The DNA binding proteins ArgR and AhrC are essential for regulation of arginine metabolism in Escherichia coli and Bacillus subtilis, respectively . A unique property of these regulators is that they form hexameric protein complexes, mediating repressionof arginine biosynthetic pathways as well as activation of arginine catabolic pathways . The gltS-argE operon of Lactococcus lactis encodes a putative glutamate or arginine transport protein and acetylornithine deacetylase, which catalyzes an important stepin the arginine biosynthesis pathway . By random integrationknockout screening we found that derepression mutants had ISS1 integrations in, among others, argR and ahrC . Single as well as double regulator deletion mutants were constructed from Lactococcus lactis subsp . cremoris MG1363 . The three arginine biosynthetic operons argCJDBF, argGH, and gltS-argE were shown to be repressedby the products of argR and ahrC . Furthermore, the argininecatabolic arcABD1C1C2TD2 operon was activated by the productof ahrC but not by that of argR . Expression from the promoterof the argCJDBF operon reached similar levels in the singlemutants and in the double mutant, suggesting that the regulatorsare interdependent and not able to complement each other . Atthe same time they also appear to have different functions,as only AhrC is involved in activation of arginine catabolism.This is the first study where two homologous arginine regulatorsare shown to be involved in arginine regulation in a prokaryote,representing an unusual mechanism of regulation.

 
Arginine, a nonessential amino acid in the lactic acid bacterium Lactococcus lactis, is synthesized de novo from glutamate in eight enzymatic steps [Fig . 1] . The recent publication of the L . lactis genome sequence [5] has revealed that the putativearginine biosynthesis genes are encoded by the three operonsargCJDBF, gltS-argE, and argGH . The products of these genesall show homology to known arginine biosynthetic enzymes, exceptfor that of gltS, which has been annotated as a putative glutamateor arginine ABC transporter [5] . While the biosynthetic geneshave been shown to be regulated by the presence of argininein other organisms, this has not been investigated in lacticacid bacteria [LAB] . The activities of the biosynthetic enzymeshave been shown to be repressed by arginine in Lactobacillusplantarum [6], but regulatory studies on the transcriptionallevel have not been performed on LAB.

 
Mechanisms for arginine catabolism vary among organisms [1]. In L . lactis, complete degradation of arginine into ornithine, ammonium, and carbon dioxide takes place via the arginine deiminase pathway [ADI pathway] in three enzymatic steps catalyzed by arginine deiminase [ArcA], ornithine carbamoyltransferase [ArcB],and carbamate kinase [ArcC] [Fig . 1] . The genes arcA, arcB,arcC1, and arcC2 encoding these enzymes are located in the arcABD1C1C2TD2gene cluster . L . lactis harbors an extra arcC homologue, calledarcC3, which is located distant from the remainder of the arginine-relatedgenes in the chromosome . The genes arcD1 and arcD2 encode antiporter proteins, allowing ATP-independent 1:1 arginine-ornithine exchange [37], while arcT specifies an aminotransferase.

It has long been known that carbon metabolism and arginine catabolism are closely connected in L . lactis [9] . However, the presenceof arginine has a higher regulatory effect than the availablecarbon source does [37] . The ADI pathway enzymes and amino acidtransport systems are more stable during starvation than areenzymes of glycolysis [23] . Thus, the ADI pathway plays an importantrole in supplying the cells with energy during recovery fromstarvation without energy expenditure . Additionally, glycolysisenzymes are more sensitive for low pH than the ADI enzymes are.Consequently, the ADI pathway represents an additional sourceof ATP production, combats acid stress by production of ammonium,and finally supplies carbamoyl phosphate, which is essentialfor de novo synthesis of pyrimidines . The identification oftwo putative cre [catabolite recognition element] sites in thearcA promoter of Lactobacillus sake [52] strongly suggests thatcarbon source-dependent regulation of the arginine catabolicgenes is mediated by the major carbon catabolite repressor CcpAin this organism.

Arginine metabolism has been shown to be regulated by a transcriptional regulator called ArgR or AhrC in several diverse organisms [10, 12, 25, 34, 41] . In this respect arginine regulation deviates from the "rule" of attenuation regulation of amino acid metabolismin prokaryotes [8, 39, 51] . Regulation of amino acid metabolismin LAB via the direct action of a DNA binding protein has beenobserved only in the case of CmbR, which activates expressionof the sulfur-related metC-cysK operon in response to acetylserinein L . lactis [13].

Several characteristic features of ArgR-AhrC-type regulatorshave been described: [i] they form hexaoligomeric complexes[12, 25], [ii] they have a winged helix-turn-helix DNA binding domain [44], and [iii] ArgR plays a role as an accessory factorin multimer resolution of ColE1 plasmids in Escherichia coli[17, 43] . ArgR and AhrC repress their own expression [25] and activate the transcription of arginine catabolic genes by interacting with other regulation factors, such as ANR and RocR of E . coli and Bacillus subtilis, respectively [14, 27, 50].

ArgR and AhrC monomers consist of two domains, an N-terminalDNA binding domain containing the winged helix-turn-helix structureand a C-terminal domain involved in arginine binding and subunit multimerization [44] . Investigation of the hexameric structureby crystallization has shown that six arginine molecules bindin the interphase between the C-terminal domains of two trimers [49] and that arginine thereby functions as a corepressor.

ArgR and AhrC homohexamers bind to operator sites [called ARG boxes] in regions of biosynthetic and catabolic arginine promoters. The ARG box is an 18-bp imperfect palindromic sequence, theconsensus of which varies slightly among organisms [11, 24,30, 33] . The number of boxes was shown to correlate with theobserved regulation . Thus, repression is stronger when two orthree ARG boxes are present, as seen in the E . coli biosyntheticpromoters, than when only a single box is present, as in theargR promoter of E . coli [10].

The publication of the entire Lactococcus lactis subsp . lactis IL1403 genome [5] has led to the identification of two ArgR-AhrCorthologues . Multiple putative arginine regulators have alsobeen found in the genomes of other bacteria [3], but the functionof these and the reason for the presence of more than one regulatorin one organism remain to be established.

In this paper we show that Lactococcus lactis subsp . cremoris MG1363 harbors two functional arginine regulators . They cooperate in the repression of arginine biosynthesis but have different functions in the activation of arginine catabolism.

 
Bacterial strains and media. Strains of L . lactis used in this study are listed in Table1 . L . lactis was grown at 30 or 37°C in M17 medium [45]with 0.5% glucose as carbon source [GM17] . A chemically definedmedium [CDM] was made as described earlier [31] with Casitone [Difco, West Molesey, United Kingdom] in concentrations of 0.1 or 4% . CDM buffer containing 15 free amino acids [CDM15] wasmade as described previously [22], omitting arginine unless stated otherwise . Arginine stock solutions were made in distilled H₂O; pH was set to 7.0 with HCl . For solid media, agar was addedto a concentration of 15 g · liter^-1 . The following componentswere added when needed: erythromycin, 4 µg · ml^-1 for selection of plasmids or 1 µg · ml^-1 for maintaining TnNuc integrations; tetracycline, 2 µg · ml^-1; chloramphenicol, 4 µg · ml^-1; and 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside [X-Gal], 40 µg · ml^-1 . Antibiotics were purchased from Sigma Chemical Co . [St . Louis, Mo.], and X-Gal was fromRoche Molecular Biochemicals [Mannheim, Germany].

 
DNA isolation and manipulations. Chromosomal and plasmid DNAs were isolated from L . lactis accordingto the methods of Johansen and Kibenich [20] and Birnboim [4], respectively . DNA was manipulated essentially as described by Sambrook et al . [40], and lactococcal strains were transformedwith plasmid DNA by electroporation [18].

Chromosomal deletion mutants were made using pVE6007 [28] ashelper plasmid for single-crossover integration of p280argR and p280ahrC in L . lactis MG1363 grown at 37°C . Excisionof pORI280, leaving the deletion constructs in the chromosomeof strain MG1363, was performed at 37°C without antibioticselection . Excissants grown on solid medium were screened byPCR, and mutants were confirmed with Southern blotting . Probelabeling, hybridization, and detection were performed usingthe ECL direct nucleic acid labeling system according to thespecifications of the manufacturer [Amersham Pharmacia Biotech,Little Chalfont, United Kingdom] . Restriction enzymes were purchasedfrom New England BioLabs [Beverly, Mass.] . DNA was amplifiedusing specific primers as listed in Table 2 . PCR products werepurified with the High Pure PCR product purification kit [RocheMolecular Biochemicals] . Taq DNA polymerase [Roche MolecularBiochemicals] was used for colony PCR, and Pwo DNA polymerase[Roche Molecular Biochemicals] was used for DNA constructs.

 
Nucleotide sequencing reactions were performed on a DNA Labstation 625 [Vistra DNA System] with the Thermo Sequenase primer cycle sequencing kit [Amersham Pharmacia] . Fragments were separatedand detected with the ALFexpress II gel system [Amersham Pharmacia].

Construction of lacZ expression plasmids. The constitutive lactococcal promoter P32 was amplified usingprimers P32-1 and P32-2 and cloned in pORI13, resulting in pORI13P32.With this plasmid as template, a PCR product containing themultiple cloning site and lacZ of pORI13 was obtained usingthe pORI13m2 and pORI13s2 primers . The PCR fragment was insertedas an MfeI/SpeI restriction fragment in the EcoRI/XbaI sites of pIL252, yielding plasmid pILORI4 . Only very low intrinsic ß-galactosidase activity could be measured in cellscarrying the empty pILORI4 vector.

The promoter fragment to be analyzed for expression was amplified from chromosomal DNA of L . lactis MG1363 by PCR with the primers listed in Table 2 and cloned in the low-copy-number expressionvector pILORI4.

Isolation of mutants derepressed in arginine metabolism. L . lactis C17 [gltS-argE::lacZ] was transformed with pGh8::ISS1 [29] and submitted to random integration screening on CDM containingerythromycin, tetracycline, X-Gal, and 4% Casitone at the nonpermissivetemperature [37°C] . Integrants showing a clear gltS-argE::lacZderepression phenotype were isolated for further characterization.pGh8::ISS1 was cured from the strains by repeated 1,000-folddilution and growth in GM17 plus erythromycin at the permissivetemperature [28°C].

Enzyme assays. ß-Galactosidase activity assays were performed oncell suspensions that were permeabilized by chloroform as describedpreviously [19].

Data analysis. The Clustal W program was used for protein sequence alignments[46] . Clone Manager 6.0 was used for free energy calculationsof palindromic DNA structures.

Nucleotide sequence accession numbers. The new sequences generated in this work have been given theaccession numbers AY518512 [argR], AY518513 [ahrC], AY518514[PargC], and AY518515 [ParcA].

 
gltS-argE derepression mutations in L . lactis target to two ArgR-AhrC-type regulators. A pTnNuc integration library of L . lactis MG1614, an isogenicL . lactis subsp . cremoris MG1363 derivative [38], was platedon GM17 plates containing erythromycin and X-Gal . Colonies werescreened by replica plating onto CDM plates containing erythromycin,X-Gal, and 4 or 0.1% Casitone . An L . lactis MG1614 strain calledC17, showing Casitone-dependent ß-galactosidase activity,had lacZ of TnNuc integrated in the C-terminal part of argE,the second gene of the arginine biosynthetic gltS-argE operon. Expression of gltS-argE was high in the 0.1% Casitone medium and low in the 4% Casitone medium . Random ISS1 transposon integration screening using pGh8::ISS1 [29] was performed in L . lactis C17,to identify genes involved in Casitone-dependent regulationof gltS-argE . Approximately 14,000 colonies were screened, and18 integrants [called gdm for gltS-argE derepression mutation]that were clearly derepressed on a rich medium containing X-Galwere isolated . Chromosomal ISS1 integration sites were determinedfor nine of the integrants by sequencing of inverse PCR products.The resultant target genes of seven of these are presented inTable 3.

 
The chromosomally integrated copy of pGh8::ISS1 was cured from the C17[gdm] strains by growing them in GM17 without tetracycline selection [29] . In this way only the ISS1 element was left atthe chromosomal integration site [strains designated "gdm-ex"],allowing for a direct comparison between the cured strains andthe parental strain L . lactis C17 under the same culturing conditions.Excision of pGh8::ISS1 was confirmed by Southern blotting, PCRon chromosomal DNA, and testing for tetracycline sensitivity.

In strains C17[gdm8] and C17[gdm29] the integration sites could be localized to an open reading frame with high homology to arcD2 of L . lactis IL1403 . This gene encodes a putative arginine-ornithineantiporter and is the last gene of the arginine catabolic pathwayoperon arcABD1C1C2TD2 . The chromosomal ISS1 targets of the sixremaining C17[gdm] strains are all located in or upstream ofeither of two open reading frames the products of which showhigh homology to ArgR-AhrC-type DNA binding proteins in otherorganisms . Growth of the integrants was found to be strongly reduced in the absence of arginine, and the experiments described below were all performed with cells grown in the presence of [different concentrations of] arginine.

The gltS-argE operon of L . lactis is strongly derepressed in both argR and ahrC mutants. ISS1 of pGh8::ISS1 had integrated in the very N-terminal partof ahrC in strains C17[gdm1ex] and C17[gdm26ex], resulting in strong derepression of gltS-argE expression . Surprisingly, the expression of gltS-argE in strains C17[gdm1ex] and C17[gdm26ex] was much higher than that observed for strain C17 even at very low arginine concentrations [Table 3] . Strain C17[gdm27ex] showedthe same derepression phenotype as that of strains C17[gdm1ex] and C17[gdm26ex], but ISS1 insertion had occurred in the yiiBgene located just upstream of ahrC [data not shown] . The genesyiiB and ahrC overlap by 4 bp, suggesting that they are transcriptionallycoupled . Homology searches predict YiiB to be a 23S rRNA methyltransferase,with some homology to an S4 RNA binding domain and to an FtsJ-likemethyltransferase [E values of 5.9e-3 and 1.8e-5, respectively],not known to have any influence on arginine metabolism . Theobserved derepression in strain C17[gdm27ex] is probably causedby a polar effect on ahrC expression rather than inactivationof the yiiB gene product . The fact that derepression reachedthe same levels as those measured for the other ahrC integrationknockouts is in accordance with this hypothesis [data not shown].The ahrC gene is followed by a terminator structure with a calculatedfree energy of -13.0 kcal . A recN homologue is present downstreamof ahrC, with an intergenic spacing of 180 bp . A weak putativepromoter [TTGTGC-18N-TATAAT] and ribosomal binding site [AGAAAGGAAAT]precede recN . Considering the genetic structure of the ahrC region, disruption of ahrC expression alone is expected to cause the derepression of gltS-argE expression in strains C17[gdm1ex] and C17[gdm26ex] and possibly also in strain C17[gdm27ex].

The C17[gdm24ex], C17[gdm25ex], and C17[gdm28ex] strains allcarry ISS1 in a 459-bp gene annotated as argR in L . lactis IL-1403.The strains differed with respect to the extent to which gltS-argEwas derepressed . Strain C17[gdm28ex], in which argR is disruptedin the start of the gene, showed a complete gltS-argE derepressionphenotype similar to that of the ahrC knockout strains C17[gdm1ex]and C17[gdm26ex] [Table 3] . In strain C17[gdm24ex] the insertion had taken place in the center of argR [Table 3] . Interestingly,disruption of argR in this region resulted in a drastic growthinhibition, with growth rates of 0.39 h^-1 in CDM15 with 0.1mM arginine to 0.31 h^-1 in CDM15 with 10 mM arginine, comparedto growth rates between 0.5 and 0.63 h^-1 for the other strains.Finally, with ISS1 insertion at the very end of argR, strainC17[gdm25ex] showed maximum derepression to a level comparableto that in strain C17[gdm28ex] but differing in that it hadmaintained the ability to sense and respond to arginine availability[Table 3] . Two transcriptional terminator structures with calculatedfree energies of -12.5 and -14.4 kcal, respectively, are locatedin the argR-murC intergenic region . The argR gene is locatedin a divergent orientation with argS [encoding arginyl-tRNA synthetase] and is separated from this gene by a putative promoter region of only 67 bp . A consensus extended -10 box [TGGTATAAT]is located upstream of argR, but no clear ribosome binding site could be identified . As argR is in opposite orientation with respect to the neighboring argS and murC genes, disruption of argR is expected to be the sole cause of gltS-argE derepressionin the strains C17[gdm24ex], C17[gdm25ex], and C17[gdm28ex].

Regulation of the arginine biosynthesis argCJDBF operon in L . lactis. A fragment of 296 bp containing the entire argC promoter [PargC]was cloned upstream of lacZ in the promoter expression vectorpILORI4 . This expression construct was introduced in the wild-typestrain L . lactis MG1363, as well as its single isogenic regulatormutants L . lactis MGargR and MGahrC and the double regulatormutant L . lactis MGargRahrC . Expression of lacZ from this promoterwas investigated during growth on CDM [CDM15] containing differentconcentrations of arginine . Clear arginine-dependent repressionwas observed in the wild-type strain MG1363 [Fig . 2] . In eachof the single regulator mutants, arginine repression was nolonger seen and ß-galactosidase expression reachedthe same levels as that in the double regulator mutant [Fig.2] . As was observed for the expression of the gltS-argE operonin the argR::ISS1 or ahrC::ISS1 knockout strains, disruptionof a single regulator gene resulted in complete derepressionof expression from PargC . Thus, it appears that the two regulators,ArgR and AhrC, have a corepressing effect rather than a cumulativeeffect on repression of the argCJDBF arginine biosynthetic operonin L . lactis.

 
ArgR and AhrC have different roles in regulation of the arginine catabolism operon of L . lactis. In the light of the regulation of the arginine biosyntheticgltS-argE and argCJDBF operons, we decided to examine the roleof the regulators in the expression of the arginine catabolicarc gene cluster . To that end, the arcA promoter region [ParcA]up to 260 bp upstream of the arcA start codon [same constructas ParcA-1 in Fig . 4] was cloned in the expression vector pILORI4, which was then introduced in L . lactis MG1363 and its isogenic regulator deletion mutants . As shown in Fig . 3, clear arginine-dependentregulation was observed in L . lactis MG1363, with expressionfrom ParcA increasing with an increase in the arginine concentration.Deletion of ahrC resulted in no or only low expression fromParcA even at a high arginine concentration in the medium . Incontrast, expression was constitutively high in the argR mutantand in the argR ahrC double deletion strain . This would suggestthat activation of arginine catabolism in L . lactis MG1363 ismediated by AhrC and that a repressing effect is exerted byArgR . Alternatively, the high ParcA expression in the argR deletionmutants could be caused by a constant high intracellular levelof arginine resulting from the derepression of arginine biosynthesis.However, in the double mutant the function of AhrC seems tobe overruled by the removal of ArgR.

 
The argS-arcA intergenic region contains several features [Fig. 4]: a putative transcription terminator with a calculated freeenergy of -9.1 kcal composed of a dyad symmetry followed bya stretch of thymidine residues, starting 19 bp downstream ofargS; two core promoter structures, P1 [5'-TTGACA-17N-TATAAT]and P2 [5'-TTGTCA-17N-TATAAA], located at 56 to 84 bp and at118 to 146 bp, respectively, from the start of arcA; and a characteristicribosomal binding site [5'-AAAGGA] 9 bp upstream of arcA . Inorder to identify possible operator sites involved in the observedregulation, deletion derivatives of the arcA promoter regionwere transcriptionally fused to lacZ in pILORI4 [Fig . 4] . ß-Galactosidase activities of these promoter fragments were measured in thewild-type strain MG1363 grown in CDM15 with 0.1 or 10 mM arginine[Table 4].

 
Removing the -35 box of P1 [Fig . 4, compare ParcA-7 to ParcA-8]resulted in a severe decrease of expression of lacZ [Table 4].Fragment ParcA-8 gave arginine-independent expression, definingP1 as the minimal promoter, lacking operators involved in arginineregulation . Partial arginine-dependent activation, comparedto ParcA-1 containing the entire promoter region, took placein ParcA-5 and ParcA-6, suggesting the presence of an operator[s]of arginine regulation in this region . Including the entireputative promoter P2 [the region up to -156 bp upstream of arcA]did not result in increased ß-galactosidase activity,questioning the functionality of P2 . However, the region includedin ParcA-4 just upstream of the P2 structure had a dramaticeffect on expression, resulting in high arginine-independentexpression . Regulation was restored only by including sequencesfurther upstream, 223 to 260 bp from arcA, with maximal arginine-dependentregulation taking place with the largest fragment, ParcA-1.

The lactococcal arginine regulators lack conserved amino acid residues. The argR gene of L . lactis subsp . cremoris MG1363 [which isisogenic to strain MG1614 [15]] encodes a putative protein of152 amino acids, called ArgR_Ll hereafter, while ahrC specifiesa putative protein of 148 amino acids, named AhrC_Ll . The tworegulators show mutual identity for 50 amino acid residues [32%]and are homologous to well-known arginine regulators, like ArgRof E . coli, AhrC of B . subtilis, and ArgR of Bacillus stearothermophilus [Fig . 5] . All these proteins contain an N-terminal DNA binding domain, a central hinge region, and a C-terminal arginine-sensing and subunit multimerization domain [Fig . 5] . Mutagenesis studiesof the arginine regulators of E . coli [ArgR_Ec] [7, 26, 48] and B . stearothermophilus [ArgR_Bst] [21] have identified amino acidresidues that are essential for regulator functionality . Ofthese residues, Ser47 and Arg48 of ArgR_Ec are conserved in bothlactococcal regulators [Fig . 5] . However, other residues knownto play a role in operator-regulator interaction have changedin ArgR_Ll and AhrC_Ll . Ser44 of ArgR_Ec has changed to Ala, Thr51is replaced by Lys or Arg, and Arg57 has changed to Lys in the regulators of the aligned gram-positive organisms in Fig . 5. A range of residues in the N-terminal part of the arginine regulators of the gram-positive bacteria is highly conserved, but lessso in the gram-negative bacterial ArgR_Ec, e.g., amino acid residues36 to 45 of AhrC_Ll show a highly conserved VTQATVSRDI motif.In the C-terminal domains of the proteins there appears to behigher similarity between the gram-negative E . coli regulatorand the gram-positive bacterial regulators, and in most cases,residues known to be essential for subunit multimerization and arginine binding have been conserved . However, it is noteworthy that, of the conserved GTI-X-GDDT motif [residues 123 to 130of ArgR_Ec], only the Ile and the double Asp residues are maintainedin AhrC_Ll . Whereas most of these residues are preserved in ArgR_Ll, it should be noted that Asp128, which is essential for arginine binding in ArgR_Ec, is replaced by an Ala residue . The possible significance of these changes will be discussed below.

 
In this work we have investigated the regulation of arginine metabolic genes in L . lactis and have shown that two ArgR-AhrC-type regulators are required for repression of the arginine biosynthetic gltS-argE operon . Chromosomal argR and ahrC deletion mutantsof L . lactis MG1363 were made to confirm that repression ofthe central arginine biosynthesis operon argCJDBF is also dependenton the presence of both regulators . Arginine-dependent regulationof the catabolic arcABD1C1C2TD2 gene cluster was also abolishedin the regulator mutants . However, in this case the mutationshad different effects, as the lack of ArgR resulted in highand arginine-independent expression while lack of AhrC resultedin constitutive low expression . Until now, the function of arginineregulators has been investigated only for organisms carryinga single arginine regulator [e.g., ArgR in E . coli and AhrCin B . subtilis] . What has mainly caught our interest is thefact that two functional, homologous regulators are involved in and necessary for arginine-dependent gene regulation in L. lactis.

The presence of two homologous regulators suggests that [i]the regulators are paralogs, able to perform the same function[s]and to complement each other, or that [ii] they have differentfunctions, e.g., one regulating arginine biosynthesis and theother regulating arginine catabolism, as proposed by Guèdonet al . [16] . Neither supposition holds true for the arginineregulators of L . lactis . The results for the regulation of thegltS-argE and argCJDBF biosynthetic operons clearly demonstratethat the two regulators are not complementary . Not only didthe ISS1 integration knockout screening allow identificationof both regulators, which would not be the case could any oneof them perform the action of the other, but also arginine-dependentregulation was abolished in both of the single regulator deletionmutants . Both regulators have different functions with respectto regulation of the arginine catabolic pathway, but neitherof the single regulators could be shown to be responsible forthe arginine-dependent regulation of arginine catabolism observedin the wild-type strain . Another surprising observation wasthat expression of gltS-argE in the wild-type strain, althoughregulated in dependence on arginine availability, was much lowerthan that in either of the regulator knockout strains . A similarobservation was made in the study of ArgR in two different E.coli strains, K-12 and B [47] . Only a single amino acid substitutiondifferentiates ArgR of E . coli K-12, which showed strong arginine-dependentregulation, from ArgR of E . coli B, which mediated only weakly arginine-dependent regulation, resulting in so-called superrepression of arginine biosynthesis [42, 47] . Both ways to regulate argininemetabolism are effective, and a mechanism of superrepressionas observed for ArgR of E . coli B might be utilized by L . lactis.This putative superrepression in the wild-type L . lactis MG1363was not observed in the promoter expression studies, but thismay be explained by the possibly low levels of ArgR_Ll and AhrC_Llin the cell: the multicopy vector situation may, to some extent,dilute the regulator proteins relative to the plasmid-locatedoperators, despite pILORI4 being a low-copy-number vector . Alternatively,the difference in the level of regulation between the argC and gltS promoters could be explained by the presence of only oneARG box upstream of the gltS operon as opposed to two in the argC operon [see below], as the number of ARG boxes is knownto correlate with the level of regulation in E . coli [10].

The three different ISS1 integration sites in argR yielded entirelydifferent growth characteristics or gltS-argE expression patterns,which allowed us to confirm the functions of the ArgR_Ll subdomains.Integration in the putative hinge region of ArgR, disruptingthe C-terminal part, caused not only arginine-independent derepressionbut also a considerable growth inhibition [Table 3] . As seenfor ArgR_Ec and AhrC_Bs, this suggests that the C terminus of ArgR_Ll is essential for arginine sensing . Additionally, the N-terminal part may have some intrinsic DNA binding capacity, disturbing other metabolic functions of the cell . The reappearanceof arginine sensing when disruption takes place in the veryC-terminal region of the regulator confirms the sensory functionof this domain . The more pronounced derepression of gltS-argEcaused by the latter mutation compared to the wild type is mostlikely the result of incorrect arginine sensing.

The arcD1 and arcD2 genes most likely encode the arginine-ornithineantiporter described by Poolman et al . [37] . The gene arcD2is the last gene in the catabolic arc operon and, therefore,the only gene the expression of which was affected by the ISS1insertion in strains C17[gdm8ex] and C17[gdm29ex] . The observedeffect on gene regulation is probably indirect: derepressionof gltS-argE expression as a result of arcD2 disruption is probablycaused by low arginine uptake rates, leading to endogenous argininedeficiency with subsequent increased expression of the argininebiosynthetic genes . In these integrants [gdm8ex and gdm29ex]gltS-argE was still regulated as a function of arginine availability,presumably via the ArgR and AhrC proteins that are present inthese strains . However, only in the highest extracellular concentrationof arginine tested was gltS-argE expression restored to wild-typelevel.

Regulation mediated by ArgR-AhrC-type regulators suggests the presence of ARG box operators . Indeed, operators similar toARG boxes of E . coli and B . subtilis, 5'-WNTGAATWWWWATTCANW[26] and 5'-CATGAATAAAAATKCAAK [32], respectively, are present in the promoter regions of the argCJDBF and gltS-argE operons:gltS_O, 5'-AATGTATAATTATACTTA [at -43 to -26 bp from the startof gltS]; argC_O1, 5'-AAAGTATAATAATACATA [at -82 to -65 bp from argC]; and argC_O2, 5'-AGTGTATAAAAATACATA [at -32 to -15 bp fromargC], where positions identical to the E . coli ARG box areunderlined . gltS_O and argC_O2 are both located in the putativecore promoters of gltS and argC, respectively . The 32-bp spacingof argC_O1 and argC_O2 is unusual, as double ARG boxes are generallyonly 3 bp apart [26] . Still, this organization would be in accordancewith repression of these promoters taking place via direct interactionbetween the arginine regulators and the ARG box operators . Thispossibility is further supported by the fact that the N-terminalDNA binding domains of both lactococcal arginine regulatorsshow high mutual similarity and similarity to those of ArgR_Ec,ArgR_Bst, and AhrC_Bsu [Fig . 5].

A catabolite-responsive element [cre site] overlaps the core promoter of arcA, which is in agreement with the previously described carbon source-dependent regulation of arginine degradation in L . lactis [9] . Subcloning of the arcA promoter allowed usto locate regions involved in the observed arginine-dependentregulation . However, in none of these regions could consensusARG boxes be identified . Regions of regulatory importance localizedto three different parts of the argS-arcA intergenic region[Fig . 4] . The region just upstream of P1 partially restoredarginine-dependent regulation of arcA, suggestive of an elementactivating expression from P1 . The high arginine-independentexpression observed by including the region upstream of P2 couldbe the result of activation via an upstream operator lackingregulatory capacity and inducing expression from P1 or P2 orboth . That the regulatory capacity was restored by including the entire promoter region points to operators being involved in arginine-dependent control by a repressing mechanism . Thispattern of regulation is intriguing and reveals a rather complexregulatory scheme, involving activation as well as repression.An A/T-rich palindromic structure [5'-TCTTTTTTAAAATATTTTGTAAAATA, 206 to 231 bp upstream of the start of arcA; nucleotides of the palindrome are underlined] that lacks features of a typical transcriptional terminator is present in the region upstreamof P2 [Fig . 4] . Approximately half of the structure is included in ParcA-3, and the complete structure is present in ParcA-1. Whether this structure in reality is involved in regulationof ParcA remains to be verified . The fact that the arginine degradative pathway is involved in a range of diverse cellular functions such as energy production, acid stress resistance,and pyrimidine biosynthesis could explain the presence of sucha complex regulatory circuit . Interestingly, O'Connell-Motherwayet al . [36] have reported on an essential two-component systemthat is involved in activation of arginine degradation . Whetherand how this system is responsible for some of the effects describedabove remain to be elucidated.

Whereas the N termini of ArgR_Ll and AhrC_Ll are highly similar,greater divergence is seen between the C-terminal domains, inparticular between those of AhrC_Ll and the other regulatorsaligned in Fig . 5 . The lack of conservation is especially intriguingfor those residues with known functions in the B . stearothermophilus,B . subtilis, and E . coli regulators [7, 21, 35, 48]: whereas,e.g., ArgR_Ll lacks one of the C-terminal Asp residues directlyinvolved in arginine binding [34], AhrC_Ll harbors an extra Aspresidue at the equivalent location [Fig . 5] . The fact that bothregulators are essential for regulation and that the missingconserved arginine-binding Asp residue of ArgR_Ll seems to becomplemented in AhrC_Ll has led us to postulate a working hypothesisin which both proteins are thought to interact to form heterohexamericcomplexes, consisting of one ArgR_Ll trimer interacting withone AhrC_Ll trimer [Fig. 6].

 
The presence of two arginine regulator homologues in Enterococcus faecalis has recently been described [2] . Only a single Aspresidue, as is the case for ArgR_Ll, is present in the putativearginine-binding region of both E . faecalis homologues, leadingto the suggestion that these regulators may bind metabolitesother than arginine . However, the functionality of the E . faecalisgene products remains to be investigated.

A gene regulatory mechanism of the type that we have describedin this paper is, to our knowledge, unprecedented in prokaryotesand is the focus of ongoing research.

 
We are grateful to Peter Ravn, Biotechnological Institute, Hørsholm, Denmark, for providing the L . lactis MG1614 TnNuc integration library used in this study.

 
* Corresponding author . Mailing address: Department of Molecular Genetics, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands . Phone: 31-50-3632111 . Fax: 31-50-3632348 . E-mail: J.Kok@biol.rug.nl .

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