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. 2026 Jun 23;17:1850263. doi: 10.3389/fmicb.2026.1850263

The LuxS/AI-2 quorum sensing system regulates osmotic adaptation, motility, and type VI secretion system in Halomonas elongata

Zhuo Wang 1,*, Yanyan Yang 2, Xin Guo 2, Chuanxu Wang 1, Jing Yang 1, Yajie Niu 1, Xin Li 3,*
PMCID: PMC13337758  PMID: 42416014

Abstract

As a widespread quorum sensing (QS) system in bacteria, the LuxS/AI-2 QS system can coordinate bacterial group behaviors to adapt to diverse environments. However, its roles in halophiles remain largely unexplored. Here, we report that the AI-2 activity of Halomonas elongata, a moderately halophilic bacterium, is closely associated with environmental salinity. Moreover, luxS deletion abolishes AI-2 production and impairs the growth of H. elongata at various salt concentrations. Transcriptomic and qRT-PCR analyses revealed that the LuxS/AI-2 QS system regulates the transcription of the compatible solute ectoine degradation genes doeCD, thereby modulating intracellular ectoine levels to facilitate osmotic adaptation. Furthermore, the LuxS/AI-2 QS system is also involved in motility and the type VI secretion system (T6SS) in H. elongata. This work provides new insights into the regulatory mechanism of LuxS/AI-2 QS system underlying osmoadaptation in halophiles.

Keywords: autoinducer-2, Halomonas elongata, LuxS, motility, osmotic adaptation, type VI secretion system

Introduction

Halophiles are a group of microorganisms that thrive in environments with high salt concentrations, such as salt lakes, saline soils, and salt mines (Yin et al., 2015; Shu and Huang, 2022; de Souza Vieira and Totola, 2025). Based on their optimal salt concentration for growth, they are generally divided into three groups: slight halophiles (0.2–0.5 M or 1–3% NaCl), moderate halophiles (0.5–2.5 M or 3–15% NaCl), and extreme halophiles (2.5–5.2 M or 15–30% NaCl; Kushner, 1978). Halomonas elongata is a moderately halophilic, Gram-negative bacterium isolated from a solar saltern (Vreeland et al., 1980; Schwibbert et al., 2011). It is a well-established model organism for studying the molecular basis of bacterial osmoadaptation, as it grows at a wide range of salinities (0.5–3.0 M NaCl; Vreeland et al., 1980; Gunde-Cimerman et al., 2018; Hobmeier et al., 2022). Moreover, H. elongata has significant potential for industrial applications, producing various bioactive metabolites (Yin et al., 2015; Benitez-Mateos and Paradisi, 2023; Yan et al., 2025).

A critical osmoadaptation strategy that enables halophiles to cope with hypersaline environments is the synthesis and accumulation of compatible solutes, which are highly water-soluble organic molecules that do not impair cell metabolism even at high intracellular levels (Gunde-Cimerman et al., 2018; Shu and Huang, 2022). H. elongata achieves a broad salt tolerance by accumulating the compatible solute ectoine in the cytoplasm (Schwibbert et al., 2011; Kindzierski et al., 2017). The metabolic pathway of ectoine has been well-characterized in H. elongata. LysC, an aspartate kinase, converts L-aspartic acid to L-4-aspartyl-phosphate, which is then converted by Asd to L-aspartate-4-semialdehyde, a key intermediate in the aspartate pathway (Schwibbert et al., 2011; Hermann et al., 2020). Subsequently, three enzymes encoded by the conserved ectABC operon are involved in ectoine de novo synthesis (Hu et al., 2024; Zou et al., 2026). The L-2,4-diaminobutyric acid (DABA) transaminase EctB converts L-aspartate-4-semialdehyde to DABA, which is acetylated by EctA (DABA-Nγ-acetyltransferase) to form Nγ-acetyl-L-2,4-diaminobutyric acid (Schwibbert et al., 2011). Finally, the cyclic condensation reaction is catalyzed by EctC (ectoine synthase) to yield ectoine. In addition to serving as a compatible solute, ectoine can also be used as a nutrient (Schwibbert et al., 2011). The gene cluster doeABCD encodes four enzymes involved in ectoine degradation (Schwibbert et al., 2011; Hermann et al., 2020; Reshetnikov et al., 2020). DoeA (ectoine hydrolase) hydrolyzes ectoine to Nα-acetyl-L-2,4-diaminobutyrate, which is deacetylated by DoeB to form DABA (Schwibbert et al., 2011; Reshetnikov et al., 2020). Then DoeD converts DABA to L-aspartate-4-semialdehyde, which is oxidized by DoeC to aspartate (Schwibbert et al., 2011; Reshetnikov et al., 2020). It has been shown that the promoter of ectA contains binding sites for sigma factor σ70 and σ38, while a σ54-controlled promoter is located upstream of ectC (Schwibbert et al., 2011; Stiller et al., 2018). The doeX gene, adjacent to the doeAB operon, encodes an AsnC/Lrp family transcriptional regulator that controls doeAB transcription (Schwibbert et al., 2011). However, the doeCD genes are not part of the doeABX operon and the underlying regulatory mechanism remains to be elucidated.

Quorum sensing (QS) is a complex cell-to-cell communication system that orchestrates bacterial group behaviors in a population density-dependent manner (Papenfort and Bassler, 2016; Fan et al., 2022). It is mediated by small signaling molecules and plays a pivotal role in bacterial adaptation to their environment (Papenfort and Bassler, 2016; Fan et al., 2022). A variety of QS signal molecules have been identified to date such as autoinducer-2 (AI-2), acyl-homoserine lactones (AHLs), and Pseudomonas quinolone signal (PQS; Papenfort and Bassler, 2016; Lin et al., 2018). LuxS is a bifunctional enzyme that plays a key role in the activated methyl cycle (AMC) and also catalyzes the production of the quorum sensing signal AI-2 from S-ribosylhomocysteine (Pereira et al., 2013). Notably, the AI-2 synthase LuxS is widespread and exists in both Gram-positive and Gram-negative bacteria, thereby mediating intra- and inter-species communication (Pereira et al., 2013). Beyond roles in bioluminescence, virulence, chemotaxis, and biofilm formation, the LuxS/AI-2 QS system has also been demonstrated to enhance bacterial stress resistance under adverse environmental conditions (Wang et al., 2018, 2019; Song and Wood, 2021; Deng et al., 2022; Cao et al., 2023; Zhang et al., 2025). Nevertheless, the characterization of the LuxS/AI-2 QS system in halophilic microorganisms remains unclear.

In the present study, the role of the LuxS/AI-2 QS system in H. elongata was investigated. Unexpectedly, we found that the AI-2 activity of H. elongata is stimulated with increasing sodium chloride concentration up to 1.5 M but decreased above this concentration. Functional analysis revealed that the LuxS/AI-2 QS system contributes to osmotic adaptation in H. elongata by regulating the transcription of the doeCD genes, thereby modulating intracellular ectoine levels. Furthermore, the LuxS/AI-2 QS system is also involved in motility and the type VI secretion system (T6SS) in H. elongata. These findings may help further our understanding of the regulatory mechanism of the LuxS/AI-2 QS in Halomonas and other bacteria.

Materials and methods

Bacterial strains and growth conditions

The bacterial strains and plasmids used in this study are listed in Supplementary Table S1. H. elongata DSM 2581 and derivatives were grown at 30 °C in LB1M medium (tryptone 1%, yeast extract 0.5%, NaCl 1 M). E. coli strains were cultured at 37 °C in LB medium (tryptone 1%, yeast extract 0.5%, NaCl 0.17 M). V. harveyi MM32 was grown at 30 °C in AB medium (Bassler et al., 1993). Antibiotics were added at the following concentrations: Kanamycin, 50 μg/ml; Chloramphenicol, 20 μg/ml for E. coli and Ampicillin, 100 μg/ml; Kanamycin, 50 μg/ml; Chloramphenicol 20 μg/ml for H. elongata.

Plasmid construction

Primers used in this study are listed in Supplementary Table S2. The suicide plasmid pK18mobsacB-ΔluxS was constructed to prepare the ΔluxS in-frame deletion mutant. Briefly, the 927-bp upstream fragment and 902-bp downstream fragment of luxS were amplified with primer pairs luxSUF-BamHI/luxSUR and luxSDF/luxSDR-SalI. Then, the upstream and downstream fragments were inserted between BamHI and SalI restriction sites of pK18mobsacB by Seamless Cloning, resulting in pK18mobsacB-ΔluxS. To express His-tagged LuxS (HELO_2343) and Pfs (HELO_2341), primers luxSF-BamHI/luxSR-SalI and pfsF-BamHI pfsR-SalI were used to amplify luxS and pfs fragments from genomic DNA of H. elongata. The PCR products of luxS and pfs were inserted into BamHI and SalI restriction sites of pET28a by Seamless Cloning, resulting in plasmids pET28a-luxS and pET28a-pfs. To complement the luxS mutant, primers luxSF-KpnI and luxSR-HindIII were used to amplify the luxS gene fragment from H. elongata genomic DNA. The PCR product of luxS was inserted into the KpnI/HindIII sites of pBBR1MCS1 by Seamless Cloning, resulting in plasmid pBBR1MCS1-luxS. pBBR1MCS2-hcp-his6 was constructed in the same way by using the primers listed in Supplementary Table S2. To construct the sfGFP fusion reporter vector PluxS-sfGFP, primers PluxSF-EcoRI and PluxSR-SalI were used to amplify the luxS promoter region from H. elongata genomic DNA. The PCR product of the luxS promoter region was inserted into the EcoRI/SalI sites of pKHGZ by Seamless Cloning, resulting in plasmid PluxS-sfGFP. The sfGFP fusion reporter vectors PectA-sfGFP, PdoeC-sfGFP, PfliF-sfGFP, and Pt6ss-sfGFP were constructed in similar manners by using the primers listed in Supplementary Table S2. The integrity of the insert in all constructs was confirmed by Sanger sequencing.

In-frame deletion and complementation in H. elongata

To construct in-frame deletion mutants, pK18mobsacB derivatives were transformed into relevant H. elongata strains through E. coli S17-1(λpir)-mediated conjugation (Grammann et al., 2002), and the transconjugants were selected by plating on LB1M agar plates supplemented with Ampicillin (100 μg/ml) and kanamycin (50 μg/ml). The in-frame deletion mutants were subsequently screened on LB1M plates containing 20% sucrose. All mutants were verified by PCR and DNA sequencing. For complementation, the pBBR1MCS1-luxS plasmid was transformed into ΔluxS mutant through E. coli S17-1(λpir)-mediated conjugation, and the transconjugants were selected by plating on LB1M agar plates containing kanamycin (50 μg/ml).

Protein expression and purification

The His6-tagged protein was expressed in E. coli strain BL21(DE3). For protein production, bacteria cultures were cultivated in LB medium at 37 °C until OD600 reached 0.5, then shifted to 22 °C and induced with 0.3 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) for an additional 12 h. Harvested cells were washed and resuspended in His binding buffer, and lysed by sonication. The His6-tagged proteins were purified with the His·Bind Ni-NTA resin (Novagen) according to the manufacturer's instructions. Purified proteins were dialyzed overnight at 4 °C and stored at −80 °C until used.

In vitro synthesis of AI-2 and bioluminescence assays

In vitro synthesis of AI-2 was performed as previously described (Zhang et al., 2020). In brief, purified LuxS protein (1 mg/ml) and Pfs protein (1 mg/ml) were added to a reaction buffer (50 mM Tris–HCl, pH 7.5, 500 mM NaCl) containing 1 mM S-adenosylhomocysteine and incubated at 30 °C for 1 h. After incubation, the mixture was filtered through Amicon Ultra-4 filters (limited 3000-molecular-weight cutoff; Millipore) to remove proteins. AI-2 levels in the filtrate were measured using Ellman's reagent (DTNB; Plummer et al., 2011). For exogenous AI-2 supplementation, the synthesized AI-2 was dissolved in sterile water to 1 mM and added to the ΔluxS mutant culture at a final concentration of 1 μM at the time of inoculation. An equal volume of sterile water was added to the control group. For bioluminescence assay, an overnight culture of the V. harveyi MM32 reporter strain in AB medium was diluted 1:5000 into fresh medium (Zhang et al., 2025). Then, 90 μl aliquots of the diluted cells were added to a white, flat-bottomed 96-well microtiter plate (Corning, USA). H. elongata strains were grown in LB medium supplemented with various concentrations of NaCl at 30 °C with shaking at 220 rpm until the culture reached an OD600 of 1.2. The culture was then centrifuged at 8000 rpm for 10 min at 4 °C to remove bacterial cells. The resulting supernatant was carefully collected and filtered through a 0.22 μm filter (Millipore). A volume of 10 μl of the cell-free supernatant was added to the wells, and the plate was incubated at 30 °C for 6 h with shaking at 150 rpm. Luminescence was detected using a chemiluminescence imaging system (Tanon 4600, China). Quantification of luminescence intensity was measured using the BioTek FLx800 Microplate Reader. AI-2 activity is reported as fold induction relative to the buffer control.

Measurement of the promoter activities by sfGFP fusions

Expression of the sfGFP-based reporters was measured as previously described (Boas Lichty et al., 2023; Zou et al., 2024). The sfGFP reporters were transformed into relevant H. elongata strains through E. coli S17-1(λpir)-mediated conjugation. The resulting strains were grown overnight in LB1M medium. The cultures were then diluted 1:100 into fresh LB1M medium and grown to an OD600 of 1.2. Subsequently, 200 μl of each culture was transferred to a black-walled, clear-bottom 96-well plate. Fluorescence (excitation/emission: 488/510 nm) was measured using the BioTek FLx800 Microplate Reader, and bacterial growth was monitored (OD600) with the BioTek Epoch 2 plate reader. Specific fluorescence is expressed as relative fluorescence intensity (RFU) divided by the optical density of each well.

RNA-seq and data analysis

The H. elongata wild-type and ΔluxS mutant strains were cultured in LB1M medium at 30 °C with shaking at 220 rpm until OD600 reached 1.2. Total RNA was extracted from each sample by using the TRIzol-based method (Tiangen, China). cDNA libraries were constructed using a SuperScript III reagent kit (Invitrogen Life Technologies) and sequenced by Shanghai Sangon Biotechnology Co., Ltd (Illumina HiSeq 2500). RNA-seq reads were mapped to the H. elongata DSM 2581 reference genome (ASM19687v2) using Bowtie 2. Differentially expressed genes (DEGs) were identified using DESeq2 (Benjamini–Hochberg adjusted P < 0.05 and |log 2 fold change| > 1) for the comparisons between H. elongata DSM 2581 wild-type and ΔluxS. The identified DEGs were enriched by Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway.

RNA extraction and quantitative real-time PCR

All strains were cultured in LB1M medium to an OD600 of 1.2. Total RNA was extracted from each sample by using the TRIzol-based method (Tiangen, China). cDNA was synthesized using PrimeScript Reverse Transcriptase (TaKaRa, China). Quantitative RT-PCR (qRT-PCR) was performed using TransStart green qPCR SuperMix (TransGen Biotech, China) on the CFX Duet Real-Time PCR System (Bio-Rad, USA). The 16S rRNA was used as the internal reference gene.

Quantification of intracellular ectoine levels

For quantification of intracellular ectoine, H. elongata strains were grown in 150 ml LB1M at 30 °C to an OD600 of 1.2. A total of 50 ml culture was centrifuged at 8,000 rpm for 10 min. The cell pellet was resuspended in 30 ml ultrapure water and lysed by sonication. After centrifugation at 12,000 rpm for 10 min, the supernatant was filtered through a 0.22 μm filter membrane for high-performance liquid chromatography (HPLC) analysis (Zou et al., 2026). Samples were analyzed using an Agilent 1260 Infinity II HPLC system equipped with a C18 reverse-phase column and a UV detector. Ectoine was eluted with 5% phase A (methanol) and 95% phase B (20 mM KH2PO4, 7 mM sodium 1-pentanesulfonate, pH 3.0), and the flow rate was 0.8 ml/min. The wavelength of the UV detector was 210 nm. Ectoine (number 81619; Sigma) was purchased from Sigma and used as a standard. The ectoine concentration of samples was determined from a standard curve established with a serially diluted ectoine solution.

Western blot analysis

Western blot analysis was performed as previously reported (Wang et al., 2015a; Li et al., 2025). Samples were separated by electrophoresis on 12% SDS–PAGE and transferred to polyvinylidene difluoride membranes (Millipore). After blocking with 5% (w/v) bovine serum albumin, the membranes were incubated with the appropriate primary antibodies: anti-His (Solarbio, China) and anti-RNAP (Solarbio, China). After five times washes with TBST buffer (50 mM Tris, 150 mM NaCl, 0.05% Tween 20, pH 7.4), the membranes were incubated with a 1:5,000 dilution of horseradish peroxidase-conjugated secondary antibody (Solarbio, China). Protein signals were detected using the ECL kit following the manufacturer's protocol.

Swimming motility assay

Swimming motility assay was performed as previously described (Wang et al., 2022). Briefly, H. elongata strains were cultured overnight in LB1M medium. 2.5 μl of bacterial suspension was injected into the center of the semi-solid agar plates (1 M NaCl, 1% tryptone, and 0.3% Difco Bacto agar). All plates were incubated at 30 °C for 32 h.

Protein Secretion Assay

Secretion of Hcp was detected according to previously reported (Song et al., 2021). Briefly, H. elongata strains were grown in 150 ml LB1M at 30 °C to an OD600 of 1.2. A 2 ml culture was centrifuged, and the cell pellet was resuspended in 100 μl SDS-loading buffer to prepare the whole-cell lysate sample. For secreted protein collection, 100 ml culture was centrifuged at 8,000 rpm. Subsequently, the culture supernatant was filtered through a 0.22-μm filter (Millipore) to remove residual bacterial cells, and the extracellular proteins were extracted by filtration over a nitrocellulose filter (BA85; Whatman, Germany) three times. Proteins bound to the nitrocellulose filter were eluted by soaking it in 100 μl of SDS sample buffer at 70 °C for 15 min. All sample preparations were normalized based on the culture OD600 and the volume processed.

Results

AI-2 is synthesized by the LuxS enzyme in H. elongata

Although the LuxS/AI-2 QS system is well characterized in Vibrio harveyi (Chen et al., 2020) and Escherichia coli (Mayer et al., 2023), it remains largely unexplored in halophiles. To test whether H. elongata produces functional AI-2 molecule, we first determined the ability of the culture supernatant of wild-type to stimulate light production in the AI-2 reporter strain Vibrio harveyi MM32 (Miller et al., 2004). The culture supernatant of wild-type promoted light production in strain MM32 at levels comparable to those induced by 1 μM AI-2, whereas the fresh medium did not induce bioluminescence in strain MM32 (Figure 1A). Sequence alignment analysis showed that H. elongata LuxS and Pfs (S-adenosylhomocysteine nucleosidase) exhibited 40.41–46.15% and 48.48–53.30% sequence identity, respectively, with their counterparts in several other bacteria, including E. coli, Salmonella enterica, Vibrio campbellii, and Bacillus velezensis, and both LuxS and Pfs contain important enzyme active sites (Plummer et al., 2011; Wang et al., 2015b; Figures 1B, C). We further constructed a ΔluxS mutant of H. elongata and confirmed that bioluminescence was abolished in the ΔluxS mutant (Figure 1D). In addition, in vitro enzymatic assays using S-adenosylhomocysteine as substrate with purified LuxS and Pfs confirmed AI-2 synthesis activity (Figure 1E). These results suggest that AI-2 is produced by LuxS in H. elongata.

Figure 1.

Panel A shows a bar graph measuring AI-2 activity with three groups: WT, LBM, and LBM plus AI-2, with significant differences indicated and a dot blot image above. Panel B displays sequence alignments of HELO_2343 and related proteins with conserved residues highlighted. Panel D features a bar graph comparing AI-2 activity in WT, ΔluxS, and ΔluxS plus AI-2 with significant differences marked and a corresponding dot blot image above. Panel C presents sequence alignments of HELO_2341 and related proteins with conserved residues highlighted. Panel E shows a bar graph of AI-2 concentration for LuxS plus Pfs, LuxS, and Pfs, indicating a significant difference.

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Confirmation of AI-2 activity in H. elongata. (A) AI-2 activity in supernatant of H. elongata wild-type. Cell supernatants were collected, filtered, and measured for AI-2 activity using the reporter V. harveyi strain MM32. AI-2 activity is reported as fold induction over the buffer control. Data are shown as mean ± SD from three independent biological replicates (n = 3). Statistical significance was determined using one-way ANOVA with Tukey's post-hoc test. (B) LuxS amino acid multiple sequence alignment. LuxS proteins used in the sequence alignment are as follows: H. elongata (HELO_2343); Escherichia coli (ECD_02542); Salmonella enterica subsp. enterica serovar Typhimurium str. SL1344 (SL1344_2802); Vibrio campbellii (VIBHAR_03484); Bacillus velezensis (RBAM_027680). The important enzyme active sites are shown in blue on a yellow background. (C) Pfs amino acid multiple sequence alignment. Pfs proteins used in the sequence alignment are as follows: H. elongata (HELO_2341); E. coli (ECD_00158); S. enterica serovar Typhimurium SL1344 (SL1344_0208); V. campbellii (VIBHAR_00923); B. velezensis (RBAM_024370). The important enzyme active sites are shown in blue on a yellow background. (D) AI-2 activity detection of H. elongata wild-type and ΔluxS mutant. Data are shown as mean ± SD from three independent biological replicates (n = 3). Statistical significance was determined using one-way ANOVA with Tukey's post-hoc test. (E) Synthesis detection of AI-2 in vitro. The products from the in vitro reaction of S-adenosylhomocysteine with LuxS and Pfs were measured using Ellman's reagent. Data are shown as mean ± SD from three independent biological replicates (n = 3). Statistical significance was determined using one-way ANOVA with Tukey's post-hoc test. ***P < 0.001; ****P < 0.0001.

The LuxS/AI-2 QS system is closely associated with salinity and contributes to osmotic adaptation

Several studies have demonstrated that the LuxS/AI-2 QS system promotes bacterial survival in stressful environments (Wang et al., 2019; Cao et al., 2023; Zhang et al., 2025). In general, high-salinity environments are harmful to bacteria, whereas H. elongata can tolerate salt concentrations well above 1.7 M NaCl (Tanimura et al., 2016). This prompted us to investigate whether the LuxS/AI-2 QS system plays a role in its osmotic adaptation. Interestingly, the AI-2 activity in the culture of wild-type H. elongata is stimulated by increasing NaCl concentration up to 1.5 M but reduced above this level (Figure 2A). Consistently, quantitative real-time PCR (qRT-PCR) analyses showed that the transcriptional levels of luxS exhibited similar trends to those of NaCl concentration (Figure 2B). Promoter activity assays were performed using the luxS-sfGFP plasmid as the reporter. The results revealed that luxS promoter activity was increased with NaCl concentration up to 1.5 M, while declined above this threshold (Figure 2C). These results indicated that the LuxS/AI-2 QS system is closely associated with salinity.

Figure 2.

Figure with three bar charts labeled A, B, and C, comparing results for 0.5 M, 1.0 M, 1.5 M, and 2.0 M NaCl conditions. A shows AI-2 activity peaking at 1.5 M NaCl. B presents relative mRNA expression, also highest at 1.5 M NaCl. C displays fold change in PluxS-sfGFP fluorescence, again peaking at 1.5 M NaCl. Panel A includes a circular dot blot image above the chart. Error bars are present on all bars.

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The LuxS/AI-2 QS system is closely associated with salinity. (A) AI-2 activity in wild-type H. elongata at 0.5–2.0 M NaCl. H. elongata was grown in LB medium supplemented with 0.5, 1.0, 1.5, and 2.0 M NaCl at 30 °C until reaching an OD600 of 1.2. Cell-free supernatants were subsequently collected and analyzed for AI-2 activity using the reporter V. harveyi strain MM32. Data are shown as mean ± SD from three independent biological replicates (n = 3). (B) The transcription levels of luxS at 0.5–2.0 M NaCl in H. elongata. H. elongata cells were cultivated under different salinity conditions, and the transcription levels of luxS were determined by qRT-PCR. Data are shown as mean ± SD from three independent biological replicates (n = 3). (C) The promoter activity of luxS in wild-type H. elongata at 0.5–2.0 M NaCl. Cultures were grown at 0.5–2.0 M NaCl and relative fluorescence intensity (RFU) was measured. Data are shown as mean ± SD from three independent biological replicates (n = 3).

To investigate whether the LuxS/AI-2 QS system contributes to osmotic adaptation in H. elongata, we monitored the growth of the wild-type, luxS mutant, and complemented strains under varying NaCl concentrations. To complement the ΔluxS mutant, the pBBR1MCS1 derivative harboring full-length luxS under a constitutive lac promoter was introduced into the ΔluxS mutant via conjugation, and the empty plasmid pBBR1MCS1 served as a vector control in all experiments. It was observed that the absence of the LuxS/AI-2 QS system resulted in severe growth inhibition at 0.5, 1.5, and 2.0 M NaCl compared to the wild-type and complemented strains (P < 0.01; Figures 3A, C, D). Additionally, under 1.0 M NaCl, the luxS mutant grew slowly during early and mid-exponential phases compared to the wild-type and complemented strains, while no significant difference was observed at stationary phase (Figure 3B). Taken together, these results demonstrated that the LuxS/AI-2 QS system contributes to osmotic adaptation in H. elongata.

Figure 3.

Set of four line graphs labeled A to D, each showing bacterial growth (OD600) over 48 hours at increasing NaCl concentrations (0.5, 1.0, 1.5, and 2.0 M) for three strains: wild-type with vector (blue), ΔluxS with vector (red), and ΔluxS with complemented luxS (yellow). Strains differ significantly at higher salt concentrations, with ΔluxS showing lower growth (** and ****, p-values indicated). Error bars represent standard deviations.

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Growth analysis of H. elongata wild-type, ΔluxS mutant, and the complementary strain ΔluxS (luxS) at various NaCl concentrations. Cells were cultivated in LB medium supplemented with 0.5 M (A), 1.0 M (B), 1.5 M (C), and 2.0 M (D) NaCl, and growth was monitored every hour for 48 h at 30 °C with shaking at 300 rpm using the MicroScreen high-throughput real-time microbial growth analysis system (Gering). Statistical significance at the 48 h time point was determined by an unpaired two-tailed Student's t-test. Mean and standard deviation of three biological replicates are shown (n = 3). **P < 0.01; ****P < 0.0001.

Transcriptomic analysis of H. elongata and its luxS mutant

To elucidate the molecular mechanism underlying the role of the LuxS/AI-2 QS system in osmotic adaptation of H. elongata, RNA-seq analysis was performed to compare the transcriptomic profiles of the wild-type and ΔluxS mutant strains. Differentially expressed genes (DEGs) between the wild-type and ΔluxS mutant strains were identified using DESeq2, with significance defined as an adjusted P < 0.05 and |log2 fold change| > 1.

A total of 351 DEGs were identified between the wild-type and ΔluxS mutant strains, including 189 upregulated and 162 downregulated genes (Figure 4A and Supplementary Table S3). The heatmap of these DEGs shows consistent expression patterns within each group and differ significantly between wild-type and ΔluxS mutant strains (Figure 4B). KEGG pathway enrichment analysis revealed that these DEGs are involved in various processes, including the two-component system, ABC transporters, flagellar assembly, and bacterial secretion system (Figure 4C). Notably, the upregulated transcripts in ΔluxS included genes associated with ectoine degradation and T6SS system, such as doeC, doeD, eutB, vasI, clpV, vipA, and vipB (Supplementary Table S3). In contrast, the repressed transcripts included genes involved in flagellar assembly, such as flhA, flhB, fliH, flgE, flgD, and flgA (Supplementary Table S3). These results suggest that the LuxS/AI-2 QS system modulates key metabolic and cellular processes in H. elongata.

Figure 4.

Panel A shows a bar chart indicating 189 upregulated and 162 downregulated differentially expressed genes (DEGs) in ΔluxS compared to wild type. Panel B displays a heat map of gene expression across biological replicates of wild type and ΔluxS strains, with expression levels ranging from blue (low) to red (high). Panel C contains a horizontal bar graph summarizing downregulated and upregulated pathways in ΔluxS versus wild type; notable changes appear in bacterial chemotaxis, flagellar assembly, bacterial secretion, and two-component system pathways.

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Transcriptome analysis of H. elongata wild-type and ΔluxS strains. (A) The number of upregulated and downregulated DEGs in wild-type and ΔluxS mutant. (B) Cluster heatmap of DEGs at the transcriptional level. (C) KEGG pathway enrichment analysis of DEGs between wild-type and ΔluxS mutant.

The LuxS/AI-2 QS system modulates intracellular ectoine levels by regulating doeCD in H. elongata

Our RNA-seq data revealed that the deletion of luxS induces expression of doeC, doeD, and eutB genes in H. elongata (Figure 5A). Previous studies have shown that the enzymes encoded by doeC and doeD, but not eutB, are involved in ectoine degradation (Schwibbert et al., 2011). To validate the transcriptome data, qRT-PCR analysis confirmed that the expression levels of doeC and doeD were significantly higher in the ΔluxS mutant compared to the wild-type and complemented strains (P < 0.01; Figure 5B). Interestingly, no significant differences were observed in the expression of the ectoine biosynthesis genes ectA, ectB, and ectC among the wild-type, ΔluxS mutant, and complemented strains (Figure 5B). In addition, the promoter activity assays demonstrated that the transcription of doeCD rather than ectA was significantly upregulated in the ΔluxS mutant (Figures 5C, D). Notably, exogenous AI-2 supplementation inhibited doeCD promoter activity to near wild-type levels in the ΔluxS mutant (Supplementary Figure S1A). To further examine the role of the LuxS/AI-2 QS system in ectoine metabolism, we measured intracellular ectoine levels in the wild-type, ΔluxS mutant, and complemented strains using high-performance liquid chromatography (HPLC). As shown in Figure 5E, the ΔluxS mutant exhibited a significant reduction in intracellular ectoine concentration relative to the wild-type and complemented strains (P < 0.01). Collectively, these results suggest that the LuxS/AI-2 QS system modulates intracellular ectoine levels by regulating doeCD in H. elongata.

Figure 5.

Five-panel bar graph figure with panels A-E analyzing gene expression and ectoine content in different bacterial strains. Panel A shows increased fold change of doeC, doeD, and eutB genes in ΔluxS compared to wild type. Panel B displays relative mRNA expression for several genes, with significantly increased doeC and doeD expression in ΔluxS(vector) compared to wild type and ΔluxS(luxS), and no significant changes for ectA, ectB, and ectC. Panel C shows higher PdoeC-sfGFP reporter activity in ΔluxS(vector) compared to the other groups. Panel D presents PectA-sfGFP reporter activity without significant differences between groups. Panel E demonstrates reduced ectoine content in ΔluxS(vector) and partial restoration in ΔluxS(luxS) compared to wild type. Statistical significance is indicated by asterisks.

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The LuxS/AI-2 QS system controls intracellular ectoine levels by regulating doeCD. (A) Fold changes of the transcriptional levels of the doeC, doeD, and eutB genes in the ΔluxS mutant vs. wild-type from RNA-seq data. (B) The relative mRNA levels of doeCD and ectABC were measured by qRT-PCR in the wild-type, ΔluxS mutant, and complemented strains. Data are shown as mean ± SD from three independent biological replicates (n = 3). Statistical significance was determined using one-way ANOVA with Tukey's post-hoc test. (C,D) The promoter activity of doeC (C) and ectA (D) in the wild-type, ΔluxS mutant, and complemented strains. Data are shown as mean ± SD from three independent biological replicates (n = 3). Statistical significance was determined using one-way ANOVA with Tukey's post-hoc test. (E) The intracellular concentrations of ectoine in the wild-type, ΔluxS mutant, and complemented strains were detected by HPLC. Data are shown as mean ± SD from three independent biological replicates (n = 3). Statistical significance was determined using one-way ANOVA with Tukey's post-hoc test. **P < 0.01; ***P < 0.001; n.s., not significant.

The LuxS/AI-2 QS system enhances swimming motility in H. elongata

RNA-seq analysis also showed that the expression levels of almost all flagellar-related genes were significantly downregulated in the ΔluxS mutant (Figure 6A and Supplementary Table S3). qRT-PCR analysis confirmed that the expression of flagella assembly genes flhA, flhB, fliE, fliF, flgG, and flgF was significantly decreased in the ΔluxS mutant compared to the wild-type and complemented strains (P < 0.01; Figure 6B). The deletion of luxS significantly represses the promoter activity of fliF compared with the wild-type and complemented strains (P < 0.001; Figure 6C). In contrast, exogenous AI-2 supplementation restored swimming motility in the ΔluxS mutant (Supplementary Figure S1B). We then sought to investigate whether the LuxS/AI-2 QS system affects motility in H. elongata. Swimming motility assays were performed by inoculating 2.5 μl of stationary-phase cultures onto the center of semisolid agar plates, and after incubation at 30 °C for 32 h, the swim diameters were measured. The ΔluxS mutant exhibited significantly reduced swimming motility compared with the wild-type strain (P < 0.001; Figures 6D, E). Expression of luxS restored swimming motility in the ΔluxS mutant (Figures 6D, E). Altogether, these results suggest that the LuxS/AI-2 QS system promotes swimming motility in H. elongata.

Figure 6.

Panel A shows a bar graph displaying fold change in gene expression (ΔluxS/WT) for multiple flagellar and motility-associated genes, all significantly downregulated. Panel B depicts a grouped bar graph of relative mRNA expression of six genes, showing significantly reduced expression in ΔluxS(vector) compared to wild-type and restored in ΔluxS(luxS). Panel C is a bar graph showing PfilF-sfGFP fold change, with ΔluxS showing reduced expression rescued by luxS. Panel D displays three circular agar plates with bacterial motility halos, corresponding to each genetic condition. Panel E is a bar graph quantifying motility halo diameters, with significant reduction in ΔluxS rescued by luxS complementation.

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The LuxS/AI-2 QS system enhances swimming motility. (A) Fold changes of the transcriptional levels of flagellar genes in the ΔluxS mutant vs. wild-type from RNA-seq data. (B) The relative mRNA levels of flhA, flhB, fliE, fliF, flgG, and flgF were measured by qRT-PCR in the wild-type, ΔluxS mutant, and complemented strains. Data are shown as mean ± SD from three independent biological replicates (n = 3). Statistical significance was determined using two-way ANOVA with Tukey's post-hoc test. (C) The promoter activity of fliF in the wild-type, ΔluxS mutant, and complemented strains. Data are shown as mean ± SD from three independent biological replicates (n = 3). Statistical significance was determined using one-way ANOVA with Tukey's post-hoc test. (D) The wild-type, ΔluxS mutant, and complemented strains were inoculated on semi-solid plates supplemented with 1.0 M NaCl and incubated at 30 °C for 32 h. (E) The swimming motility was assessed by measuring the sizes of swimming zones. Data are shown as mean ± SD from three independent biological replicates (n = 3). Statistical significance was determined using one-way ANOVA with Tukey's post-hoc test. **P < 0.01; ***P < 0.001.

The LuxS/AI-2 QS system represses T6SS in H. elongata

According to the RNA-seq data, we found that the deletion of luxS promotes T6SS expression in H. elongata (Figure 7A and Supplementary Table S3). qRT-PCR analysis confirmed that the expression of T6SS-related genes vgrG1, clpV, vipA, and vipB was significantly increased in the ΔluxS mutant compared with the wild-type and complemented strains (P < 0.05; Figure 7B). Exogenous AI-2 supplementation restored T6SS gene expression to levels comparable to the wild-type in the ΔluxS mutant (Supplementary Figure S1C). Additionally, the loss of luxS significantly enhanced the promoter activity of T6SS in the ΔluxS mutant (P < 0.01; Figure 7C).

Figure 7.

Four-panel scientific figure analyzing gene expression and protein secretion in different bacterial strains. Panel A shows a bar graph of fold changes for various genes in ΔluxS compared to wild type. Panel B presents a bar graph comparing relative mRNA expression levels for selected genes between WT(Vector), ΔluxS(Vector), and ΔluxS(luxS), with statistical significance indicated. Panel C displays a bar graph of Pt6ss-sfGFP reporter activity in the three strains, showing significant differences. Panel D contains Western blot images for Hcp secretion and cellular levels, using anti-His and anti-RNAP antibodies, across the three strains.

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The LuxS/AI-2 QS system represses T6SS. (A) Fold changes of the transcriptional levels of T6SS genes in the ΔluxS mutant vs. wild-type from RNA-seq data. (B) The relative mRNA levels of vgrG1, clpV, vipA, and vipB were measured by qRT-PCR in the wild-type, ΔluxS mutant, and complemented strains. Data are shown as mean ± SD from three independent biological replicates (n = 3). Statistical significance was determined using one-way ANOVA with Tukey's post-hoc test. (C) The promoter activity of t6ss in the wild-type, ΔluxS mutant, and complemented strains. Data are shown as mean ± SD from three independent biological replicates (n = 3). Statistical significance was determined using two-way ANOVA with Tukey's post-hoc test. (D) Western blot detection of Hcp secretion in H. elongata wild-type, ΔluxS mutant, and complemented strains. His-tagged proteins were detected with an anti-His antibody. RNA polymerase (RNAP) was used as a loading control. *P < 0.05; **P < 0.01; ***P < 0.001.

To further validate this finding, we assessed the secretion of Hemolysin co-regulated protein (Hcp), which is both a structural protein and a secretion protein of T6SS (Basler, 2015; Manera et al., 2021), in the wild-type, ΔluxS mutant, and complemented strains. In order to detect the secretion of Hcp, a plasmid expressing Hcp-His6 was introduced into indicated H. elongata strains via conjugation to produce the His-tagged Hcp. For the pellet fraction, RNA polymerase (RNAP) was used as a loading control. Western blotting analysis confirmed that Hcp secretion was elevated in ΔluxS mutant relative to the wild-type and complemented strains (Figure 7D). Collectively, these results suggest that the LuxS/AI-2 QS system downregulates T6SS in H. elongata.

Discussion

Previous studies have shown that the LuxS/AI-2 QS system plays an essential role in bacterial adaptation and survival in their environment (Wang et al., 2018, 2019; Cao et al., 2023; Zhang et al., 2025). However, the roles of the LuxS/AI-2 QS system in halophiles are largely unknown. In this work, we report that the LuxS/AI-2 QS system regulates doeCD transcription to modulate intracellular ectoine levels, thus facilitating osmotic adaptation in H. elongata. Additionally, the LuxS/AI-2 QS system regulates motility and T6SS in H. elongata. Exogenous addition of AI-2 to the ΔluxS mutant rescued these phenotypes, confirming that they are specifically mediated by AI-2 signaling rather than metabolic disruption of the activated methyl cycle (Supplementary Figure S1).

H. elongata can tolerate salt concentrations well above 1.7 M NaCl, relying on the synthesis and accumulation of compatible solutes, especially ectoine (Kindzierski et al., 2017; Hermann et al., 2020; Hobmeier et al., 2022). Previous studies have demonstrated that the ectoine-deficient ΔectABC mutant strain is unable to tolerate salt concentrations above 0.5 M NaCl (Goller et al., 1998; Grammann et al., 2002), emphasizing the critical role of ectoine for osmotic adaptation in H. elongata. Intriguingly, our results indicate that the AI-2 activity of H. elongata is closely related to salinity and the deletion of luxS impairs the growth of H. elongata at various salinities, suggesting the LuxS/AI-2 QS system contributes to osmotic adaptation in H. elongata (Figures 2, 3). The intracellular concentration of ectoine in H. elongata is mainly controlled by its synthesis and degradation. The transcript level of the ectABC operon is controlled by σ70 and σ38 promoters upstream of ectA and a σ54 promoter upstream of ectC (Schwibbert et al., 2011; Stiller et al., 2018). However, the transcript level and promoter activity of the ectABC operon are not affected by luxS deletion (Figures 5B, D). The genes doeABCD encodes four enzymes for degrading ectoine to aspartate (Hermann et al., 2020). DoeX regulates expression of the doeAB operon but not the doeCD genes (Schwibbert et al., 2011). Our data indicate that the LuxS/AI-2 QS system represses the transcription of doeCD genes, and the deletion of luxS decreases the intracellular ectoine levels (Figure 5). These findings reveal that H. elongata employs the LuxS/AI-2 QS system to fine-tune intracellular ectoine concentrations by repressing doeCD transcription, thereby achieving precise osmotic adaptation to fluctuating salinity. Although complementation was mediated by a constitutive promoter, confirming that luxS is required for the observed adaptations, whether the native salt-dependent transcriptional dynamics of luxS are physiologically necessary remains to be determined. Furthermore, the molecular mechanism by which the LuxS/AI-2 QS system represses doeCD transcription remains to be elucidated. Whether the downstream effector of the LuxS/AI-2 QS system directly or indirectly regulates the doeCD promoter remains unclear.

Motility is a key behavior enabling bacteria to find an optimal environment (Mitchell and Kogure, 2006; Moreno-Gamez, 2022). Previous studies have revealed diverse regulatory relationships between the LuxS/AI-2 QS system and motility (Pereira et al., 2013; Wang et al., 2018; Song and Wood, 2021; Zhang et al., 2025). In agreement with these reports, our findings show that the LuxS/AI-2 QS system enhances motility by upregulating nearly all flagellar-related genes in H. elongata (Figure 6). Notably, genes involved in flagellar assembly and function in H. elongata are closely associated with salinity (Hobmeier et al., 2022; Yu et al., 2022). These genes have higher transcription levels at high salt concentrations than at low salt concentrations (Hobmeier et al., 2022; Yu et al., 2022). Here, we show that the AI-2 activity of H. elongata exhibits similar trends at varying salt concentrations (Figure 2A), suggesting that H. elongata may employ the LuxS/AI-2 QS system to coordinate motility with salinity changes, enabling it to navigate toward optimal osmotic conditions.

T6SS is a widely distributed transmembrane macromolecular apparatus employed by many Gram-negative bacteria to translocate effectors into adjacent cells or the extracellular environment (Basler, 2015; Singh and Kumari, 2023). Traditionally, T6SS is recognized as an anti-bacterial weapon for interspecies competition (Allsopp and Bernal, 2023; Singh and Kumari, 2023). However, in some species, T6SS is also involved in combating diverse stresses (Lin et al., 2017; Si et al., 2017; Li et al., 2021; Lin et al., 2021; Yu et al., 2021). For example, in Yersinia pseudotuberculosis, OxyR, HpaR, and ZntR coordinately activate the expression of T6SS4 to acquire Zn2+ by secreting a Zn2+-binding effector YezP, thereby alleviating oxidative stresses (Wang et al., 2015a, 2017, 2020). A previous study showed that H. elongata T6SS is upregulated after 2.2 M NaCl (13%) shock treatment (Yu et al., 2024). In Pseudomonas syringae B728a, 10 genes in the T6SS are increased with the osmotic pressure increasing (Freeman et al., 2013). Intriguingly, we observed that the LuxS/AI-2 QS system represses T6SS expression and effector secretion in H. elongata. Whether the LuxS/AI-2 QS-mediated regulation of T6SS has any adaptive role in osmotic adaptation remains to be determined and is a subject for future investigation.

In summary, we revealed that the LuxS/AI-2 QS system regulates osmotic adaptation, motility, and T6SS in H. elongata. These findings shed light on the functional roles of the LuxS/AI-2 QS system in H. elongata, providing new insights into the regulatory mechanism underlying osmoadaptation in halophiles.

Funding Statement

The author(s) declared that financial support was received for this work and/or its publication. This work was supported by grants from the National Natural Science Foundation of China (32300111), the Fundamental Research Program of Shanxi Province (202303021212258), the Support Project for Research Innovation Capability of Young Faculty in Shanxi Higher Education Institutions (2025Q035), and the Yuncheng Science and Technology Foundation (YCKJ-2024022, YCKJ-2023030).

Footnotes

Edited by: Ram Karan, University of Delhi, India

Reviewed by: Luis Mario Hernandez-Soto, Autonomous Metropolitan University, Lerma, Mexico

Houda Zouagui, Mohammed V. University, Morocco

Data availability statement

The RNA-seq data from this study is available in the NCBI Sequence Read Archive (SRA) under the accession number PRJNA1427421.

Author contributions

ZW: Investigation, Methodology, Software, Writing – review & editing, Conceptualization, Project administration, Formal analysis, Funding acquisition, Writing – original draft. YY: Investigation, Writing – review & editing, Formal analysis, Software, Data curation. XG: Formal analysis, Investigation, Writing – review & editing, Data curation. CW: Writing – review & editing, Formal analysis, Methodology. JY: Methodology, Formal analysis, Writing – review & editing. YN: Formal analysis, Writing – review & editing, Methodology. XL: Supervision, Conceptualization, Funding acquisition, Writing – review & editing, Formal analysis, Project administration.

Conflict of interest

The author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Generative AI statement

The author(s) declared that Generative AI was not used in the creation of this manuscript.

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Supplementary material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb.2026.1850263/full#supplementary-material

Supplementary_file_1.docx (229.7KB, docx)

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary_file_1.docx (229.7KB, docx)

Data Availability Statement

The RNA-seq data from this study is available in the NCBI Sequence Read Archive (SRA) under the accession number PRJNA1427421.

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