The repression of the carAB operon encoding carbamoyl phosphate synthase leads to Lactobacillus plantarum FB331 growth inhibition in the presence of arginine . This phenotype was used in a positive screening to select spontaneous mutants deregulated in the arginine biosynthesis pathway . Fourteen mutants were genetically characterized for constitutive arginine production . Mutations were located either in one of the arginine repressor genes [argR1 or argR2] present in L . plantarum or in a putative ARG operator in the intergenic region of the bipolar carAB-argCJBDF operons involvedin arginine biosynthesis . Although the presence of two ArgR regulators is commonly found in gram-positive bacteria, only single arginine repressors have so far been well studied in Escherichia coli or Bacillus subtilis . In L . plantarum, argininerepression was abolished when ArgR1 or ArgR2 was mutated in the DNA binding domain, or in the oligomerization domain or when an A123D mutation occurred in ArgR1 . A123, equivalent tothe conserved residue A124 in E . coli ArgR involved in arginine binding, was different in the wild-type ArgR2 . Thus, corepressor binding sites may be different in ArgR1 and ArgR2, which haveonly 35% identical residues . Other mutants harbored wild-typeargR genes, and 20 mutants have lost their ability to grow innormal air without carbon dioxide enrichment; this revealeda link between arginine biosynthesis and a still-unknown CO₂-dependent metabolic pathway . In many gram-positive bacteria, the expressionand interaction of different ArgR-like proteins may imply acomplex regulatory network in response to environmental stimuli.

 
Lactobacillus plantarum, a gram-positive bacterium found in nutritionally rich biotopes like plants and the intestines of mammals, is used in a wide variety of fermentation processes[e.g., meat, vegetables, and dairy products] . Among the lacticacid bacteria, L . plantarum is found in the most diverse biotopes. Its metabolic flexibility allows it to adapt to a variety of environments . Genome analysis of L . plantarum revealed a high proportion of regulatory proteins [8.5%], which is a typicalfeature of bacteria that can be found in diverse environments,such as Listeria monocytogenes and pseudomonads [17, 34] . Amongthe regulatory proteins, two putative arginine repressor geneswere identified in the genome of strain WCFS1 . This was surprising,in view of the paradigm that arginine repression is mediatedby a single arginine repressor, as found in Escherichia coli[22] and Bacillus subtilis [8] . Moreover, an analysis of sequencedgenomes revealed the frequent presence of several potentialarginine regulator genes in the genomes of gram-positive bacteria[5] . These observations led us to investigate the putative rolesof the two ArgR repressors found in L . plantarum.

Arginine-mediated regulation is remarkably well conserved invery divergent bacteria, i.e., gram-negative bacteria, suchas E . coli [22], Salmonella enterica serovar Typhimurium [21], Thermotoga [24], and Moritella profunda [38], and gram-positivebacteria, such as B . subtilis [8], Geobacillus stearothermophilus[previously called Bacillus stearothermophilus] [11], and Streptomyces clavuligerus [31] . The resolved ArgR structures from E . coli,G . stearothermophilus, and B . subtilis have similar foldingpatterns, despite only 27% amino acid identity between the enterobacterialand the bacillus arginine repressors [10, 26, 35, 37] . The argininerepressor subunit consists of a basic N-terminal DNA binding domain [DBD], which belongs to the winged helix-turn-helix family and is connected through a flexible linker to an acidic C-terminal domain responsible for oligomerization and arginine binding. The active regulator consists of an ArgR hexamer formed by the dimerization of two trimers stabilized by the fixation of six L-arginine molecules at the trimer-trimer interface, which resultsin the allosteric activation of the regulator [15] . The activehexamer recognizes one or more "ARG box" DNA sequences madeof 18-bp imperfect palindromes [22-24] . The activated transcriptionalregulator can act as a transcriptional inhibitor and/or activator,depending on the prokaryote studied . In B . subtilis in the presenceof arginine, the arginine repressor AhrC inhibits arginine biosynthesisand activates the arginine catabolic arginase pathway [23].It is also an obligate accessory protein for the cer/Xer site-specific recombination mechanism resolving multimers of ColE1-like plasmids in E . coli [33] . Unlike other lactic acid bacteria, most L.plantarum organisms have no known arginine catabolic pathways[1], and no genes of the arginine deiminase [ADI] pathway werefound in the genome of the WCFS1 strain [17] or strain CCM 1904used in this study [27] . The genes encoding arginine biosynthetic enzymes were characterized [Fig . 1 shows the gene organizationof the arginine biosynthetic pathway] . The peculiar presenceof two arginine repressors in an organism that synthesizes butdoes not catabolize arginine prompted us to investigate the role of each argR in arginine biosynthesis in L . plantarum. The predicted ArgR1 [153 residues] and ArgR2 [152 residues] have 35% identical amino acids . Thirty-one and 39% of ArgR1and ArgR2 residues, respectively, are identical to those ofthe B . subtilis arginine repressor AhrC, and 26 and 21% of theresidues are identical to those of the E . coli ArgR protein.We selected spontaneous mutants with altered arginine regulationin order to investigate arginine-dependent regulation in L.plantarum and, in particular, the functions of argR1 and argR2in this regulation.

 
Bacterial strains and culture conditions. A list of the strains studied is given in Table 1 . CCM 1904is auxotrophic for 11 amino acids and six vitamins but prototrophicfor both arginine and pyrimidines . L . plantarum strains weregrown at 30°C without agitation on MRS medium [Difco Laboratories]or the defined rich medium DLA [lactobacillus rich defined medium]free of arginine and pyrimidines [3] . Nutritional requirements were tested at 30°C on agar plates either unsupplementedor supplemented with 50 µg of uracil [DLAU] or of arginine/mlin aerobiosis or CO₂-enriched air . Aerobiosis was obtained by incubation in ordinary air . To calibrate the gas phase CO₂ concentrationat 4%, a water-jacketed CH/P incubator [Forma Scientific] wasused . The ability of L . plantarum to grow in the presence ofthe toxic arginine analogue canavanine in final concentrationsranging from 5 to 200 µg/ml was tested in CO₂-enriched air in DLAU medium . Liquid cultures were performed in a CO₂-enriched atmosphere without agitation in 250-ml Erlenmeyer flasks containing 50 ml of DLAU supplemented with arginine or unsupplemented.For RNA extracts used in reverse transcription [RT]-PCR, cellswere cultivated in the water-jacketed CH/P incubator with theCO₂ concentration calibrated at 4% . For RNA extracts used inNorthern blots, cells were cultivated in Erlenmeyer flasks closedwith gas-tight corks to prevent CO₂ loss during incubation,and 10.8 ml of pure CO₂ gas was injecting through the cork with a sterile syringe to obtain 4% in the gas phase.

 
Positive screening for arginine-deregulated mutants. Spontaneous mutants deregulated in arginine biosynthesis andderived from independent mutational events were isolated fromstrain FB331 . Isolated colonies grown on MRS agar plates froma FB331 frozen stock culture were suspended in 1 ml of sterilephysiological water [NaCl, 9 g/liter] . Fifty microliters ofeach suspension was spread on DLA plates or DLA plates supplementedwith arginine [50 µg/ml] and incubated for 3 days at 30°Cin CO₂-enriched air . Under these conditions, the wild-type strainFB331 was unable to grow, but a few spontaneous mutants derivedfrom strain FB331 were obtained . A single mutant clone fromeach plate was transferred to MRS plates, cultivated in MRSmedium, and then stored at –80°C in the presence of 20% glycerol.

Determining arginine excretion. Arginine excretion was estimated by measuring the arginine presentin stationary-phase cell culture supernatants by using a bioassay.The indicator bacterium was L . plantarum HN217 [27] . This strainhas no arginine-regulated carbamoyl phosphate synthetase [CPS-A]and pyrimidine-regulated CPS [CPS-P], so that its growth requires arginine and uracil [Fig . 1A] . HN217 growth was assessed at 30°C without shaking in liquid DLAU medium supplementedwith 4-day-old filter-sterilized [Millipore 0.2-µm-pore-sizefilters] culture supernatants of arginine-excreting strainspreviously grown in 2 ml of liquid DLAU medium . For each testedstrain, 60 and 300 µl of this sterilized medium were addedto 1.94 and 1.7 ml of new DLAU medium, respectively, and strainHN217 was grown for 4 days . The turbidity [optical density [OD]at 600 nm] of each culture was directly proportional to thearginine concentration in the culture medium . This turbiditywas compared to the OD obtained with the sterilized supernatantof the parental arginine-regulated strain FB331 with added arginineconcentrations ranging from 0 to 5 µg/ml . Two referencecurves were obtained by mixing 60 [or 300] µl of sterilizedused FB331 medium with 1.93 [or 1.69] ml of new DLAU mediumand 10 µl of new DLAU medium at 0 to 0.1% arginine [data not shown] . The minimum detectable arginine concentrations using this method were 0.2 µg/ml.

DNA techniques. Four genetic loci were PCR amplified and sequenced using theprimers described in Table 2 . PCR amplifications were done ona Peltier Thermal Cycler PTC-200 [MJ Research] with Taq polymerase[Sigma] . The PCR amplifications consisted of denaturation at95°C for 1 min, followed by 35 cycles of denaturation for20 s at 94°C, hybridization for 20 s at 56°C, and elongationfor 5 min at 72°C . Finally, the PCR was completed by a 10-minpostelongation treatment at 72°C . Prior to sequencing of the PCR products, the unincorporated primers and deoxynucleoside triphosphates were eliminated by passage on a Microspin S-400 HR column [Amersham Biosciences] . The argR2 gene of L . plantarum CCM 1904 was sequenced after amplification using inverse PCR. In our study of arginine synthesis, we fortuitously found thatprimer argCP2 not only hybridized to argC but also to the argR2 gene . This primer, in combination with primers argR5 [5'-GGTAACGGACCAGCACTAGC-3']and argRd4 [5'-CKWGAWAYNGTNGCYTGTGT-3', where K is G or T, Wis A or T, Y is C or T, and N is A, C, G, or T] were used toamplify the entire argR2 gene and its flanking regions [datanot shown] in a 2,402-bp sequence [GenBank accession no. AF451891].

 
RNA extraction. L . plantarum organisms grown until mid-exponential growth phase[OD at 600 nm, 0.4] were harvested by centrifugation at 4°C.The pellet was suspended in 400 µl of suspension solution[10% glucose, 12.5 mM Tris, pH 8, 5 mM EDTA] and 60 µlof 0.5 M EDTA . The cell suspension was then transferred to a 1.5-ml Eppendorf tube containing 500 µl of acidic phenoland 0.4 g of glass beads [average diameter, between 0.25 and0.5 mm] . The cells were broken by four series of shaking [Retschapparatus at maximal speed for 30 s] with 1-min pauses at 4°C.The cell debris was pelleted by centrifugation [5 min at 4°C;10,000 x g] . The supernatant was mixed with 1 ml of Trizol reagent[Invitrogen] and incubated for 5 min at room temperature, andthen 100 µl of chloroform-isoamyl alcohol [24/1 [vol/vol]]was added for 3 min at room temperature . After centrifugation[5 min at 4°C; 10,000 x g], the upper phase was treatedagain with 200 µl of chloroform-isoamyl alcohol . To precipitatethe nucleic acids in the upper phase, 500 µl of isopropanolwas added . After centrifugation [15 min at 4°C; 10,000 xg], the pellet was washed once with 70% ethanol, dried, solubilizedin 50 µl of Tris-EDTA, and treated for 20 min at 37°Cwith DNase I [10 U; Amersham Biosciences] . The DNase I was heatinactivated [65°C for 10 min].

Transcription analysis. RT-PCR was performed with the Invitrogen SuperScript one-stepRT-PCR with platinum Taq kit as recommended by the manufacturer.After a 5-min denaturation at 65°C, 25 ng of RNA templatewas used to detect transcripts specific to the carA, argG, andrrn genes . The argC transcripts were detected using 60 ng ofRNA template . RT was performed at 50°C for 30 min, and theresulting cDNA was PCR amplified . Denaturation at 94°C for2 min was followed by 30 cycles of denaturation at 94°Cfor 15 s, hybridization at 54°C for 30 s, and elongationat 68°C for 50 s . The program ended with a postelongation step at 72°C for 10 min . The primers used in RT-PCR are described in Table 2 . The RT-PCR products were stained with ethidium bromide and detected by UV after electrophoresis ina gel [2% Nusieve agarose gel; FMC BioProducts] separating smallDNA fragments . To control the absence of contaminant DNA inRNA preparations, the RT step was omitted, and no PCR productswere amplified.

The transcripts of the arginine-regulated operons [carAB, argCJBDF, and argGH] were quantified using Northern hybridization DNA probes specific to carA, argC, and argG, respectively . The probeswere obtained after PCR amplification [95°C for 1 min, followedby 35 three-step cycles of 94°C for 40 s, 50°C for 40s, and 72°C for 2 min; a postelongation at 72°C for 10 min completed the program] . The primers are listed in Table 2 . The PCR products were digoxigenin [DIG] labeled, and the concentrations of DIG-labeled probes were estimated by comparison with the labeled DNA control from the DIG DNA labeling and detection kit [Roche] . For each probe, the optimal amount of RNA to be used for quantification was tested in the range of 0.5 to 10µg of total RNA and was found to be different for thecarA [2.5 µg], argC and argG [10 µg], and rrn [100to 250 ng] probes . RNAs [25 µl] were heat denatured [10min at 65°C] in the presence of 75 µl of denaturationmixture [prepared by mixing 500 µl of formamide with 162µl of formaldehyde and 100 µl of 10x MOPS [morpholinepropanesulfonicacid]] . After 5 min on ice, 100 µl of cold 20x SSC [1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate] was added, and the RNAs were transferred onto positively charged Hybond nylon membranes [Amersham Biosciences] using the Bio-Dot SF microfiltration apparatus [Bio-Rad] . The air-dried RNAs were fixed on the membranes by UV irradiation . A temperature of 50°C was used for prehybridization and overnight hybridization with 10 ng of DIG-labeled probes/ml. Nucleic acid hybrids were detected with the nonradioactive DIG DNA labeling and detection kit using the alkaline phosphatase chemiluminescent substrate CDP-star [Roche] . Image acquisitionwas performed with the ChemiDoc XRS camera [Bio-Rad], and thesignals were quantified with Quantity One software [Bio-Rad].The background level was subtracted from the measured signal.To calculate the relative signal, the measured signal was dividedby the quantity of RNA put on the membrane . This relative signalwas then divided by the relative signal obtained with the rrnprobe to obtain the relative amount of each arg or car gene.

Computer analysis. Sequences were compared using the BLAST program [http://www.ncbi.nlm.nih.gov/BLAST/], and the ClustalX program was used to analyze protein alignments. Three-dimensional visualization of proteins was done with Swiss-Pdb viewer software [14].

Nucleotide sequence accession number. The DNA sequence of argR2 from L . plantarum CCM 1904 has beendeposited in the GenBank database under accession no. AF451891.

 
In L . plantarum, the two argR-like genes are adjacent to genes also found in other gram-positive bacteria. The complete genome of L . plantarum WCFS1 harbors two argR genesnamed argR1 and argR2 [17] . Since the sequenced strain is auxotrophicfor arginine when grown in air [4], the prototroph strain CCM1904 was chosen to study arginine regulation . CCM 1904 has anargR2 gene identical to the sequenced strain but has three silentmutations in the argR1 locus [1292191CT, 1292389AG, and 1292419CT, according to sequence accession no. NC_004567] . The genes adjacentto argR1 and argR2 were also found in other gram-positive bacteria,which highlights synteny group conservation around argR genes[Table 3].

 
Selection and phenotype of spontaneous arginine-deregulated mutants Arginine biosynthesis-deregulated mutants were obtained fromL . plantarum FB331, a strain with CPS-P deleted in which theonly active CPS is CPS-A [27] [Fig . 1A] . Thus, in the presenceof arginine, no carbamoyl phosphate [CP] was available for pyrimidinebiosynthesis, since the carA-carB gene transcription was argininerepressed [FB331 is arginine inhibited as long as no pyrimidinesare present in the medium] [27] . This property was exploitedin a positive screening to isolate deregulated mutants ableto grow in the presence of arginine in pyrimidine-free medium.Moreover, FB331 is unable to grow in the absence of arginineand pyrimidines in the medium . Apparently, the wild-type CPS-Aexpression level is not sufficient to provide CP at a levelrequired for both arginine and pyrimidine biosynthesis . This observation was exploited in a second screening procedure to isolate deregulated mutants able to grow in DLA medium without pyrimidines and arginine . All these mutants were isolated underCO₂-enriched growth conditions [aerobiosis enriched with 4%CO₂], since CPS-A activity depends on CO₂ concentrations higherthan those found in air [27] . Forty-four spontaneous independent mutants were isolated on DLA and DLA-plus-arginine media . The growth of these mutants was then tested under different conditions [aerobiosis and aerobiosis enriched with CO₂] and on different media; the results are listed in Table 4 . Three different phenotypeswere characterized [Table 4, classes 1, 2, and 3] . Unlike theparental strain, in pyrimidine-free media all mutants grew inCO₂-enriched air . The presence of arginine in the selectionprocess had no detected effect on the mutant phenotypes.

 
All 44 mutants had higher resistance to canavanine [canavanine minimal inhibitor concentrations were >200 µg ·ml^–1 compared to 25 µg · ml^–1 observedwith the parental FB331 strain [Table 4]] . The amounts of arginineexcreted by the mutants varied from 0.4 to 90 µg ·ml^–1 and were higher than that for the regulated parent[Table 4] . Class 1 mutants were the most frequent: 20 out of44 strains . These mutants displayed the characteristic growthdependency on the CO₂ supply; on DLA medium, no growth in aerobiosiswas observed even when both arginine and uracil were supplied.Analogous to the parental strain, other mutants grew on DLAcomplemented with arginine and uracil in aerobiosis . Class 3mutants [12 mutants] presented the same phenotype as class 2mutants, except for their extremely high excretion of arginine[averaging 75 µg · ml^–1] [Table 4] . The higherlevel of arginine excretion suggests that class 3 mutant regulationof arginine biosynthesis was the most impaired . We hypothesizedthat class 3 mutants were good candidates to search for arginineregulator mutants.

Characterization of arginine-deregulated mutants: argR1 and argR2 mutants had the class 3 phenotype. To characterize the mutation loci, the sequences of the parentalstrain and of its derivative mutant in four genetic loci werecompared: [i] the carA-carB genes encoding CPS-A, [ii] the carA-argCinteroperonic sequence, [iii] the pyrR gene encoding the pyrimidine-regulated transcriptional regulator, and [iv] the argR1 and argR2 genes[see Materials and Methods] . The 12 mutants of class 3 [Table 4] contained mutations in either the argR1 or the argR2 geneanalyzed [Table 1 and Fig. 2], and no mutations were found inthe other loci analyzed . In mutant FB331-1, a GT substitutionwas identified 42 nucleotides upstream of the argR1 initiationcodon . The localization of this mutation within the argR1 promoterregion suggests that the argR1 transcription is impaired inthis mutant . All other class 3 mutations were found in the ArgRcoding sequences . Mutations in the ArgR proteins were foundin the two functional domains of arginine repressors, the DBDin the N-terminal domain [37] and the C-terminal oligomerizationand arginine-binding domain [26].

 
Mutations in the DBD were mapped in ArgR1 [A39E, S42L, and D44G; mutants FB331-2 to -4] and in ArgR2 [R6H, R43C, and R43H; mutants FB331-7 to -9] [Fig . 2] . The high homology between the G . stearothermophilusArgR and L . plantarum ArgR2 proteins [38% identity] suggeststhat these domains are structurally similar . Such structuralsimilarities are also found for ArgR N- and C-terminal domainsof E . coli [35, 37] and G . stearothermophilus, while these proteins show only 28% sequence identity . The corresponding residues mutated in the L . plantarum ArgR1 and ArgR2 proteins were analyzed in view of the G . stearothermophilus ArgR N-terminal region [accession no . 1B4A] [26] . By comparison with the three-dimensional representation of the G . stearothermophilus crystallized ArgR, the L . plantarum ArgR mutated residues R43, A39, S42, and D44 are found in the helix 3, which has been described as makingdirect contacts with DNA [data not shown] . In mutant FB331-7,the R6H amino acid substitution conferred the fully derepressedphenotype [Tables 1 and 4], confirming that the argR2 translation initiation codon is GTG and not the downstream ATG . Mutationof residue R6 has never been described before [16], although adjacent residues altered E . coli ArgR activity [37] . The R6Hmutation is located in the ArgR2 1 helix, which is spatiallyclose to the 3 helix [data not shown] . In this mutant, the lossof a potential hydrogen bond between residues R6 and D44 maybe altered.

In the C-terminal domain, three mutations were found, includingan amino acid substitution [A123D] in ArgR1 and two deletions[Q140-D153 in ArgR1 and L135-H152 in ArgR2] . These deletionsin the oligomerization domain of the protein may generate incorrectfolding or oligomerization, which may make them nonfunctionalrepressors . In conclusion, class 3 mutants harbored mutationsin one of the argR genes, and these mutations appeared to bedominant and to alter arginine regulation . Moreover, arginineregulation depended on functional binding of the repressorsto DNA and the corepressor and also on the integrity of therepressor oligomerization domain.

Mutations in argR1 or argR2 resulted in loss of arginine repression. The arginine biosynthetic operons [argGH, carAB, and argCJBDF[Fig . 1B]] may be regulated differently by ArgR1 or ArgR2 . Thus,the transcription of argG, carA, and argC, which are the firstgenes of these operons, was analyzed by RT-PCR [Fig . 3] and Northern hybridization [Fig . 4] in cells grown in the presenceor absence of arginine in three genetic contexts . Arginine repressionwas compared in strain FB331, harboring two wild-type argR genes,and the mutants FB331-4 and FB331-10, which harbor single-amino-acidsubstitutions in ArgR1 [D44G] and ArgR2 [R43H] DBDs, respectively[Fig . 2] . In the presence of both wild-type argR alleles, argininebiosynthetic gene transcription was repressed by arginine [Fig.3 [compare lanes 1 and 2] and 4] . Complete arginine repressionwas observed for the carA and argG genes but not for the argC gene [Fig . 3, argC, compare lanes 1 and 2] . This was quantifiedusing Northern hybridization: cultivating the wild-type strainin the presence of arginine drastically inhibited transcriptionof the carA and argG genes [<1% of the nonrepressed level],and only half of the argC transcripts were detected [Fig . 4].On the other hand, in the presence of arginine, carA, argC,and argG transcripts were more abundant in the ArgR1 mutantor the ArgR2 mutant than in the wild-type strain [RT-PCR data[Fig . 3, compare lanes 4 and 6 to lane 2] and Northern blots[Fig. 4]] . This demonstrates that a mutation in either ArgR1 or ArgR2 led to the loss of arginine repression of the arginine biosynthetic genes . Since a mutation in the DNA binding siteof only one L . plantarum argR gene is sufficient to alter arginine regulation, we propose that both ArgR proteins bind to targetDNA sequences, which we refer to as the ARG box by analogy towhat has been described in other eubacteria [24].

 
Class 2 mutants with impaired ARG operators. Class 2 mutants harbored wild-type argR1 and argR2 gene sequences[data not shown] . Compared to the class 3 mutants, class 2 mutantsexcreted less arginine and may be the result of mutations presentin the operators of the arginine regulons . Putative ARG boxeslocated in the bipolar carA-argC promoter region have been determinedby homology with the E . coli ARG box consensus sequence [3]. Among the 10 tested class 2 mutants, two clones presented mutations in the carA-argC intergenic sequence [Table 1], but not withinthe CPS-A structural genes . Mutant FB331-13 harbored a TC pointmutation located in the previously proposed R9 ARG box, 5'-ATTGAAAAAAtATTCACT-3'[accession no. X99978; nucleotides 4773 to 4756] [Fig . 1C].In mutant FB331-14, a deletion of 48 nucleotides including theR9 ARG box was found . Despite the fact that the deletion siteis located within the argC promoter, the argC promoter sequencein FB331-14 was unchanged [the T base found in the wild-type–35 box was replaced by another T located upstream ofthe deletion] [Fig. 1C] . The effects of these mutations on arginine-dependent transcription of the bipolar car and arg operons were tested using RT-PCR [Fig . 3] and Northern blotting [Fig. 4] . Both mutantshad constitutive carA transcription, but a higher level of transcriptionwas observed with the deletion mutation [in FB331-14] than withthe point mutation [in FB331-13] [Fig . 4] . Although these mutationshad a minor effect on argC transcription compared to carA transcription, the 48-nucleotide deletion clearly impaired argC arginine repression[Fig. 4].

 
Like many other gram-positive bacteria, L . plantarum harbors several ArgR-like homologues [5] . In order to study the roles of ArgR1 and ArgR2 in L . plantarum, arginine biosynthesis mutants were isolated . The 44 independent spontaneous arginine-deregulated mutants had common physiological traits . Compared to the parental strain FB331, all of the mutants [i] were excreting more arginine; [ii] were more resistant to canavanine, a toxic analogue of arginine; and [iii] were able to grow in carbon dioxide-enrichedair either in the absence of both uracil and arginine or inthe presence of arginine only [Table 4] . These common traits suggest that no mutation hindered arginine transport . A mutationin arginine transport would have conferred higher resistanceto canavanine and allowed growth in the presence of arginine.However, such a mutation in arginine transport would not explainarginine excretion and growth in the absence of both uraciland arginine . Instead, the observed canavanine resistance wouldresult from deregulation of arginine biosynthesis with overproductionof CP and arginine, as discussed below.

FB331 growth is limited by carAB expression. In the genetic context of strain FB331, which lacks a functionalCPS-P, CP synthesis is solely dependent on CPS-A [Fig . 1A]. Previous data suggested that CPS-A cannot provide enough CPfor both arginine and pyrimidine synthesis, since FB331 is unableto grow in the absence of added uracil [27] . This phenotype may be due to low carAB expression . All the selected spontaneous arginine-deregulated mutants acquired the ability to grow on minimal media . Increased expression of the carAB operon was clearly identified in mutants with mutations in the carA operator [transcription studies [Fig . 3 and 4]] . As a conclusion, enhancedcarAB operon transcription would generate sufficient CPS-A activityto supply CP synthesis for both arginine and pyrimidine biosyntheticpathways . Thus, wild-type carAB transcription is growth limitingin minimal media in L . plantarum strains that lack CPS-P.

Evidence of a regulation link between arginine metabolism and another yet-uncharacterized carbon dioxide-dependent metabolism. CPS-A activity is dependent on high concentrations of carbondioxide [27] . CPS-A may have low affinity for its substratecarbon dioxide or its dissolved form, bicarbonate . Another possibilityis that other systems regulate CPS-A activity or the CP poolwhen L . plantarum is grown in CO₂-enriched air . In this work,we found that high carAB expression did not alleviate CPS-Adependence on higher CO₂ concentrations; mutants with constitutively high carAB expression did not grow in ordinary air in defined medium [DLA] without arginine and uracil [Fig . 3 and 4 [carAprobe] and Table 4] . Moreover, half of the mutants unexpectedlyacquired a strict CO₂ dependence on DLA [Table 4, class 1 mutants]but not on the rich undefined medium MRS [which also containsarginine and uracil] . These mutants had the phenotype of arginine-deregulated mutants, since they [i] resisted high canavanine concentrations,[ii] excreted arginine, and [iii] grew on DLA and DLA complementedwith arginine under carbon dioxide-enriched growth conditions.Wild-type argR1 or argR2 genes, as well as the wild-type carA-argC intergenic region, were found in 11 out of 20 class 1 mutants. Thus, even if the mutated genes have not been characterized,the phenotype of these class 1 mutants demonstrates that regulationof CPS-A activity is linked to another metabolism, which isalso dependent on carbon dioxide concentrations . Additionalphysiologic and genetic experiments are required to identifythe metabolic pathways that are altered in class 1 mutants.

Two arginine repressors control transcription of arginine biosynthetic genes in L . plantarum. One-third of the isolated mutants excrete large amounts of arginine[Table 4, class 3 mutants] and were shown to be mutated in oneof the two argR genes present in L . plantarum . Transcriptional studies [Fig . 3 and 4] confirmed that mutation in one of thetwo regulators resulted in a complete loss of arginine repression,so the activity of any of these proteins is dependent on theactive presence of the other one . Single-amino-acid substitutionmutations were obtained for both ArgR copies, which providesvaluable information about the domains of these molecules thatare involved in arginine repression . [i] Integrity of the DBDs of both ArgR1 and ArgR2 was necessary for arginine repression, so we conclude that both ArgR proteins bind to target DNA sequences. Loss of arginine repression was correlated with mutations foundin the 3 helix, a structural motif of the DBD in ArgR1 [A39Ein FB331-2; S42L in FB331-3; D44G in FB331-4] and in ArgR2 [R43Cin FB331-9; R43H in FB331-10] . [ii] The last C-terminal residuesof both ArgR1 and ArgR2 are essential for arginine repression.Arginine regulation was abolished when the last 18 ArgR1 residuesin mutant FB331-6 and the last 14 ArgR2 residues in mutant FB331-12were deleted . These residues belong to a predicted helix structure[data not shown], despite the fact that their ArgR C-terminalpeptides are not homologous . This 6 helix may facilitate dimerizationof the two repressor trimers . [iii] ArgR1 and ArgR2 may havedifferent corepressor binding sites . The conserved motif GTIAGDDT[Fig . 2] is required for arginine binding and dimerization ofthe two ArgR trimers of the well-studied single-copy repressorsfound in E . coli and B . subtilis . Within this motif, residueA123 seems to be essential for ArgR1 activity [mutant FB331-5].This motif is not conserved in ArgR2 and other regulators [Fig.2], including the two Lactococcus lactis repressors [20] . This observation suggests a novel arginine regulation mechanism in gram-positive bacteria harboring several argR genes.

A sequence resembling an E . coli ARG box mediates carA arginine repression but is not found in the argG operator. The proposed L . plantaraum ARG box consists of two 18-bp imperfect palindromes separated by three A bases [mismatched bases comparedto the consensus E . coli ARG box shown in Fig . 1C] . The TC mutationoccurred in the highly conserved half-site ARG box that harboredonly one mismatch compared to the consensus sequence . When theproposed ARG box is mutated by complete deletion or by a point mutation [mutants FB331-13 and FB331-14], carA transcription is constitutive . These two mutations did not deregulate argC transcription to the same extent as carA transcription [Fig. 3 and 4] . This suggests that the proposed ARG box is not commonto the bipolar operons . Alternatively, since wild-type argininerepression of argC is weaker than that of carA [Fig . 3 and 4, half versus total repression, respectively], one would expect less effect of mutations impairing the arginine operator inthe less tightly regulated operon . The proposed L . plantarumARG box is localized 5 bp before the argC –35 box andat least 61 bp from the –35 box of the carA promoter [Fig.1C] . This organization is different from that of other reported arginine-repressed bipolar genes . The transcription start sitesare adjacent in M . profunda [38], overlap by 13 nucleotidesin E . coli [18], and are separated by 162 nucleotides in L.plantarum . Even though the argGH operon was arginine regulatedby ArgR1 and ArgR2, no ARG box-like sequence highly similarto known ARG boxes was found in the argG operator . Therefore,ARG boxes highly similar to the E . coli ARG box consensus, likethe one found in the bipolar carA-argC operators, appear notto be a prerequisite . Electrophoretic mobility shift assaysand footprinting need to be performed to test whether ArgR1,ArgR2, or both repressors bind to the proposed ARG box or othersequences of the bipolar operators.

A novel arginine repression mechanism in L . plantarum? In most of the bacteria in which regulation of arginine biosynthesis has been studied in detail, repression of the arginine regulonis exerted by the binding of a hexameric repressor molecule.In fact, the ArgR active form found in E . coli, B . subtilis,and G . stearothermophilus consists of a dimer of homotrimers[10, 26, 35, 37] whose interactions are strengthened by thebinding of six L-arginine molecules . This is the first reportof two different ArgR transcription regulators that repressarginine biosynthesis operons . The L . plantarum active repressormay be a heterohexamer of ArgR1 and ArgR2 monomers . Mutationsin one of those monomers would lead to an inactive repressorform . This mutual dependence and the fact that, compared tothe characterized ArgR molecules, L . plantarum ArgR1 and ArgR2have lost conserved residues of the arginine-binding sites [seeabove] favor the hypothesis of cooperative binding of ArgR1and ArgR2 to ARG boxes . Further experiments are needed to elucidatearginine repression in L . plantarum, to clarify whether thefunctional repressors are constituted of mixtures of ArgR1 andArgR2 molecules, and to identify the ArgR1-ArgR2 DNA bindingto cognate and heterologous operators.

What is the physiological impact of argR gene duplication in gram-positive bacteria? More than a single copy of argR is frequently encountered ingram-positive bacteria [5] . The number of argR paralogs varieswithin a given genus, such as within Bacillus [two copies inBacillus cereus and only one in the other sequenced Bacillaceae],Lactobacillus [two homologs in L . plantarum and none in Lactobacillus johnsonii], Staphylococcus [two homologs in Staphylococcus aureus versus three copies in Staphylococcus epidermidis], and Clostridium[two homologs in Clostridium perfringens versus a single copyin Clostridium tetani] . Only Enterococcus faecalis, formerlycalled Streptococcus faecalis, harbored four paralogs, whilethe other entirely sequenced Streptococcaceae harbored threecopies [5; H . Nicoloff, F . Arsène-Ploetze, and F . Bringel,unpublished data] . The fact that up to four different ArgR/AhrC-likeproteins in some bacteria, and in other prokaryotes a singleregulator, regulate both arginine biosynthesis and argininecatabolism raises the question of the roles of multiple regulatorsin a cell.

Gene duplication generates functional redundancy, which is often not advantageous [39] . Theoretical population genetics predictsthat both duplicates can be stably maintained when some aspectsof their functions differ [29], which can occur by subfunctionalization.In this case, each paralog would mutate up to the point at whichits total capacity would be reduced to the level of the single-copyancestral gene, and it would adopt part of the function of theancestor . In the two cases where the functions of duplicatedargR genes were genetically investigated, both gene productswere active and required for active transcriptional regulation[reference 20 and this work] . The subfunctionalization of thedifferent paralogs can be at the level of protein function orexpression . This hypothesis is in agreement with results observedin E . faecalis, where glucose and arginine influenced argR1and argR2 transcription differently [2], and in L . lactis, where only one argR gene was implicated in the regulation of arginine catabolism [20] . Thus, the presence of several argR genes mayfacilitate microbial response to variations of environmentalgrowth conditions . In L . lactis, mutation in one of its twoargR genes conferred improved acid resistance upon enhancedADI arginine catabolism [30] . The ADI system plays a role inthe acid resistance of a number of gram-positive bacteria [fora review, see reference 7] and acts as a virulence factor inStreptococcus pyogenes [9] . Unlike L . lactis, most L . plantarumorganisms do not catabolize arginine, and no difference in thefunction of L . plantarum argR genes was found under the testedconditions, which does not exclude the possibility that thesegenes have a different pattern of function or expression . Whetheronly one ArgR protein is also sufficient to activate the argininecatabolic genes in the few L . plantarum strains described asharboring an ADI pathway [32] remains to be tested . Since genesthat are functionally related are sometimes organized in operons,L . plantarum argR gene linkage was examined and compared tothat in other organisms . L . plantarum and Leuconostoc mesenteroidesboth harbor two copies of argR that are found in their genomesadjacent to similar genes. argR1 is adjacent to pbp2A, whichencodes a putative membrane carboxypeptidase [penicillin-bindingprotein] [National Center for Biotechnology Information conserveddomain COG0744] [Table 3, group 1] . argR2 is bordered by yqxC, which encodes a putative FtsJ-like methyltransferase whose substrate is the 23S rRNA [listed in the National Center for Biotechnology Information conserved domains as COG1189], and recN, which encodes a DNA repair protein in E . coli [12] [Table 3, group 2] . E.faecalis also contains both synteny groups but contains twoadditional argR copies [argR1 and argR2], which are linked tothe ADI arginine catabolic operon [2] . Unlike L . plantarum andL . mesenteroides [13], E . faecalis not only synthesizes arginine[25] but also degrades it . L . lactis harbors two genes withahrC linked to yqxC-recN [Table 3, group 2] and argR linkedto the arginine catabolic arc genes [20] . AhrC, but not ArgR, is involved in activation of the arc genes [20] . The conservationof argR genes in synteny groups may be correlated with the differentphysiological roles of ArgR proteins in gram-positive bacteria.We suggest that argR duplication generated complex argR regulationnetworks in some gram-positive bacteria in response to specificniche adaptation.

 
We thank Jean-Claude Hubert for fruitful discussion about thiswork and Isabelle Guillouard for providing an RNA extractionprotocol.

 
* Corresponding author . Mailing address: Laboratoire de Dynamique, Evolution et Expression de Génomes de Microorganismes, Université Louis Pasteur/CNRS FRE 2326, 28 rue Goethe, 67083 Strasbourg, France . Phone: 33 3 90 24 18 15 . Fax: 33 3 90 24 20 28 . E-mail: bringel@gem.u-strasbg.fr.

1. Arena, M . E., F . M . Saguir, and M . C . Manca de Nadra. 1999 . Arginine dihydrolase pathway in Lactobacillus plantarum from orange . Int . J . Food Microbiol . 47:203-209.
2. Barcelona-Andres, B., A . Marina, and V . Rubio. 2002 . Gene structure, organization, expression, and potential regulatory mechanisms of arginine catabolism in Enterococcus faecalis . J . Bacteriol . 184:6289-6300 .
3. Bringel, F., L . Frey, S . Boivin, and J . C . Hubert. 1997 . Arginine biosynthesis and regulation in Lactobacillus plantarum: the carA gene and the argCJBDF cluster are divergently transcribed . J . Bacteriol . 179:2697-2706.
4. Bringel, F., and J . C . Hubert. 2003 . Extent of genetic lesions of the arginine and pyrimidine biosynthetic pathways in Lactobacillus plantarum, L . paraplantarum, L . pentosus and L . casei: prevalence of CO₂-dependent auxotrophs and characterization of deficient arg genes in L . plantarum. Appl . Environ . Microbiol . 69:2674-2683 .
5. Bringel, F., H . Nicoloff, F . Arsene-Ploetze, and J . C . Hubert. 2002 . Phylogenetic analysis and gene linkage of ArgR/AhrC orthologs revealed groups of synteny in Gram-positive bacteria . Sci . Aliments 22:133-142.
6. Burke, M., A . F . Merican, and D . J . Sherratt. 1994 . Mutant Escherichia coli arginine repressor proteins that fail to bind L-arginine, yet retain the ability to bind their normal DNA-binding sites . Mol . Microbiol . 13:609-618.
7. Cotter, P . D., and C . Hill. 2003 . Surviving the acid test: responses of gram-positive bacteria to low pH . Microbiol . Mol . Biol . Rev . 67:429-453 .
8. Czaplewski, L . G., A . K . North, M . C . Smith, S . Baumberg, and P . G . Stockley. 1992 . Purification and initial characterization of AhrC: the regulator of arginine metabolism genes in Bacillus subtilis . Mol . Microbiol . 6:267-275.
9. Degnan, B . A., J . M . Palmer, T . Robson, C . E . Jones, M . Fischer, M . Glanville, G . D . Mellor, A . G . Diamond, M . A . Kehoe, and J . A . Goodacre. 1998 . Inhibition of human peripheral blood mononuclear cell proliferation by Streptococcus pyogenes cell extract is associated with arginine deiminase activity . Infect . Immun . 66:3050-3058 .
10. Dennis, C . C., N . M . Glykos, M . R . Parsons, and S . E . Phillips. 2002 . The structure of AhrC, the arginine repressor/activator protein from Bacillus subtilis . Acta Crystallogr . D 58:421-430.
11. Dion, M., D . Charlier, H . Wang, D . Gigot, A . Savchenko, J . N . Hallet, N . Glansdorff, and V . Sakanyan. 1997 . The highly thermostable arginine repressor of Bacillus stearothermophilus: gene cloning and repressor-operator interactions . Mol . Microbiol . 25:385-398.
12. Dunman, P . M., L . Ren, M . S . Rahman, V . A . Palejwala, H . S . Murphy, M . R . Volkert, and M . Z . Humayun. 2000 . Escherichia coli cells defective for the recN gene display constitutive elevation of mutagenesis at 3,N[4]-ethenocytosine via an SOS-induced mechanism . Mol . Microbiol . 37:680-686.
13. Garvie, E . I. 1986 . Genus Leuconostoc van Tieghem 1878, 198^AL emend mut . char . Hucker and Pederson 1930, 66^AL, p . 1071-1075 . In P . H . A . Sneath, N . S . Mari, M . E . Sharpe, and J . G . Holt [ed.], Bergey’s manual of systematic bacteriology, vol . 2 . Williams and Wilkins, Baltimore, Md.
14. Guex, N., and M . C . Peitsch. 1997 . SWISS-MODEL and the Swiss-PdbViewer: an environment for comparative protein modeling . Electrophoresis 18:2714-2723.
15. Holtham, C . A., K . Jumel, C . M . Miller, S . E . Harding, S . Baumberg, and P . G . Stockley. 1999 . Probing activation of the prokaryotic arginine transcriptional regulator using chimeric proteins . J . Mol . Biol . 289:707-727.
16. Karaivanova, I . M., P . Weigel, M . Takahashi, C . Fort, A . Versavaud, G . Van Duyne, D . Charlier, J . N . Hallet, N . Glansdorff, and V . Sakanyan. 1999 . Mutational analysis of the thermostable arginine repressor from Bacillus stearothermophilus: dissecting residues involved in DNA binding properties . J . Mol . Biol . 291:843-855.
17. Kleerebezem, M., J . Boekhorst, R . van Kranenburg, D . Molenaar, O . P . Kuipers, R . Leer, R . Tarchini, S . A . Peters, H . M . Sandbrink, M . W . Fiers, W . Stiekema, R . M . Lankhorst, P . A . Bron, S . M . Hoffer, M . N . Groot, R . Kerkhoven, M . de Vries, B . Ursing, W . M . de Vos, and R . J . Siezen. 2003 . Complete genome sequence of Lactobacillus plantarum WCFS1 . Proc . Natl . Acad . Sci . USA 100:1990-1995 .
18. Krin, E., C . Laurent-Winter, P . N . Bertin, A . Danchin, and A . Kolb. 2003 . Transcription regulation coupling of the divergent argG and metY promoters in Escherichia coli K-12 . J . Bacteriol . 185:3139-3146 .
19. Kueh, R., N . A . Rahman, and A . F . Merican. 2003 . Computational docking of L-arginine and its structural analogues to C-terminal domain of Escherichia coli arginine repressor protein [ArgRc] . J . Mol . Model . 9:88-98 . [Online.]
20. Larsen, R., G . Buist, O . P . Kuipers, and J . Kok. 2004 . ArgR and AhrC are both required for regulation of arginine metabolism in Lactococcus lactis . J . Bacteriol . 186:1147-1157 .
21. Lu, C . D., J . E . Houghton, and A . T . Abdelal. 1992 . Characterization of the arginine repressor from Salmonella typhimurium and its interactions with the carAB operator . J . Mol . Biol . 225:11-24.
22. Maas, W . K. 1994 . The arginine repressor of Escherichia coli . Microbiol . Rev . 58:631-640.
23. Miller, C . M., S . Baumberg, and P . G . Stockley. 1997 . Operator interactions by the Bacillus subtilis arginine repressor/activator, AhrC: novel positioning and DNA-mediated assembly of a transcriptional activator at catabolic sites . Mol . Microbiol . 26:37-48.
24. Morin, A., N . Huysveld, F . Braun, D . Dimova, V . Sakanyan, and D . Charlier. 2003 . Hyperthermophilic Thermotoga arginine repressor binding to full-length cognate and heterologous arginine operators and to half-site targets . J . Mol . Biol . 332:537-553.
25. Murray, B . E., K . V . Singh, R . P . Ross, J . D . Heath, G . M . Dunny, and G . M . Weinstock. 1993 . Generation of restriction map of Enterococcus faecalis OG1 and investigation of growth requirements and regions encoding biosynthetic function . J . Bacteriol . 175:5216-5223.
26. Ni, J., V . Sakanyan, D . Charlier, N . Glansdorff, and G . D . Van Duyne. 1999 . Structure of the arginine repressor from Bacillus stearothermophilus . Nat . Struct . Biol . 6:427-432.
27. Nicoloff, H., J . C . Hubert, and F . Bringel. 2000 . In Lactobacillus plantarum, carbamoyl phosphate is synthesized by two carbamoyl-phosphate synthetases [CPS]: carbon dioxide differentiates the arginine-repressed from the pyrimidine-regulated CPS . J . Bacteriol . 182:3416-3422 .
28. Niersbach, H., R . Lin, G . D . Van Duyne, and W . K . Maas. 1998 . A superrepressor mutant of the arginine repressor with a correctly predicted alteration of ligand binding specificity . J . Mol . Biol . 279:753-760.
29. Nowak, M . A., M . C . Boerlijst, J . Cooke, and J . M . Smith. 1997 . Evolution of genetic redundancy . Nature 388:167-171.
30. Rallu, F., A . Gruss, S . D . Ehrlich, and E . Maguin. 2000 . Acid- and multistress-resistant mutants of Lactococcus lactis: identification of intracellular stress signals . Mol . Microbiol . 35:517-528.
31. Rodriguez-Garcia, A., M . Ludovice, J . F . Martin, and P . Liras. 1997 . Arginine boxes and the argR gene in Streptomyces clavuligerus: evidence for a clear regulation of the arginine pathway . Mol . Microbiol . 25:219-228.
32. Spano, G., G . Chieppa, L . Beneduce, and S . Massa. 2004 . Expression analysis of putative arcA, arcB, and arcC genes partially cloned from Lactobacillus plantarum isolated from wine . J . Appl . Microbiol . 96:185-193.
33. Stirling, C . J., G . Szatmari, G . Stewart, M . C . Smith, and D . J . Sherratt. 1988 . The arginine repressor is essential for plasmid-stabilizing site-specific recombination at the ColE1 cer locus . EMBO J . 7:4389-4395.
34. Stover, C . K., X . Q . Pham, A . L . Erwin, S . D . Mizoguchi, P . Warrener, M . J . Hickey, F . S . Brinkman, W . O . Hufnagle, D . J . Kowalik, M . Lagrou, R . L . Garber, L . Goltry, E . Tolentino, S . Westbrock-Wadman, Y . Yuan, L . L . Brody, S . N . Coulter, K . R . Folger, A . Kas, K . Larbig, R . Lim, K . Smith, D . Spencer, G . K . Wong, Z . Wu, I . T . Paulsen, J . Reizer, M . H . Saier, R . E . Hancock, S . Lory, and M . V . Olson. 2000 . Complete genome sequence of Pseudomonas aeruginosa PA01, an opportunistic pathogen . Nature 406:959-964.
35. Sunnerhagen, M., M . Nilges, G . Otting, and J . Carey. 1997 . Solution structure of the DNA-binding domain and model for the complex of multifunctional hexameric arginine repressor with DNA . Nat . Struct . Biol. 4:819-826.
36. Tian, G., and W . K . Maas. 1994 . Mutational analysis of the arginine repressor of Escherichia coli . Mol . Microbiol . 13:599-608.
37. Van Duyne, G . D., G . Ghosh, W . K . Maas, and P . B . Sigler. 1996 . Structure of the oligomerization and L-arginine binding domain of the arginine repressor of Escherichia coli . J . Mol . Biol . 256:377-391.
38. Xu, Y., Y . Sun, N . Huysveld, D . Gigot, N . Glansdorff, and D . Charlier. 2003 . Regulation of arginine biosynthesis in the psychropiezophilic bacterium Moritella profunda: in vivo repressibility and in vitro repressor-operator contact probing . J . Mol . Biol . 326:353-369.
39. Zhang, J. 2003 . Evolution by gene duplication: an update . Trends Ecol . Evol . 18:292-298.

Free Online Full-text Article

What Is Dna?, What Is Bioremediation?, What Is MIC?, What Is Anthrax?, What Is Pcr?, i, Microbe, c, Microbiology, r, Bacteriology, a, Microbes, a, Microorganism, o, Escherichia coli, o, S. cerevisiae, o, Salmonella, o, Pichia, c, Bacillus, a, Antimicrobial, a, Microbiological, s, Nitrifying, e, Bacteriological, s, Proteus, i, Antimicrobial, o, Microorganism, a, Proteus, s, Vibriosis, s, Escherichia coli, c, Bacillus, o, Salmonella, n, Microbiological, r, Phage, a, Haemophilus, i, Escherichia coli




 

© 2005 Transgalactic Ltd (manufacturer of Bioscreen C software) | Privacy Statement | P.O. Box 1393, 00101 Helsinki, Finland, phone: +358 9 85172920, fax: +358 9 8749481, e-mail: microbiology@bionewsonline.com
 

Last modified: May 25, 2005