH11. Furthermore, we tested whether KLH11 can produce the novel para-coumaroyl-HSL (pC-HSL) molecule that was originally discovered in Rhodopseudomonas palustris and requires the substrate para-courmarate for synthesis by the LuxI homologue RpaI (Schaefer et al., 2008).

Methods

Bacterial strains, oligonucleotides and growth conditions.

Bacterial strains and plasmids used in this study are listed in Table 1. Oligonucleotides were obtained from Integrated DNA Technologies (Coralville, IA, USA). Unless stated otherwise, E. coli strains were grown in Luria–Bertani (LB) broth at 37 °C with aeration, Ruegeria sp. KLH11 strains were grown in marine broth 2216 at 28 °C (MB 2216; BD) and Agrobacterium tumefaciens strains were grown in AT minimal salt medium supplemented with 0.5 % (w/v) glucose and 15 mM (NH4)₂SO₄ (ATGN; Tempé et al., 1977). Rhodopseudomonas palustris CGA814 was grown in LB broth. Antibiotics were used at the following final concentrations (μg ml⁻¹): (i) E. coli (ampicillin, Ap, 100; gentamicin, Gm, 25; kanamycin, Km, 25; spectinomycin, Sp, 100; tetracycline, Tc, 5). (ii) KLH11 (Km, 100; rifampicin, Rif, 200; Gm 25; Sp, 100; Tc, 5). (iii) A. tumefaciens (Gm, 300; Sp, 200). (iv) Rh. palustris CGA814 (Km, 50).

Table 1. Bacterial strains and plasmids used in this study.

Bacteria/plasmid	Relevant feature	Reference
E. coli DH5α/λpir	Strain for propagating R6K suicide plasmids	Lab collection
E. coli S17-1/λpir	IncP conjugal donor	Kalogeraki & Winans (1997)
E. coli XL-1 Blue	Standard alpha-complementation strain	Lab collection
E. coli MC4100	K-12 derivative, ΔlacZ	Lab collection
A. tumefaciens NTL4	Ti plasmid-less derivative, nopaline chromosomal background	Zhu et al. (1998)
KLH11	WT	Mohamed et al. (2008)
KLH11-EC1	RifR	Zan et al. (2012)
KLH11-SK01	ΔssaI, RifR	Zan et al. (2012)
KLH11-SK02	ΔssaI ΔssbI, RifR	Zan et al. (2012)
KLH11-OKC2	sscI-lacZ, RifR KmR	This study
KLH11-OKC6	ΔssaI ΔssbI ssc-lacZ, RifR KmR	This study
CGA814	Rh. palustris; rpaI-lacZ, KmR	Schaefer et al. (2008)
pCR2.1-TOPO	PCR fragment cloning vector, Ap/KmR	Invitrogen
pBBR1-MCS5	BHR Plac expression vector, GmR	Kovach et al. (1995)
pSRKTc	BHR expression vector containing lac promoter and lacIq, TcR	Khan et al. (2008)
pVIK112	R6K-based lacZ transcriptional fusion, KmR	Kalogeraki & Winans (1997)
pRA301	BHR lacZ translational fusion vector	Akakura & Winans (2002)
pEC108	pBBR1-MCS5 derivative carrying full-length Plac-ssaI, GmR	Zan et al. (2012)
pEC109	pBBR1-MCS5 derivation carrying full-length Plac-ssbI, from pEC110, GmR	Zan et al. (2012)
pEC112	pBBR1-MCS5 derivative, carrying full-length Plac-ssaR, from pEC106, GmR	Zan et al. (2012)
pEC116	pRA301 derivation, PssaI-lacZ, SpR	Zan et al. (2012)
pEC121	pRA301 derivative, PssbI-lacZ, Sp/SmR	Zan et al. (2012)
pOKC1	pCR2.1-TOPO, carrying internal fragment of sscI, KmR	This study
pOKC2	pVIK112 derivative, carrying internal fragment of sscI, KmR	This study
pOKC3	pCR2.1-TOPO, carrying full-length sscI, KmR	This study
pOKC4	pSRKTC derivative, carrying full-length Plac-sscI, TcR	This study
pOKC8	pRA301 derivation, PsscI-lacZ, SpR	This study

Sm, Streptomycin; Tc, tetracycline.

Plasmid construction for sscI null mutation, expression and lacZ-fusion.

Several regions around the sscI gene were isolated by PCR amplification from KLH11 genomic DNA. The method used to construct the sscI Campbell insertion mutant was similar to that described by Zan et al. (2012). Briefly, an internal fragment of the sscI gene was amplified using forward primer 5′-GAATTCATGTTTCGCGATCGAGCAGAT-3′ (the EcoRI recognition site is underlined) and reverse primer 5′-GGTACCTCTTGATACTCCCGCTC-3′ (the KpnI recognition site is underlined). The PCR amplicon was gel-purified and cloned into pCR 2.1-TOPO vector (Invitrogen) to create pOKC1 and the insert was confirmed by sequencing. For recombinational mutagenesis, pOKC1 was digested with EcoRI and KpnI, and the resulting sscI fragment was ligated to a similarly digested R6K replicon, the pVIK112 suicide vector (Kalogeraki & Winans, 1997), creating pOKC2. pOKC2 was conjugated into KLH11 and Km-resistant (Km^R) transconjugants were selected and confirmed by sequencing using the forward primer 5′-ATTAACCATAATCAAGCATCTCTT-3′. To construct double and triple AHL synthase gene mutants, pOCK2 was conjugated into ΔssaI and ΔssbI, and ΔssaI ΔssbI strains, respectively, and the transconjugants were selected and confirmed as described for the sscI single mutant.

A controlled expression construct of sscI was generated by PCR amplification of the coding regions using the forward primer 5′-TCTAGACTGAAACAGGAAACAGCTATGCTCCGTTATGTTTTTGCA-3′ (the XbaI recognition site is underlined, the stop codon TGA and the start codon ATG are in bold type and the E. coli lacZ ribosome-binding site is in italics) and the reverse primer 5′-CTCGAGTCAAGCGGTTCTTTGAAACTT-3′ (the stop codon is in bold type and the XhoI recognition site is underlined). The PCR products were ligated into pCR2.1-TOPO vector (Invitrogen) to create pOKC3 and confirmed by sequencing. pOKC3 was digested by XbaI and XhoI and the insert was subcloned into the vector pSRKTc (Khan et al., 2008) to create pOKC4. The insert carried by the construct was confirmed by sequencing.

In order to generate a plasmid-borne P_sscI-lacZ fusion, the presumptive promoter sequences were PCR amplified. The forward primer 5′-GAATTCGCCGAGATGAACTGTTCAAAGAAC-3′ (the EcoRI recognition site is underlined) and the reverse primer 5′-GGATCCGAGCATTTTTAACCTCTTGTTCAC-3′ (the BamHI recognition site is underlined) annealing 255 bp upstream and 3 bp downstream of the sscI translational start site were used to amplify its promoter. The PCR products were cloned into pCR2.1-TOPO vector and the inserts were confirmed by DNA sequencing. The pCR2.1-TOPO derivatives were digested with EcoRI and PstI and the resulting fragments were ligated with pRA301 vector digested with the same restriction enzymes (Akakura & Winans, 2002) to create pOKC8 with the P_sscI-lacZ translational fusion.

Organic extraction, TLC and MS analysis of AHLs.

As described previously, organic extraction of KLH11-derivative cultures, followed by reverse-phase TLC of organic extracts and AHL bioassay analysis with an ultrasensitive AHL bioreporter derived from A. tumefaciens was used to characterize the AHLs specified by SscI (Zan et al., 2012; Zhu et al., 2003).

Identification of SscI AHLs by LC-MS-MS was also performed. KLH11 derivatives were grown in MB 2216 with appropriate antibiotics (and 0.5 mM IPTG to induce the P_lac promoter) at 28 °C to stationary phase (OD₆₀₀~2.0) in the presence of 5 g Amberlite XAD 16 resin l⁻¹ for 36 h. Cells and resin were separated by centrifugation and extracted with 50 ml methanol and dried to 2 ml. Three nanomoles of D₃-C₆-HSL (D₃ indicates that there are three deuterium atoms at the terminal position of the acyl chain in the AHL molecule) was added to each sample as an internal standard and a volume of 0.2 ml of each extract was purified using solid phase extraction methods as described previously (Gould et al., 2006).

Extracts were dried down and resuspended in 38 µl solvent A (8.3 mM ammonium acetate, pH 5.7) and 2 µl solvent B (methanol). This solution was injected on to a 50×3.00 mm 2.6 µ C18 Kinetex (Phenomenex) column. A mobile phase gradient was generated from 5 % B to 65 % B in 5 min, then B was increased to 95 % in 15 min and held for 8 min at a flow rate of 250 µl min⁻¹. The HPLC system was interfaced to the electrospray source of a triple quadrupole mass spectrometer (Sciex API2000, PE Sciex). Precursor ion-scanning experiments were performed in positive-ion mode with the third quadrupole set to monitor m/z 102.3 and the first quadrupole set to scan a mass range of 170 to 700 over 9 s. The collision cell and instrument parameters were as follows: ion spray voltage of 4200 V, declustering potential of 50 V and collision energy of 25 V with nitrogen as the collision gas.

Preparation of extracts and bioassay for pC-HSL.

KLH11 and derivative strains were grown to stationary phase in MB 2216 supplemented with or without 1 mM p-coumaric acid. The cultures (acidified by 0.01 % acetic acid) were extracted twice with an equal amount of acetyl acetate and the extracts were dried in a rotary evaporator under vacuum. Each extract was concentrated 1000-fold, dissolving in 50 % (v/v) methanol. Fifty microlitres of the enriched extract was added to 1 ml of Rh. palustris CGA814 culture grown in LB. β-Galactosidase assays were performed as described previously (Zan et al., 2013).

Motility assays.

Bacterial swim assays were performed using MB 2216 with 0.25 % (w/v) agar. Plates were inoculated at the centre with freshly isolated KLH11 colonies. KLH11 crude organic extract (0.5 %, v/v) was added to MB 2216 agar. Plates were placed in an airtight container with a beaker containing 15 ml K₂SO₄ to maintain constant humidity, and incubated for 5–7 days at 28 °C. Photos were taken by using a Nikon D90 camera.

Results and Discussion

Identification of sscI

Genome sequencing of KLH11 revealed the presence of a luxI homologue, designated sscI, encoded on a large assembled sequence scaffold (>700 kb) that contains neither the ssaRI nor the ssbRI operons (Zan et al., 2011). The sscI and ssbI genes and their translation products are highly similar (70 % and 76 % nucleotide and amino acid identity, respectively) (Fig. 1), and much less similar to those of ssaI (52 % and 27 %, respectively). The sequence conservation between the ssbI and sscI genes is strikingly confined to the coding sequences, without significant similarity in their flanking regions (Fig. 1). This strongly suggests that sscI arose from a gene duplication event with ssbI. The closely related Ruegeria pomeroyi DSS-3 genome encodes ssaRI and ssbRI regions that are highly homologous and syntenic to KLH11, but does not encode an sscI gene (Moran et al., 2004). This indicates that sscI was either generated by duplication in KLH11 or, conversely, lost in DSS-3 since the time of their most recent common ancestor in the Ruegeria lineage. The region downstream of sscI in KLH11 is conserved with Ru. pomeroyi DSS-3, particularly linkage to a large gene encoding a predicted type I secretion target repeat protein (SPO2401). Upstream of sscI both genomes are chequered with several transposase and phage integrase gene remnants (Fig. 1) with a large number of frame-shift mutations, suggesting a high level of chromosomal rearrangement.

Fig. 1.

Genetic map of KLH11 sscI region and comparison with the ssbR-ssbI region. The sscI region shows homology to an analogous region of Ru. pomeroyi DSS-3 on the downstream side of the gene, and upstream of the gene there is a similar yet not identical area with several transposase and phage integrase partial gene fragments.

Solo luxR-type genes are common in bacterial genome sequences, but there are very few reported intact luxI-type solos. One exception is in another roseobacter, Dinoroseobacter shibae DFL 12^T, an algal symbiont that also has two sets of luxR-luxI QS systems and one luxI solo, designated luxI3 (Wagner-Döbler et al., 2010). The genomic location of this luxI-type solo is not recognizably similar to that of sscI in KLH11 and the LuxI3 protein is not particularly similar to SsbI or SscI (~30 % identity). The luxI3 solo in D. shibae is therefore distinct from sscI in KLH11.

SscI-derived AHL production

A targeted sscI mutation using a 514 bp internal fragment of sscI and the pVIK112 suicide plasmid (Kalogeraki & Winans, 1997) was made, generating a sscI null mutant (OKC2), with a sscI-lacZ transcriptional fusion. β-Galactosidase assays of OKC2 revealed significant levels of sscI-lacZ expression that were unaffected by the addition of KLH11 whole culture extracts containing AHLs (Miller units: 189.6±20.2 and 199.8±7.9; P>0.05; unpaired Student’s t-test) that strongly activate expression of ssaI. This level of expression, although AHL-independent, was ~200-fold higher than the expression of an ssbI-lacZ fusion generated in an analogous manner with the pVIK112 plasmid (Zan et al., 2012). This difference in expression probably reflects the lack of identity in the regions immediately upstream of these coding sequences. It is worth noting that neither a Lux box nor the previously defined ssa box (Zan et al., 2012) was found in either the ssbI or sscI promoter regions (data not shown).

A triple mutant, ΔssaI ΔssbI sscI⁻ (OKC6, with sscI disrupted using the pVIK112 derivative), was analysed for AHL production in whole-cell extracts using TLC overlaid with agar containing an A. tumefaciens AHL reporter as described by Zan et al. (2012). No AHL production was observed for this mutant (Fig. 2a). Quantitative MS, as previously described (Gould et al., 2006; Zan et al., 2012), also failed to detect AHLs in this mutant (Fig. 2b). Provision of plasmid-borne copies of each AHL synthase gene individually in this triple mutant resulted in AHL synthesis (Fig. 2a); the ssaI plasmid is weakly active and its AHLs were difficult to detect by this bioassay, but clearly detected by MS (Zan et al., 2012). MS analysis of the sscI-expressing derivative revealed high-level synthesis of several hydroxylated AHLs (Fig. 2c), consistent with our findings on its expression in E. coli (Zan et al., 2012). The high levels of AHL driven by sscI suggest that it encodes a highly active enzyme.

Fig. 2.

(a) Reverse-phase (RP)-TLC analysis of AHLs from KLH11 and QS mutants. TLC plate was overlaid with the A. tumefaciens ultrasensitive AHL reporter strain (Zhu et al., 2003). Organic extracts of cultures of strain OKC6 containing vector pSRKTc (Khan et al., 2008) were used as negative control. The concentration of X-Gal in the agar was 40 µg ml⁻¹. (b, c) MS analysis of purified samples. The products of reverse-phase chromatographic separation of AHLs extracted and purified from (b) OKC6 (ΔssaI ΔssbI sscI⁻) and (c) OKC6 (ΔssaI ΔssbI sscI⁻/P_lac-sscI) were examined using the precursor ion-scanning mode (transitions were monitored for precursor [M+H]⁺, →m/z 102.1). The peaks in the chromatograms are labelled with upper-case lettering and include the AHLs noted and as described by Zan et al. (2012).

SscI-derived AHLs are involved in ssaI expression and influence swimming motility

SsaR responds to SsaI-directed 3-oxo-HSL derivatives, and also, but more weakly, to those synthesized by SsbI (Zan et al., 2012). To test whether SsaR can also respond to SscI-derived AHLs, a plasmid-borne copy of ssaR (pEC112) or a vector control were paired with a compatible plasmid carrying the P_ssaI-lacZ fusion (pEC116), in an AHL⁻, plasmid-less derivative of A. tumefaciens NTL4. Cultures of A. tumefaciens derivatives were grown with 2.5 % (v/v) culture extracts containing SscI-derived AHLs (whole culture dichloromethane extracts from an A. tumefaciens NTL4 derivative grown with IPTG to induce expression of the P_lac-sscI plasmid). Expression of ssaI increased ~fourfold in response to the extracts compared with the negative control (P<0.01) (Fig. 3a; as in prior studies, the P_lac-ssaR plasmid modestly stimulates AHL-independent ssaI expression). This response of SsaR to SscI-directed AHLs adds another layer of complexity to the QS network in KLH11.

Fig. 3.

(a) SscI-derived AHLs stimulate ssaI expression and the sscI mutant is decreased for motility. Promoter fusion of ssaI with lacZ reporter (pEC116) paired with P_lac-ssaR (pEC116) (Zan et al., 2012) or empty vector (pBBR1-MCS5; Kovach et al., 1995) were transformed into A. tumefaciens NTL4. β-Galactosidase assay was used to monitor the lacZ activity with and without organic extracts. Error bars represent the standard deviation from three replicates. (b) sscI is involved in motility control. WT KLH11 and derivatives were inoculated on MB 2216 (supplemented with 0.25 %, w/v, agar) swim agar plates for 1 week at 28 °C. The results shown are representatives of several independent experiments each with three biological replicates.

Flagellar motility is strictly dependent on activation by the ssaRI system through the CckA-ChpT-CtrA motility regulators (Zan et al., 2012, 2013). The sscI null mutant (OKC2) consistently showed a 20 % decrease in swim ring diameter relative to WT KLH11 (P<0.05) and plasmid-borne sscI complemented this defect (Fig. 3b). We hypothesize that this mild effect on motility is most likely due to the impact of SscI-derived AHLs on the ssaI gene expression through SsaR.

Production of pC-HSL independently from LuxI homologues

Several novel types of AHL molecules have been reported recently (Ahlgren et al., 2011; Lindemann et al., 2011; Schaefer et al., 2008). Rh. palustris produces pC-HSL, which is an arylhomoserine lactone incorporating a coumaroyl group rather than an acyl chain. Remarkably, pC-HSL is only produced in the presence of para-coumarate, a compound synthesized by plants and certain algae, and directly incorporated into the signal molecule via the LuxI-type protein RpaI. In the absence of para-coumarate, RpaI does not synthesize a product. Ru. pomeroyi DSS-3, a relative of KLH11, was also found to produce pC-HSL in cultures grown with para-coumarate (Schaefer et al., 2008). We used the pC-HSL reporter strain Rh. palustris CGA814 that cannot synthesize pC-HSL but directs RpaR-dependent expression of a target rpaI-lacZ fusion (Hirakawa et al., 2011) to examine KLH11 culture extracts. KLH11 grown in the presence of 1 mM para-coumarate can activate the expression of rpaI-lacZ to a level equivalent to 1 µM pC-HSL, suggesting the presence of pC-HSL or a structurally similar molecule. Surprisingly, a KLH11 mutant disrupted for all three luxI-type genes (ssaI, ssbI and sscI) retained this activity (Fig. 4), suggesting the existence of a novel enzyme(s) in KLH11 responsible for its synthesis.

Fig. 4.

β-Galactosidase assay of the expression of an rpaI-lacZ fusion. Rh. palustris CGA814 was used as the reporter strain. Organic extracts of KLH11 strains were prepared from cultures grown in MB 2216 supplemented with 1 mM para-coumarate. An extract of MB 2216 plus para-coumarate was used as the negative control and 1 µM pC-HSL was used as the positive control. Bars represent the mean of three biological replicates and the error bars represent the sd of triplicates.

The roseobacter Phaeobacter gallaeciensis BS107 can respond to the presence of para-coumarate produced by the microalga Emiliania huxleyi potentially via pC-HSL (Seyedsayamdost et al., 2011). Novel signal molecules are synthesized by other roseobacters, including Silicibacter sp. TM1040, which does not encode luxI homologues or luxM (Cao & Meighen, 1989; Ng & Bassler, 2009), the gene encoding an alternative AHL synthase that directs the synthesis of 3-OH-C₄ HSL in Vibrio harveyi and exists in several Vibrio species, but rather produces the Roseobacter Motility Inducer (RMI) that can be induced by addition of para-coumarate (Sule & Belas, 2013). Several roseobacters produce the antibiotic and novel QS molecule tropodithietic acid (TDA), which regulates its own synthesis. Our findings contribute to the emerging impression that the roseobacter group may be an underexplored and rich source of novel signalling molecules.

Abbreviations:

AHL

acylhomoserine lactone

Ap

ampicillin

Gm

gentamicin

HSL

homoserine lactone

Km

kanamycin

pC

para-coumaroyl

QS

quorum sensing

Rif

rifampicin

Sp

spectinomycin