Listeria monocytogenes 10403S was used as the wild-type strain. Escherichia coli DH5α was employed for cloning experiments and as the host strain for plasmids pET30a(+; Merck, Darmstadt, Germany), pERL3 and pKSV7. E. coli Rosetta (DE3) was used for prokaryotic protein expression. L. monocytogenes was cultured in brain heart infusion (BHI) medium (Oxoid, Hampshire, England). DH5α and Rosetta (DE3) cells were grown at 37°C in Luria-Bertani broth (LB; Oxoid). Stock solutions of ampicillin (50 mg/ml), erythromycin (50 mg/ml), kanamycin (50 mg/ml), or chloramphenicol (50 mg/ml) were added to media when necessary. All chemicals were obtained from Sangon Biotech (Shanghai, China), Merck or Sigma-Aldrich (St. Louis, MO, USA) and were of the highest available purity.

Alignment of nucleotide and deduced amino acid sequences was performed with MUSCLE by using Geneious software (Edgar, 2004). The amino acid sequences of ArgR of L. monocytogenes 10403S strain and its homologs in other microbial species were obtained from the National Centre for Biotechnology Information database (NCBI). The known crystal structure of B. subtilis ArgR (PDB: 1F9N) was acquired from the Protein Data Bank (PDB). A putative model of L. monocytogenes ArgR was constructed using SWISS-MODEL Workspace (Arnold et al., 2006; Bordoli et al., 2009; Bordoli and Schwede, 2012). Promoters of genes of interest from the L. monocytogenes 10403S genome sequence were identified using the BPROM modules of the Softberry website¹. This program gives output scores from -1 to ∼25 to estimate the likelihood that a predicted promoter is functional and a higher score indicates that the prediction is more likely to be correct. ArgR binding sites composed of two palindrome sequences, known as ARG boxes, have been identified previously in several bacteria species (Larsen et al., 2005; Kloosterman and Kuipers, 2011; Perez-Redondo et al., 2012). Promoter/operator elements containing ARG box motifs were identified by searching the L. monocytogenes genome with a position weight matrix derived from known E. coli ArgR recognition elements (Chen et al., 1997), using the Virtual Footprint software program² (Munch et al., 2005).

The temperature-sensitive pKSV7 shuttle vector was used for generating mutations in L. monocytogenes 10403S. A homologous recombination strategy with the splicing by overlap extension (SOE) PCR procedure was used for in-frame deletion to construct gene deletion mutants (Monk et al., 2008). DNA fragments containing homologous arms upstream and downstream of the gene of interest were obtained via amplification of 10403S genomic DNA using the primer pairs listed in Supplementary Table S1. The obtained fragment was then cloned into pKSV7 and transformed into DH5α. After confirmation by sequencing, the recombinant vector containing the target gene deletion cassette was electroporated into the competent L. monocytogenes cells. Transformants were selected on BHI agar plates containing chloramphenicol (10 μg/ml). A single transformant was serially passaged at a non-permissive temperature (41°C) in BHI medium containing chloramphenicol to promote chromosomal integration. A single colony with chromosomal integration was successively passaged in BHI medium without chloramphenicol at a permissive temperature (30°C) to enable plasmid excision and curing (Camilli et al., 1993). Recombinants were identified as chloramphenicol-sensitive colonies, and mutagenesis was further confirmed by PCR and DNA sequencing. The single mutant strain was used in a second round of mutagenesis to construct double deletion mutants.

To complement the L. monocytogenesΔArgR strain, the complete argR open reading frame (ORF) along with its promoter was amplified from genomic DNA using primer pairs listed in Supplementary Table S1. After digestion with appropriate enzymes, the PCR product was cloned into pERL3, a plasmid capable of replication in Gram positive bacteria. The resulting plasmid was then electroporated into the L. monocytogenes ΔArgR strain. Plasmid-containing cells were selected on BHI agar plates containing erythromycin (10 μg/ml). The complemented strain was designated as CΔArgR.

The recombinant proteins used in this study were expressed as fusion proteins to the N-terminal His-tag using pET30a(+) as the expression vector. Rosetta (DE3) was used as the expression host. The full-length ORF of the gene of interest from the 10403S genome was amplified with the primer pair listed in Supplementary Table S1 and inserted into the pET30a(+) vector, and finally transformed into Rosetta competent cells. E. coli cells harboring recombinant plasmids were grown in 250 mL LB supplemented with 50 μg/mL kanamycin at 37°C until cultures reached 1.2–1.4 at OD_{600 nm}. Isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to a final concentration of 0.4 mM to induce expression of interest proteins for an additional 3–4 h at 30°C in the form of soluble protein. His-tagged fusion proteins were purified using nickel-chelated affinity column chromatography (Weishi-Bohui Chromtotech Co., Beijing, China). Specifically, IPTG-induced cell pellets were collected, re-suspended in 50 mM PBS (pH 7.4) and disrupted by sonication. After centrifugation at 12,000 g for 30 min, the soluble protein samples were collected and loaded onto a 1 ml pre-packed nickel-chelated agarose gel column according to the manufacturer’s instructions. Finally, expression and purification of recombinant proteins were analyzed via 10% SDS-PAGE followed by Coomassie brilliant blue staining and protein concentration was quantified with the Bradford method.

Rabbits were initially immunized via subcutaneous injection of 500 μg protein with an equal volume of Freund’s complete adjuvant (Sigma). After 2 weeks, rabbits were given subcutaneous booster injections of 250 μg protein each in incomplete Freund’s adjuvant (Sigma) three times at 10-day intervals. Rabbits were bled ∼10 days after the last injection.

To identify the predicted active sites of ArgR, a double mutant (S42AR43A) was generated using the original vector template, pET30a-ArgR, and the QuikChange Site-Directed Mutagenesis kit (Agilent, Santa Clara, CA, USA) with the primer pairs described in Supplementary Table S1. Template DNA was removed via digestion with DpnI (TOYOBO, Osaka, Japan) for 2 h at 37°C. The mutant construct was sequenced to ensure that only the desired single mutations had been incorporated correctly. The corresponding mutant protein was designated ArgR_S42AR43A, and expressed and purified as described above.

The purified N-terminal 6-histidine-tagged ArgR proteins were crosslinked with various amounts of glutaraldehyde (Sigma) in 50 mM HEPES (pH 8.0), containing 150 mM KCl and 1 mM L-arginine. The reaction mixture was incubated with or without 1% β-mercaptoethanol at room temperature for 2 h and the cross-linked ArgR complexes were analyzed by 10% SDS-PAGE, and stained with Coomassie Brilliant Blue.

DNA binding of ArgR and its mutant ArgR_S42AR43A was investigated in vitro by using electrophoretic mobility shift assay (EMSA). The DNA fragment of the promoter region of argC, argG, arcA, or sigB containing the putative ARG box was generated by PCR with the specific primer pairs (Supplementary Table S1). The DNA fragments were purified with a PCR Purification Kit (Sangon). Then 200 ng DNA was incubated with varying concentrations of purified recombinant ArgR or ArgR_S42AR43A in binding buffer (50 mM Tris-HCl, pH 8.0, 250 mM NaCl, 5.0 mM MgCl₂, 2.5 mM DTT, 2.5 mM EDTA, and 20% glycerol) for 30 min at room temperature. Protein-DNA complexes were separated electrophoretically on a native 5% polyacrylamide gel at 80 V with 0.5 x Tris-acetate-EDTA (TAE) buffer and visualized using ethidium bromide staining.

For transcriptional fusion of the argG promoter (P_argG) to the GFP reporter protein, the fragment containing the promoter-operator region of the argGH operon was amplified with the primer pair listed in Supplementary Table S1 using genomic DNA from L. monocytogenes 10403S as template. In parallel, the promoterless gfp allele gfpmut3^∗ was amplified from the Listeria shuttle vector pAMGFP3 using primers listed in Supplementary Table S1. The two fragments were fused by using overlapping PCR. The resulting fragment containing the promoter-gfp fusion was cloned into vector pERL3 to generate the reporter plasmid which was then electroporated into the wild-type 10403S or the ΔArgR strain. Transformants were selected by plating onto erythromycin-containing BHI agar plates. For promoter studies, L. monocytogenes was grown to stationary phase (OD_{600 nm} = 1.2) in BHI broth at 37°C, and then exposed to acidic (pH 5.5) or neutral (pH 7.0) conditions for an additional 60 min. Bacteria in 1 mL of culture were harvested by centrifugation, the cell pellets were washed once with 10 mM PBS (pH 7.4) and resuspended in 1 mL of 10 mM PBS. One hundred microliters of the suspension was used for gfp measurements and fluorescence observation. For the former, relative fluorescence unites (RFU) were measured in a fluorescence reader (BioTek Synergy H1, Winooski, VT, USA) with excitation at 485 nm and emission at 535 nm. Relative fluorescence values were calculated by subtracting extinction from the PBS background. For the latter, fluorescence intensity was observed by using confocal laser scanning microscopy (FLV 1000; Olympus, Japan).

Cells from stationary phase cultures of L. monocytogenes 10403S, mutants (ΔArgR, ΔSigB, and ΔArgRΔSigB) and complemented strain CΔArgR were harvested, washed in PBS and re-suspended in BHI (adjusted to pH 3.5 with 3M lactic acid). After 30, 60, 90, 120,160, or 200 min of incubation at 37°C, the surviving cells were plated onto BHI agar after appropriate dilutions. The plates were incubated at 37°C for 24 h and survival rates are reported as the mean of three independent experiments, which were performed in duplicate.

Listeria monocytogenes wild-type 10403S and its mutant strain ΔArgR were grown to the stationary phase (OD_{600 nm} = 1.2) in BHI broth at 37°C, and then exposed to acidic (pH 5.5) and neutral (pH 7.0) conditions, respectively, for additional 1 h. Total RNA was extracted using the Column Bacterial total RNA Purification Kit (Sangon), according to the manufacturer’s instructions, genomic DNA removed using DNase I (TaKara, Japan) and cDNA synthesized with reverse transcriptase (TOYOBO, Osaka, Japan). Real-time quantitative PCR was performed in a 20 μL reaction volume containing 200 ng cDNA, 10 μL SYBR quantitative PCR mix (TOYOBO), and 1 μL gene-specific primers (200 nM, Supplementary Table S1) to measure the transcriptional levels of arcA, sigB, argC, and argG using the Mx3000P PCR detection system (Agilent). The housekeeping gene, gyrB, was used as an internal control for normalization in each sample as previously described (Chen et al., 2011). Relative transcription levels were quantified using the 2^-ΔΔCT method and shown as relative fold changes (Livak and Schmittgen, 2001). Triplicate assays were performed for each gene.

Bacteria were grown in BHI broth to the stationary growth phase and lysates were prepared as described before (Ryan et al., 2009). Specifically, the stationary bacteria exposed to acidic (pH 5.5) and neutral (pH 7.0) conditions, respectively, for additional 1 h. Bacterial pellets were then re-suspended in 1 mL of extraction solution (2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris-HCl, 1 mM EDTA, pH 8.0). One gram of glass beads (G8772, Sigma) was added and samples lysed using the homogenizer Precellys 24 (Bertin, Provence, France) at 6000 rpm for 30 s with intermittent cooling for 30 s (two cycles in total), followed by centrifugation at 12,000 g for 15 min. Supernatants were retained as cell-free extracts. Samples containing equal amounts of protein were subjected to 12% SDS-PAGE and the separated proteins were blotted onto 0.22 μm polyvinylidene difluoride (PVDF) membranes (Merck Millipore). Membranes were blocked for 1 h with 5% skimmed milk, and incubated for 1 h with polyclonal antisera against recombinant recombinant ArgR, ArgG, SigB, or ArcA in 0.5% skimmed milk. Next, membranes probed with anti-interest protein were developed using HRP-conjugated goat anti-rabbit IgG (Santa Cruz, California, CA, USA) as the secondary antibody. Chemiluminescence was detected via a bio-imaging system (UVP EC3 Imaging System, UVP Inc., Upland, CA, USA), and the densities of the interest protein bands were normalized to the GAPDH signal and quantified using Quantity One software (Bio-Rad).

All data comparisons were analyzed using the two-tailed Student t-test. Differences with P-values of 0.05 were considered statistically significant, and those with P-values of 0.01 were considered markedly statistically significant.

The best characterized ArgR homolog from Gram-positive bacteria is AhrC from B. subtilis, as its crystal structure was determined in 2002 (PDB ID: 1F9N; Garnett et al., 2007). To better characterize L. monocytogenes ArgR, the amino acid sequence of this protein was aligned with those from seven other bacterial species. The alignment showed sequence identities ranging from 22 to 65%, the highest percentage was exhibited between ArgR from L. monocytogenes and AhrC from B. subtilis (Figure 1A). Based on the amino acid sequence analysis, the L. monocytogenes ArgR monomer appears to possess two highly conserved motifs, the “SR” motif for DNA binding (residues 42–43) in the N-terminal region, and the “GTICGDDT” motif for arginine binding (residues 120–127) and oligomerization (residues 125–126) located in the C-terminal region (Figure 1A). Furthermore, we modeled L. monocytogenes ArgR in the SWISS-MODEL Workspace using the crystal structure of B. subtilis AhrC as the template. The predicted structure L. monocytogenes ArgR is of high similarity to that of AhrC. Specifically, the monomer ArgR also associates via its C-terminal (core) domain to form a hexamer, probably as a result of the face-to-face association of a pair of trimers (Dennis et al., 2002). The six C-terminal domains strictly follow the 32 non-crystallographic symmetry (NCS) and domains from each trimer locate above another one, giving the hexameric core a stacked configuration when viewed along the threefold axis. The DNA-binding domains (DBD) adopt slightly different positions around the periphery of the core and deviate from strict NCS (Figure 1B). The recombinant ArgR protein was expressed in E. coli, purified and subjected to crosslinking analysis using glutaraldehyde. The his-tagged ArgR protein had a molecular weight of about 23 kDa, and was able to form higher order multimeric complexes (mainly formed as the trimers and hexamers) in the presence of 0.05% glutaraldehyde (Figure 1C). These results suggest that ArgR of L. monocytogenes is a typical arginine repressor which might contribute to transcriptional regulation of arginine metabolism.

We further analyzed the sequences of the promoter regions of argCJBD, argGH, sigB, and arcA genes for possible ArgR binding sites using a virtual footprint promoter analysis program (see Materials and Methods; Munch et al., 2005). The ARG box consensus was described as TNTGAATWWWWATTCANW in E. coli (Maas, 1994), CATGAATAAAAATKCAAK in B. subtilis (Miller et al., 1997), and AWTGCATRWWYATGCAWT in Streptomyces (Rodriguez-Garcia et al., 1997; where W = A or T, K = G or T, R = A or G, Y = T or C, N = any base). Five putative ARG boxes were identified in each putative promoter region upstream of these five genes from L. monocytogenes on the basis of similarity with the B. subtilis (Makarova et al., 2001) and B. licheniformis (Maghnouj et al., 1998) and there were 1–3 bp mismatch with respect to the consensus sequence (Figure 1D).

To confirm that ArgR directly binds to the respective ARG boxes identified above, the EMSA was performed. The promoter regions containing the putative ARG boxes were generated and incubated with recombinant ArgR, and the protein-DNA complexes were assayed by native gel electrophoresis. Figure 2A shows that recombinant ArgR was able to bind to DNA oligonucleotides of each promoter of argCJBD, argGH, sigB, or arcA. More significantly, ArgR shows stronger binding capacity to argCJBD and argGH promoters than to those of sigB and arcA under the experimental conditions we studied. These results indicate that L. monocytogenes ArgR contributes to overall regulation of the argCJBD, argGH, sigB, and arcA promoter activities although the degree of regulation could be different. More importantly, we found that ArgR protein completely lost the binding ability to ARG boxes when the two residues Ser42 and Arg43 (SR motif) were mutated to alanine (Figure 2A), strongly suggesting that these two sites are critical amino acids for ArgR-DNA binding.

To further analyze the regulatory function of ArgR in expression of its target genes, we cloned a DNA fragment covering the promoter-operator region of the argGH operon (as the representative gene cluster involved in arginine anabolism) into the gfp reporter vector which was transformed into ΔArgR mutant and its parent strain. Data show that GFP expression was significantly elevated in the ΔArgR mutant under neutral and acidic conditions while GFP was barely detectable in the wild-type strain (Figures 2B,C). Thus, ArgR is shown to repress the arginine biosynthetic pathway by interacting with the promoter of the argGH operon.

Since the putative ARG boxes are present in the promoter regions of argC, argG, arcA, and sigB, transcription of these genes might be regulated by ArgR. To find out if such regulation occurs, real-time quantitative PCR was performed using total RNA isolated from the wild-type strain L. monocytogenes 10403S and the argR deletion mutant ΔArgR in the presence (10 mM) or absence of arginine. We found that expression of argR was significantly induced in response to acidic pH at 5.5 regardless of arginine supplementation (Figures 3A,D). The transcriptional levels of two representative genes (argC and argG) involved in arginine anabolism were significantly increased in the ΔArgR mutant under neutral or acidic conditions (Figures 3B,C), and such effects were augmented by addition of exogenous arginine (Figures 3E,F). These findings indicate that L. monocytogenes ArgR plays a classical role of ArgR/AhrC family in feedback inhibition of the arginine biosynthetic pathway using arginine as a corepressor. Transcription of arcA was downregulated in the ΔArgR mutant under neutral pH (Figure 3B), which is consistent with findings from the previous study by Ryan et al. (2009), whereas sigB was slightly upregulated (Figure 3B). However, these two genes were markedly repressed by ArgR when bacterial cells were exposed to acidic pH in the absence of arginine (Figure 3C), but addition of arginine weakened the effect of ArgR on transcription of arcA and sigB regardless of pH conditions (Figures 3E,F). Therefore, L. monocytogenes ArgR appears to be a functional transcriptional regulator that modulates the expression of the arc operon positively and negatively under neutral and acidic pH conditions, respectively, by employing arginine as a cofactor.

Immuno-blotting was used to determine the relevance of ArgR to the expression of arginine metabolism operon proteins ArgG and ArcA as well as SigB under neutral and acidic pH (5.5) conditions. Expression of ArgG in ΔArgR strain was significantly higher under neutral and acidic environments than that of the wild-type strain. (Figures 4A,B). When exogenous arginine was added, expression of ArgG was not detected in the wild-type strain, but expression strongly increased when ArgR was absent (Figures 4C,D), further indicating that arginine cooperates with ArgR to repress the arginine biosynthetic pathway in Listeria. In addition, ArgR can regulate the expression of ArcA and SigB in an arginine-dependent and independent manner (Figure 4), which is consistent with the results from transcriptional analysis mentioned above.

In order to investigate the contribution of ArgR to the survival of the bacterium at lethal pH values, acid tolerance experiments were carried out on the mutants in complex medium adjusted to a lethal pH of 3.5 using 3 M lactic acid. Data show that deletion of argR exhibited no significant difference in the rate of survival relative to the parent strain at the early time points (30 and 60 min; Figure 5). However, a notable increase in the number of surviving cells was observed for the ΔArgR from minutes 90 onward, as compared to those of the wild-type strain (Figure 5). Conversely, constitutive overexpression of ArgR compromised bacterial survival under the same pH conditions (Figure 5). It’s worth noting that these data are contradictory to findings by Ryan et al. (2009) who reported that L. monocytogenes ΔArgR had defect in acid resistance at both sublethal and lethal pH levels (Ryan et al., 2009). Nonetheless, based on our findings for ArgR involved in regulations on arcA and sigB, we speculated that increasing of the acidic survival in the absence of ArgR was most probably due to the activation arcA and sigB. In addition, consistent with our previous studies (Cheng et al., 2015), survival of L. monocytogenes was markedly compromised in the absence of sigB in the lethal acidic conditions (Figure 5).