The bacterial strains used in this study are listed in Table 1. Unless otherwise stated, all strains were routinely grown at 37°C in Tryptic Soy broth (TSB) (BD), with shaking (240 rpm) in a TC-7 roller drum (New Brunswick). B. ambifaria HSJ1 cultures were inoculated from freshly grown colonies on TSB plates solidified with 1.5% agar and containing 0.1% Congo Red to avoid picking phase variants (Vial et al., 2010). Tetracycline was used at 15 and 200 μg/mL for Escherichia coli and B. ambifaria HSJ1, respectively, while trimethoprim was included at 100 μg/mL for both species. TSB was supplemented with 62.5 μg/mL diaminopimelic acid (DAP) for growth of auxotrophic E. coli χ7213.

The plasmids used in this study are listed in Table 2. All primers used for the constructions were purchased from Alpha DNA (Montreal, QC, Canada) and are listed in Supplementary Table 1.

The cepI2- marked mutant of B. ambifaria strain HSJ1 was constructed essentially using the method described by Barrett et al. (2008). Briefly, one upstream and one downstream fragments of the Bamb_6053 locus (AMMD strain sequence, assembly GCF_000203915.1, from www.burkholderia.com) were amplified by PCR using the Bamb6053_02F/Bamb6053_02R and Bamb6053_03F/Bamb6053_03R2 primer pairs, respectively (Supplementary Table 1). A FRT-flanked trimethoprim resistance cassette was amplified by PCR from the pFTP1 donor plasmid (Choi et al., 2005) using primers Bamb6053_Trim01F2 and Bamb6053_Trim01R2. The three PCR products were joined together using the Bamb6053_02F/Bamb6053_03R2 primer pair and Taq DNA polymerase (Feldan). The resulting fragment was digested using FastDigest HindIII (ThermoScientific) and cloned into pEX18Tet-pheS (Barrett et al., 2008). The resulting pAC1 construction was introduced into E. coli SM10 (λpir) allowing conjugation with B. ambifaria HSJ1. Transformants were selected onto agar plates supplemented with tetracycline and trimethoprim. Merodiploids were resolved by successive subcultures in TSB with trimethoprim only. Tetracycline-sensitive/trimethoprim-resistant colonies were selected on TSB agar plates supplemented with appropriate antibiotics and then PCR-confirmed. The pAC1 construct was introduced in the HSJ1 cepI- background (ED372) to generate the cepI-cepI2- marked double mutant.

The cepR2- mutant was constructed using the same methodology. Primers Bamb6040_02F and 02R and Bamb6040_03F and _03R were used to respectively amplify regions upstream and downstream of the Bamb_6040 locus in strain HSJ1. The trimethoprim resistance cassette from pFTP1 was amplified with primers Bamb6040_01F2 and 01R. The three fragments were joined together by amplification using Bamb6040_02F and Bamb6040_03R. The resulting fragment was purified and digested with HindIII and ligated in pEX18Tet-PheS digested with the same enzyme, to generate the pMCG27 construct. Double crossing-over in B. ambifaria HSJ1 was performed using subcultures on M9 agar plates with 0.1% p-chlorophenylalanine, 0.2% dextrose and 100 μg/mL trimethoprim, then verified as described above.

Amplification of the intergenic region upstream of the hmqA gene was carried out using primers hmqAA-L and hmqAA-R containing respectively EcoRI and BamHI restriction sites. The PhmqA PCR product was ligated with T4 DNA ligase (BioBasic, Inc.) in EcoRI/BamHI-digested mini-CTX-lacZ (Becher and Schweizer, 2000) to generate pJPD01. Chromosomal integration of the mini-CTX-hmqA-lacZ reporter at the attB locus in B. ambifaria strain HSJ1, and in hmqA-, cepI-, cepR-, cepI2-, cepR2-, and cepI-cepI2- mutants was performed by mating with donor strain E. coli χ7213 harboring pJPD01. An overnight culture of each mutant (recipient) was diluted in fresh broth and incubated with agitation at 37°C until an OD₆₀₀ of 0.5 was reached. An overnight culture of donor E. coli χ7213 (pJPD01) was also diluted in fresh DAP-containing TSB and statically grown at 37°C to an OD₆₀₀ of 0.5. Volumes of 1.5 mL from each culture were centrifuged at 8,000 ×g and both pellets were resuspended together in 100 μL PBS. The whole volume was then spotted onto an LB agar plate containing 100 μg/mL DAP and incubated overnight at 30°C. The bacteria were then suspended in 1 mL PBS and spread on TSB agar plates containing tetracycline. Tetracycline-resistant conjugants were selected and insertion of the hmqA-lacZ fusion was confirmed by PCR. Finally, an unmarked insertion was obtained by Flp-mediated excision of the tetracycline cassette, using pFLPe4 (Choi et al., 2005).

To obtain a quantity of 4-hydroxy-3-methyl-2-heptenylquinoline (HMAQ-C₇:2′), the main HMAQ produced by B. ambifaria (Vial et al., 2008), 6 L of B. ambifaria HSJ1 were grown until stationary phase in TSB. Three liters of methanol were then added to the culture and cells were removed by centrifugation at 6,000 ×g for 25 min. After methanol evaporation using a Rotovapor R110 (Büchi, Switzerland), the supernatant was extracted three times with 1.5 L ethyl acetate. The extracts were pooled, dehydrated with Na₂SO₄ and filtered on 6 μm glass fiber. Complete evaporation was obtained using the Rotovapor and the residue was solubilized in 10 mL methanol. Purification was performed by HPLC (Waters Delta Prep 4000) on a Gemini C18 column (10 μm, 110 Å, 50^∗21.2 mm, Phenomenex) with a linear gradient of acetonitrile/water (neutralized to pH 7.0 with NH₄OH). The chromatographic fractions containing HMAQ-C₇:2′ were pooled, evaporated and suspended in 1 mL isopropanol. Twenty mL pentane was added for overnight precipitation at 16°C. The mixture was filtered on 6 μm glass fiber filter and air dried completely. LC/MS was used to assess purity during the process.

The samples were prepared and analyzed as previously described (Chapalain et al., 2013), with the following differences: the positive electrospray ionization (ESI+) mode, supplemented by the multiple reactions monitoring (MRM) mode were used and the following transitions were monitored: HHQ-d4: 148→163; C₆-HSL: 200→102; C₈-HSL: 228→102; 3OH-C₈-HSL: 244→102; C₁₀-HSL: 256→102; HMAQ-C₇:2′: 256→172; 3OH-C₁₀-HSL: 272→102; HMAQ-C₉:2′: 284→172; 3OH-C₁₂-HSL: 300→102. AHL concentrations were calculated from integration of peak areas, expressed in relative value compared to the internal standard 5,6,7,8-tetradeutero-4-hydroxy-2-heptylquinoline (HHQ-d4) as before (Lepine and Deziel, 2011).

Levels of expression from the hmqABCDEFG promoter were assessed using strains carrying the chromosomal lacZ transcriptional fusion. β-Galactosidase assays were performed as described (Miller, 1972). For experiments with addition of AHLs, C₈-HSL (Sigma-Aldrich), 3-OH-C₈-HSL and 3-OH-C₁₀-HSL (Nottingham) stocks were prepared in HPLC-grade acetonitrile. B. ambifaria HSJ1::hmqA-lacZ cultures were grown in TSB from freshly picked colonies. Cultures were then diluted to an OD₆₀₀ = 0.1 in TSB and 10 μM AHL of interest were added. Acetonitrile only was added in controls. For experiments with addition of HMAQs, cultures were prepared as above and HMAQs were added to cultures to a final concentration of 50 μM from stocks prepared in HPLC-grade methanol. Methanol was added in controls. β-Galactosidase activity was measured at various time intervals during growth. All experiments were performed with four replicates and repeated at least twice.

Samples were prepared as previously described (Chapalain et al., 2013). Primers used for cepI, cepI2, and hmqA mRNA quantification are shown in Supplementary Table 1, using the method described before (Chapalain et al., 2013). The reference gene was ndh (Subsin et al., 2007). Gene expression differences between HSJ1 WT and cepR and cepR2 or hmqA- mutants were calculated using the 2^-ΔΔCT formula (Livak and Schmittgen, 2001).

Unless otherwise stated, data are reported as mean ± standard deviation (SD). Statistical analyses were performed with the R software v.3.3.3¹ using one-way analysis of variance (ANOVA). Probability values less than 0.05 were considered significant.

In P. aeruginosa, the HAQs HHQ and PQS are able to activate the transcription of the pqsABCDE operon via their binding to the MvfR (PqsR) regulator (Xiao et al., 2006). In B. ambifaria HSJ1, no such regulator has been identified (Vial et al., 2008). However, we have reported that an hmqG- mutant, which is only able to produce non-methylated HAQs, produces lower concentrations of total HAQs compared to the WT strain (only 25–30% of WT), thus raising the possibility of a positive regulation of HMAQs on their own production, likely via hmqABCDEFG regulation (Vial et al., 2008).

To verify this hypothesis, the expression of the hmqABCDEFG operon was monitored using the hmqA-lacZ reporter gene in WT and hmqA- mutant strains cultures, supplemented or not with HMAQ-C₇:2′, the principal HMAQ produced by B. ambifaria HSJ1. The β-galactosidase activity in the hmqA- mutant was not abolished, and was even significantly higher than in the WT strain on all time points (p < 0.05) (Figure 1). Supplementation with HMAQ-C₇:2′ had no effect on transcription from the hmqA promoter in the WT strain, whereas it significantly (p < 0.05) restored activity to the WT level in hmqA- for the late time points (Figure 1). The same results were obtained with addition of HMAQ-C₉:2′, the second most abundant congener produced in our strain (data not shown). Collectively these results indicate that, unlike in P. aeruginosa, HMAQs do not activate transcription of the operon responsible for their synthesis in B. ambifaria HSJ1, and at best are poor negative signals on the hmq system.

In a precedent study, we had demonstrated that the hmqA- mutant of B. ambifaria HSJ1 overproduces C₈-HSL, the main QS molecule that was then known in Bcc species (Vial et al., 2008). Although C₈-HSL is the most abundant AHL produced by Bcc bacteria, other AHLs can also be produced by Burkholderia. Based on the reported AHLs produced by Burkholderia species (Suarez-Moreno et al., 2010; Majerczyk et al., 2013) and since it was previously reported, however using only TLC assays, that B. ambifaria produces more diversified AHLs than simply C₆-HSL and C₈-HSL (Lutter et al., 2001), the following AHLs were investigated: C₄-HSL, C₆-HSL, C₈-HSL, C₁₀-HSL, 3OHC₈-HSL, 3OHC₁₀-HSL, 3OHC₁₂-HSL, 3oxoC₈-HSL, 3oxoC₁₂-HSL, and 3oxoC₁₄-HSL. Under our conditions, HSJ1 indeed mostly produces C₈-HSL (Table 3). However, unexpectedly and interestingly, the next more abundant AHLs are 3-hydroxylated, especially 3OHC₁₀-HSL. We saw neither C₄-HSL, nor any oxo-substituted AHLs. In agreement with our previous data, levels of all these new AHLs were also significantly higher in cultures of the hmqA- when compared to the WT (p < 0.05) (Figure 2 and data not shown).

When production of these AHLs in the cepI- mutant was investigated we confirmed that while C₆-HSL, C₈-HSL, and C₁₀-HSL production was clearly abolished and thus directly depend on this synthase (Chapalain et al., 2013), 3OHC₁₀-HSL and small concentrations of 3OHC₈-HSL were still detected (Table 3). The same results were obtained with the cepR- mutant (data not shown).

The above results pointed to the presence of at least a second AHL synthase in our strain. We have previously used the B. ambifaria strain AMMD sequenced genome to identify genes in strain HSJ1 (Winsor et al., 2008; Chapalain et al., 2013). In AMMD, predictions indicate that the Bamb_6053 locus on the third chromosome encodes a putative AHL synthase, with homology to bviI from B. vietnamiensis G4 (54.92% identity) and to btaI2 from B. thailandensis E264 (65.54% identity). A multiple sequence alignment of Bamb_6053 with various AHL synthases shows that the predicted synthase possesses eight residues that are conserved within the LuxI AHL synthase family and are needed for AHL synthesis (Watson et al., 2002) (Supplementary Figure 1).

We knocked-out the Bamb_6053 gene in our HSJ1 strain, which we named cepI2, resulting in the almost complete loss of 3OHC₁₀-HSL production, while 3OHC₈-HSL remained unaffected (Table 3). On the other hand, the cepI2- mutant still showed C₆-HSL, C₈-HSL, and C₁₀-HSL levels similar to the WT strain (Table 3). Furthermore, a cepI-cepI2- mutant does not produce any known AHLs, confirming that there is no other synthase in this strain (data not shown). We conclude that the clinical B. ambifaria HSJ1 strain expresses two AHL synthases: the canonical CepI responsible for C₈-HSL, C₆-HSL, C₁₀-HSL and 3OH-C₈-HSL, and CepI2 which is mainly responsible for 3OH-C₁₀-HSL and 3OH-C₁₂-HSL synthesis.

Different types of organization of the QS luxI/R family genes are reported, including in Burkholderia (Choudhary et al., 2013). In strain AMMD, no cognate luxR-type gene was identified associated with cepI2 (Choudhary et al., 2013). To identify such cognate regulator for cepI2, the genetic proximity between luxI and luxR genes often is a clue. However, the closest luxR-type genes are located either 13 genes upstream (Bamb_6040) or 11 genes downstream (Bamb_6064) from the cepI2 locus. Using the InterPro database, we quickly discarded Bamb_6064 as a potential candidate, since it possesses only the DNA binding domain of a LuxR-type regulator (IPR000792 entry) and not the autoinducer binding domain (IPR005143 entry), characteristic of a LuxR-type regulator that interacts with AHLs (Subramoni et al., 2015). Moreover, nine residues are conserved in the LuxR family (Egland and Greenberg, 2001; Koch et al., 2005); six of them involved in ligand binding. The remaining three are involved in DNA binding. Bamb_6040 possesses the nine residues conserved in the LuxR family (Supplementary Figure 2), while in Bamb_6064 some key residues are replaced. A phylogenetic analysis of described LuxR from the Burkholderia genus, including Bamb_6040 and LuxR references from other species is presented in Supplementary Figure 3. This analysis shows that Bamb_6040 is clustered with and closer to BviR from B. vietnamiensis G4 and B. cepacia DB01 (with whom it shares 41% of identity) than to BtaR2 from B. thailandensis. However, these three are more closely related than to CepR or BtaR1 (Supplementary Figure 3).

In order to confirm that Bamb_6040 encodes the cognate LuxR of cepI2, a mutant was constructed in our strain HSJ1 and studied for AHL production. The mutant still produced similar concentrations of C₈-HSL compared to the WT, but drastically reduced concentrations of 3OHC₁₀-HSL (Supplementary Figures 4A,B). Complementation with a plasmid-borne cepR2 restored 3OHC₁₀-HSL production to WT levels (data not shown). Our qRT-PCR experiments confirmed that expression of the cepI2 gene was lowered in the Bamb_6040 mutant compared to the WT, compatible with the level of decrease of 3OHC₁₀-HSL (Figure 3). Since these elements support Bamb_6040 as the cognate LuxR-type regulator of cepI2 we named it CepR2.

Several phenotypes previously reported to be affected in the cepI- mutant (Chapalain et al., 2013) have been evaluated in the cepI2- and cepR2- mutants, such as protease production, hemolytic and antifungal activities. Other known phenotypes were also included in the characterization, such as resistance to antibiotics, biofilms formation, and virulence against macrophages or amoeba (Vial et al., 2010). We observed that the cepR2- mutant displayed a higher proteolytic activity than the WT (Supplementary Figure 5A), while it was significantly reduced in cepI- and in the AHL-defective cepI-cepI2- mutant. For cytotoxicity against macrophages (Supplementary Figure 5B), cepR2- mutant displayed a moderate but significant decrease in cytotoxicity compared to the WT against a human monocytes/macrophages cell line, whereas cepI- and cepI-cepI2- were both importantly affected. For the other phenotypes tested, cepI2- and cepR2- mutants displayed no significant differences with the WT. In all our assays, cepR- displayed the same pattern as cepI- (data not shown).

In order to more globally evaluate the involvement of the second QS system in strain HSJ1, we undertook competition assays. We used the cepR- and cepR2- mutants to avoid chemical complementation with AHLs produced by the WT strain. The competition conducted in human monocytes/macrophages THP-1 cell line revealed that the cepR- mutant displayed the same ability as the WT to enter in macrophages (2 h post-infection), while it was less competitive in replication (evaluation at 8 h post-infection) (Supplementary Figure 5C). The cepR2- mutant displayed a slight decrease in competitive index to enter in macrophages, and was also less competitive to replicate (Supplementary Figure 5C, left panel). The defect was more pronounced in the cepR- mutant than in the cepR2- mutant, nevertheless both systems seemed to be required in this environment. As for the competition in pea rhizosphere, only the cepR- mutant was less recovered from roots compared to the WT, providing the evidence that only the first QS system is essential in this environment (Supplementary Figure 5, right panel).

As the hmqA- mutant strain overproduces AHLs synthetized by both CepI and CepI2, we wondered whether the production of HMAQs was reciprocally affected in QS mutants of B. ambifaria, as seen for HAQs in P. aeruginosa (Déziel et al., 2004). Strain HSJ1 produces a mix of HMAQs, the most abundant being HMAQ-C₇:2′, while the polar hmqA- mutant produces no HMAQ (Vial et al., 2008). We thus determined the kinetic of production of HMAQ-C₇:2′ in the different mutants described above vs. the WT strain. The cepI- (Figure 4A) and cepR- (data not shown) mutants no longer produce HMAQs. On the other hand, the cepI2- mutant displays only a moderate, although statistically significant decrease in HMAQ-C₇:2′ production (p < 0.05) (Figure 4A). AHL-mediated QS affect directly and indirectly the transcription of the pqsABCDE operon in P. aeruginosa (McGrath et al., 2004; Xiao et al., 2006). An hmqA-lacZ reporter allowed us to follow the transcription from the hmqABCDEFG operon promoter in the different strains described above (Figure 4B). Expression of hmqA-lacZ is reduced in cepI2- mutant background, and is even lower in the cepI- mutant (Figure 4B), which is consistent with the HMAQ measurements (Figure 4A). Taken together, these results suggest an influence of the AHL-based regulon on HMAQ production.

To further discriminate the role of both AHL QS systems in the transcription of hmqABCDEFG, we then assessed the activity of our hmqA-lacZ reporter in the AHL-defective cepI-cepI2- double mutant upon supplementation with one of the two main AHL produced by each synthase, namely C₈-HSL and 3OHC₁₀-HSL. We measured no effect of 3OHC₁₀-HSL on the activity of hmqA-lacZ, whereas C₈-HSL strongly induced expression above the level of the control (p < 0.05) (Figure 4C). Accordingly, supplementation of the cepI- mutant with C₈-HSL restored HMAQ production (Supplementary Figure 6A). This was also confirmed at the transcriptional level as the hmqA gene is almost 50 times downregulated in the cepI- mutant strain compared to the WT (Supplementary Figure 6B). The expression was partially restored if the mutant culture is supplemented with C₈-HSL. Although these results collectively indicate that the CepIR QS system is essential for the expression of the hmqABCDEFG operon, and that C₈-HSL is the most potent AHL for this induction, we have been unable to show a direct interaction between CepR and the hmqABCDEFG promoter when co-expressed together in a heterologous host system (data not shown).

Like the other Bcc species, B. ambifaria possesses a canonical CepI synthase responsible for C₈-HSL and C₆-HSL production (Zhou et al., 2003; Venturi et al., 2004; Chapalain et al., 2013). Other AHLs produced by B. ambifaria, then known as genomovar VII, were detected previously using TLC plates, but they could not be identified (Lutter et al., 2001). Performing an exhaustive search using LC/MS–MS analyses, we have found that strain HSJ1 produces additional AHLs, namely C₁₀-HSL, 3OH-C₈-HSL, 3OH-C₁₀-HSL, and 3OHC₁₂-HSL. We have also identified a second AHL synthase gene that we have named cepI2 primarily responsible for production of the latter two.

The two AHL synthases produce sets of mostly non-overlapping AHLs (Table 3). Although 3OHC₈-HSL seems produced by both LuxI homologues, CepI is clearly the main synthase for this AHL. At first glance it was surprising that CepI produces a mixture of carbonyl- and hydroxyl-AHL. If a synthase could theoretically produce a variety of AHLs, they often differ by the length of their acyl chain but not by the third carbon-borne substitution (Watson et al., 2002). Nevertheless, there are examples in the literature reporting synthases able to produce a mix of AHLs harboring different substitutions (Wisniewski-Dye et al., 2002; Niu et al., 2008). The mechanism by which a synthase recognizes and discriminates its favorite acyl-ACP is far to be fully understood, even if recent study reports progress in this respect for B. mallei BmaI1 (Montebello et al., 2014). For example the 140th amino acid of the synthase sequence was reported to determine the substitution borne by the third carbon, as a threonine appeared required to produce 3-oxo-AHL (Watson et al., 2002). Neither CepI nor CepI2 has a threonine at this position in their sequence; accordingly, we did not find any oxo-AHL in supernatants of strain HSJ1.

As the 3OHC₁₀-HSL levels were similar between the cepI and cepR mutant strains (data not shown), we suspected that there was a LuxR-type transcriptional regulator other than CepR involved with cepI2 regulation. A previous study on the QS genes organization in Burkholderia has reported that the AMMD strain possesses a potential synthase, but no cognate LuxR-type homolog was identified (Choudhary et al., 2013). Indeed, while no obvious LuxR-type transcriptional regulator is found in the close vicinity of the Bamb_6053 locus, our in silico analysis predicted Bamb_6040 to be the best candidate for cepI2 regulation. The inactivation of this gene, renamed cepR2, confirmed this hypothesis.

As 3OH-C₁₀-HSL production in the cepI mutant is lowered compared to the WT (Table 3), we propose a hierarchical influence of CepR on cepI2 (Figure 5), CepI being the producer of the main AHLs recognized by CepR (Weingart et al., 2005). The kinetics of C₈-HSL and 3OH-C₁₀-HSL productions in the WT strain displayed in Figure 2 supports this hypothesis, showing that C₈-HSL accumulation starts earlier and faster than 3OH-C₁₀-HSL. Such hierarchical relationships between two or more AHL-based QS systems have already been described in other Gram-negative bacteria, such as LasIR and RhlIR systems in P. aeruginosa, or CciIR in B. cenocepacia (O’Grady et al., 2009; Jimenez et al., 2012). We identified a similar organization in B. thailandensis E264 (Le Guillouzer et al., unpublished). The observation that the CepIR system is well-conserved in Bcc species suggests its early acquisition (Suarez-Moreno et al., 2012a); we could thus expect CepIR to be the primary system modulating additional AHL-based QS modules. However, examples in other Bcc suggest that the second QS system can be integrated in a species or even strain-dependent manner, rendering any hierarchical relationship difficult to predict (Malott and Sokol, 2007; O’Grady et al., 2009).

Accordingly, it was difficult to identify phenotypes clearly attributed only to the CepI2R2 system. The competition assay in macrophages revealed conditions where the second QS system seems to be required. Interestingly, the CepIR system appeared to be essential only in the rhizosphere. Consistently with this result, we did not observe antifungal activities depending on the second QS system, while the first system is clearly essential (data not shown) (Zhou et al., 2003; Chapalain et al., 2013). It is possible that the implication of the CepI2R2 system is condition-dependent. The determination of genes and phenotypes controlled either by CepIR or CepI2R2, cooperatively, independently or in opposition will require a global and comprehensive approach, such as those undertaken for B. cenocepacia or B. thailandensis (O’Grady et al., 2009; Majerczyk et al., 2014a).

To study the reciprocal effect of AHL on the hmqABCDEFG operon transcription in HSJ1, we performed experiments using diverse tools and mutants, including hmqA-lacZ reporter assays correlated to LC/MS measurements of AHL and HMAQ production.

We verified whether HMAQs can act as classical signaling molecules implicated in an autoinducing loop in B. ambifaria HSJ1, similarly to the situation in P. aeruginosa. Indeed, this bacterium also possesses two AHL-based QS systems intertwined with a HAQ-based QS system. The pqsABCDE operon is directly and indirectly influenced by the two AHL-based QS systems, but is also positively autoinduced via the binding of HHQ or PQS ligands to the LysR-type regulator MvfR (PqsR) (McGrath et al., 2004; Xiao et al., 2006). While no such regulator has yet been identified for B. ambifaria (Vial et al., 2008), we investigated whether HMAQ-C₇:2′ induces the transcription of the hmqABCDEFG operon in strain HSJ1. The transcriptional activity from the hmq promoter did not decrease in the hmqA- mutant. Actually, especially considering the negative effect of the exogenous addition of HMAQ-C₇:2′ at certain time points in the cultures, it even seems that HMAQs and/or the hmq system slightly downregulates its expression, likely indirectly. It is noteworthy that we observe the same scenario in an hmqA- mutant from HMAQ-producer strain B. thailandensis E264 (Le Guillouzer et al., unpublished). This result was unexpected because in complete contrast with the situation seen with the pqs system in P. aeruginosa, where a pqsA- mutant strain exhibits a very low pqsABCDE operon transcriptional activity, which is induced by addition of HHQ, or even better PQS (Xiao et al., 2006). It is possible that other regulatory components present in the operon are responsible for the observed effect on the transcription in the hmqA- mutant. In P. aeruginosa, the pqsABCDE operon is upregulated in a pqsE- mutant (Hazan et al., 2010). It is possible HmqE might have a similar impact in B. ambifaria since both proteins seem to be functionally complementary (Diggle et al., 2006).

We knew that the hmqA- mutant of B. ambifaria HSJ1 overproduces C₈-HSL, the main AHL then known, already revealing a link between QS and HMAQ production (Vial et al., 2008). Now that the QS circuitry is better understood, we checked whether production of other AHLs is affected by the absence of hmqA and, indeed, we found that all AHLs from both synthases are overproduced in the hmqA- mutant. Accordingly, we observed an upregulated expression of cepI2 in the hmqA- mutant using qRT-PCR (Supplementary Figure 7). However, expression of cepI was similar to the WT in both exponential and stationary growth phases. This was surprising considering the observed effect on C₈-HSL levels.

One explanation could be that HMAQs act as inhibitors of CepI activity or C₈-HSL function, which would then affect cepI2 transcription via diminished CepR activity (Figure 5). Further supporting this model, the addition of HMAQ-C₇:2′ in the AHL-defective double cepI-cepI2- mutant had no effect on hmqA-lacZ activity (data not shown), while it downregulated transcription from the hmqA promoter in a HMAQ-negative background, where CepI is overproducing C₈-HSL (Figure 1). These results confirm that even if the hmqABCDEFG operon impacts its own expression, HMAQs do not have autoinducing properties in Burkholderia. Since the hmqA- mutant is polar on downstream genes (Vial et al., 2008), we do not know if what we observed is due to the absence of HMAQ molecules or of another gene of the hmq operon. In P. aeruginosa HAQs do not impact AHL production (Déziel et al., 2005), highlighting another difference between systems.

Recent transcriptomic data suggest that globally QS downregulates the expression of the hmqABCDEFG operon in two other HMAQ-producing Burkholderia species, B. pseudomallei and B. thailandensis (Majerczyk et al., 2014a,b). In B. ambifaria, the CepIR QS system acts as a positive regulator of the hmqABCDEFG operon (Figure 5), highlighting an intriguing difference in HMAQ regulation between Bcc and pseudomallei-thailandensis groups. In the cepI-cepI2- background of B. ambifaria HSJ1, the main product of CepI C₈-HSL very efficiently induces transcription from the hmq promoter. Still, despite the clear implication of the CepIR system in the induction of the operon, a direct regulation of CepR on the hmqABCDEFG promoter was not observed using an heterologous host strategy, presumably because QS impact on HMAQ production implicates additional regulatory elements. Investigating HMAQ production and hmqABCDEFG transcription revealed that the cepI2- mutant displays a statistically significant lower transcriptional activity than the WT strain, with a well-correlated decreased HMAQ production (Figures 4A,B). However, 3OHC₁₀-HSL produced via CepI2 did not increase the transcriptional activity above the level of the control in the cepI-cepI2- mutant. Together with the observed upregulation of cepI2 expression in the hmqA- mutant, these results suggest that the CepI2R2 system would affect indirectly the transcription of hmqABCDEFG via an effect on the CepIR system.

Some other observations lead to suggest that QS is not the only regulon controlling the production of HMAQs in B. ambifaria. First, although phase variants of HSJ1 express a functional CepIR system and produce similar amount of C₈-HSL compared to the WT, no HMAQ production is observed (Vial et al., 2010). Secondly, under our experimental conditions, environmental strains which possess the operon, such as the AMMD strain, do not produce HMAQs (Vial et al., 2010). However, another team has demonstrated that AMMD can produce some HMAQs, using experimental conditions that also favor antifungal and antimicrobial molecules production (Mahenthiralingam et al., 2011). This collectively suggests the presence of additional regulatory levels on HMAQ production dependent on the environmental conditions, maybe at a post-transcriptional level or via another regulator yet to be discovered. Supplemental experiments are thus needed to completely decipher the regulation of HMAQ biosynthesis. Since the hmqABCDEFG operon seems to be present in many now sequenced Bcc strains (burkholderia.com), there is still a lot to be learned on the functionality and regulation of this operon. Even if we have contributed to better understand HMAQ regulation, their role in B. ambifaria HSJ1 is still largely unknown. It has been proposed that HMAQs are QS-controlled secondary metabolites (Mahenthiralingam et al., 2011; Majerczyk et al., 2014b). Indeed, in another Bcc species, namely B. cepacia, HMAQs have been described as antifungals (Kilani-Feki et al., 2011). This property cannot be easily verified in HSJ1 as the hmqA- mutant overproduces all the AHLs, resulting in an overexpression of all the QS-regulated phenotypes including antifungal compounds, masking the antifungal contribution of HMAQs.