AcrR1, a novel TetR/AcrR family repressor, mediates acid and antibiotic resistance and nisin biosynthesis in Lactococcus lactis F44

Lactococcus lactis , widely used in the manufacture of dairy products, encounters various environmental stresses both in natural habitats and during industrial processes. It has evolved intricate machinery of stress sensing and defense to survive harsh stress conditions. Here, we identified a novel TetR/AcrR family transcription regulator, designated AcrR1, to be a repressor for acid and antibiotic tolerance that was derepressed in the presence of vancomycin or under acid stress. The survival rates of acrR1 deletion strain ∆AcrR1 under acid and vancomycin stresses were about 28.7-fold (pH 3.0, HCl), 8.57-fold (pH 4.0, lactic acid) and 2.73-fold (300 ng/mL vancomycin) greater than that of original strain F44. We also demonstrated that ∆AcrR1 was better able to maintain intracellular pH homeostasis and had a lower affinity to vancomycin. No evident effects of AcrR1 deletion on the growth and morphology of strain F44 were observed. Subsequently, we characterized that the transcription level of genes associated with amino acids bio-synthesis, carbohydrate transport and metabolism, multidrug resistance, and DNA repair proteins significantly upregulated in ∆AcrR1 using transcriptome analysis and quantitative reverse transcription-PCR assays. Additionally, AcrR1 could repress the transcription of the nisin post-translational modification gene, nisC , leading to a 16.3% increase in nisin yield after AcrR1 deletion. Our results not only refined the knowledge of the regulatory mechanism of TetR/AcrR family regulator in L. lactis , but presented a potential strategy to enhance industrial production of nisin.


INTRODUCTION
Lactococcus lactis, a gram-positive bacterium, holds significant economic importance as part of the lactic acid bacteria (LAB).Widely used in producing fermented foods such as cheese, yogurt, and kimchi, it has gained recognition as a safe microbial strain approved for food production by the US Food and Drug Administration (Mao et al., 2016).In the fermentation and maturation process of dairy products, L. lactis acts as a starter culture, converting lactose to lactic acid, thus imparting the desired sour taste and texture.Its high lactic acid production capacity and acid tolerance make it an ideal choice for dairy fermentation.Additionally, L. lactis could produce bacteriocins, which have antimicrobial properties, helping to preserve dairy products and preventing spoilage.Lactococcus lactis can also serve as a probiotic within the human body, fostering the inhibition of pathogenic bacteria, balancing gastrointestinal flora, combating infections, and regulating immunity, making it a valuable addition to dairy products for enhanced nutritional value (Cavanagh et al., 2015).Overall, L. lactis is an essential component in dairy production, contributing to the flavor, texture, safety, and nutritional value of various dairy products.Additionally, L. lactis has great potential as a cell factory for the large-scale production of biobased chemicals such as polyols, vitamins, exopolysaccharides and vaccines (Sauer et al., 2017).
Lactococcus lactis encounters various environmental stresses during its growth and application.The acidification caused by the production and accumulation of lactic acid is the most important environmental stress during L. lactis fermentation.This self-imposed stress brings about many challenges, such as inhibition of glycolytic process, disruption of cell membrane structure, reduction of denaturation, and DNA stability.Ultimately, it is auto acidification, rather than nutrient depletion, that results in cessation of L. lactis growth (Papadimitriou et al., 2016).Additionally, the dissemination of antibiotic resistance through the food chain has become a critical public health challenge, and development of antibiotic resistance at the genetic level in the dairy starter L. lactis forms a significant aspect of this problem (Mathur and Singh, 2005).Therefore, investigating the mechanisms behind the response and tolerance to environmental stresses holds significant importance.In recent years, the main acid-tolerance mechanisms in bacteria, such as proton pump, macromolecular protection and DNA repair, cell wall and cell membrane protection, and metabolic pathway regulation have been deeply investigated.The drug resistance mechanisms mainly involve antibiotic efflux, modification of antibiotic targets, and protection of cell walls and cell membranes (Papadimitriou et al., 2016).
Bacteria have evolved intricate machinery to sense extracellular signals and activate the above-mentioned resistance mechanisms under diverse environmental stresses.Transcriptional regulators are a type of protein molecule that can bind to specific DNA base sequences and regulate target genes at the transcription level under specific conditions.Bacterial transcription regulators have been found to play an important role in the sensing, response, and regulation to acid and antibiotic stresses.For example, GadE, a LuxR family activator in Escherichia coli, can induce the expression of lysine decarboxylase-and arginine decarboxylase-related genes (gadA, gadB, and gadC), bacterial envelope biosynthesis genes (ompC, rfaQ, and rcsA), DNA repair and chaperone genes (hdeA, hdeB, and hdeD) to achieve acid tolerance (Hommais et al., 2004).In gram-positive bacteria, the ClpXP proteolytic enzyme complex of the Clp family maintains intracellular pH dynamic equilibrium by regulating the transcription and expression of stress genes.It was found that the deletion of clpX or clpP could result in slow growth, decreased autolysis, and decreased viability under acid stress in Streptococcus mutans (Kajfasz et al., 2009).In Bacillus subtilis, SpxA can directly regulate oxidative stress genes (dpr, nox, and sodA).The enzyme SpxB may regulate DNA repair-related genes (mutY and smxA) and cell envelope-and fatty acid metabolism-related genes (era, cshA, fabK, fadD, and fabM; Nakano et al., 2002).CodY, a global regulator of L. lactis, regulates a variety of cellular processes, including nitrogen metabolism, nutrient transport, branched-chain amino acid metabolism, and stress response (Belitsky et al., 2015;Wang et al., 2017b).Loss of CodY can cause changes in the transcriptional levels of the genes in the arginine deiminase pathway, and affect the resistant ability of the strain to acid environment (den Hengst et al., 2005).RcfB, from the Crp-Fnr family of transcription regulators, plays a role in acid adaptation because the survival rate of the rcfB mutant after acid challenge was 130 times less than that of the wild-type strain L. lactis MG1363 (Madsen et al., 2005).As reported in our previous study, TCSR7, a 2-component system transcriptional regulator, regulates the transcription level of DLT operon genes under acid stress, increasing the positive charge on the cell membrane surface and thus increasing the acid resistance of L. lactis F44 (Wu et al., 2022b).
Certain L. lactis strains showcase their ability to produce the bacteriocin nisin, renowned for its efficacy against a broad spectrum of gram-positive bacteria.As a natural and safe antibacterial peptide, nisin has been approved by the US Food Drug Administration for use as food additive.Additionally, it has shown efficacy in inhibiting the growth of cancer cells, treating diarrhea, oral health care, and immunomodulation (Azari-Anpar et al., 2023;Chan et al., 2023;Wu et al., 2023).The methods and technologies adopted to improve nisin yield and reduce production cost are mainly focused on fermentation optimizing, random mutation and selection, and gene engineering (Özel et al., 2018).Our recent studies discovered several transcriptional regulators including CodY (Wu et al., 2022a), ComX (Yuan et al., 2021), LssR (Song et al., 2022), and YthA (Wu et al., 2018) positively regulating nisin biosynthesis and immunity, and demonstrated the feasibility of improving nisin yield through manipulating these regulators.
The TetR family transcriptional regulators, distributed in a variety of prokaryotes, are a large and important single-component signal transduction system (Deng et al., 2013;Ramos et al., 2005).They consist of a conserved helix-turn-helix DNA-binding domain and a larger C-terminal domain.The most common function of TetR family regulators is to regulate the transcription of genes encoding efflux pumps and transporters involved in antibiotic resistance and toxic compound resistance.The TetR family proteins have also been reported to control the genes involved in environmental stresses, antibiotic biosynthesis, division, metabolism, virulence, and pathogenicity (Cuthbertson and Nodwell, 2013).In LAB, most of the reported TetR family regulators function as activators.For example, in Lactobacillus plantarum, the TetR-type regulator Lp_2642 positively regulates the production of plantaricin EF (Zhao et al., 2022), and AcrR enhances ethanol tolerance by positively regulating fatty acid synthesis (Yang et al., 2019).In L. lactis, DhaS positively regulates the expression of dihydroxyacetone kinase, thus affecting glycerol catabolism (Christen et al., 2006).LnqR expression promotes the production of bacteriocin lacticin Q (Iwatani et al., 2016), and LssR positively regulates the resistance to acid and nisin stresses (Song et al., 2022).Despite the above-mentioned TetR family activators, the identification and understanding of TetR repressors participating in environmental stresses-sensing, response, and regulation are still lacking in L. lactis.
In this study, we characterized a novel TetR/AcrR family transcriptional regulator, AcrR1, to be a repressor for acid and vancomycin tolerance and nisin production in L. lactis F44.AcrR1 was significantly downregulated under acid and vancomycin stresses.The possible regulatory mechanism of AcrR1 was explored by comparative transcriptome analysis and quantitative real-time (qRT)-PCR verification.To our knowledge, AcrR1 is the first TetR/AcrR family repressor reported in L. lactis to be involved in environmental stress resistance and bacteriocin biosynthesis.

Strains, Plasmids, and Culture Conditions
All of the strains and plasmids used in this study are listed in Supplemental Table S1 (see Notes).The parent strain in this study was L. lactis F44 (genome number GCA_002804185.1), a nisin-producing strain derived from L. lactis YF11 (accession number CGMCC7.52) in our previous study (Zhang et al., 2014).Lactococcus lactis F44 and the ∆AcrR1 were cultured in GM17 medium at 30°C without shaking.Escherichia coli TG1, used to propagate the plasmids, was grown in LB medium with 100 μg/mL erythromycin at 37°C with shaking at 250 rpm.The fermentation medium was formulated as follows (per liter): 42.3 g M17 powder, 15 g sucrose and 0.26 g cysteine.Micrococcus luteus ATCC 10240, an indicator strain for the nisin titer assay, was grown in nutrient broth medium.Its agar-diffusion bioassay medium (pH 7.0) contained (per liter) 5 g glucose, 5 g yeast extract, 8 g tryptone, 5 g NaCl, 2 g Na 2 HPO 4 , and 15 g agar.All chemicals and reagents were purchased from Sigma Chemical Company (Shanghai, China).
The primers used for cloning and qRT-PCR were synthesized in GENEWIZ Inc. (Suzhou, China) and listed in Supplemental Table S2 (see Notes).Genomic DNA of L. lactis strains were isolated using the TIANamp Bacteria DNA Kit (Tiangen, China).For knockout of acrR1 in L. lactis F44, a 2-step homologous recombination was employed via a plasmid pCS1966 containing the counterselection procedure using 5-fluoroorotate (Solem et al., 2008).Plasmids were constructed by ligating the upstream and downstream homologous sequences of acrR1 gene into plasmid pCS1966 using EasyGeno Assembly Cloning kit (Tiangen, China) to yield plasmid pCS1966-∆AcrR1.Polymerase chain reaction amplification and DNA sequencing were used to verify the constructed plasmids.The plasmids were transformed into E. coli TG1 for enrichment using the heat shock transformation method.The plasmids were extracted with TIANprep Mini Plasmid Kit (Tiangen, China) after antibiotics selection.To obtain L. lactis ∆AcrR1, the plasmid pCS1966-∆AcrR1 was first transformed into the L. lactis F44 and the successful integration was selected by erythromycin resistance.Subsequently, the first crossover recombinant was grown in GM17 medium supplemented with 1 μg/ mL Em for generations, diluted, and spread on SA medium containing 5-fluoroorotate.The growing colony was selected and verified by colony PCR.Eventually, the acrR1-deleted strain ∆AcrR1 was gained.

Cell Morphology Analysis
Lactococcus lactis F44 and ∆AcrR1 cells, grown to the midexponential phase (7 h), were collected and observed using an optical microscope (XSP-35-1600, Phenix).The cells were washed 3 times with PBS buffer and fixed overnight with 4% paraformaldehyde solution at 4°C.After 3 times washing with pure water, the samples were resuspended with water.The samples were obtained by dripping the suspension on coverslips and then dried with gradient concentrations (50%, 70%, 80%, 90%, and 100%) of ethanol.The morphology of the cell was observed using a scanning electron microscope (ScEM, JSM-7610F JEOL Ltd., Japan).

Acid-and Antibiotic-Tolerance Assays
Activated strains of L. lactis were cultured for 7 h to midexponential phase, and stationary-phase cultures of L. lactis were harvested by centrifugation at 7,104 × g and 4°C for 5 min and washing twice with 0.9% NaCl.Then L. lactis were suspended in equal volumes of GM17 medium with pH 3.0 (HCl), pH 4.0 (lactic acid), and 300 ng/mL vancomycin, respectively, and incubated for 3 h at 30°C.Suspension aliquots before and after stress were serially diluted and then plated on GM17 plates for colony counting to calculate the survival ratio.For the acid (lactic acid and HCl)-tolerance assay, the serially diluted samples after stress were simultaneously dripped on GM17 medium every 8 µL, and the number of living cells was observed for 36 h.For the vancomycintolerance assay, the serially diluted samples before stress were simultaneously dripped on GM17 medium with 300 ng/mL vancomycin every 8 µL, and the number of living cells was observed for 36 h.
To evaluate the tolerance to other types of antibiotics, activated strains of L. lactis were cultured for 7 h to midexponential phase, then inoculated into GM17 medium containing gradient concentrations of different antibiotics including streptomycin, erythromycin, ampicillin, tetracycline, and lincomycin.After 10 h of culture at 30°C, the growth state of the strains under different concentrations of antibiotics was measured.

Nisin Yield Assay
The activated strain was inoculated in 100 mL fermentation medium with 5% inoculum volume and incubated at 30°C.The nisin yield of the fermentation broth was tested by sampling every 2 h through the plate-diffusion method.The standard nisin (40,000 IU/mg) was purchased from Chihonbio Co. Ltd. (Luoyang, China).A series of nisin standard solutions (200, 100, 50, and 25 IU/mL) were prepared with 0.02 M HCl solution.An agar-diffusion bioassay medium of 26 mL with 1.5% agar was heated until dissolution of the agar, and 390 μL Tween 20 was added to each medium at 70°C.After cooling to 50°C, 100 μL of Micrococcus luteus ATCC 10240 suspension was added, and then the mixture was poured into the culture dish and set.Four wells were drilled using a 7-mm-diameter hole punch on each assay agar plate.Fermentation broth, 500 mL, was mixed with 0.02 M HCl solution and boiled for 5 min at 100°C.After centrifugation at 7,104 × g and 4°C for 5 min, the supernatant was diluted with 0.02 M HCl solution to make 3 parallel samples.Then 100 μL of standard solution or sample was added into each well of the plate and cultured at 37°C for 24 h until the bacteriostatic zone was clear.The diameter of the bacteriostatic ring was measured with a Vernier caliper.The standard curve was drawn according to the diameter of the bacteriostasis circle of the standard solution.The standard equation was obtained to calculate the nisin titer of the samples.

Determination of Extracellular pH (pHe) and Intracellular pH (pHi)
The pHe value was detected using a pH meter (FE20, Mettler Toledo).The pHi value was measured using the fluorescence method as described in our previous report (Song et al., 2022).Briefly, L. lactis, cultured for 8 h, was collected and washed twice in PBS by centrifuging at 7,104 × g and 4°C for 5 min.Then the bacterial precipitate was resuspended in PBS buffer containing a fluorescent probe.The fluorescence signal was measured using a fluorescence microplate reader (Varioskan Lux, Thermo Fisher Scientific) after 30°C for 30 to 60 min with excitation and emission wavelengths of 488 nm and 528 nm, respectively.A series of standard curves of pH 5.5, 6.0, 6.5, 7.0, and 7.5 were prepared.Cell precipitates were resuspended in standard solutions with different pH gradients containing fluorescent probes, respectively, and then valinomycin (1 μM) and Nigeriumin (1 μM) were added to disrupt cell membrane permeability, after incubation at 30°C for 10 min.Then the fluorescence signal intensity was detected.

Vancomycin Fluorescence Confocal Detection
The vancomycin fluorescent derivative (Van-FL) was prepared according to the method described in the literature (Daniel and Errington, 2003).Five hundred microliters of vancomycin solution with a concentration of 10 mg/mL was incubated with 50 mL 5(6)-carboxyfluorescein-N-hydroxysuccinimide ester (5 mg/mL) overnight at 4°C.The mixture was then mixed with 450 μL of 0.1 M Tris (pH = 8.0) and incubated at 22°C for 1 h.The activated L. lactis was incubated for 7 h to midexponential phase.Then the Van-FL (5 mg/mL) was added into the medium at the final concentration of 0.3 μg/mL and incubated at 30°C for ~15-20 min.Subsequently, 500 μL of bacterial solution was collected in a 1.5-mL centrifuge tube, and the bacteria were centrifuged (8,000 rpm, 5 min) and washed with PBS solution 3 times.The 20-μL culture sample was placed on the microscope slide, and 30 μL of anti-fluorescence quenching agent was dripped on it.The cover slide was covered and placed upside down for 3 min.The fluorescence intensity was observed by a confocal fluorescence microscope (Leica TCS SP5).

RNA Extraction, Sequencing, and Transcriptome Analysis
Precultures of strain F44 and ∆AcrR1 were grown for 7 h at 30°C in GM17 medium, then harvested by centrifugation at 8,000 rpm for 5 min.Each strain was set in parallel with 3 groups.Transcriptome sequencing was performed by Bioengineering Co. Ltd. (Shanghai, China).The total RNA from L. lactis F44 and ∆AcrR1 was prepared with a ZR RNA MiniPrep Kit (Zymo Research, Irvine, CA).The quality and quantity of RNA were determined by Agilent 2100 Bioanalyzer (Agilent, Santa Clara, CA).The first-strand cDNA was synthesized through the reverse transcription with ProtoScript II reverse transcriptase (New England BioLabs, Ipswich, MA).Then second strand synthesis enzyme mix was used for the synthesis of the second strand of the cDNA.The resulting double-stranded cDNA was purified using the AxyPrep Mag PCR clean-up kit (Axygen Scientific, Union City, CA).Strand-specific libraries were constructed with the second strand of cDNA, with a library size of 300-400 bp, sample quality was examined using Agilent 2100 Bioanalyzer, and total library concentration was detected by fluorescence quantitative PCR and sequenced on the HiSeq 4000 system (TruSeq SBS KIT-HS V3, Illumina).Raw reads generated by Illumina HiSeq TM 4000 were subjected to quality control by analyzing base composition and quality distribution (Q20 > 95% and Q30 > 90%) to ensure accuracy.Gene expression was determined using a standard calculation of reads per kilobase of gene per million mapped reads (RPKM).The corresponding gene expression ratios of samples were calculated based on RPKM.Significantly differentially expressed genes (DEG) were defined as: |log 2 foldchange (F44/∆AcrR1)| ≥ 0.5 and P < 0.05, and classified according to clusters of orthologous genes (COG) classification (www .ncbi.nlm.nih.gov/COG/ ).

qRT-PCR Analysis
Specific DNA primers of the target genes were designed to amplify qRT-PCR products (Supplemental Table S2).
To verify the DEG identified through transcriptome data, L. lactis F44 and ∆AcrR1 were incubated at 30°C for 7 h in GM17 medium and the total RNA was extracted by using the SPARKeasy improved bacteria RNA kit (SparkJade, China).The total bacterial RNA samples were analyzed by agarose gel electrophoresis to determine whether the RNA bands were clear and whether there were DNA tails.The qRT-PCR was performed on a QuantStudio 1 Real-Time PCR System (Applied Biosystems).The procedure and reaction system were described in FastStart Universal SYBR Green Master (Roche, Switzerland).The concentration of cDNA as the template for 16S rRNA amplification was diluted × 1,000-fold than that for the target gene.The concentration of template cDNA for target gene amplification was 20 ng/mL, and the concentration for 16s rRNA gene amplification was 1,000× dilution.Similarly, qRT-PCR was also performed to determine the mRNA levels of acrR1 from the cells after exposure at pH 3.0 and in the presence of 300 ng/ mL vancomycin for 3 h.Three parallel samples per group were conducted.

Sequence Analysis of AcrR1
The basic local alignment search tool (BLAST; https: / / blast .ncbi.nlm.nih.gov/Blast .cgi)was used to search homologous proteins of AcrR1 (WP_010905240.1) in Lactococcus species.The phylogenetic tree of AcrR1 homologue protein sequences with an E value cutoff of less than 1e-10, identity of more than 40%, and coverage of more than 40% was constructed using the MEGA tool with the LG amino acid substitution model and gamma distributed with invariant sites (G+1; Kumar et al., 2018).Then sequence alignment was generated from sequences of AcrR1 and the best-matched proteins in 4 extensively researched L. lactis strains including QOK50430.1 from L. lactis N8, UCS90511.1 from L. lactis LAC460, AYV52019.1 from L. lactis IL1403, and ADA64032.1 from L. lactis KF147.The picture of the alignment was displayed by ESPript 3.0 (http: / / espript .ibcp.fr/ESPript).
The domains of AcrR1 were predicted by InterPro annotation (Mitchell et al., 2019)

Statistical Analysis
To determine the differences for cell density, survival rate, pH value, and nisin titer between control and experimental groups, a t-test was performed.Experimental data were expressed as means ± SD.

AcrR1 Belongs to TetR/AcrR Family Transcriptional Regulators and Is Conserved in L. lactis ssp. lactis
The sequence of AcrR1 (ID: WP_010905240.1)contained 215 amino acids and was encoded by the gene acrR1 (ID: CV702_RS01165) in L. lactis F44.It shares 40% identity with AcrR from E. coli (AcrR EC , ID: CAD6020473.1).AcrR EC , belonging to TetR-type regulator, negatively regulates the multidrug resistance through binding to the 24 bp sequence (5′-TACATACATTTGT-GAATGTATGTA-3′) in acrAB promoters (Touzé et al., 2004;Deng et al., 2013).The BLAST search results showed that AcrR1 had high similarities to several proteins belonging to Lactococcus ssp., all of which were annotated as TetR/AcrR family transcriptional regulators (Figure 1A).Structures obtained with AlphaFold and Phyre suggested that AcrR1 contains 11 α-helices, which was slightly different from the extensively investigated TetR-type regulators (Figure 1B).The multiple sequence alignment analysis showed that the sequence of AcrR1 was highly conserved with an identity above 96% in L. lactis ssp.lactis and relatively conserved with an identity above 40% in several other species in the genus Lactococcus including L. raffinolactis, L. allomyrinae, L. fujiensis, L. protaetiae, L. nasutitermitis, L. insecticola, and L. laudensis.Interestingly, although AcrR1 was highly conserved in L. lactis ssp.lactis, no AcrR1-homologue protein was attained in other L. lactis subspecies such as cremoris and hordniae through the BLAST search.

Construction of Strain ∆AcrR1 and Its Influence on Growth and Morphology
Seamless knockout of the gene acrR1 encoding AcrR1 was achieved in L. lactis F44 with plasmid pCS1966 through double-crossover recombination.The time profile of cell density of ∆AcrR1 and F44 cultured in GM17 medium is shown in Figure 2A.No significant difference in growth performance was detected between ∆AcrR1 and F44.The morphology of ∆AcrR1 and F44 cells in logarithmic phase was observed by optical microscope (Figure 2B) and ScEM (Figure 2C).The ScEM images showed that both the cells from F44 and ∆AcrR1 displayed a regular coccoid shape and a flat, smooth, and compact surface.However, a remarkable difference under optical microscope observation was recognized: ∆AcrR1 cells seemed to be more reluctant to form long chains, probably due to the reduced adhesion.The underlying reason needs to be explored further.

AcrR1 Contributes to Acid and Antibiotic Stresses Tolerance of L. lactis F44
We evaluated the effects of AcrR1 on the acid and vancomycin resistance on L. lactis F44.As shown in Figure 3A and 3B, the survival rate of ∆AcrR1 was 28.7 times that of F44 under hydrochloric acid stress (pH 3.0, 3 h).Under the condition of lactic acid stress (pH 4.0, 3h ), an 8.57 times increase in survival rate was achieved by ∆AcrR1 compared with F44.A stable pHi value ensured normal physiological activities of bacteria cells.We observed that ∆AcrR1 was better able to maintain pHi homeostasis than F44 at the late fermentation stage (Figure 3E), in which the broth was acidified to pH 4.6.Furthermore, similar results were obtained after 3 h of exposure to pH 3.0 in GM17 medium.
The survival rate of ∆AcrR1 was 2.73 times that of F44 under vancomycin stress at 300 ng/mL for 3 h (Figure 3C).Vancomycin is an antibiotic with important clinical applications, which can specifically bind to d-alanyl-dalanine at the fourth and fifth ends of the nascent peptidoglycan.It has been reported that fluorescent derivatives of vancomycin could be used to detect the synthesis pattern of the nascent cell wall of Bacillus subtilis (Lam et al., 2009).Therefore, we speculated that their method could be extended to evaluate the vancomycin tolerance by observing the fluorescence intensity, and the weaker the fluorescence intensity, the more resistant to vancomycin the cell is.After incubation of the derivatives at 300 ng/mL with the bacteria for 3 h, it can be seen in Figure 3D that the fluorescence intensity of ∆AcrR1 was generally weaker than that of F44, suggesting that ∆AcrR1 was less likely to bind with vancomycin, and thus exhibiting a higher vancomycin resistance, which is consistent with the results of the survival experiment.In summary, the transcription regulator AcrR1 could negatively regulate the resistance of L. lactis F44 to acid and vancomycin stress.
Because one of the most common regulatory functions of TetR family regulators is the regulation of enzymes involved in antibiotic resistance (Ramos et al., 2005), several other types of antibiotics, including streptomycin, belonging to aminoglycoside group; erythromycin, belonging to macrolide group; penicillin, belonging to β-lactam group; lincomycin, belonging to lincosamide group; and tetracycline, belonging to tetracycline group, were selected.The range of antibiotic concentrations under which F44 and ∆AcrR1 exhibited obvious growth differences is presented in Supplemental Figure S1 (see Notes).Overall, strain ∆AcrR1 showed better growth performance than F44, suggesting the detrimental role of AcrR1 in antibiotic resistance.

Transcriptional Analysis of the ∆AcrR1
To explore the regulation mechanism of the transcription regulator AcrR1, the difference of gene expression at midexponential phase between F44 and ∆AcrR1 was analyzed by transcriptome analysis.A total of 48 significant DEG regulated by AcrR1 (|log 2 foldchange (F44/∆AcrR1)| ≥ 0.5; P < 0.05) were identified, most of which were upregulated after deletion of AcrR1, indicating that AcrR1 principally functions as a repressor in L. lactis.The DEG involved in environmental stress resistance and nisin biosynthesis were listed in Tables 1 and 2. According to the COG classification, the DEG were mainly involved in the pathways of amino acid biosynthesis, carbohydrate transport and metabolism, transcription, and inorganic ion transport and metabo-lism (Figure 4A).We also selected 12 of the DEG to validate their transcription levels by qRT-PCR, and the results confirmed a consistency between qRT-PCR and transcriptomic results (Figure 4B).In our previous study, the comparative transcriptome analysis of F44 after exposure to pH 7.0 and pH 4.0 conditions revealed that the mRNA level of acrR1 was downregulated under acid stress (Tian et al., 2019).To further investigate the effect of environmental stresses on acrR1 expression, we compared the transcription levels of acrR1 of F44 before and after 3 h exposure to pH 3.0 and 300 ng/mL vancomycin in GM17 medium.As shown in Figure 4C, both acid and vancomycin challenges could significantly downregulate the transcription level of acrR1.
Effects of AcrR1 on Multidrug Efflux Pump.Significantly, the transcription levels of ycfB and ycfC in ∆AcrR1 were increased by 10.67-and 9.43-fold compared with F44.The ABC transporter protein encoded by ycfB and ycfC belongs to ABC-type multidrug efflux pump.The multidrug efflux pump recognizes different classes of antibiotics and some small molecules and exports them from the inside to the outside of bacterial cells, facilitating multidrug resistance (Van Bambeke et al., 2000).Many studies proved the effectiveness of increasing the expression level of multidrug efflux pump proteins in improving antibiotic resistance in bacteria.For example, overexpression of the ABC transporter LmrA could render E. coli resistant to various antibiotics, including aminoglycosides, quinolones, tetracycline, and chloramphenicol (Putman et al., 2000).Regulation of multidrug efflux pumps is one of the best-known functions of the members of TetR/AcrR family.The TetR family transcriptional regulator EmhR represses the expression of EmhABC efflux pump by binding to the promoter region in Pseudomonas fluorescens.When indoles are present, indoles bind to EmhR and induce dissociation of the DNA-EmhR complex, activating efflux pump emhABC expression, and thus the strain was highly resistant to ampicillin (Han et al., 2021).It is worth noting that AcrR1 was located 87 bp upstream of the translation initiation codon of ycfB, implying that there might be a specific mechanism to control the transcriptional activities of downstream genes.
Effects of AcrR1 on Glycopeptide Biosynthesis.We found the transcription level of vanY (zinc d-alanyl-d-alanine carboxypeptidase, 1.2-fold) was obviously increased in ∆AcrR1.Vancomycin, a kind of glycopeptide antibiotic targeting the bacterial cell envelope, was recognized as the last line of defense to treat severe infections caused by gram-positive bacteria such as Clostridium difficile, Staphylococcus aureus, and Enterococcus species.Inducible vancomycin resistance has been discovered in the above species (Hong et al., 2008;Moscoso et al., 2011;Stogios and Savchenko, 2020).Vancomycin can bind to the D-alanyl-D-alanine C-terminus of the peptidoglycan precursor, leading to failure to form mature peptidoglycan, thereby affecting or even destroying cell wall synthesis (Tynkkynen et al., 1998).VanY is a d, d-carboxypeptidase that dissociates the C-terminal d-alanine residue of the peptidoglycan precursor.The transcriptome analysis identified that the gene vanY was  Strains F44 and ∆AcrR1 were grown for 7 h at 30°C in GM17 medium, then harvested for transcriptome sequencing.The significantly downregulated genes involved in environmental stress resistance and nisin biosynthesis are listed. 2 BaseMean indicates the homogenization result of the gene read count of the 3 parallel samples in the group. 3 Values are expressed as the fold change (F44/∆AcrR1) in a log obviously upregulated after deletion of acrR1.Therefore, VanY contributes to altering the structure of the pentapeptide precursor by changing its terminal from d-alanyl-d-alanine to d-alanyl-d-lactate, which enriches vancomycin-resistant peptidoglycan precursors and reduces the affinity to vancomycin by about 1,000 times (Bugg et al., 1991).It was found that overexpression of vanY gene in Enterococcus faecalis JH2-2 resulted in a 4-fold increase in vancomycin minimal inhibitory concentration (Arthur et al., 1994).Revealing the antibiotic resistance mechanism of bacteria not only facilitates the development of strategies to potentiate the efficacy of antibiotics but also provides guidance for minimizing the emergence of antibiotic resistance.Additionally, antibiotic stress is also relevant for probiotic LAB in the intestine due to orally administered antibiotics.In addition, we noted that VanY is a zinc-dependent enzyme.Previous studies identified the zinc-binding residues in VanY by site-directed mutagenesis, which was later confirmed by crystal structure analysis (Binda et al., 2012;Kim et al., 2018).It has also been found that low concentrations of Zn 2+ can reactivate VanY that was inactivated by EDTA (Podmore and Reynolds, 2002).These results all prove that Zn 2+ plays an important role in the activity of VanY.
In the ∆AcrR1, adcA (2.18-fold), encoding zinc transport system substrate-binding protein, was significantly upregulated, which might further increase vancomycin resistance through enhancing VanY activity.
Effects of AcrR1 on DNA Repair.DNA damage is a common phenomenon for bacteria exposed to environmental stresses such as heat, acidic or alkaline pH, osmotic shock, and oxidative agents.Not only is the  DNA repair process essential for correcting replicative mistakes and maintaining cell homeostasis (Friedberg, 2003;Friedberg et al., 2006), but the repair process also plays critical roles in preventing damage from environmental stresses such as acidic pH (Faustoferri et al., 2005), UV (Martinez et al., 2022), and reactive oxygen (Poetsch, 2020).DNA repair proteins, responsible for repair of base excision, nucleotide excision and mismatch, and homologous recombination, are vital for restoring genomic stability (Friedberg, 2003).In ∆AcrR1, the transcription levels of Y-family DNA polymerase gene umuC (1.68-fold),DNA topology modulation protein-encoding gene flaR (1.50-fold),DNA repair protein-encoding genes radC (1.48-fold) and adaA (1.46-fold), recombinase gene recT (1.45-fold), were obviously increased.Additionally, there was also slight increase of transcription levels of another 2 DNA repairrelated genes recR (1.15-fold) and recN (1.1-fold) after acrR1 deletion although not statistically significant.Homologous recombination is a common method of gene recombination, which can make cells accurately repair the damage caused by double-stranded DNA break (Li and Heyer, 2008).In prokaryotic cells, the rec family genes encode the proteins associated with homologous recombination (Wigley, 2013;Vlašić et al., 2014).RecT protein is a single-stranded DNA-binding protein that can bind to oligonucleotide to prevent nuclease degradation and promote the annealing between oligonucleotide and template strands (Court et al., 2002).It was found that overexpression of the recT gene in Lactobacillus acidophilus NZ9000 could increase the survival rate by 10.4 times under lactic acid stress (Zhu et al., 2018).The over-production of DNA repair protein RecO in Lactobacillus acidophilus NZ9000 was also studied and the recombinant strain showed higher tolerance to lactic acid (Wu et al., 2013).The role of DNA repair proteins in drug resistance has also been studied.For example, the phosphorylation of RecA at the serine 207 site was in Mycobacterium after DNA damage, and the antibiotic resistance was inhibited by selectively inhibiting the LexA coprotease function of recA without affecting its ATPase or chain-exchange function (Wipperman et al., 2018).The DNA repair proteins RadA and RadC play important roles in response to the DNA damage agents methyl methanesulfonate, hydroxyurea, and UV radiation (Burghout et al., 2007;Liang et al., 2013).Our previous study also found the transcription level of DNA repair genes, including dprA, radA, radC, recA, recQ, and ssbA, was upregulated when L. lactis F44 was exposed to erythromycin stress (Wang et al., 2017a).
Effects of AcrR1 on Carbohydrate Transport and Metabolism.When encountered with environmental stress conditions, bacteria tend to use alternative carbon sources by changing the metabolic and energy flux (Ko-ponen et al., 2012).The transcription levels of several genes, including ugpC (1.68-fold), encoding multiple sugar transport system ATP-binding protein involved in carbohydrate transport and metabolism, were significantly upregulated in ∆AcrR1.UgpC is involved in the transport of a variety of mono-and oligosaccharides, such as maltose, galactose oligomer, raffinose, sorbitol, α-glucoside, cellobiose, chitobiose, and arabino oligosaccharide.We compared the growth performance of F44 and ∆AcrR1 using the carbon sources that could be transported by UgpC.As shown in Supplemental Figure S2 (see Notes), the strains could hardly utilize arabinose for growth, probably due to the lack of arabinose metabolic pathway.Cellobiose, galactose, and maltose could support the growth but there was a certain delay in the early growth stage.The growth rate of ∆AcrR1 was slightly higher than that of F44, suggesting the inhibition effect of AcrR1 on carbohydrate transport.Because the transcription level of UgpC was significantly upregulated in ∆AcrR1, we hypothesized that it is the metabolic ability rather than the transport ability that accounts for the ratelimiting step for the utilization of these carbon sources.
The transcription level of spxB (pyruvate oxidase) in the pyruvate metabolic pathway was significantly reduced by 1.53 times, which resulted in a decrease in acetyl phosphate production.Thus, the level of conversion of pyruvate to acidic products such as acetic acid is reduced, which can contribute to maintaining pHi stability.In addition, the transcription level of the pentose phosphate pathway rbsK (ribokinase, 1.78-fold) also increased significantly.
Effects of AcrR1 on Amino Acid Synthesis.Amino acids are involved in protein synthesis, ATP production, and regulation of pHi homeostasis, and have been shown to be closely related to resistance to environmental stresses including acid stress (Batista-Silva et al., 2019) and antibiotic stress (Harms et al., 2016) in microorganisms.The transcription level of the serA (D-3-phosphoglycerate dehydrogenase, 1.67-fold) in the l-serine synthesis pathway and hisF (imidazole glycerol-phosphate synthase subunit, 1.5-fold) in the lhistidine synthesis pathway was significantly increased in ∆AcrR1, suggesting the enhanced synthetic ability of l-serine and l-histidine after acrR1 deletion.The regulation of intracellular amino acid metabolism is a common mechanism adopted by microorganisms to cope with environmental stresses.Broadbent et al. (2010) found that Lactobacillus casei ATCC 334 accumulated a large amount of histidine at pH 4.5, and the cell survival at pH 2.5 was at least 100-fold increased when 30 mM histidine was added to acidic medium, which suggested that the accumulation of histidine may be an important mechanism of LAB defense against lethal conditions.It has also been revealed that l-serine supplementation could significantly improve the cell wall and membrane integrity, and increase the intracellular ATP availability in yeast under lactic acid stress (Wei et al., 2023).A recent study demonstrated the importance of histidine transport in the acid stress adaptation of Staphylococcus aureus (Beetham et al., 2024).
Other Effects of AcrR1 on Environmental Stresses Resistance.Lipid metabolism is involved in many biological functions, and essential for bacteria's survival and homeostasis maintenance in adverse environments.The transcription level of dhaL (2.37-fold) and dhaK (2.31fold) related to lipid metabolism increased significantly in ∆AcrR1, which encodes phosphoenolpyruvate-glycerone phosphotransferase.Additionally, the transcriptional levels of coaE, encoding dephospho-CoA kinase CoaE involved in CoA biosynthesis pathway, were increased by 1.53-fold after acrR1 deletion.The CoaE protein is one of the key enzymes in CoA biosynthesis pathway.CoA is involved in synthesis and metabolism of fatty acids, which are crucial for bacterial survival under environmental stresses through enhancing the rigidity and impermeability of cell membranes (Martín Mauricio et al., 2004;Muller et al., 2011).In ∆AcrR1, the mRNA level of gene purA, encoding adenylosuccinate synthase, involved in purine metabolism, was 1.54-fold compared with that in F44.The pathways related to purine were considered the target of antibiotic development because they directly affect the synthesis of DNA and RNA, and further have an effect on the ability of protein biosynthesis.Numerous studies have revealed the role of purine metabolism in antibiotic resistance of bacteria (Yee et al., 2015;Lin et al., 2019;Wang et al., 2021).
Effects of AcrR1 on Nisin Biosynthesis.Nisin was produced by certain L. lactis strains possessing nisin cluster, such as N8, F44, IO-1, and PLAC7 (Özel et al., 2018).Transcriptomic data and qRT-PCR analysis revealed that the expression level of the nisC gene was remarkably increased after the knockout of AcrR1 in F44.The post-translational modification gene nisC encodes cyclase NisC which catalyzes the cyclization of dehydrated amino acids.Thus, the result drove us to determine whether the deletion of acrR1 could enhance nisin biosynthesis ability of the strain.As shown in Figure 5, the highest nisin titer of strain ∆AcrR1 was up to 3,369 IU/mL, increased by 16.3% compared with strain F44.Additionally, the production rate in the early exponential phase was obviously improved, suggesting that NisC might be the rate-limiting enzyme for nisin biosynthesis in the fermentation process.The 14 kb nisin cluster carries 11 genes (nisZBTCIPRKFEG).Overexpressing these genes individually or in combination proved effective in increasing nisin production.For example, increasing the copy number of the nisin resistance gene nisI resulted in a 20% increase in nisin production of the wild producer (Kim et al., 1998).Co-overexpression of the nisin regulation and immunity genes, including nisRKFEG in L. lactis LL27, improved nisin yield by 45% (Şimşek et al., 2009).Another study showed that through co-overexpressing nisA, nisRK, and nisFEG, the maximum nisin yield in a shake flask increased by approximately 66.3% (Ni et al., 2017).Our previous study demonstrated that the higher productive ability of L. lactis F44 than YF11 was mainly due to the increased transcription levels of structural gene nisZ and resistance gene nisI (Zhang et al., 2014).However, to the best of our knowledge, there has been no report of increased nisin yield through manipulating the modification gene nisC.Indeed, we have attempted to increase NisC expression level in F44.It was found that overexpression of nisC could inhibit the growth of strain, resulting in a significantly decreased nisin yield (data not shown).We speculated that this was because NisC was located on the cell membrane and thus excessive NisC expression might be toxic to the cell.Therefore, mining the transcriptional repressors of nisC and eliminating the repression could be particularly meaningful to achieve an ideal nisin yield in industry.
Based on the above comparative transcriptome analysis between F44 and ∆AcrR1, we proposed the regulation mechanism of AcrR1, as shown in Figure 6.In brief, under acid and vancomycin stress, AcrR1 is inhibited, thereby relieving inhibition of multiple pathways such as sugar transporters, CoA biosynthesis, amino acid biosynthesis, DNA repair, drug transporters, peptidoglycan modification, nisin biosynthesis, and other pathways.

CONCLUSIONS
This study discovered AcrR1 as the first example of a TetR/AcrR family repressor involved in environmental stress resistance and nisin biosynthesis of L. lactis.Based on comparative transcriptome analysis combined with qRT-PCR validation, we demonstrated the participation of AcrR1 in several diverse cellular processes including multidrug efflux pumps, amino acid biosynthesis, carbohydrate transport and metabolism, nisin post-translation modification, DNA repair, and peptidoglycan biosynthesis.We further found that AcrR1 expression was significantly downregulated under acid and vancomycin stresses, leading to the depression of transcription of its target genes.Toward revealing the complete and precise environmental stress-sensing and regulation system mediated by AcrR1, further studies should be done to discover the signal transduction system adopted by AcrR1 to sense the extracellular environment.Additionally, this study provides an effective strategy to improve industrial nisin production by manipulating the regulators of nisin post-translational modification genes.

NOTES
This research was financially supported by the National Natural Science Foundation of China (31900029; Beijing, China), the Natural Science Foundation of Sichuan Province (2023NSFSC1228; Chengdu, Sichuan, China), and Sichuan University-Luzhou Science and Technology Innovation Platform Construction Project (2022CDLZ-20; Luzhou, Sichuan, China).Supplemental material for this article is available at https: / / doi .org/ 10 .17632/bpz6s9f45h .1.The data that support the findings of this study are available from the corresponding author upon reasonable request.No human or animal subjects were used, so this analysis did not require approval by an Institutional Animal Care and Use Committee or Institutional Review Board.The authors have not stated any conflicts of interest.

Figure 1 .
Figure 1.Sequence alignment of AcrR1 from L. lactis F44.(A) Phylogenetic tree alignment of the protein sequences of AcrR1 homologues.The clades of the L. lactis species are indicated by the red lines.(B) Sequence alignment of AcrR1 from L. lactis F44, QOK50430.1 from L. lactis N8, UCS90511.1 from L. lactis LAC460, AYV52019.1 from L. lactis IL1403, and ADA64032.1 from L. lactis KF147.The secondary structure of AcrR1 was predicted by Phyre 2.0.The sequence logo is shown below the alignment and represents the conservation degree of the residue.

Figure 3 .
Figure 3. Acid and vancomycin stress responses of L. lactis F44 and the acrR1 deletion strain ∆AcrR1.(A) Spot dilutions (i) and relative survival (ii) of the F44 and ∆AcrR1 in GM17 medium containing HCl at pH 3.0 for 3 h.(B) Spot dilutions (i) and relative survival (ii) of F44 and ∆AcrR1 in GM17 medium containing lactic acid at pH 4.0 for 3 h.(C) Spot dilutions (i) of the F44 and ∆AcrR1 in the presence of 300 ng/mL vancomycin; (ii) relative survival of F44 and ∆AcrR1 in GM17 medium containing 300 ng/mL vancomycin for 3 h.(D) Fluorescence detection of F44 (i) and ∆AcrR1 (ii) in GM17 medium containing 300 ng/mL Van-FL for 3 h; (iii) fluorescence intensity statistics of F44 and ∆AcrR1.(E) The pHe and pHi values of F44 and ∆AcrR1 after growth for 8 h (i) and 14 h (ii); the pHi value of F44 and ∆AcrR1 stress at pH = 3.0 (iii) for 3 h.The error bars indicate the SD from 3 replicate experiments.

Figure 4 .
Figure 4. (A) Distribution of DEG (|log 2 -fold change (F44/∆AcrR1)| ≥ 0.5, P-value ≤ 0.05) according to COG classification.The y-axis indicates the number of genes in various COG categories.(B) Relative mRNA level changes of 12 genes in ∆AcrR1 compared between transcriptome data and qRT-PCR results.(C) Relative mRNA level change of acrR1 in F44 after treatment with GM17 medium acidified with HCl to pH 3.0 and containing 300 ng/mL vancomycin, respectively, for 3 h at 30°C.The error bars indicate the SD from 3 replicate experiments.

Figure 5 .
Figure 5. Effects of deletion of gene acrR1 on nisin titer.(A) Nisin titer of L. lactis F44 and ∆AcrR1 during fermentation.Samples were taken every 2 h, ranging from 2 to 16 h.(B; inset) Results of the agar-diffusion experiment with the fermentation broths of F44 and ∆AcrR1 at 4 and 8 h.Error bars indicate the SD from 3 independent experiments.

Figure 6 .
Figure 6.Proposed regulatory mechanism of the transcriptional regulator AcrR1 of L. lactis F44.According to transcriptome analysis, it is assumed that AcrR1 can regulate the amino acids biosynthesis, carbohydrate transport and metabolism, multidrug resistance, DNA repair proteins, CoA biosynthesis and nisin biosynthesis.

Table 2 .
Jian et al.: AcrR1 REGULATES ACID AND ANTIBIOTIC RESISTANCE Significantly upregulated genes BaseMean indicates the homogenization result of the gene read count of the 3 parallel samples in the group.2Valuesare expressed as the fold change (F44/∆AcrR1) in a log 2 scale.