S. suis strain 10 (Smith et al., 1999), a highly virulent serotype 2 strain, was used in this study. Bacteria were routinely grown on blood agar plates (BD) at 37°C with 5% CO₂, or cultivated in liquid TSB (BD, Heidelberg, Germany) medium under the same conditions. Following day, bacteria were adjusted to an optical density at 600 nm of 0.05 in a tryptone-yeast-based medium supplemented with 10 mM glucose or galactose, respectively. If indicated, 50 mM arginine was supplemented (Burne et al., 1987; Zeng et al., 2006). Auxotrophy studies were performed in a chemically defined medium (CDM) in the presence of absence of arginine essentially as described elsewhere (van de Rijn and Kessler, 1980; Hitzmann et al., 2013). Growth was monitored every hour using a Nova Spec II Photometer (Pharmacia, Freiburg, Germany). Assays were performed in triplicates and repeated at least four times.

To determine the transcriptional organization of the ADS, bacteria were grown in TY medium supplemented with 50 mM arginine and 10 mM glucose or galactose, respectively, to an OD₆₀₀ of 0.2. Then, 10 ml of bacterial culture were harvested by centrifugation. Pellets were resuspended in 1 ml of Trizol (Invitrogen/Life Technologies, Carlsbad, California, USA) and immediately snap-frozen in liquid nitrogen.

If not stated otherwise, all enzymes and reagents were purchased from Invitrogen (Life Technologies, Carlsbad, California, USA) and NEB (New England Biolabs, Frankfurt am Main, Germany). Chromosomal DNA was prepared using the Qiagen's DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer's recommendations. Plasmid DNA was purified with the NucleoSpin® Plasmid Kit (Macherey-Nagel, Dueren, Germany) according to manufacturer's instructions. RNA was prepared as described by Hitzmann et al. (2013) using the Ambion's RiboPure™-Bacteria Kit. Residual DNA was digested with the Ambion TURBO DNA-free™-kit. Complementary DNA (cDNA) was synthesized from 1 μg total RNA using random primers (3 μg). Primers and RNA were heated for 10 min at 70°C in 12 μl of dH2O and then chilled on ice. Eight μl of master mix, consisting of 4 μl 5x first strand buffer, 2 μl 10 mM dNTP mix, 1 μl RNase Inhibitor and 1 μl 100 mM DTT, were added and incubated for 5 min at 25°C. Then, 1 μl of Reverse Transcriptase (SuperScript II) was added and another 10 min incubation-step at 25°C followed. Then the reaction was incubated at 42°C for 1 h and the Reverse Transcriptase was inactivated. cDNA was purified using the Qiagen PCR-purification kit. To analyse the transcriptional organization of the ADS, RT-PCR was performed using following intergenic primer pairs: flpS-arcA (CGA TGG TCT TGT TTG AAA CCT/ACA CCA GCC ATC GTT TTC TC), arcD-arcT (CTC CAC ATG GGT GAA GAA GG/CGC CAT CGA AGG ACC TTT A), arcT-arcH (CTG CGG ATA AAG AAG CCC TA/CTG ATG CTG GCT GTT GGT TA).

ArcD was inactivated by insertion mutagenesis as described earlier (Fulde et al., 2011). Briefly, the gene arcD was amplified from the streptococcal genome with the primers arcDKOfor (CCG TTA CTG TGG CTG AAT TGG) and arcDKOrev (CCT TGC AAT CCT TCT TCA CC) and subsequently introduced into the cloning vector pGEM®-T Easy (Promega, Mannheim, Germany). The resulting plasmid pGEM-arcD was linearized by HpaI. Then, the PvuII released erythromycin resistance cassette derived from vector pICerm (kindly provided by Christoph Baums, Institute for Microbiology, University of Veterinary Medicine, Hannover) was introduced to disrupt arcD. Electroporation was essentially performed as previously described (Smith et al., 1995). Mutants were tested for integrity by PCR using the primer pair arcDKOfor/arcDKOrev.

A method to determine arginine in the supernatant of bacterial cultures was developed based on the Sakaguchi reaction (Sakaguchi, 1925). Briefly, bacteria were grown in TY medium in the presence of 10 mM arginine. At an OD₆₀₀ of 0.2, streptococci were harvested by centrifugation and the resulting supernatant was filtrated using the Millex® Syringe Filters with pore size of 0.22 μm (Merck Millipore, Schwalbach, Germany). Then, 100 μl of bacterial supernatant was mixed with 100 μl reagent A (0.05% (w/v) chloronaphthol, 5% urea (m/v) in 95% EtOH). After extensive shaking, 200 μl reagent B (0.7% Brom (v/v), 5% NaOH (w/v) in H₂O) was added. A change in color was determined spectro-photometrically by an OD of 500 nm. Quantification was done along a calibration curve with different concentrations of arginine diluted in TY medium. Non-inoculated TY medium served as control.

In all labeling experiments, a CDM overnight culture of the indicated S. suis strain was harvested by centrifugation, washed twice in PBS, and then inoculated in fresh CDM to an OD₆₀₀ of 0.002. In experiments determining the ¹³C-label in proteinogenic amino acids, bacteria were grown in CDM containing 2.5 mM [U-¹³C₆]arginine (Campro Scientific) at 37°C and harvested at an OD₆₀₀ of 0.2 by centrifugation at 4000 × g at 4°C for 5 min. The bacterial cells were washed twice in ice-cold PBS, immediately autoclaved at 120°C for 15 min, and lyophilized. Samples were hydrolyzed under acidic conditions. The resulting amino acids were purified using a cation exchange column, converted into TBDMS derivatives (except for arginine, see below), and analyzed by GC/MS as described earlier (Eylert et al., 2008). For the [U-¹³C₆]arginine uptake experiments, bacteria were first grown in CDM with ¹²C-arginine to an OD₆₀₀ of 0.2, then harvested by centrifugation, washed twice in PBS, and afterwards transferred to CDM without any arginine to induce arginine starvation for 15 min at 37°C. The bacteria were concentrated by centrifugation and then incubated in CDM containing 2.5 mM [U-¹³C₆]arginine at 37°C for 30 min. Bacteria were then pelleted by centrifugation, washed twice in ice-cold double-distilled water, and immediately disrupted by ultrasonic disintegration in a Branson Sonifier with continuous water cooling for 15 min at 4°C, and output control of 8. The lysates were cleared by centrifugation at 10,000 × g at 4°C for 30 min and the remaining supernatant lyophilized for further analysis.

Arginine does not form trimethylsilyl (TMS) and tertbutyldimethylsilyl (TBDMS) derivatives (Halket et al., 2005) and can be analyzed as arginine- trifluoroacetic acid (TFA)-methylester (Darbre and Islam, 1968). An aliquot of the cation exchange eluate described above was dried under a stream of nitrogen and dissolved in 200 μl of methanolic HCl (3N). The mixture was heated to 70°C for 30 min and then dried under a stream of nitrogen. The residue was dissolved in 50 μl of TFA and heated to 140°C for 10 min. The mixture was dried again, dissolved in 100 μl of anhydrous ethylacetate and subjected to GC/MS analysis. General GC/MS conditions were the same as described for amino acid TBDMS derivatives (Eylert et al., 2008). For TFA-methylester derivatives the column was kept at 70°C for 3 min and then developed with a temperature gradient of 10°C min⁻¹ to a final temperature of 200°C that was kept for 3 min. The retention time for the arginine-TFA-methylester was 17.2 min. The molecular mass of the arginine derivative was 476. ¹³C-excess calculations were performed with m/z 407 [M-CF₃]^+., a fragment still containing all C atoms of arginine.

Ornithine was determined from the freeze dried supernatant and derivatized as described for arginine. Under identical GC/MS conditions, ornithine was analyzed as ornithine-TFA-methylester (M: 338) at R_t 14.0 min. The observed fragment m/z 306 corresponds to [M-CH₃OH]. The ¹³C/¹²C ratio was calculated with the relative intensities of m/z 306 (¹²C-ornithine) and m/z 311 ([U-¹³C₅]ornithine).

Determination of ammonia in the culture supernatant of WT strain 10 and its arcD-deficient mutant strain was performed using the ammonia assay kit (Sigma, Munich, Germany) as described previously (Fulde et al., 2011). pH values in the bacterial culture supernatant were determined using a specific electrode (pH 197, WTW, Weilheim, Germany).

AD activity was determined according to the protocol of Oginsky (1957) and Degnan et al. (1998) as described previously (Gruening et al., 2006; Winterhoff et al., 2002). Briefly, bacteria were grown in CDM medium as in the [U−¹³C₆]arginine uptake assays and harvested by centrifugation. Then, bacteria were lysed and the respective lysates were incubated for 2 h in a 0.1 M potassium phosphate buffer containing 10 mM L-arginine at 37°C. The supplementation of 250 μl of an acidic solution (1:3, 96% sulfuric acid and 85% orthophosphoric acid) stopped enzymatic reactions. After addition of 31.3 μl of a 3% diacetyl monoxime solution, the suspension was incubated for 15 min at 100°C. Production of citrulline was determined colorimetrically at an OD₄₅₀. Results are given in nmol citrulline produced in 1 h per mg whole cell protein

Experiments were performed essentially as described earlier (Benga et al., 2004; Gruening et al., 2006). Briefly, WT strain 10 and its isogenic mutant strain 10ΔarcD were grown overnight in TSB. Then, bacteria were harvested by centrifugation and resuspended in a buffer containing 20 mM Na₂HPO₄, 1 mM MgCl₂, 25 mM arginine-HCl adjusted to pH 5, 6, or 7, respectively. Bacteria were incubated at 37°C for the indicated intervals and survival was monitored by plating. Results represent means and standard deviations of one experiment performed in triplicates. Experiments were repeated at least three times.

The ability of the wild-type strain 10 and the arcD deficient mutant strain to survive in HEp-2 epithelial cells was determined as described previously with some modifications (Benga et al., 2004; Fulde et al., 2011). Briefly, in addition to untreated HEp-2 cells, parallel assays were done with HEp-2 cells that had been pre-treated with bafilomycin (200 nM) for 1 h to inhibit endosomal acidification. HEp-2 cells were then infected with 100 bacteria per cell (MOI 100:1) for 2 h and afterwards washed thrice with PBS. In parallel, cells were incubated in DMEM containing 31.25 μg ml⁻¹ Daptomycin (Cubicin®) for 90 and 210 min, respectively, at 37°C with 8% CO₂ to kill extracellular bacteria. The monolayers were washed three times with PBS and 100 μl trypsin-EDTA solution was added to each well. After 5 min, 900 μl of 1% sterile saponin was added and the lysates were plated in triplicates on blood agar and incubated at 37°C for 24 h. The number of CFU was determined at 90 and 210 min post-infection of the cells and expressed as percentage of intracellular bacterial survival after 2 h. Thus, one hundred percent indicates that no difference in intracellular CFU was detected after two hours. The experiments were repeated three times.

Prediction of localization and topology of ArcD was performed using the SignalP 4.1 Server and the TMHMM Server v. 2.0 available at: http://www.cbs.dtu.dk.

The ADS is a highly conserved cluster of seven genes encoding the most important arginine-catabolizing pathway in S. suis and two flanking genes encoding for transcriptional regulators (Figure 1A, Gruening et al., 2006). The core ADS, facilitating the degradation of arginine to ATP, is composed of three genes: arcA, encoding for an arginine deiminase; arcB, an ornithine-carbamoyltransferase, and arcC, a carbamate kinase. These genes are in close proximity to the putative arginine-ornithine antiporter gene arcD and a potential Xaa-His dipeptidase gene arcT, as well as arcH, a putative endo-β-galactosidase C. Thus, we performed RT-PCR analysis using intergenic primer pairs from RNA of bacteria grown under inducing (TY medium supplemented with 50 mM arginine and 10 mM galactose) and repressive (50 mM arginine and 10 mM glucose) conditions. As depicted in Figure 1B, a positive PCR signal indicating polycistronic transcription was only detected for the arcD-arcT intergenic region (primer pair C/D) indicating expression of arcD and arcT from an operon. Interestingly, similar to what is known for the arcABC operon (Gruening et al., 2006), the transcription of arcDT was significantly increased when galactose was present as the sole carbon source. In contrast, the regulatory gene flpS (intergenic primer pair A/B) located upstream of arcA as well as the accessory gene arcH (primer pair E/F) were transcribed monocistronically.

In silico analysis of the S. suis ArcD revealed significant homologies to transmembranal proteins with arginine-ornithine antiporter function of other streptococci and other arginine-fermenting bacteria (Table 1). However, functional studies on this topic are rare. Therefore, we inactivated arcD by insertion mutagenesis and characterized the phenotype of the mutant by growth kinetics. As depicted in Figure 2A, a comparable growth of WT strain 10 and its isogenic mutant strain 10ΔarcD was observed in the first hours of growth with a mean OD₆₀₀ ranging between 0.0279 ± 0.008 (WT, with arginine supplementation) and 0.0315 ± 0.0055 (10ΔarcD, without arginine supplementation). After 4 h, the growth of WT strain 10 (red lines) was higher as compared to the arcD-deficient mutant strain (blue lines). Interestingly, supplementation of arginine (solid lines) did not lead to a higher growth, neither of WT strain 10 nor of the mutant until 5 h. After 6 h, WT strain 10 reached an OD₆₀₀ of 0.2253 ± 0.047 (broken red line) without arginine supplementation, and an OD₆₀₀ of 0.3983 ± 0.12 (solid red line) when arginine had been supplemented. In contrast, significantly lower OD values were detected for the arcD-deficient mutant strain, with (0.1343 ± 0.025, solid blue line) and without arginine supplementation (0.093 ± 0.001, broken blue line). Nevertheless, though less prominent the mutant strain 10ΔarcD showed an arginine-dependent phenotype similar to the WT strain. Overall, differences in bacterial numbers and arginine availability increased over time between both strains.

Next we monitored changes in the pH of the medium during growth of WT strain 10 and its arcD-deficient mutant. As depicted in Figure 2B, pH values of the culture medium decreased similarly for strain 10 and 10ΔarcD without arginine supplementation. Nevertheless, a slight difference between strain 10 (6.855 ± 0.065) and 10ΔarcD (6.655 ± 0.005) was detected at 8 h of growth. This difference was even more prominent when external arginine was supplemented to the growth medium (solid lines). WT strain 10 was able to antagonize growth-dependent acidification of the culture medium resulting in an increase in pH from 6.905 ± 0.059 at the time of inoculation to 8.063 ± 0.2 (solid red line) after 24 h, whereas the pH values detected for the arcD-deficient strain (blue lines) dropped similarly to those monitored without arginine supplementation from 6.8425 ± 0.03 (0 h) to 5.54 ± 0.07 (24 h).

Our previous studies showed that ADS-dependent ammonia production as a by-product of arginine catabolism is essentially involved in environmental pH homeostasis (Fulde et al., 2011). To investigate whether similar effects hold true for the ArcD-deficient mutant strain, we determined ammonia production of WT strain 10 and its arcD-deficient mutant strain grown under conditions with and without supplementation of arginine (Figure 2C). As expected, supplementation led to a more than 10-fold increase of ammonia production (0.11 ± 0.08 mg ml⁻¹ vs. 1.37 ± 0.36 mg ml⁻¹) for WT strain 10. Interestingly, although strain 10ΔarcD was also able to increase the amount of ammonia in the presence of arginine (0.084 ± 0.04 mg ml⁻¹ vs. 0.026 ± 0.001 mg ml⁻¹), this effect was comparable to that seen in growth of WT strain without arginine supplementation.

Since ArcD is predicted to be an arginine-ornithine antiporter, we wondered whether a deletion in the respective gene would lead to deficiencies in arginine uptake. For this, we adapted the method described by Sakaguchi (1925) to determine extracellular arginine concentrations. As depicted in Figure 2D, WT strain 10 was able to completely deplete free arginine from the bacterial culture medium (red bar). In contrast, the medium inoculated with strain 10ΔarcD (blue bar) still contained significantly higher amounts of arginine (10.371 ± 0.18 mM) at the same OD.

In summary, these results indicate that ArcD is involved in the arginine uptake which is necessary to support the central functions of the ADS. Furthermore, they show that extracellular arginine is important for bacterial growth and a substrate for the arginine deiminase system in S. suis.

In order to demonstrate the contribution of ArcD to arginine uptake we performed growth experiments in a chemically defined medium (CDM) containing all amino acids including or excluding arginine. These experiments revealed that S. suis strain 10 and strain 10ΔarcD were not able to grow in CDM medium containing all amino acids except arginine (Figure 3A). Supplementation of arginine restored growth of both strains, even though the growth of strain 10ΔarcD was remarkably diminished when compared to that of the parental strain. These data indicate that S. suis strain 10 is auxotrophic for arginine and that ArcD contributes to arginine uptake.

In order to verify that S. suis is capable to take up arginine by ArcD and incorporate arginine in newly synthesized proteins, we performed labeling experiments in CDM supplemented with [U-¹³C₆]arginine that were followed by the detection of the ¹³C-label in protein derived amino acids by GC/MS analysis. As depicted in Figure 3B, [U-¹³C₆]arginine was taken up and used for protein biosynthesis in both strains. It is important to note that in this experiment the ¹³C excess in arginine is no quantitative value for uptake, because [U−¹³C₆]arginine is the sole arginine source which is also taken up by the arcD-deficient strain (Figure 3A). However, high levels of ¹³C-enrichment were not detected in any other proteinogenic amino acid excluding de novo biosynthesis of these amino acids from [U-¹³C₆]arginine as a precursor. Nevertheless, ¹³C-excess below 1 mol% was found as a ¹³C₁-labeled isotopolog in aspartate. This indicates that ¹³CO₂, formed as a by-product of ADS mediated [U-¹³C₆]arginine catabolization, is used as a precursor in a carboxylation reaction required for aspartate biosynthesis. However, the overall ¹³C excess in arginine did not differ between the WT strain and strain 10ΔarcD. Since bacteria were harvested at the same optical density this may be an explanation for that, and these results emphasize that arginine uptake is the growth limiting step for strain 10ΔarcD in CDM.

To further elucidate if 10ΔarcD has a reduced capacity to take up arginine, we reduced the [U-¹³C₆]arginine labeling time to 30 min and determined the ¹³C/¹²C ratio of free intracellular ornithine, a product of the arginine deiminase pathway, since the free arginine levels were under the detection limit. The efficiency of [U-¹³C₆]arginine derived ¹³C incorporation in intracellular [U-¹³C₅]ornithine was approximately 15-fold higher in the WT strain when compared to strain 10ΔarcD (Figure 3C, left panel). The arginine deiminase activity did not differ between both strains under these conditions (Figure 3C, right panel) which excluded a different arginine consumption of the strain. Taken together, these results indicate that ArcD is an arginine transporter.

The above data indicate a central role of arginine and arginine uptake for the metabolism of S. suis. Therefore, we next analyzed the relevance of ArcD for bacterial survival. As shown in Figure 4A survival of WT strain 10 (black bars) and its isogenic, arcD-deficient mutant strain 10ΔarcD (white bars) differed when incubated in an arginine-containing buffer with pH values adjusted to 5.0, 6.0, and 7.0, respectively. Bacteria were replica-plated after 4 h to monitor survival. No significant differences were observed at pH values of 7.0 and 6.0. However, at pH 5.0, the survival rate of the WT strain was 65.5% ± 6.5, whereas the arcD-deficient mutant strain was almost completely killed (0.5% ± 0.5). As a control, bacteria were incubated in buffer adjusted to pH 5.0 without the supplementation of arginine. Under these conditions, strain 10 and 10ΔarcD were similarly affected in survival emphasizing the important role of ArcD as an arginine supplier for ADS-mediated resistance under acidic conditions.

In order to elucidate the relevance of ArcD for biological fitness of S. suis in a biological model, we performed infection experiments with the epithelial cell line HEp-2. As shown in Figure 4B, the WT strain 10 was able to survive intracellularly at a rate of about 70%, whereas significantly lower survival rates (approximately 35%) were determined for the arcD mutant strain. To analyse whether reduced survival correlated with acidification and, thus, the inability of strain 10ΔarcD for efficient arginine supply to generate ammonia via the ADS and prevent acidification, HEp-2 cells were treated with bafilomycin to inhibit endosomal acidification before infection. Compared with the infection of untreated cells, the pretreatment of the cells with bafilomycin significantly increased the survival rate of strain 10ΔarcD. These data suggest that ArcD substantially contributes to efficient arginine uptake in S. suis and, thereby, to its resistance against endosomal acidification in HEp-2 cells.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.