The recombination regulator RecX negatively regulates heat-stable antifungal factor (HSAF) biosynthesis in Lysobacter enzymogenes

Abstract

Bacteria often use multiple transcription factors to regulate specific biological processes. Biosynthesis of heat-stable antifungal factor (HSAF) is regulated by multiple factors in Lysobacter enzymogenes. However, the mechanism of HSAF biosynthesis regulation remains largely unknown. In this study, we screened a potential HSAF biosynthesis regulator, RecX, by a DNA pull-down assay. Deletion of recX resulted in a significant increase in the production of HSAF, and overexpression of recX significantly suppressed HSAF production. Importantly, our results showe that RecX directly binds to the promoter region of the lafB gene to inhibit its transcription and thus decreases HSAF production in L. enzymogenes. These findings reveal the novel mechanism of RecX regulation of antifungal antibiotic production in L. enzymogenes.

Background

Lysobacter is a group of gram-negative bacteria belonging to the family Xanthomonadaceae (Christensen and Cook 1978). A representative and well-studied species of this genus is L. enzymogenes, which is a biocontrol bacterium that has been used to control crop fungal diseases (Qian et al. 2009; Odhiambo et al. 2017; Zhao et al. 2017, 2019a). This bacterium is able to achieve such broad-spectrum antifungal activity by secreting an antifungal antibiotic called HSAF (heat-stable antifungal factor), which is a polycyclic tetramate macrolactam and is regarded as an attractive agent for the biological control in fungal diseases of agriculturally important plants (Li et al. 2006, 2018; Yu et al. 2007; Lou et al. 2011). HSAF inhibits the growth of a wide range of fungal species, and its chemical structure and mode of action are distinct from those existing antifungal drugs and fungicides (Li et al. 2006; Zhao et al. 2019a).

Previous studies have shown that the production of HSAF by the L. enzymogenes wild-type strain is low. Understanding the biosynthetic and regulatory mechanisms of HSAF is important to improve its yield in L. enzymogenes strains. The HSAF biosynthesis operon constitutes a single transcription unit (ORF1–ORF7) (Wang et al. 2017a). The single-module hybrid PKS/NRPS protein lafB (ORF6) is the key enzyme for HSAF biosynthesis in L. enzymogenes (Lou et al. 2011; Xu et al. 2018, 2021a, 2021b; Li et al. 2021). Our previous work revealed that the biosynthesis of HSAF is regulated by multiple factors. Our collaborators discovered several small-molecule signal factors, such as diffusible signal factor (DSF) and diffusible factor (DF), that positively regulate the biosynthesis of HSAF in L. enzymogenes (Qian et al. 2013; Han et al. 2015). In particular, 4-hydroxybenzoic acid (4-HBA) serves as a DF that connects the metabolic shikimate pathway to the HSAF biosynthetic pathway in L. enzymogenes (Zhou et al. 2013a, 2013b; Su et al. 2017). Cyclic dimeric GMP (c-di-GMP) is a ubiquitous dinucleotide secondary messenger in a diverse range of bacteria (Romling et al. 2005). This compound hinders HSAF production by decreasing the expression of the HSAF biosynthesis gene (Chen et al. 2017; Xu et al. 2018). Spermidine also has also been shown to play a significant role in the regulation of HSAF biosynthesis (Chen et al. 2018; Zhao et al. 2019b). In addition to chemical signals, other factors also affect the biosynthesis of HSAF in L. enzymogenes. The global transcriptional regulator Clp is a direct regulator of HSAF biosynthesis and positively regulates HSAF production in a c-di-GMP-dependent manner (Xu et al. 2018). The TetR family protein LetR is involved in the transcriptional repression of HSAF production in L. enzymogenes (Wang et al. 2017a). LysR-family transcription factor (LysR_Le) can bind to the promoter of the HSAF biosynthesis gene (plafB) and positively regulate HSAF production (Qian et al. 2014; Su et al. 2017). Previous results showed that LarR, a member of the MarR family, is involved in interplay with LysR_Le during 4-HBA-regulated HSAF production (Su et al. 2018). In addition, evidence has shown that other response regulators mediate the regulation of HSAF biosynthesis, such as PilG, PilR, Lsp, and TonB-dependent receptors (Zhou et al. 2015; Wang et al. 2016, 2017b; Chen et al. 2017). RpfB1 and RpfB2 increase the metabolism of free fatty acids by inducing the activity of fatty acyl-CoA ligase which blocks the biosynthesis of HSAF in L. enzymogenes (Li et al. 2020a). Recent evidence indicates that the DSF type-based quorum-sensing (QS) system component RpfG interacts with three hybrid two-component system proteins (HtsH1, HtsH2, and HtsH3) to regulate the biosynthesis of HSAF in L. enzymogenes (Li et al. 2021). However, the mechanism underlying the regulation of HSAF biosynthesis in L. enzymogenes remains largely unknown.

In this study, we describe the role of the recombination regulator RecX in HASF biosynthesis. Deletion of recX increased production of the antifungal antibiotic HSAF in L. enzymogenes. We also demonstrate that RecX directly bound to the promoter of the HSAF biosynthesis gene (plafB) to negatively regulate HSAF biosynthesis. Overall, the results of the present study facilitate the current understanding of RecX-mediated regulation of HASF biosynthesis in L. enzymogenes.

Results

DNA pull-down screening for potential regulators of HSAF biosynthesis in L. enzymogenes

To investigate the novel genes that regulate HSAF biosynthesis in L. enzymogenes, we performed DNA pull-down analyses using biotin-conjugated plafB as a probe. A total of 340 proteins capable of directly binding plafB were identified (Fig. 1a). These proteins include the canonical HSAF synthesis regulator protein Clp (Xu et al. 2018), other regulator proteins, such as LysR_Le (Su et al. 2018) and some two-component system regulators (Chen et al. 2017). Furthermore, we identified 8 uncharacterized regulator proteins that may regulate HSAF synthesis by directly binding plafB (Fig. 1b). To identify the physiological functions of these 8 proteins in HSAF production, the genes Le2680, Le3484, Le3720, Le4060, Le4256, Le4312, Le5100, and Le5153 were deleted using a two-step homologous recombination approach to construct the corresponding single knockout strain, respectively. We compared the growth rates of the generated knockout mutants in the medium used to measure HSAF production (0.1% TSB) and found that none of the mutants showed significant growth defects when compared to the wild-type strain (Additional file 1: Figure S1a). As a control, all test mutants and the wild-type strain were grown in nutrient-rich conditions (LB), and we found no significant differences in their colony morphologies (Additional file 1: Figure S1b). When cultured on media used to indicate protease and cellulase production, all knockout mutants produced wild-type levels of these two extracellular enzymes (Additional file 1: Figure S1c, d), suggesting that these 8 genes are not involved in regulating the production of extracellular enzymes in L. enzymogenes.

Fig. 1

DNA pull-down screen to identify potential transcription factors the regulate HSAF biosynthesis. a The Venn diagrams show the potential lafB promoter-binding proteins in the total protein extracts that were pulled down using the 5-biotinylated probe. b Eight potential transcription factors were identified that might regulate HSAF biosynthesis

To evaluated the physiological functions of the L. enzymogenes genes Le2680, Le3484, Le3720, Le4060, Le4256, Le4312, Le5100, and Le5153 in HSAF production, we quantified HSAF production in the corresponding mutant strains by high-performance liquid chromatography (HPLC). The Δ3484 strain had lower HSAF levels and deletion of Le5100 resulted in 1.75-fold increase in HSAF production compared to the wild-type (Fig. 2).

Fig. 2

Quantification of HSAF produced by the Lysobacter enzymogenes knockout mutants of the 8 potential transcription factors. Error bars, means ± standard deviation (n = 3). ***P < 0.001, ****P < 0.0001, assessed by one-way ANOVA. All experiments were repeated three times with similar results

The recX gene in the L. enzymogenes genome is conserved among different bacteria species.

In this study, we focused on one of the newly identified regulators, Le5100, that was found to be involved in the regulation of HSAF biosynthesis; Le3484 will be investigated in a separate study. The Le5100 gene is located downstream of the rexA gene (Fig. 3a). The Le5100 gene encodes a typical RecX family protein with pfam-RecX domains (Fig. 3b). To investigate the function of the Le5100 gene in L. enzymogenes, protein sequence alignments of L. enzymogenes OH11 Le5100 with RecX proteins from Xanthomonas campestris pv. campestris, X. oryzae pv. oryzae, Stenotrophomonas maltophilia, and L. enzymogenes C3 were performed (Fig. 3c). The results showed that the L. enzymogenes OH11 Le5100 protein shares 30.5, 31.1, 33.5, and 89.4% identity residues with X. campestris pv. campestris RecX, X. oryzae pv. oryzae RecX, S. maltophilia RecX, and L. enzymogenes C3 RecX, respectively (Fig. 3c). Based on these criteria, it seems reasonable that Le5100, namely, RecX, could be a functional RecX family member that plays a crucial role in HSAF production in L. enzymogenes.

Fig. 3

Identification and sequence characterization of RecX in Lysobacter enzymogenes. a Genomic localization of recX. csrA: carbon storage regulator; alaS: alanyl-tRNA synthetase; recX: regulatory protein; recA: recombination protein; lexA: repressor LexA; HP: hypothetical protein; marR: MarR family N-acetyltransferase transcriptional regulator; cinA: competence damage-inducible protein A. b Bioinformatics analyses of the domain organization of RecX. c Alignment of L. enzymogenes, Xanthomonas campestris pv. campestris, X. oryzae pv. Oryzae, and Stenotrophomonas maltophilia RecX sequences. The alignment was performed with Clustal W based on identical residues

RecX is involved in HSAF biosynthesis

To ascertain the role of RecX in the regulation of HSAF biosynthesis, we constructed a recX-overexpressing strain in a wild-type OH11 background and complemented the recX mutant with plasmid-borne recX. HSAF production was significantly decreased in the recX-overexpressing strain (OH11/recX) when compared with the wild-type (Fig. 4). The complemented strain ΔrecX/recX, containing a plasmid-borne recX, had a similar level of HSAF production as the wild-type (Fig. 4). Importantly, the recX mutation did not impair bacterial growth (Additional file 1: Figure S1a, b), implying that L. enzymogenes RecX plays a specific role in regulating HSAF production.

Fig. 4

Quantification of HSAF produced by the Lysobacter enzymogenes ∆recX mutant. Error bars, mean ± standard deviation (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, assessed by one-way ANOVA. All experiments were repeated three times with similar results

RecX directly binds to the promoter of the HSAF biosynthesis gene (plafB)

As mentioned earlier, deletion of recX resulted in increased HSAF production (Fig. 2) and DNA pull-down assays confirmed that RecX directly binds to the promoter of the HSAF biosynthesis gene. Thus, we wondered whether RecX might directly target HSAF biosynthesis gene promoters. To test this hypothesis, we performed an Escherichia coli-based one-hybrid assay and found that RecX could directly bind to the promoter of the L. enzymogenes HSAF biosynthesis gene lafB (Fig. 5a). To further verify this binding, an electrophoretic mobility shift assay (EMSA) was performed. The RecX protein had a monomeric molecular weight of 35.8 kDa and N-terminal-His 6-tagged version of the protein was purified by nickel chelate chromatography and showed a single band when run on an SDS-gel (Fig. 5b). A PCR-amplified 590 bp DNA fragment from plafB was used as a probe. The addition of purified RecX protein, ranging in concentration from 0 to 4 µM, to the reaction mixtures (20 µL at 28°C for 25 min) caused a mobility shift of the plafB DNA fragment. EMSA indicated strong RecX binding with the plafB probe in a dose-dependent manner (Fig. 5c). These results suggest that RecX directly binds to the promoter region of the HSAF biosynthesis gene lafB in L. enzymogenes.

Fig. 5

RecX directly bound the promoter of the HSAF biosynthesis gene (plafB). a Direct physical interaction between RecX and the lafB promoter region was detected in E. coli. Experiments were performed according to the procedures described in the ‘Experimental procedures’ section. + , cotransformant containing pBX-R2031 and pTRG-R3133, used as a positive control; − , cotransformant containing pBXcmT-plafB and the empty pTRG, used as a negative control; L. enzymogenes RecX and plafB, cotransformant harboring pTRG-RecX and pBXcmT-plafB. -3AT-Str, plate with no selective medium (3AT 3-amino-1,2,4-triazole and Str streptomycin) and + 3AT + Str, plate with M9-based selective medium. b The purified protein was analyzed by 12% SDS-PAGE. Lane 1, molecular mass markers; Lane 2, RecX protein. c Gel shift assay showing that RecX directly bound plafB. RecX (0, 0.5, 1, 2, or 4 μM) was added to reaction mixtures containing 50 ng of probe DNA, and the reaction mixtures were separated on polyacrylamide gels

RecX negatively regulates the expression of the HSAF biosynthesis gene

Our results confirmed that deletion of recX resulted in an increase in the production of HSAF in L. enzymogenes (Fig. 4). Therefore, we hypothesized that L. enzymogenes RecX negatively regulates the expression of the HSAF biosynthesis gene to decrease HSAF production. To test this hypothesis, we performed reverse transcription-quantitative PCR (RT-qPCR) and found that the expression of the HSAF biosynthesis gene lafB in the ΔrecX mutant strain was increased compared with that in the wild-type L. enzymogenes strain, indicating that RecX negatively regulates the expression of lafB (Fig. 6a). To further study the regulation of lafB by RecX, we constructed a plafB-lacZ reporter systems in the recX mutant strain. Consistent with the RT-qPCR results, deletion of recX resulted in increased expression levels of the HSAF biosynthesis gene lafB (Fig. 6b). Taken together, these results showed that RecX can directly target the promoter of the HSAF biosynthesis gene to decrease the expression and production of HSAF in L. enzymogenes.

Fig. 6

Lysobacter enzymogenes RecX negatively regulated lafB expression. a RT-qPCR analyses of lafB mRNA in the wild-type and recX mutants grown in 10% TSB. b The effect of RecX on lafB gene expression was measured by assessing the β-galactosidase activity of the lafB-lacZ transcriptional fusion in the L. enzymogenes wild-type and ΔrecX strains. Error bars, means ± standard deviation (n = 3). ***P < 0.001, ****P < 0.0001, assessed by one-way ANOVA. All experiments were repeated three times with similar results

Discussion

Previous studies have indicated that the biosynthesis of HSAF is regulated by multiple factors, including small-molecule compounds, transcription factors and other proteins. However, the mechanism underlying the regulation of HSAF biosynthesis remains largely unknown in L. enzymogenes. In this study, we screened a novel transcription factor, RecX, that can regulate HSAF biosynthesis in L. enzymogenes. This study provides biochemical, genetic, and physiological evidence to demonstrate that RecX directly binds the promoter of the HSAF biosynthesis gene (plafB) to negatively regulate HSAF biosynthesis in L. enzymogenes (Fig. 7).

Fig. 7

Model of speculation about RecX regulation mechanism. This study revealed the role of RecX in negatively regulating the production of HSAF in Lysobacter enzymogenes using a DNA pull-down screening approach

Several studies have demonstrated an alternative role for recX in the DNA repair of recombination performed by RecA (Pages et al. 2003; Ragone et al. 2008; Prasad et al. 2019). The absence of RecX severely impaired the natural transformations in both plasmid and chromosomal DNA. A previous study indicated that RecX suppresses the negative effect exerted by RecA during plasmid transformation, preventing RecA missense recombination in single-stranded DNA tracts, and modulating DNA strand exchange (Cardenas et al. 2012). RecX abrogates RecA·ATP-hydrolysis, and strand exchange promoted by RecA and RecX might act as an anti-recombinase to inhibit inappropriate recombination repair during normal DNA metabolism (Venkatesh et al. 2002; Yakimov et al. 2017). Previous work revealed that RecX is also involved in regulating the Mycobacterium smegmatis stress response (Prasad et al. 2019). However, the regulatory mechanisms of RecX have been less studied in the biocontrol bacterium L. enzymogenes.

Our data show that the in-frame deletion of the recX coding sequence significantly increases HSAF production (Fig. 4), and the overexpression of recX in the wild-type OH11 strain and the ΔrecX mutant strain significantly suppressed HSAF production (Fig. 4). Moreover, a DNA pull-down assay confirmed that RecX directly binds plafB (Fig. 1). To confirm this result, we performed an E. coli-based one-hybrid assay and EMSA. The results demonstrate that RecX could directly target the promoter of the HSAF biosynthesis gene (Fig. 5). We further analyzed the transcription level of the HSAF biosynthesis-related gene lafB in the recX mutant. Knockout of recX significantly increased the transcription level of lafB (Fig. 6). These results suggest that RecX can directly regulate the expression of the HSAF biosynthesis gene and decrease the production of the antifungal factor HSAF in L. enzymogenes. However, this study investigated the role of RecX only in relation to HSAF biosynthesis in L. enzymogenes. To determine whether RecX regulates recombination DNA repair in L. enzymogenes, we will need additional research.

Conclusions

In this study, we screened a potential HSAF biosynthesis regulator, RecX, by a DNA pull-down assay. One of the notable results of this study is that the recombination regulator RecX negatively regulated the expression of the HSAF biosynthesis gene by directly binding to its promoters to block the biosynthesis of HSAF in L. enzymogenes.

Methods

Bacterial strains, plasmids, and growth conditions

The strains and plasmids used in this study are shown in Additional file 2: Table S1. E. coli strains were grown in Luria–Bertani medium (10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl, pH 7.0) at 37°C. L. enzymogenes strains were grown in Luria–Bertani medium and 10% TSB at 28°C. For the preparation of the culture media, tryptone, peptone, beef extract, and yeast extract were purchased from Sangon Biotech (Shanghai, China). When needed, antibiotics were added (30 μg/mL kanamycin sulfate, 50 μg/mL gentamycin) to the E. coli or L. enzymogenes cultures.

DNA pull-down assays

The promoter of the lafB gene was amplified by PCR using the 5-biotin-labeled primers listed in Additional file 2: Table S2. The 5-biotinylated DNA of the promoter of the lafB gene was immobilized to streptavidin beads using Dynabeads™ M270 Streptavidin based on the manufacturer’s protocol (Thermo Fisher Scientific Inc., San Jose, CA, USA). Proteins in the nuclear fraction were incubated with 5-biotinylated DNA beads on a rotating shaker at 4°C overnight in 500 μL protein binding buffer containing 5% glycerol, 0.05% Triton X-100, 25 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1 mM dithiothreitol, and 1 mM EDTA. Following this incubation, the supernatant was removed. The beads were washed three times with cold protein binding buffer. After the last wash, the pull-down mixture was resuspended in distilled water at 90°C for 2 min to break the streptavidin and biotin bond. The eluted samples containing the bound proteins were subjected to SDS-PAGE followed by Coomassie blue staining and LC-MS/MS analyses. The proteins that were eluted from the beads without the biotinylated DNA probe were used as controls.

Gene deletion and complementation

The in-frame deletions in L. enzymogenes OH11 were generated via double-crossover homologous recombination (Xu et al. 2018; Li et al. 2020a) using the primers listed in Additional file 2: Table S2. In brief, the flanking regions of each gene were PCR-amplified and cloned into the suicide vector pEX18Gm (Additional file 2: Table S1). The deletion constructs were transformed into the wild-type strain by electroporation, and gentamycin was used to select for integration of the nonreplicating plasmid into the recipient chromosome. A single-crossover integrant colony was spread on LB medium without gentamycin and incubated at 28°C for 3 days, and after appropriate dilution, the culture was spread on LB plates containing 15% sucrose. Colonies sensitive to gentamycin were screened by PCR using the primers listed in Additional file 2: Table S2, and the gene deletion strains were obtained.

For gene complementation constructs, DNA fragments containing the full-length genes along with their promoters were PCR-amplified and cloned into the versatile plasmid pBBR1MCS5 (Kovach et al. 1995). The resulting plasmids were transferred into the L. enzymogenes strain by electroporation, and the transformants were selected on LB plates containing gentamycin.

HSAF extraction and quantification

HSAF extraction and quantification were performed as follows (Tang et al. 2018; Li et al. 2021). HSAF was extracted from 4 mL L. enzymogenes cultures grown in 10% TSB for 48 h at 28°C with shaking (at 180 rpm) and adjusted to pH 2.5 with HCl. Same volume of ethyl acetate was added to the acidified broth and the mixture was shaken in a vortexer at 2000 rpm for 1 min. The ethyl acetate fractions were collected, and ventilated to dryness in a fume hood. The residue was dissolved in 100 µL of methanol. The crude extract was subjected to 0.22-µm Mini-star filtration, and the filtrate was concentrated to 100 µL. The extract (20 µL) was used for HPLC analysis using a C18 reversed-phase HPLC column (4.6 × 250 mm, Agilent Technologies Inc.) with detection at 318 nm. Pure water and acetonitrile containing 0.04% (v/v) Trifluoroacetic acid (TFA) were used as the A and B mobile phases, respectively. The gradient program used a flow rate of 1 mL/min. The experiments were performed three times, and three replicates were used in each sample.

Reverse transcription-quantitative PCR (RT-qRCR)

The bacterial cells were collected when the optical density (OD₆₀₀) reached 1.0 in 10% TSB. Total RNA was extracted using a TRIzol-based method (Life Technologies, CA, USA). RNA quality control was performed via the following several steps: (1) the degree of RNA degradation and potential contamination were monitored on 1% agarose gels; (2) the RNA purity (OD₂₆₀/OD₂₈₀, OD₂₆₀/OD₂₃₀) was checked using a NanoPhotometer^® spectrophotometer (IMPLEN, CA, USA); and (3) the RNA integrity was measured using a Bioanalyzer 2100 (Agilent, Santa Clara, CA, USA). The primers used in this assay are listed in Additional file 2: Table S2. cDNA was synthesized from each RNA sample (400 ng) using the TransScript^® All-in-One First-Strand cDNA Synthesis SuperMix for qPCR (One-Step gDNA Removal) Kit (TransGen Biotech, Beijing, China) according to the manufacturer’s instructions. RT-qPCR was performed using TransStart Top Green qPCR SuperMix (TransGen Biotech) on a QuantStudio TM 6 Flex Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) with the following thermal cycling parameters: denaturation at 94°C for 30 s, followed by 40 cycles of 94°C for 5 s and 60°C for 34 s. Gene expression analyses were performed using the 2^–∆∆CT method with 16S rRNA as the endogenous control, and the expression level in the wild-type was set to a value of 1. The experiments were performed three times, and three replicates were used in each run.

Bacterial one-hybrid assays

Bacterial one-hybrid assays were performed as previously reported (Xu et al. 2016; Wang et al. 2018; Li et al. 2021). In brief, the bacterial one-hybrid reporter system contained the following three components: the plasmids pBXcmT and pTRG, which are used to clone the target DNA and to express the target protein, respectively, and the E. coli XL1-Blue MRFˊ kan strain, which is the host strain used for the propagation of the pBXcmT and pTRG recombinants (Guo et al. 2009). In this study, the promoter of the HSAF biosynthesis gene (plafB) was cloned into the pBXcmT plasmid to generate the recombinant vector pBXcmT-plafB. Similarly, the coding regions of recX were cloned into the pTRG plasmid to create the final construct pTRG-recX. The two recombinant vectors were transformed into the XL1-Blue MRFˊ kan strain. If direct physical binding occurred between RecX and plafB, the positive-transformant E. coli strain containing both pBXcmT-plafB and pTRG-RecX would grow well on the selective medium, that is, minimal medium containing 6 mM or 10 mM 3-amino-1,2,4-triazole, 8 μg/mL streptomycin, 12.5 μg/mL tetracycline, 34 μg/mL chloramphenicol, and 30 μg/mL kanamycin. Furthermore, transformants containing both pBX-R2031/pTRG-R3133 served as a positive control (Guo et al. 2009), and co-transformants containing either empty pTRG and pBXcmT-plafB were used as negative controls. All co-transformants were spotted onto selective medium, grown at 28°C for 3–4 days, and then photographed.

Protein expression and purification

Protein expression and purification were performed as previously reported (Li et al. 2017, 2021, 2022). To clone the recX gene, genomic DNA extracted from L. enzymogenes was used for PCR amplification using Pfu DNA polymerase, and the primers are listed in Additional file 2: Table S2. The PCR products were inserted into pET-28b ( +) to produce the plasmid pET-recX. The recX gene was verified by nucleotide sequencing by GenScript (Nanjing, Jiangsu, China). L. enzymogenes recX with a vector-encoded His₆-tagged N-terminus was expressed in E. coli BL21 (DE3) and purified with Ni-nitrilotriacetic acid (NTA) agarose (Qiagen, Chatsworth, CA, USA) using a nickel-ion affinity column (Qiagen, Chatsworth, CA, USA). The protein purity was monitored by SDS-PAGE.

Electrophoretic mobility gel shift assays (EMSAs)

Electrophoretic mobility gel shift assays were performed as previously described (Hirakawa et al. 2015; Shao et al. 2018; Li et al. 2021). For RecX gel shift assays, we used DNA fragments that included plafB as a probe. The probe DNA (50 ng) was mixed with protein in a 20 μL reaction mixture containing 10 mM Tris-HCl (pH 7.5), 50 mM KCl, 1 mM dithiothreitol, and 0.4% glycerol. After incubation for 30 min at 28°C, samples were electrophoresed on a 5% nondenaturing acrylamide gel in 0.5 × TBE buffer at 4°C. The gel was soaked in 10,000-fold-diluted SYBR Green I nucleic acid dye (Sangon Biotech, Shanghai, China), and the DNA was visualized at 300 nm.

Construction of reporter strains and β-galactosidase measurement assays

The plafB-lacZ reporter was introduced into the L. enzymogenes wild type and recX mutant strains by electroporation. The transconjugants were selected on LB agar plates supplemented with gentamycin, and X-Gal. To measure β-galactosidase activities, the overnight cultured bacteria were diluted to the same cell density (OD₆₀₀ ≈ 0.01) in 10% TSB medium. The inoculated cultures were then incubated at 28°C and 180 rpm. The cells were harvested to assess β-galactosidase activities according to previous studies (Wang et al. 2019; Li et al. 2020b, 2022; Wang et al. 2021).

Statistics and reproducibility

The experimental datasets were subjected to analyses of variance using GraphPad Prism 7.0. The significance of the treatment effects was determined by the F value (P = 0.05). If a significant F value was obtained, separation of means was accomplished by Fisher’s protected least significant difference at P ≤ 0.05.