Quinolone-mediated metabolic cross-feeding develops aluminium tolerance in soil microbial consortia

Bacterial growth conditions

The bacterial species used in this study, R. erythropolis and P. aeruginosa, were isolated from the rhizosphere soil of japonica rice, Nanjing46, grown in acidic red soil. R. erythropolis and P. aeruginosa were shaken overnight (28 °C, 180 rpm) using Luria–Bertani (LB) media (5 g L^–1 yeast extract, 10 g L^–1 tryptone, 10 g L^–1 sodium chloride). When the optical density at 600 nm (OD₆₀₀) reached 0.6–0.8 during the exponential growth phase, single colonies were isolated by streaking on plates into a pure culture. These colonies were then used for subsequent culture experiments.

For further observations and analysis, minimal media and modified minimal media (MM) supplemented with different forms of P were used. The minimal media was composed of 2 g L^–1 (NH₄)₂SO₄, 1.2 mM K₂HPO₄ ∙ 3H₂O, 0.8 mM KH₂PO₄, 1 g L^–1 trisodium citrate, 80 nM MnCl₂ ∙ 4H₂O, 1 μM FeSO₄ ∙ 7H₂O, 0.5 mM MgSO₄ ∙ 7H₂O, and 2% D-glucose. The MM included 0.5 g L^–1 (NH₄)₂SO₄, 0.3 g L^–1 KCl, 0.3 g L^–1 NaCl, 0.03 g L^–1 MnSO₄·H₂O, 0.03 g L^–1 FeSO₄·7H₂O, and 0.03 g L^–1 MgSO₄·7H₂O, supplemented with 1 g L^-1 sodium phytate or 1 g L^-1 tricalcium phosphate as the sole P source, respectively (Supplementary Method 1 and 2). Note that the Al stress conditions in this study were established by addition of AlCl₃ to the culture medium, with the specified concentrations referring to the added concentrations of Al³⁺. AlCl₃ was filtered through a 0.22 μm filter for sterilization before being added to the media with a final pH of 4.0. This approach to establishing Al stress environment has been validated in previous studies^18,29.

Synthetic microbial community preparation for inoculation

Individual colonies of R. erythropolis and P. aeruginosa were inoculated into 50 mL of liquid LB media and incubated at 28 °C with shaking at 180 rpm for 24 h, reaching a density of ~2.3 × 10⁸ CFU mL^–1. Then, the two strains were propagated in a fermentation tank for 18 h. The resultant bacteria biomass was harvested using a disk centrifuge method (10,000 × g, 5 min) to remove any residual LB medium. The SynCom formulations consisted of an equimolar mixture of the strains suspended in ultrapure water with a final concentration of 1 × 10⁸ CFU mL⁻¹. Three days following the transplantation of rice seedlings, each plant was treated with 25 mL of the SynCom solution.

Experimental site description and field experiments

The field experiment was established at the Yingtan National Agricultural Ecosystem Observation and Research Station in Jiangxi Province (28°12′N, 116°55′E), China. The experimental area has a typical subtropical climate with an average annual precipitation of 1881.8 mm and an average annual temperature of 18.4 °C. The field soil used in the experiment was Quaternary red clay with a soil content of 36%, which is strongly weathered and has high Al and iron oxide contents. It is classified as a typical Plinthosol by the US soil classification system, and its relevant physical and chemical properties are shown in Supplementary Data 2. In accordance with local traditional farming methods, the experimental field was managed using a single rice crop system per year, employing furrow irrigation and consistent weed management throughout the growing season.

Rice seeds from japonica Nanjing 46 were surface sterilized in 75% ethanol for 30 s and 2.5% sodium hypochlorite three times for 15 min and germinated on Murashige and Skoog (MS) agar media for 15 days (25 °C). Rice plants in similar growth stages were subsequently transplanted into field plots, with 40 plants per plot. The size of each plot was 1.5 m × 6.0 m, with furrows serving as boundaries. The field experiment included four treatments: non-inoculation (CK), mono-inoculation with R. erythropolis (Rh), mono-inoculation with P. aeruginosa (Ps), and inoculation with co-cultured R. erythropolis and P. aeruginosa (RP), with each treatment having one plot (Supplementary Fig. 1). During the heading and maturity stages of rice, photos were taken using a digital camera (RICH GRIII, R02010, Vietnam), and the leaf chlorophyll content (SPAD-502-plus, Konica Minolta, Japan) and rice plant height were measured. A total of 40 rice plants were harvested at 110 days after transplanting. Shoot biomass and panicle weight per plant were measured.

Pot experiments

The soil used for the pot experiment was collected from the field site. The soil was sieved through a 10-mesh screen to remove stones and impurities. A total of 145.6 g of red soil was transferred to a 160 mL root box and compacted to ensure a bulk density of 1.3 for each root box. Surface-sterilized rice seedlings from Nanjing46 were transplanted with one plant per root box. The pot experiment included four treatments: non-inoculation, mono- and co-cultures of R. erythropolis and P. aeruginosa. Each treatment was replicated five times. The leaf chlorophyll content and rice plant height were assessed at seven-day intervals while documenting the growth status of the plants. At maturity, shoot and grain samples were collected from the rice plants. The roots were shaken to remove loosely adherent soil, followed by collection of adhering soil (rhizosphere soil) from the root surface³⁰. Root samples were dried to a constant weight in an oven at 65 °C and then Al concentration in rice roots was measured (Supplementary Method 3). Soil samples were immediately stored in a −80 °C freezer for subsequent determination of the absolute abundance of 16S rRNA genes.

Reverse-Raman-D₂O for assessing the bacterial metabolic activity

Reverse-Raman-D₂O was used to measure the metabolic activity of the bacteria³¹. In brief, single strains of R. erythropolis and P. aeruginosa and their co-cultures were initially cultured in LB media supplemented with 50% heavy water (99.9 atomic% D, CIL, Inc., USA) for 24 h in triplicate. After labeling the cells with heavy atoms D, the cultures were transferred to minimal media without D₂O. Different concentrations of AlCl₃ (0, 0.1, and 1 mM, pH 4.0) were added under sterile conditions. The cultures were then incubated at 28 °C and 180 rpm for 5 h. Cells were harvested by centrifugation (13,600 × g, 10 min) at room temperature, washed twice with ultrapure water, and resuspended in deionized water to disperse cell clusters.

For Raman measurement, 2 μL of cell suspension was loaded on Al foil and air-dried. Raman spectra were acquired using a LabRAM HR Evolution microscope (HORIBA Scientific, France) with a 532 nm Nd:YAG laser (laser Quantum), a 100× objective lens (Olympus, NA = 0.9), and a spectral range of 400–3200 cm⁻¹. A total of 30 individual bacteria were randomly selected from each treatment for Raman measurement. Baseline correction, normalization, and subsequent Fourier transformation were consistently performed on all measurements using LabSpec6 software (Horiba Jobin-Yvon). The C–D _ratio was calculated to assess D assimilation, using the integral intensity of the C–H peak (2800–3100 cm⁻¹) and C–D peak (2040–2300 cm⁻¹), with lower C–D _ratio indicating higher metabolic activity under Al stress. In co-culture, the resonance Raman peak of cytochrome C (heme group, 749.95 cm⁻¹) was selected as the basis for distinguishing between P. aeruginosa and R. erythropolis³².

FISH labeling of R. erythropolis

The fresh rhizosphere soil samples were fixed with 320 μL of a 25% (w/v) particle-free paraformaldehyde solution (4% final concentration) supplemented with 1× phosphate-buffered saline (PBS). The mixed suspension was subsequently fixed at 4 °C for 5 h, washed twice with 1× PBS, centrifuged at 10,000 × g for 5 min at 4 °C, and stored in PBS/ethanol (1:1) at −20 °C for further processing. Then, 100 μL of the stored sample was diluted with 900 μL of PBS/ethanol and dispersed by ultrasonication for 30 s.

Subsequently, 30 μL of the dispersed sample was mixed with 60 μL of 1× PBS, 10 μL of 0.01% SDS (w/v) and 10 μL of 1% (w/v) low melting point agarose at 55 °C. 10 μL of the sample suspension was pipetted onto epoxy-coated glass slides (Thermo Fisher Scientific, Wilmington, USA). The slides were dried in an incubator at 37 °C and dehydrated using a graded series of ethanol (50% for 5 min, 80% for 1 min, and 98% for 1 min). For permeabilization of the cell walls, each well of the slides with agarose-embedded samples was treated with 10 μL of a lysozyme solution (10 mg of lysozyme, 100 μL of 0.5 M EDTA (pH 8.0), 100 μL of 1 M Tris–HCl (pH 8.0), and 800 μL of ultrapure H₂O). After incubation in a humidified PE tube (50 mL) for 1 h at 37 °C, the slides were washed using ultrapure water, and dehydrated. Endogenous peroxidase activity was inactivated by the addition of 0.15% H₂O₂ in methanol.

For in situ labeling, an oligonucleotide probe (sequence: 5′-CY3-CACCTGCCAGAAAATCCTTGGATCAACTG-3′) was used of R. erythropolis bacteria³³. The hybridization buffer was set to a formamide concentration of 55% (0.9 M NaCl, 20 mM Tris–HCl (pH 8.0), 10% (w/v) dextran sulfate, 2% (w/v) blocking reagent (Roche, Mannheim, Germany), 0.1% (w/v) sodium dodecyl sulfate, and 55% (v/v) formamide). Hybridization was performed at 37 °C for 2 h, followed by washing in prewarmed buffer. The slides were transferred to a tube containing 50 mL of prewarmed washing buffer (3 mM NaCl, 5 mM EDTA (pH 8.0), 20 mM Tris–HCl (pH 8.0), and 0.01% (w/v) SDS). The slides were then treated with 0.05% (v/v) Triton X-100 (Solarbio Science & Technology Co., Ltd, Beijing, China)-amended PBS for 15 min at room temperature, rinsed with ultrapure water, and dehydrated with ethanol.

Finally, 10 μL of 4,6-diamido-2-phenylindole (DAPI) was added to each well and incubated for 8 min in the dark. The labeled sections were observed using a fluorescence microscope (Nikon Ti-S, Nikon Co., Ltd, Tokyo, Japan) with excitation at 510–560 nm and emission at 590 nm. Twenty different regions were collected from each sample and probe, with more than 1000 cells per region.

Soil DNA extraction

Soil DNA was extracted using a previously described method³⁴. Briefly, extractions were performed from 0.5 g of well-mixed soil from each sample by combining freeze grinding and sodium dodecyl sulfate for cell lysis. The crude DNA was further purified by agarose gel electrophoresis, followed by consecutive extractions with phenol, chloroform, and butanol. The quality of the extracted DNA was assessed based on the absorbance ratio at 260/280 nm and 260/230 nm using a Nanodrop 2000 (Thermo Fisher Scientific, Wilmington, DE, USA) and a Qubit 3.0 spectrophotometer (Thermo Fisher Scientific, USA). All DNA samples were stored at −80 °C.

Absolute quantification of R. erythropolis, P. aeruginosa, and rhizosphere bacteria in soil

qRT‒PCR was performed in a volume of 20 μL, which contained 10 μL of 2× SG Fast qPCR Master Mix (Sangon Biotech Co., Ltd, Shanghai, China), 0.4 μL of 10 μM forward and reverse primers, and 2 μL of template DNA diluted in 7.2 μL of enzyme-free water³⁵. The qRT‒PCR primers were designed and assessed using Primer Premier 6.00 (Premier Biosoft) software with a melting temperature of 80 ± 5 °C. The primer pairs used for each gene are detailed in Supplementary Data 3. The amplified DNA fragments ranged in size from 100 to 300 bp. Using an external standard method, quantitative data were analysed using StepOne software (version 2.3, Applied Biosystems, CA, USA). The abundances of R. erythropolis, P. aeruginosa, and total soil bacteria were calculated as the average fold difference between the samples and the respective 10-fold serial dilutions of plasmid standards in their respective standards³⁶. The bacterial 16S rRNA gene copies number calculated in this study had been adjusted using the rrnDB database (https://rrndb.umms.med.umich.edu/)³⁷. The bacterial abundance is expressed as the number of gene copies per gram of soil.

Inference of potential metabolic pathways for HHQ

Based on previous literature, we established a potential metabolic pathway for R. erythropolis to degrade the quinolone compound HHQ, which involves multiple steps and a series of enzymes, including quinolone monooxygenase, dioxygenase hydrolase, carboxylesterase, and ribotransferase (Supplementary Fig. 7). Among them, alkylquinolone-specific catabolic enzymes (AqdB1, AqdC1, and AqdA1) are key genes involved in the synthesis of the tryptophan precursor AA³⁸. Ribotransferase genes (TrpD and TrpF) participate in the subsequent biosynthesis of tryptophan³⁹. At the same time, tryptophan or its derivatives play an important role in the synthesis of microbial cell walls, especially peptidoglycan⁴⁰. The main skeleton structure of the cell wall peptidoglycan is formed by the polymerization of amino sugars and muramic acid⁴¹. The key functional genes PGAM and UAGCVT participate in the synthesis of N-acetylmuramic acid⁴². N-acetylmuramic acid and N-acetylglucosamine are linked by β-1,4 glycosidic bonds under the regulation of MltG, and DacD can regulate the composition of oligopeptide chains to form a tetrapeptide tail and further bind to form a peptide bridge structure, making peptidoglycan have a mesh structure⁴³. The cell wall formed by the aggregation of peptidoglycan is crucial for maintaining the normal physiological functions and structural integrity of cells under Al stress⁴⁴.

Relative quantification of functional genes in R. erythropolis

Nine functional genes and the housekeeping gene gyrB, were identified to elucidate the impact of tryptophan and HHQ on the metabolism of quinolone substances, tryptophan production, peptidoglycan synthesis, and cell wall peptidoglycan crosslinking in R. erythropolis. The sequences of the primers used for amplification of related genes are listed in Supplementary Data 3. The results of primer specificity validation are provided in Supplementary Figs. 20–23 and Supplementary Data 4. The nucleotide sequences are presented in Supplementary Data 5.

Four experimental groups are included: Rh, RP, a mono-culture of R. erythropolis supplemented with tryptopha (Rh+Trp), and a mono-culture of R. erythropolis supplemented with HHQ (Rh+HHQ). These groups were cultivated in minimal media supplemented with 0, 0.1, and 1 mM AlCl₃, with three replicates for each group at 28 °C and 180 rpm for 24 h. After incubation, the bacterial solution was centrifuged at 13,400 × g and 25 °C, and the cell precipitates were washed in 100 μL cold TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) and stored at −80 °C.

Total RNA extraction, reverse transcription, and qRT‒PCR were performed using previously reported methods³⁸. Briefly, the cell suspension was thawed and resuspended in 1 mL of TE buffer containing 3 mg mL⁻¹ lysozyme for 15 min. Total RNA was extracted using the RNAprep Pure Cell/Bacteria Kit (TIANGEN Biotech Co., Ltd, Beijing, China). The remaining DNA impurities were digested with 80 μL of DNase I without RNA, and then, 1 μg of sample RNA was used to synthesize first-strand DNA at 42 °C through a FastKing RT kit (TIANGEN Biotech Co., Ltd, Beijing, China). The fold change in target gene expression was analysed by the comparative threshold cycle (CT) method⁴⁵. The calculation formula is:

where sample A is the cDNA sample from each treatment group, and sample B is the control group. The amplification efficiency of each primer was detected using LinRegPCR (version 2013.1, Amsterdam, Netherlands).

Prokaryotic chain-specific RNA-seq analysis

R. erythropolis and P. aeruginosa strains were cultured individually or co-cultured in minimal media supplemented with 0 and 0.1 mM AlCl₃ (pH 4.0) for 24 h. Three parallel experiments were conducted for each group. After incubation, the cell suspensions were collected by centrifugation at 13,400 × g for 5 min, and all the samples were immediately transferred to −80 °C for total RNA extraction. The supernatants were passed through a 0.22 μm filter membrane, and 1 mL of each liquid was stored at −20 °C for metabolite identification. Total RNA was extracted from each sample using TRIzol reagent (Merck KGaA, Darmstadt, Germany) according to the manufacturer’s instructions.

The quality of the extracted RNA was assessed using a Nanodrop 2000 (Thermo Fisher Scientific, Wilmington, USA) and an Agilent 4200 Tape Station bioanalyzer (Agilent Technologies, CA, USA). Only RNA samples with an RNA integrity value (RIN) ≥ 7 were selected for cDNA library construction. The quality-controlled RNA samples were further processed by using a Ribo-Zero rRNA removal kit (Bacteria; Epicenter, WI, USA). First-strand cDNA was synthesized using random hexamers, followed by RNA strand degradation using RNase H. Second-strand cDNA was synthesized using DNA polymerase I and dNTPs. The remaining overhangs were converted to blunt ends by exonuclease/polymerase activity, and enzymes were removed using the NEBNext Ultra IITM directional RNA library Prep Kit for Illumina. The 3′ ends of the DNA fragments were adenylated, and Illumina PE adapter oligonucleotides were ligated for hybridization.

The cDNA library fragments were purified using the AMPure XP system (Beckman Coulter, Beverly, USA) to ensure a preferred length of 400–500 bp. The number of PCR cycles was adjusted to 15, and the final amplified library was quality checked using a Bioanalyzer 2100 system (Agilent Technologies, CA, USA). An equimolar library was constructed using the Kapa-sybr FAST qPCR Kit Light Cycler 480 (KK4610) and a reference standard from Kapa Biosystems. Each library was sequenced in paired-end mode using the TruSeq SBS kit v3-HS with a read length of 2 × 76 bp on the HiSeq2000 instrument (Illumina) according to the manufacturer’s protocol for mRNA sequencing experiments.

The R. erythropolis and P. aeruginosa genomes (GenBank assembly accession numbers: GCA_001715845.1 and GCA_016743035.1) were used as the reference genome. FastQC was used to evaluate the quality of the RNA sequencing reads. The raw data in Fastq format were preprocessed using sickle (version 1.2) by removing adapter sequences, poly-N, and low-quality reads to obtain clean data⁴⁶. The expression levels of transcripts and genes were calculated based on the expected fragments per kilobase per million reads (FPKM) model of exons. Differentially expressed mRNAs were detected using DESeq (v1.30.0), defined as transcripts with a fold change > 1.5 and a FDR < 0.05. P-values were calculated using a negative binomial distribution and corrected for multiple testing by Benjamini‒Hochberg (B&H). Weighted gene co-expression network analysis (WGCNA)⁴⁷ was performed using the “clusterProfiler” package in R software 4.0.5. Note that the cell wall components pathway includes peptidoglycan (ko00550), arabinogalactan (ko00572), and lipopolysaccharide (ko00571) biosynthesis pathway. Network analysis and visualization were carried out using Gephi software (version 0.9.2).

Metabolite assays

The metabolic compounds produced by R. erythropolis and P. aeruginosa in mono-cultures and co-culture were identified using a single quadrupole GC‒MS system. The bacteria were cultivated in minimal media supplemented with 0 or 0.1 mM AlCl₃ (pH 4.0) for 24 h in triplicate. After incubation, the cell suspensions were collected by centrifugation at 13,400 × g. The supernatants were transferred evenly to 2 mL Eppendorf centrifuge tubes. Then, 0.5 mL of 3:1 (v/v) methanol:water was added to each tube, and the mixture was vortexed. Quality control samples were prepared with equal volumes of minimum medium. Subsequently, the supernatants were centrifuged at 12,000 × g for 15 min at 4 °C, and 350 μL was transferred to glass sampling bottles. After adding 50 μL of BSTFA (containing 1% TMCS), derivatization was carried out for 60 min at 70 °C. The derivatized sample was dried under nitrogen, reconstituted in 500 μL of hexane (chromatographically pure), filtered through a 0.22 μm membrane, and stored at 4 °C.

The derivatives were analysed on an Agilent 7890B gas chromatography system coupled with an Agilent 5977 A single quadrupole system (Agilent Technologies Inc., CA, USA). The separation of the derivatives was performed using a DB-5 MS fused silica capillary column (30 m × 0.25 mm × 0.25 μm, Agilent JW Scientific, Folsom, CA, USA). The injector temperature was maintained at 250 °C, and a sample volume of 1 μL was injected in splitless mode. The following GC temperature program was used: the initial temperature was set to 80 °C and held for 1 min, then increased at a rate of 5 °C min⁻¹ over 40 min to 280 °C, held for 10 min at 280 °C, and finally decreased to 80 °C and held for 2 min. The temperatures of the ion source and connector MS were set to 200 °C and 285 °C, respectively. Mass spectra data were acquired in Q3 scan mode (m/z 45–800) with a solvent delay time of 2.5 min. The raw GC‒MS data (D format) were converted to a general format (CDF format) using ChemStation analysis software (version E.02.1431, Agilent, CA, USA). Then, the preprocessed data were analysed using Chroma TOF (version 4.34, LECO, St Joseph, MI, USA). The metabolites were characterized using the National Institute of Standards and Technology (NIST) database.

Production and degradation of HHQ

To examine the production of HHQ by P. aeruginosa and its degradation by R. erythropolis under both aqueous culture and soil conditions, five experiments were conducted. In summary, water-based experiments included: (1) mono- and co-cultures of R. erythropolis and P. aeruginosa in minimal media supplemented with 0.1 mM AlCl₃ (pH 4.0); (2) mono-culture of R. erythropolis and mono-culture with an initial concentration of 20 μM HHQ in minimal media, supplemented with 0, 0.1, and 1 mM AlCl₃ (pH 4.0); (3–5) pot experiments in natural and sterilized acidic soil, as well as in a sterilized clay-based system, with the following treatments: non-inoculation, mono- and co-cultures of R. erythropolis and P. aeruginosa, inoculation with R. erythropolis and 20 μg L⁻¹ HHQ. Additionally, an initial HHQ concentration of 20 μg L⁻¹ was established to mimic natural soil levels in sterilized conditions.

In experiment 1 and 2, each group was conducted in triplicate, incubated at 28 °C and 180 rpm for 24 h. The bacterial solution was centrifuged at 13,400 × g. The HHQ concentration was determined using a relative quantification method (Supplementary Method 4). For pot experiment 3–5, three replicates were also maintained, and the HHQ concentration was measured using an absolute quantification method (Supplementary Method 5).

Muramic acid concentration in R. erythropolis

The experimental groups included mono-culture of R. erythropolis, co-culture of R. erythropolis and P. aeruginosa, mono-culture of R. erythropolis supplemented with HHQ, and a mono-culture of R. erythropolis supplemented with tryptophan. These groups were cultivated in minimum medium supplemented with 0, 0.1, and 1 mM AlCl₃, with three replicates for each group at 28 °C and 180 rpm for 24 h. The cultured bacterial solution was centrifuged for 10 min at 13,600 × g to remove the supernatant and obtain the bacterial precipitate.

The bacterial precipitate was dehydrated using a freeze dryer (FreeZone 4.5, Labconco, USA) to obtain a bacterial powder. Subsequently, 0.5 g of bacterial powder was placed in a hydrolysis bottle, and 10 mL of 6 M hydrochloric acid was added and swirled evenly. The mixture was hydrolyzed at 105 °C (in an oven) for 8 h and then vortexed and allowed to stand overnight. 0.5 mL of the supernatant was carefully transferred to a glass test tube, and 100 µL of N-methylglucosamine solution (1 mg mL⁻¹) was added as an internal standard. The mixture was dried under a stream of nitrogen, then diluted to 1 mL with methanol for analysis. After filtration through a 0.22 μm membrane, the concentration of muramic acid in the cell wall was detected using an AB 5500 liquid chromatography-tandem mass spectrometry (LC-MS/MS) system (AB SCIEX LLC, MA, USA).

Separation was performed using a Waters Hillc column (100 mm × 2.1 mm, 1.7 µm, Waters, Milford, MA, USA) with a mobile phase consisting of 0.1% formic acid in water (A) and acetonitrile (B) at a flow rate of 0.3 mL min⁻¹ and an injection volume of 1 μL. The elution program was as follows: initially 90% phase B was maintained for 0.5 min, then decreased from 90% phase B to 50% within 6.5 min, followed by a maintenance period of 1.5 min. Subsequently, it increased from 50% phase B to 90% within 0.5 min and balanced for 4 min.

The limit of detection limit (LOD) of the instrument was determined by external standards (muramic acid) for quantitative analysis, while internal standard 1 was used for recovery rate and concentration correction of the samples. The mass spectrometry information of the external standard muramic acid was as follows: Q1 = 252.2, Q3 = 126.3, DP = 60 V, and CE = 25 eV (quantification); Q1 = 252.2, Q3 = 216.1, DP = 60 V, and CE = 18 eV (qualitative). The mass spectrometry information of the internal standard N-methylglucosamine solution was as follows: Q1 = 196.4, Q3 = 58.2, DP = 70 V, and CE = 25 eV (quantification); Q1 = 196.4, Q3 = 74.1, DP = 70 V, and CE = 25 eV (qualitative). The method LOD refers to a signal peak that is three times greater than the background noise of the instrument. In this study, the LOD of muramic acid was 0.1 μg g^-1, and the recovery rate was 82.55%–102%.

Simulation of semiflexible molecular docking of the detected substance

QsdR is the only key transcription factor found in R. erythropolis that can regulate quorum-quenching (QQ)-lipase, and it can regulate the expression of pyridine ring oxidation-related enzymes in quinolones²⁰. MvfR is a typical LysR-type transcriptional regulatory factor in the P. aeruginosa strain that can regulate the expression of pqsA-E genes to adjust QS and the expression of multiple virulence factors¹⁹. We obtained the protein structures of 4ZA6 (QsdR; accession A0A0C2W9F0) and 6b8a (MvfR; accession Q9I4X0) from the UniProt database. The obtained structures were optimized, dehydrated, and hydrogenated using PyMOL 2.5 software (Schrodinger Inc., NY, USA). The 3D structure PDB file of metabolic detection products was generated using ChemDraw 20.0 (PerkinElmer Inc., CT, USA), followed by the semiflexible docking mode⁴⁸ between ligands and receptors performed with AutoDockTools 1.5.7 software. Docking simulations were conducted a minimum of 100 times using the genetic algorithm to obtain the predicted binding free energy results. It is generally believed that a binding free energy less than −4 is the threshold for ligand binding to receptor proteins, and the lower the free energy is, the greater the probability of binding. The results of the molecular docking simulation showed that 2-heptyl-1H-quinolin-4-one (HHQ) could form stable hydrogen bonds with the amino acid residue TYR-159 of the QsdR protein, with a binding energy of −7.39 kcal mol^-1, and hydrogen bonds with the amino acid residues ASP-264 and LYS-266 of the mvfR protein, with a binding energy of −6.15 kcal mol^-1.

Cell wall morphology observation

The experimental groups included mono-cultures of R. erythropolis and P. aeruginosa, and mono-cultures of R. erythropolis supplemented with 20 μg L⁻¹ HHQ, cultured in minimal media supplemented with 0, 0.1, and 1 mM AlCl₃ (pH 4.0) at 28 °C and 180 rpm for 24 h. After incubation, the bacterial suspensions were centrifuged at room temperature (13,400 × g), and the cell pellets were washed in 100 μL of cold TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) for AFM and SEM observation.

For AFM observation, the pretreatment method used for the cell samples was described previously⁴⁹. In brief, the cleaned bacterial samples were fixed in 2.5% glutaraldehyde for 4 h, centrifuged at 6000 × g for 5 min, after which the glutaraldehyde was removed, and the samples were washed with PBS three times. Then, 50 μL of bacterial suspension was transferred onto silicon substrates and dried under flowing nitrogen for testing. The TESPA-V2 probes (Bruker) with a standard spring constant of 37 N m⁻¹ and resonance frequency in air of 320 kHz were used as imaging probes for AFM experiments in air. High-resolution scanning probe microscopy (MultiMode 8, Bruker, Karlsruhe, Germany) was used to observe the samples in tapping mode, with an average tip sample force of 260–450 pN. The scanning range was set as 100 nm × 100 nm. The thickness and roughness rate (R_a) of each cell wall were determined by height histograms, and the mean values and standard deviations of each group were calculated using paired t tests for data comparison between groups. All 3D images were created using Nanoscope Analysis software.

For SEM observation, bacterial cells were fixed with 10% formaldehyde solution overnight. During the fixation process, the bacterial suspension was shaken constantly to ensure sufficient contact between the formaldehyde solution and the bacterial sample. Then, the samples were centrifuged at 6000 × g for 5 min, and the bacterial samples were dehydrated in gradually increasing ethanol solutions (30%, 50%, 70%, 85%, 90%, and 100%). Each bathing step lasted 10 min, and the samples were dried until use in a freeze dryer. The samples were sputter-coated with gold using a JEOL coating machine (JFC-1100E, JEOL Co., Ltd, Japan), vacuum-dried, observed and photographed under 30,000x magnification using a field-emission scanning electron microscopes (Regulus SU8100, Hitachi High-Tech Corporation, Tokyo, Japan).

Statistics and graphics

The normality assumption and equal variance assumption were evaluated using the Kolmogorov‒Smirnov test and Levene test, respectively. One-way ANOVA and Tukey’s multiple comparison tests were performed using GraphPad Prism 9 (GraphPad Software Inc., CA, USA). Figures were generated using the R 4.2.1 statistical environment (https://cran.r-project.org/), GraphPad Prism 9, OriginLab 2016, and Microsoft PowerPoint (Microsoft Office Home and Student 2019).

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

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