ArcD1 and ArcD2 Arginine/Ornithine Exchangers Encoded in the Arginine Deiminase Pathway Gene Cluster of Lactococcus lactis

ABSTRACT

The arginine deiminase (ADI) pathway gene cluster in Lactococcus lactis contains two copies of a gene encoding an l-arginine/l-ornithine exchanger, the arcD1 and arcD2 genes. The physiological function of ArcD1 and ArcD2 was studied by deleting the two genes. Deletion of arcD1 resulted in loss of the growth advantage observed in the presence of high l-arginine in different growth media. Uptake of l-arginine and l-ornithine by resting cells was reduced to the low level observed for an ArcD1/ArcD2 double deletion mutant. Deletion of the arcD2 gene did not affect the growth enhancement, and uptake activities were slightly reduced. Nevertheless, recombinant expression of ArcD2 in the ArcD1/ArcD2 double mutant did recover the growth advantage. Kinetic characterization of ArcD1 and ArcD2 showed high affinities for both l-arginine and l-ornithine (K_m in the micromolar range). A difference between the two transporters was the significantly lower affinity of ArcD2 for the cationic amino acids l-ornithine, l-lysine, and l-histidine. In contrast, the affinity of ArcD2 was higher for the neutral amino acid l-alanine. Moreover, ArcD2 efficiently translocated l-alanine, while ArcD1 did not. Both transporters revealed affinities in the mM range for agmatine, cadaverine, histamine, and putrescine. These amines bind but are not translocated. It is concluded that ArcD1 is the main l-arginine/l-ornithine exchanger in the ADI pathway and that ArcD2 is not functionally expressed in the media used. ArcD2 is proposed to function together with the arcT gene that encodes a putative transaminase and is found adjacent to the arcD2 gene.

IMPORTANCE The arginine deiminase (ADI) pathway gene cluster in Lactococcus lactis contains two copies of a gene encoding an l-arginine/l-ornithine exchanger, the arcD1 and arcD2 genes. The physiological function of ArcD1 and ArcD2 was studied by deleting the two genes. It is concluded that ArcD1 is the main l-arginine/l-ornithine exchanger in the ADI pathway. ArcD2 is proposed to function as a l-arginine/l-alanine exchanger in a pathway together with the arcT gene, which is found adjacent to the arcD2 gene in the ADI gene cluster.

INTRODUCTION

The arginine deiminase (ADI) pathway catalyzes the degradation of l-arginine to l-ornithine, ammonia, and carbon dioxide and generates 1 mol of ATP per mol of l-arginine consumed. The pathway is widely distributed among bacteria and serves as a source of energy, carbon, and/or nitrogen (1, 2) and as a mechanism for survival in acidic environments (3). The pathway consists of three metabolic steps (Fig. 1A): l-arginine is converted into citrulline and ammonia, a reaction catalyzed by arginine deiminase (ADI; encoded by arcA); citrulline is metabolized into l-ornithine and carbamoyl-P, a reaction catalyzed by ornithine transcarbamylase (OTC; encoded by arcB); and carbamoyl phosphate is used to phosphorylate ADP, yielding ATP, carbon dioxide, and ammonia, a reaction catalyzed by carbamate kinase (CK; encoded by arcC) (2, 4). An essential role in the ADI pathway is played by the l-arginine/l-ornithine exchanger ArcD (encoded by arcD), which catalyzes electroneutral exchange between l-arginine and l-ornithine and is responsible for the concomitant uptake of the substrate l-arginine and excretion of the end product l-ornithine (5, 6). Since no metabolic energy is needed for the transport reaction, ATP produced by the ADI pathway can be entirely used for other energy-demanding purposes.

FIG 1.

Schematic representations of the ADI pathway (A) and the ADI gene cluster in L. lactis (B). ADI, arginine deiminase; OTC, ornithine transcarbamoylase; CK, carbamate kinase. The arcD1 and arcD2 genes encode the l-arginine/l-ornithine exchangers.

In the lactic acid bacterium Lactococcus lactis, the genes encoding the ADI pathway are found in a rather unusual cluster of 9 genes (Fig. 1B). Four genes essential for building the pathway are located in the center in the order arcA-arcB-arcD1-arcC1. Upstream of arcA and transcribed in the same direction are argS, encoding a putative arginyl-tRNA synthetase, and the divergently transcribed gene argR, encoding an arginine repressor that is involved in arginine-dependent regulation of expression of the ADI pathway in L. lactis (7, 8). Downstream of arcC1, there are additional copies of arcC and arcD and arcT, encoding a putative aminotransferase (8), in the order arcC2-arcT-arcD2. The physiological function of the duplications of the arcC and arcD genes and of the arcT gene product are not known.

The genes llmg_2311 (arcD1) and llmg_2307 (arcD2) of L. lactis MG1363 encode membrane proteins containing 526 (ArcD1) and 497 (ArcD2) amino acid residues, respectively. The proteins share 63% sequence identity and are members of the APC (amino acid-polyamine-organocation) superfamily of transporters (9). The longer ArcD1 sequence is predicted to contain 14 transmembrane segments versus 13 segments predicted for ArcD2. Recently, the arcD1 and arcD2 genes were cloned and expressed in L. lactis MG1363, resulting for both genes in higher transport activities with l-arginine and l-ornithine relative to the wild-type background (10). Inhibition studies revealed a difference between the substrate specificity of the two transporters. ArcD1 was inhibited by l-lysine and l-histidine and ArcD2 by l-lysine and l-alanine. It was concluded that both ArcD1 and ArcD2 function as l-arginine/l-ornithine exchangers. Here, the physiological role of ArcD1 and ArcD2 in L. lactis was investigated by growth and transport studies using ΔarcD1 and ΔarcD2 deletion strains and recombinant strains expressing the two transporters in a double deletion mutant background. In contrast to what was suggested by the earlier study described above (10), it is demonstrated here that ArcD1 is the main l-arginine/l-ornithine exchanger in the ADI pathway. ArcD2 appears not to be functionally expressed in the different media used in this study. A physiological function for ArcD2 is proposed based on the different substrate specificities of the transporters.

MATERIALS AND METHODS

Strains, media, and growth conditions.

Lactococcus lactis JP9000 (referred to as the wild type), derived from strain MG1363 and carrying the nisRK genes in the pseudo-10 locus (11), was used as the parent for construction of the ΔarcD1, ΔarcD2, and ΔarcD1D2 deletion mutants. The ΔarcD1D2 double mutant was used as the host for the nisin-inducible expression of the ArcD1 and ArcD2 transporters in the D1ΔarcD1D2 and D2ΔarcD1D2 strains, respectively. L. lactis was grown at 30°C in M17 medium supplemented with 28 mM glucose or 28 mM galactose and in the presence of 3 μg/ml erythromycin or 5 μg/ml chloramphenicol, when appropriate, or in chemically defined SA medium containing 19 free amino acids and supplemented with 56 mM glucose or galactose and nisin at the indicated concentrations when appropriate (12). Escherichia coli DH5α, used for routine cloning to create the deletion constructs, was grown in LB medium containing 150 μg/ml erythromycin. Bacterial strains are summarized in Table 1.

TABLE 1.

L. lactis strains

Strain	Description	Reference
JP9000	MG1363 with nisRK in pseudo-10 locus	11
JP9000 ΔarcD1	JP9000 ΔarcD1	This paper
JP9000 ΔarcD2	JP9000 ΔarcD2	This paper
JP9000 ΔarcD1D2	JP9000 ΔarcD1 ΔarcD2	This paper
JP9000 D1ΔarcD1D2	ΔarcD1D2/pNZarcD1	This paper
JP9000 D2ΔarcD1D2	ΔarcD1D2/pNZarcD2	This paper

Growth curves of L. lactis strains were recorded using a Biotek Powerwave 340 96-well plate reader. Overnight cultures in M17 with glucose were washed and diluted to an optical density at 600 nm (OD₆₀₀) of 0.05 in 200 μl of the indicated medium and covered with 50 μl of silicon oil (1:4 ratio of silicon oil M20 and M200) to prevent evaporation. The optical density at 600 nm was measured for 20 h every 10 min with 30 s of shaking before each measurement.

Construction of deletion and expression mutants.

The L. lactis JP9000 genes llmg_2311 (arcD1) and llmg_2307 (arcD2) were deleted with the two-step integration-and-excision system using plasmid pCS1966 (13), creating the single-knockout ΔarcD1 and ΔarcD2 strains and, subsequently, the double-knockout ΔarcD1D2 strain. Two 800-bp fragments, corresponding in sequence to the up- and downstream regions of the genes with 130 to 240 bp overlapping with the genes, were amplified by PCR using specific primer pairs and subsequently digested with specific restriction endonucleases. The amplicons were ligated into pCS1966 digested with the same endonucleases, yielding pCSdelarcD1 and pCSdelarcD2. The resulting plasmids were transformed to L. lactis JP9000, generating the markerless ΔarcD1 and ΔarcD2 deletion mutants. The ΔarcD1D2 mutant was produced by transforming the ΔarcD1 mutant with pCSdelarcD2 (10, 13).

Expression strains D1ΔarcD1D2 and D2ΔarcD1D2 were constructed by transformation of plasmids pNZarcD1 or pNZarcD2 (10), encoding ArcD1 and ArcD2 under the control of the nisin-inducible promoter (P_nisA), respectively, to L. lactis ΔarcD1D2 using a standard electroporation protocol.

Amino acid transport assays.

Cells were grown in M17 medium with glucose containing nisin when appropriate (see below). Cells were harvested, washed, resuspended to an OD₆₀₀ of 2 in ice-cold 100 mM potassium phosphate (pH 6.0) buffer containing 0.2% glucose, and kept on ice until use. An aliquot of 100 μl of cells was preincubated for 5 min at 30°C with continuous stirring, followed by the addition of ¹⁴C-labeled l-arginine or l-ornithine to final concentrations of 1.3 μM and 10 μM, respectively. Uptake was stopped by addition of 2 ml ice-cold 0.1 M LiCl, and the suspension was filtered over a 0.45-μm-pore-size nitrocellulose filter (BA85; Schleicher & Schuell GmbH). The filter was washed once with 2 ml of ice-cold 0.1 M LiCl and submerged in Emulsifier Scintillator Plus scintillation fluid (Packard Bioscience). Radioactivity was measured by scintillation counting with a Tri-Carb 2000CA liquid scintillation analyzer (Packard Instruments).

Strains D1ΔarcD1D2 and D2ΔarcD1D2 were used for the determination of kinetic parameters. The cells were grown in M17 with glucose to an OD₆₀₀ of 0.5, after which expression was induced by adding 0.5 ng/ml of nisin to the D1ΔarcD1D2 and D2ΔarcD1D2 cultures, followed by an additional 30 min of incubation. The nisin concentration and induction time were adjusted to obtain a maximal uptake after 10 s of approximately 10 to 20% of total radiolabel in the experiments. Initial rates were inferred from the 10-s time points. The K_m for l-arginine was determined from the initial uptake rates measured in the concentration range of 1.3 to 21.3 μM. The K_m for l-ornithine was determined in the range of 0.5 to 5 μM and 10 to 60 μM for ArcD1 and ArcD2, respectively.

RESULTS

Growth enhancement by the ADI pathway in ArcD1 and ArcD2 deletion mutants.

The ΔarcD1, ΔarcD2, and ΔarcD1D2 strains were derived from strain L. lactis JP9000 by deletion of the arcD1 and/or arcD2 genes, using a two-step integration-and-excision system (13). The role of the putative l-arginine/l-ornithine transporters ArcD1 and ArcD2 in the arginine deiminase pathway (ADI) was studied by its impact on growth in four different media. Rich M17 medium containing approximately 2 mM free l-arginine (14) and chemically defined SA medium containing 1.1 mM l-arginine (12) were supplemented with glucose or galactose as the carbon and energy source. In line with previous observations (15) the parent strain JP9000 showed a higher biomass yield in all four media when the medium contained an additional 25 mM l-arginine. Also, in the presence of additional l-arginine, a higher specific growth rate was observed in the presence of galactose but not in the presence of glucose (Fig. 2A to D).

FIG 2.

(A to D) Growth of the wild type in the absence (■) and presence (□) of 25 mM l-arginine in rich M17 medium and chemically defined SA medium supplemented with glucose and galactose as indicated. (E to H) Growth of the ΔarcD1 (▲), ΔarcD2 (●) and ΔarcD1D2 (△) deletion strains in the presence of 25 mM l-arginine in the same media. Values are means from at least two independent experiments.

Growth of the ΔarcD1, ΔarcD2, and ΔarcD1D2 mutants was not significantly different from growth of the parent strain in all four media when no additional l-arginine was provided (data not shown). In contrast, in the presence of additional l-arginine, the ΔarcD1 and ΔarcD1D2 strains showed the same behavior as the wild type in the absence of additional l-arginine, while the ΔarcD2 strain showed the same growth enhancement as the wild type (Fig. 2E to H). On all media, the growth rate of the ΔarcD1 and ΔarcD1D2 mutants was slightly reduced in the presence of extra l-arginine. It follows that the growth enhancement by the ADI pathway observed in all four media relies fully on the arcD1 gene, while the arcD2 gene is dispensable. Therefore, ArcD1 appears to be the main l-arginine/l-ornithine exchanger in the ADI pathway under these conditions.

Uptake of l-arginine and l-ornithine in ArcD1 and ArcD2 deletion mutants.

The L. lactis wild type and the ΔarcD1, ΔarcD2, and ΔarcD1D2 deletion mutants were grown to mid-exponential growth phase in M17 medium supplemented with glucose, and uptake of l-[¹⁴C]arginine and l-[¹⁴C]ornithine was measured by resting cells energized by glucose (Fig. 3A and B). Uptake was too fast to measure initial rates by the filtration assay used. Wild-type cells took up l-arginine to 1.3 nmol/mg protein at 10 s, after which uptake leveled off rapidly. The rate of uptake was reduced by over 90% in the ΔarcD1D2 mutant. The low residual uptake indicates the presence of an l-arginine uptake system(s) other than ArcD1 and ArcD2 in the membrane of L. lactis. The decrease in uptake by the double mutant could be attributed to ArcD1, since uptake by the ΔarcD1 mutant was similar to uptake by the ΔarcD1D2 mutant. The ΔarcD2 mutant took up l-arginine to 0.94 nmol/mg protein at 10 s, which was 72% of the wild-type uptake (Fig. 3A). A similar pattern was observed for l-ornithine uptake. Here, uptake was almost completely abolished for the ΔarcD1 and ΔarcD1D2 strains, showing that ArcD1 is responsible solely for l-ornithine uptake. Mutant ΔarcD2 took up l-ornithine to 2.8 nmol/mg protein at 10 s, which was around 60% of the uptake by the wild type (Fig. 3B).

FIG 3.

(A and B) Uptake of l-[¹⁴C]arginine (A) and l-[¹⁴C]ornithine (B) at concentrations of 1.3 and 10 μM, respectively, by resting cells of the wild-type (■), ΔarcD1 (▲), ΔarcD2 (□), and ΔarcD1D2 (△) strains grown in M17-glucose medium. (C to F) Uptake of l-[¹⁴C]ornithine at 10 s and at a concentration of 10 μM by resting cells of the wild-type, ΔarcD1, ΔarcD2, and ΔarcD1D2 strains grown in M17 and SA media supplemented with glucose and galactose as indicated and in the absence (gray bars) and presence (black bars) of 25 mM l-arginine. Cells were collected in mid-exponential phase (E) (OD₆₀₀ of 0.4) or early stationary phase (S).

The transport activities of ArcD1 obtained in the above-described experiment demonstrated that the transporter is functionally expressed in M17 medium supplemented with glucose and without additional l-arginine, conditions that did not result in growth enhancement (Fig. 2A). Ornithine uptake activity by the wild-type and mutant strains was determined in the media used for the growth experiments whose results are shown in Fig. 2 in the mid-exponential and early stationary growth phases (Fig. 3C to F). The uptake activity of the wild type increased slightly when the cells entered the stationary phase in M17-glucose medium without additional l-arginine. In the presence of additional l-arginine, the activity was higher and showed a similar increase between the exponential and stationary phases. The same pattern was observed for the ΔarcD2 mutant, while no activity was observed for the ΔarcD1 and ΔarcD1D2 mutants under any of these conditions (Fig. 3C). While the pattern for the wild type may be different, the latter was also observed after growth in the other media. Activities of the ΔarcD2 mutant were similar to those of the wild type, and no activity for the ΔarcD1 and ΔarcD1D2 mutants was observed (Fig. 3D to F). At a glance, the activities of the ΔarcD2 mutant were somewhat lower than those of the wild type. It follows that ArcD1 was consistently responsible for the uptake of l-ornithine in the wild-type cells and that no ArcD2 activity was observed in any of these conditions or growth stages. The expression pattern of ArcD1 in the different media is discussed in more detail in Discussion.

Recombinant expression of ArcD1 and ArcD2.

The ΔarcD1D2 double mutant is the perfect host for the recombinant expression of ArcD1 and ArcD2 to determine the characteristics of both transporters independently of each other. Uptake of l-[¹⁴C]arginine and [l-¹⁴C]ornithine was measured by resting cells of strains D1ΔarcD1D2 and D2ΔarcD1D2, producing ArcD1 and ArcD2, respectively, using the nisin-controlled expression (NICE) system (16). Both strains showed significant uptake of both substrates above the level of the double mutant, demonstrating functional expression (Fig. 4A and B). The cells expressing ArcD2 accumulated the substrates to a higher level than the cells expressing ArcD1. The initial rate was extremely high for both l-arginine and l-ornithine uptake by D1ΔarcD1D2. In line with this observation, the D1ΔarcD1D2 strain took up l-arginine and l-ornithine to a 2.3-fold-higher level at 10 s than the corresponding ΔarcD2 strain (Fig. 3A and B), indicating higher levels of expression of ArcD1 in the recombinant strain. The initial rate was similarly high for l-arginine uptake by ArcD2 but considerably lower for l-ornithine uptake by this transporter.

FIG 4.

(A and B) Uptake of l-[¹⁴C]arginine (A) and l-[¹⁴C]ornithine (B) at concentrations of 1.3 and 10 μM, respectively, by resting cells of ΔarcD1D2 (■), D1ΔarcD1D2 (▲), and D2ΔarcD1D2 (●) grown in M17 plus glucose in the presence of 0.5 ng/ml nisin. (C) Growth of the wild-type (□), ΔarcD1D2 (■), D1ΔarcD1D2 (▲), and D2ΔarcD1D2 (●) strains in M17 supplemented with glucose and 25 mM l-arginine and in the presence of 0.5 ng/ml nisin. Values are means from at least two independent experiments.

In spite of lacking an apparent role in the growth enhancement by the ADI pathway shown above (Fig. 2), ArcD2 appeared to share similar transport activities with ArcD1. Recombinant strain D2ΔarcD1D2 was grown in M17 with glucose in the presence of 25 mM l-arginine to demonstrate recovery of a functional ADI pathway in vivo. Functional expression of ArcD2 in the double mutant resulted in an increased biomass yield, as was observed with the wild type and the strain expressing ArcD1 (Fig. 4C). This strongly suggests that in the wild type, the lack of function of ArcD2 in the ADI pathway is due to the lack of expression of the arcD2 gene. The ADI activity of the recombinant D1ΔarcD1D2 and D2ΔarcD1D2 strains showed that all metabolic enzymes of the ADI pathway were expressed and active in the ΔarcD1D2 strain. Therefore, deleting arcD1 or arcD2 did not prevent the expression of the other genes located in the ADI gene cluster.

Substrate specificity of ArcD1 and ArcD2.

Kinetic constants of ArcD1 and ArcD2 for uptake of different substrates by the D1ΔarcD1D2 and D2ΔarcD1D2 strains are summarized in Table 2. Nisin inducer concentrations and induction times were adjusted to allow for initial rate measurements (see Materials and Methods). Both ArcD1 and ArcD2 transport l-arginine with high affinity (K_m of 5 and 4 μM, respectively). The maximal rates of uptake by the two recombinant strains were also similar, with a maximal rate (V_max) of 30 and 22 nmol/min · mg, respectively. Clear differences between the two transporters were observed with l-ornithine as the substrate. ArcD1 showed a 5-fold-higher affinity for l-ornithine than for l-arginine (K_m of 1 versus 5 μM), while the affinity of ArcD2 was approximately 7-fold lower (K_m of 29 versus 4 μM). The lower affinity for l-ornithine was compensated for by a higher V_max of 128 nmol/min.mg. The higher maximal rate may be a consequence of a higher level of expression or a higher turnover number of ArcD2. Previously, inhibition studies identified l-lysine and l-histidine as additional substrates of ArcD1 and l-lysine of ArcD2 (10). Inhibition studies with the D1ΔarcD1D2 recombinant strain presented here showed that ArcD1 has affinities for l-lysine and l-histidine in the same order of magnitude as observed for l-arginine and l-ornithine, with inhibition constants (K_i) of 2 and 16 μM, respectively. Remarkably, the affinities of ArcD2 for these cationic amino acids were 50- to 100-fold lower (K_i of 106 and 1,750 μM, respectively). In the previous study (10), neutral l-alanine was identified as a potential substrate of ArcD2. l-Ornithine uptake by D2ΔarcD1D2 was significantly inhibited by l-alanine with a K_i of 609 μM. While the affinity of ArcD1 was 30- to 50-fold higher for l-ornithine, l-lysine, and l-histidine, the affinity for l-alanine was 6-fold lower than that observed for ArcD2 (Table 2).

TABLE 2.

Kinetic parameters of the amino acid transporters ArcD1 and ArcD2^a

Substrate	ArcD1			ArcD2
	Km (μM)	Vmax (nmol/mg · min)	Ki (μM)	Km (μM)	Vmax (nmol/mg · min)	Kib (μM)
l-Arg	5 ± 1	30 ± 23		4 ± 2	22 ± 17
l-Orn	1 ± 1	45 ± 32		29 ± 5	128 ± 6
l-Lys			2 ± 0.1b			106 ± 61
l-His			16 ± 2c			1,750 ± 100
l-Ala			3,500 ± 700b,c			609 ± 192
Agm			2,250 ± 1,300c			481 ± 191
Cad			5,500 ± 2,800c			3,900 ± 1,600
Hist			3,100 ± 1,800c			805 ± 164
Putr			4,100 ± 1,000c			9,400 ± 3,600

^aValues are means and standard deviations for at least two independent replicates. Agm, agmatine; Cad, cadaverine; Hist, histamine; Putr, putrescine.

^bInferred from the inhibition of l-ornithine uptake at 10 μM.

^cInferred from the inhibition of l-ornithine uptake at 2.5 μM.

ArcD1 and ArcD2 showed affinities in the mM range for the decarboxylation products of the cationic amino acid substrates, i.e., agmatine, cadaverine, histamine, and putrescine, derived from l-arginine, l-lysine, l-histidine and l-ornithine, respectively. The affinity of ArcD2 for agmatine and histamine was found to be significantly higher (∼4-fold) than that of ArcD1 (Table 2).

Inhibition does not necessarily reflect transport. The ability of the transporters to translocate a substrate was demonstrated by chase experiments. Cells of D1ΔarcD1D2 were allowed to take up ¹⁴C-labeled l-ornithine initially present at a concentration of 10 μM until a steady-state level was reached. Subsequently, unlabeled l-arginine, l-ornithine, l-histidine, or l-lysine was added at a 100-fold excess of 1 mM. The immediate release of all internalized labeled l-ornithine demonstrates efficient translocation of l-histidine and l-lysine (and l-arginine and l-ornithine) into the cell in the exchange process catalyzed by ArcD1 (Fig. 5A). The same experiment using D2ΔarcD1D2 cells showed rapid release upon addition of l-lysine and l-ornithine, while l-histidine and, surprisingly, l-arginine resulted in much slower exchange (Fig. 5B). Apparently, l-arginine/l-ornithine exchange is catalyzed less efficiently by ArcD2 than by ArcD1. The lower exchange rate with l-histidine is partly due to the lower affinity of ArcD2 for l-histidine. To compensate for the lower affinities, the concentrations of the amines agmatine, cadaverine, histamine, and putrescine in the chase experiments were raised to 4 mM. Neither addition of the amines resulted in the release of accumulated ornithine, for ArcD1 or for ArcD2 (Fig. 5C and D). It follows that the amines do bind to the transporters, but they are not or very poorly translocated.

FIG 5.

Uptake of l-[¹⁴C]ornithine by resting cells of D1ΔarcD1D2 (A and C) and D2ΔarcD1D2 (B and D) (■). At the 60-s time point (arrow), a 1 mM concentration of unlabeled l-alanine (□), l-arginine (▲), l-histidine (△), l-lysine (●), and l-ornithine (○) (A and B) or a 4 mM concentration of unlabeled agmatine (□), cadaverine (▲), histamine (△), and putrescine (●) (C and D) was added to the cell suspension. The initial concentration of l-ornithine was 10 μM.

Chasing cells expressing ArcD1 with 1 mM unlabeled l-alanine did not result in release of label (Fig. 5A). Raising the concentration to 4 mM showed slow release, down to 70% at 120 s (data not shown). Apparently, ArcD1 only poorly translocates l-alanine. Cells expressing ArcD2 rapidly released accumulated l-ornithine upon addition of 1 mM l-alanine, but only partly (Fig. 6A). In contrast to exchange of l-ornithine with the cationic amino acids shown above, l-ornithine/l-alanine exchange is an electrogenic process that is affected by the membrane potential across the membrane. The cells were routinely incubated with glucose which generates a membrane potential that favors l-ornithine in the cytoplasm (positive out). Leaving glucose out of the mixture did not affect the chase by ornithine, as expected, but the addition of l-alanine resulted in continued release of label. The release is likely to slow down because the exchange process itself generates membrane potential (Fig. 6B).

FIG 6.

Uptake of l-[¹⁴C]ornithine by resting cells of D2ΔarcD1D2 preincubated for 5 min in the presence (A) or absence (B) of 10 mM glucose (■). Uptake was plotted as the percentage of the amount of l-ornithine taken up after 120 s (■). At the 60-s time point (arrow), a 1 mM concentration of unlabeled l-alanine (△) and l-ornithine (▲) was added to the cells. The initial concentration of l-ornithine was 10 μM.

DISCUSSION

The ADI gene cluster of Lactococcus lactis contains two homologous genes, arcD1 and arcD2, that encode proteins of the APC superfamily of transporters (9). Previously, the genes were overexpressed in a wild-type background, which resulted in enhanced uptake activity of l-arginine and l-ornithine (10), strongly suggesting that both ArcD1 and ArcD2 function as the postulated l-arginine/l-ornithine exchanger in the pathway (5, 17). Here, it is shown that in fact ArcD1 is the only one of the two that functions as the transporter in the ADI pathway. Deletion of the arcD1 gene reduced the transport activity of l-arginine and l-ornithine to the level observed in an arcD1 arcD2 double mutant (ΔarcD1D2 strain) and completely abolished the growth enhancement by the ADI pathway observed in four different media (Fig. 3C to F and Fig. 2E to H, respectively). While deletion of the second gene, arcD2, did not affect the growth enhancement, recombinant expression of the gene could recover the phenotype in the ΔarcD1D2 double mutant, suggesting l-arginine/l-ornithine exchange activity by ArcD2, which was confirmed by transport studies of the D2ΔarcD1D2 recombinant strain. The lack of ADI pathway activity and transport activity in the ArcD1 deletion mutant suggests that the arcD2 gene is not transcribed during growth in the media used in this study. Possibly, ArcD2 has a different physiological function in L. lactis under yet different conditions.

ArcD1 appears to be the main l-arginine/l-ornithine exchanger in the ADI pathway. The transporter has affinity for l-arginine (and l-ornithine) in the micromolar range (Table 2). ADI activity during growth is not observed in standard media containing millimolar concentrations of l-arginine (2 mM and 1.1 mM for M17 and SA, respectively) but requires l-arginine at 10-fold-higher concentrations (typically 25 mM). The high concentrations of l-arginine may be required to efficiently compete with the other cationic amino acids for which ArcD1 has equally high affinities. Both M17 and SA media contain free l-lysine at concentrations of 2.5 and 1.4 mM, respectively, and l-histidine at 0.09 and 0.3 mM, respectively (12, 14). While this explains why a high concentration of l-arginine is needed, it does not explain why the affinity of ArcD1 for l-arginine is so high.

The correlation between growth enhancement by ADI pathway activity and uptake activity of ArcD1 by resting cells was rather poor. No growth enhancement was observed in the standard media, while addition of 25 mM l-arginine resulted in maximal enhancement (Fig. 2). In contrast, cells grown in standard medium showed significant ArcD1 transport activity that increased, for instance, only 2-fold in M17 medium plus glucose when additional l-arginine was present (Fig. 3C). It follows that in the absence of activity of the ADI pathway as a whole, ArcD1 was present at significant levels. Most likely this reflects the complex regulation of expression of the ADI gene cluster involving differential regulation of expression of the genes that was observed before (1, 8, 18). The ADI pathway in L. lactis is believed to be induced by the substrate l-arginine (1, 8, 15) and under the control of carbon catabolite repression (CCR) (8, 15, 19). At the level of enzyme synthesis, it was shown that activities of ADI and OTC but not CK (Fig. 1) were higher when galactose, rather than glucose, was used and when arginine was added to the medium (1). At transcript level, the coexistence of multiple transcripts of the ADI gene cluster and the presence of nine potential stem-loop structures suggested that expression of the ADI operon involves mRNA processing and/or premature termination (8). Here, induction of expression of the arcD1 gene by l-arginine was apparent from the higher transport activities of resting cells grown in the presence of additional l-arginine in all four media and in both the exponential and stationary growth phases (Fig. 3C to F, compare gray and black bars). CCR of the pathway as a whole is apparent from the increased growth rate on galactose in the presence of additional l-arginine that is not observed on glucose (Fig. 2). Alternatively, the difference may be explained by growth on glucose not being energy limited. Repression by CCR is expected to be most effective in exponentially growing cells in M17 medium supplemented with glucose and absent when supplemented with galactose. Nevertheless, a significant transport activity of ArcD1 was observed for cells grown on glucose that was half of the activity of cells grown on galactose (Fig. 3C and D). Apparently, CCR-mediated repression of arcD1 is rather weak (see also reference 15). CCR of the arcD1 gene is evident from the increase in expression levels between the exponential and stationary growth phases in M17 medium plus glucose (Fig. 3C). With glucose as the carbon and energy source, repression is relieved due to depletion of glucose. In contrast, on galactose the expression levels decrease between the two growth phases, which is due to decreased induction by l-arginine that is cometabolized with galactose from the start of the culture (Fig. 3D). The latter pattern was not observed for chemically defined SA medium with galactose, which might be caused by the poor growth of the cells in this medium. It is concluded that the differential regulation of expression of the ADI pathway genes involves a less strict CCR control of the expression level of the transporter gene arcD1.

A putative physiological function of ArcD2 is likely to be related to the different substrate specificities of the arcD1 and arcD2 gene products. One prominent difference is the activity of ArcD2 with l-alanine as the substrate. l-Alanine is the only amino acid for which ArcD2 has a higher affinity than ArcD1, and in contrast to ArcD1, l-alanine is rapidly translocated by ArcD2. l-Alanine is an unexpected substrate of these transporters, because all the other substrates are cationic. Moreover, l-alanine is the only noncationic amino acid for which ArcD2 has significant affinity (10). Looking at the genetic organization, the physiological function of the arcD2 gene product is likely to be linked to the function of the adjacent arcT gene, which encodes an amino acid transaminase. In the order Lactobacillales, close homologues of arcT are not widespread, but they are found mostly in species that also harbor the ADI pathway genes and that have a second arcD gene on their chromosomes. The gene cluster found in all sequenced strains of L. lactis is unique to the species, but in Lactococcus garvieae, the arcT gene is found downstream of the arcABDC operon as well and a second arcD gene is located distantly on the chromosome. In Lactobacillus reuteri and Enterococcus faecium, the arcD and arcT genes form a pair that is distant from the ADI operon, while in Lactobacillus brevis and Lactobacillus sakei, the pair is inserted in the ADI operon. Yet in other species like Lactobacillus fermentum, the two genes are located elsewhere on the chromosome as well but not paired. The arcT gene product of Lactobacillales has never been studied experimentally, but the genetic analysis suggests that the transaminase is involved in l-arginine metabolism. In Pseudomonas aeruginosa and other pseudomonads the l-arginine aminotransferase pathway (ATA) is the second major pathway for l-arginine utilization under aerobic conditions (20). The first metabolic step in this pathway is the conversion of the substrates l-arginine and pyruvate into 2-ketoarginine and l-alanine, catalyzed by an l-arginine aminotransferase (21). Subsequently, the keto-acid is converted to succinate in a series of reactions. The responsible transaminase, AruH, is homologous to L. lactis ArcT. The two proteins share 23% sequence identity. A gene encoding a putative transport protein was identified in the locus encoding the enzymes of the ATA pathway in pseudomonads that is likely to be responsible for the uptake of l-arginine from the medium. The transporter is in the same APC superfamily of transporters as ArcD transporters, but the sequence identity is low (<20%). We hypothesize a (partial) ATA pathway in L. lactis that consists of ArcD2 and ArcT. The transaminase would convert cytoplasmic l-arginine and pyruvate into 2-ketoarginine and l-alanine, and the transporter would catalyze uptake of the external substrate l-arginine in exchange for the end product l-alanine, which is excreted from the cell.