Transcriptome Analysis of the Lactococcus lactis ArgR and AhrC Regulons

In previous studies, we have shown that direct protein-proteininteraction between the two regulators ArgR and AhrC in Lactococcuslactis is required for arginine-dependent repression of thebiosynthetic argC promoter and the activation of the catabolicarcA promoter. Here, we establish the global ArgR and AhrC regulonsby transcriptome analyses and show that both regulators arededicated to the control of arginine metabolism in L. lactis.

INTRODUCTION

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INTRODUCTION

In many bacteria, arginine metabolism is controlled by transcriptionalregulators of the ArgR family, including the well-characterizedArgR of Escherichia coli (13), AhrC of Bacillus subtilis (5),and ArgR of Bacillus stearothermophilus (7). Regulation is mediatedthrough the interaction of the regulators with operators designatedARG boxes, which consist of 18-bp palindromic structures overlappingthe core promoters of target genes (4, 5, 17). Low-G+C-contentgram-positive bacteria often harbor more than one ArgR-typehomologue (see reference 2 for an overview), and separate studiesrecently demonstrated that two arginine regulators are necessaryfor the regulation of arginine metabolism in the lactic acidbacteria Lactococcus lactis and Lactobacillus plantarum (11,15). The deletion or mutation of any single arginine regulatorin these organisms results in the complete disruption of arginine-mediatedregulation. L. lactis AhrC is known to facilitate the bindingof ArgR to ARG boxes in the biosynthetic argC promoter and tosequester and prevent ArgR from binding to ARC boxes (ARG boxhalf sites) in the catabolic arcA promoter (12). In this way,arginine leads to the repression of arginine biosynthesis andthe activation of arginine catabolism.

Here, we establish the complete regulons of ArgR and AhrC inL. lactis and show that these regulators are dedicated to thecontrol of arginine metabolism.

ArgR and AhrC are dedicated regulators of arginine metabolism.

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ArgR and AhrC are...

The global regulatory effects of ArgR and AhrC of L. lactiswere studied by DNA microarray-based transcriptome analysesas described previously, by using amplicon-based L. lactis IL-1403slides (6, 10). IL-1403 amplicon sequences and the microarraydata used are available online at http://molgen.biol.rug.nl/publication/arg_data/.RNA was isolated from cells grown to mid-exponential phase inchemically defined medium (CDM) containing 10 mM arginine (11).The transcription profile of L. lactis MG1363 was compared tothose of its two single regulator mutants MG ${Delta}$ argR and MG ${Delta}$ ahrCand the double regulator mutant MG ${Delta}$ argRahrC (11). Significantderepression of all the genes in the argCJDBF, gltS-argE, andargGH operons of the arginine biosynthesis pathway in all threemutants was observed (Fig. 1). These results extend the knownArgR/AhrC regulons considerably and also establish the operonstructures of the regulated genes, in agreement with the predictedpromoter and putative terminator structures (Fig. 1). Whereasthe levels of derepression of all biosynthesis genes in MG ${Delta}$ argR,as well as those in MG ${Delta}$ argRahrC, varied between 5- and 15-fold,the levels of derepression of these genes in MG ${Delta}$ ahrC never exceeded6-fold (Fig. 1). The lower mutant-versus-wild-type ratios obtainedfor MG ${Delta}$ ahrC than for MG ${Delta}$ argR and MG ${Delta}$ argRahrC can be explained bythe previous observation that ArgR, independently of AhrC, canweakly bind to the argC promoter in the presence of arginine(12). Still, both arginine regulators are essential for thecomplete repression of arginine bio-synthesis, as the loss ofeither of the two regulators leads to an increase in the expressionof all involved genes.

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FIG. 1. Derepression of the arginine biosynthesis operons of L. lactis, as measured by DNA microarray analyses. Cells were grown to mid-exponential phase in CDM with 10 mM arginine (11). The argCJDBF (A), gltS-argE (B), and argGH (C) operons and their neighboring genes, predicted promoters, and putative stem-loop (terminator) structures (calculated as ${Delta}$ G° in kilocalories per mole) are drawn schematically, not to scale. The bars in the graphs show the ratios of expression in L. lactis strains MG ${Delta}$ argR, MG ${Delta}$ ahrC, and MG ${Delta}$ argRahrC relative to that in L. lactis MG1363. The change in expression (n-fold) is shown on the y axis, with positive and negative values indicating up- and downregulation, respectively; if the expression of a gene in the mutant was equal to that in the wild-type strain, this resulted in a value near the x axis (+1/–1). The combined P values for the respective operons, thus excluding the values for surrounding genes, are given in parentheses next to the symbols.

Regulation of the arginine catabolic gene cluster.

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Regulation of the arginine...

The transcriptome analyses demonstrated a complex pattern ofregulation of the arc gene cluster encoding the catabolic argininedeiminase (ADI) pathway (Fig. 2). The deletion of argR had nosignificant effect on arc expression, while the deletion ofahrC resulted in general downregulation, and the expressionof arc genes in MG ${Delta}$ argRahrC was increased relative to that inthe wild-type strain (Fig. 2A). The arcB gene did not show significantchanges in expression in any of the regulator mutants (Fig.2A), which seems surprising considering that the changes inthe expression of the other arc genes were highly significant.L. lactis arc is recognized as one of the largest and most complexof bacterial ADI pathway operons (19). The ADI operon arcDABCof Pseudomonas aeruginosa is subject to posttranscriptionalregulation via mRNA processing and transcription termination,resulting in differential gene expression according to the requirementof the gene products in the cell (8, 9). A similar process maybe in effect in L. lactis, although the role therein, if any,of the putative stem-loop structures downstream of arcB (Fig.2A) remains to be investigated.

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FIG. 2. (A) Deregulation of the L. lactis arginine catabolic arcABD1C1C2TD2-yvaD gene cluster (not drawn to scale). Refer to the legend to Fig. 1 for details. Numbered horizontal bars refer to regions examined by RT-PCR. (B) Results from RT-PCR investigation of the arc operon structure. Primer pairs were designed to amplify the intergenic regions indicated by numbered bars in panel A. Regions 1 and 2 correspond to RT-PCR probes of the arcA promoter region (negative control) and an intragenic arcA region (positive control), respectively. "DNA" indicates a positive PCR control with chromosomal DNA as the template. "RT" and "RNA" indicate PCRs performed with total RNA with and without reverse transcriptase treatment, respectively, the latter acting as a negative control. M, molecular size markers.

RT-PCR analysis of the arc gene cluster.

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RT-PCR analysis of the...

To verify the arc operon structure as determined by transcriptomeanalyses, reverse transcription (RT)-PCR analyses of total RNAfrom cells grown in CDM with 10 mM arginine were carried outusing primer pairs spanning all intergenic and neighboring regions.Only a little RT-PCR product corresponding to the arcT-arcD2and arcD2-yvaD regions was formed, while no product was obtainedby probing between argS and arcA and between yvaD and dinP,defining the arginine catabolic operon to comprise arcABD1C1C2TD2-yvaD(Fig. 2B). The lower level of transcription of promoter-distalgenes than of genes further upstream in the operon is likelycaused by the possible terminator ( ${Delta}$ G = –19.2 kcal/mol)in the arcT-arcD2 intergenic region, which would allow onlypartial transcription elongation (Fig. 2). The putative yvaDproduct does not show homology to any known protein, and itsfunction was not investigated further.

ARG box operators in L. lactis.

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ARG box operators in...

Overrepresented sequence motifs in the regions upstream of theArgR and AhrC target genes were identified using the MotifSampleronline tool (18). Considering the regulatory patterns and theresulting operon structures (Fig. 1 and 2), only the regionsupstream of the first genes of the operons were subjected tothe motif search. By employing the Genome2D software tool (1),an 18-bp motif was found to be present in all three argininebiosynthetic promoters (Fig. 3). A weight matrix based on thelactococcal ArgR motif was applied to all the promoter regionsof L. lactis by using Genome2D (1). The obtained motif sequenceswere ranked according to their levels of similarity to the consensussequence. By using the matrix, 8 of the 10 targets with thehighest levels of similarity to the consensus sequence weredetermined to be present in the promoters of the arginine biosyntheticgenes of L. lactis MG1363 (data not shown). The identified motifhas a high degree of similarity to the 5'-WnTGnATWWWWATnCAnW-3'consensus ARG box motif of E. coli (uppercase letters representhighly conserved residues) (13) (Fig. 3A). However, the typical2- to 3-bp spacing between double ARG boxes in E. coli and otherorganisms was not observed in L. lactis, where the spacing (inmultitudes of 10 to 11 bp) suggests the ARG boxes to be presenton the same face of the helix but separated by one, two, orthree helical turns (Fig. 3B). Note that no clear ARG box wasobtained for the arcA promoter region, which is in agreementwith results obtained previously by DNase I footprinting (12).

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FIG. 3. L. lactis ARG operators. (A) Consensus logo of ARG box operators in the arginine biosynthetic promoters of L. lactis, created using the WebLogo tool (http://weblogo.berkeley.edu/) (3, 16). (B) Schematic comparison of ARG box organization patterns in different organisms (S. enterica, Salmonella enterica serovar Typhimurium; M. profunda, Moritella profunda). Core promoter –35 (left) and –10 (right) sequences are indicated as black boxes. White boxes represent 18-bp ARG boxes. The numbers below the horizontal lines (indicating chromosomal DNA) refer to the lengths of the spacer regions between ARG boxes.

Indirect effect on the pyrimidine genes of arginine regulator mutants.

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Indirect effect on the...

The levels of expression of pyrimidine de novo biosynthesispathway genes (pyr) in MG ${Delta}$ argR and MG ${Delta}$ argRahrC were between 1.3-and 1.7-fold higher than those in strain MG1363, whereas nosignificant effects in MG ${Delta}$ ahrC were observed (Fig. 4; data areshown only for the pyrR-carA operon). To assess the validityof these DNA microarray results, the activities of orotate phosphoribosyltransferase(PyrE, the gene for which was upregulated 1.7- and 1.5-foldin MG ${Delta}$ argR and MG ${Delta}$ argRahrC, respectively) and orotidine-phosphatedecarboxylase (PyrF, the gene for which was upregulated 1.5-foldin both MG ${Delta}$ argR and MG ${Delta}$ argRahrC) were determined (Table 1). Theactivities of these two enzymes of the pyrimidine de novo pathwayconfirmed the results of the transcriptome analyses, showingthat the impaired arginine regulation in the two mutants lackingArgR did have a significant albeit weak effect on downstreampyrimidine metabolism (Table 1). The pyr gene expression profilescorrelated well with the expression profiles of the argininebiosynthesis genes and not with those of the catabolic genes.Thus, in MG ${Delta}$ argR and MG ${Delta}$ argRahrC, the flux toward arginine wasmost likely increased due to the high-level expression of thearginine biosynthetic genes. As a consequence, carbamoylphosphate,which is required for the synthesis of both arginine and pyrimidine,was directed toward arginine, resulting in carbamoylphosphateand pyrimidine depletion. To counteract this effect, the pyrimidinebiosynthetic genes, including both carA and carB (the latteris upregulated 1.9- and 1.5-fold in MG ${Delta}$ argR and MG ${Delta}$ argRahrC, respectively),were induced, possibly via the known transcriptional regulatorPyrR (14).

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FIG. 4. Deregulation of pyrimidine de novo biosynthesis genes in the pyrRPB-carA operon of L. lactis. See the legend to Fig. 1 for details.

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TABLE 1. Specific activities of orotate phosphoribosyltransferase (PyrE) and orotidine-phosphate decarboxylase (PyrF) in MG1363 and its isogenic regulator deletion derivatives^a

Concluding remarks.

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Concluding remarks.

The presence of two to four ArgR-type regulators in many low-G+C-contentgram-positive bacteria led us to speculate as to possible globaltarget genes of the two homologues in L. lactis. In supportof the findings of our previous mechanistic studies (11, 12),we here show that both regulators are dedicated to the functionalcontrol of arginine metabolism. Additionally, the data extendthe known ArgR/AhrC regulons of L. lactis, define the biosynthetic(arg) and catabolic (arc) operon structures, and show that evenweak indirect effects can be significantly measured for thede novo pyrimidine genes in response to disrupted arginine regulation.

ACKNOWLEDGMENTS

We thank Anne de Jong and Aldert Zomer for advice on and assistancewith the DNA microarray studies.

FOOTNOTES

* Corresponding author. Mailing address: Department of Molecular Genetics, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Kerklaan 30, NL-9751 NN Haren, The Netherlands. Phone: 31-50-3632111. Fax: 31-50-3632348. E-mail: jan.kok{at}rug.nl

Published ahead of print on 6 June 2008.

Present address: Inflammation Group, Instituto Gulbenkian deCiência, Rua da Quinta Grande 6, 2780-156 Oeiras, Portugal.

REFERENCES

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