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J Bacteriol. Apr 2007; 189(7): 2720–2733.
Published online Jan 26, 2007. doi:  10.1128/JB.01876-06
PMCID: PMC1855810

The IclR-Type Transcriptional Repressor LtbR Regulates the Expression of Leucine and Tryptophan Biosynthesis Genes in the Amino Acid Producer Corynebacterium glutamicum[down-pointing small open triangle]

Abstract

The transcriptional regulator Cg1486 of Corynebacterium glutamicum ATCC 13032 is a member of the IclR protein family and belongs to the conserved set of regulatory proteins in corynebacteria. A defined deletion in the cg1486 gene, now designated ltbR (leucine and tryptophan biosynthesis regulator), led to the mutant strain C. glutamicum IB1486. According to whole-genome expression analysis by DNA microarray hybridizations, transcription of the leuB and leuCD genes encoding enzymes of the leucine biosynthesis pathway was enhanced in C. glutamicum IB1486 compared with the wild-type strain. Moreover, the genes of the trpEGDCFBA operon involved in tryptophan biosynthesis of C. glutamicum showed an enhanced expression in the cg1486 mutant strain. Bioinformatics pattern searches in the upstream regions of the differentially expressed genes revealed the common 12-bp motif CA(T/C)ATAGTG(A/G)GA that is located downstream of the −10 region of the mapped promoter sequences. DNA band shift assays with a streptavidin-tagged LtbR protein demonstrated the specific binding of the purified protein to 40-mers containing the 12-bp motif localized in front of leuB, leuC, and trpE, thereby confirming the direct regulatory role of LtbR in the expression of the leucine and tryptophan biosynthesis pathway genes of C. glutamicum. Genes homologous with ltbR were detected upstream of the leuCD genes in almost all sequenced genomes of bacteria belonging to the taxonomic class Actinobacteria. The ltbR-like genes of Corynebacterium diphtheriae, Corynebacterium jeikeium, Mycobacterium bovis, and Bifidobacterium longum were cloned and shown to complement the deregulation of leuB, leuCD, and trpE gene expression in C. glutamicum IB1486.

Corynebacterium glutamicum is a gram-positive soil bacterium that was discovered in the 1950s as a natural producer of l-glutamic acid (65). Nowadays, it is widely used for the industrial production of economically important amino acids, especially l-glutamic acid and l-lysine (22, 32). In addition to these traditional products, other amino acids such as l-leucine and l-tryptophan can be produced by specially developed mutant strains of C. glutamicum (64, 16). The biosynthesis pathways of both the branched-chain amino acid leucine and the aromatic amino acid tryptophan have been investigated in detail in recent years (27, 71). The genes involved in leucine biosynthesis were localized in different regions of the C. glutamicum genome (29). The leuA gene encodes isopropylmalate (IPM) synthase, catalyzing the first step in leucine biosynthesis by converting 2-oxoisovalerate to 2-IPM (45, 46). The two other enzymes that are unique to the leucine biosynthesis pathway are IPM dehydratase, encoded by the leuCD gene cluster, and IPM dehydrogenase, encoded by the leuB gene. IPM dehydratase is involved in the conversion of 2-IPM to 3-IPM, whereas IPM dehydrogenase catalyzes the oxidative decarboxylation of 3-IPM to yield α-ketoisocaproate (45, 46). The amination of α-ketoisocaproate to leucine is carried out by the branched-chain amino acid aminotransferase IlvE, encoded by the ilvE gene. The transaminase IlvE also participates in the synthesis of the amino acids isoleucine, valine, and phenylalanine (40, 35). Regulation of the leucine biosynthesis pathway is apparently multivalent in C. glutamicum. The IPM synthase is subject to strong feedback inhibition by leucine, and the leuA gene is under negative transcriptional control, probably by an alternative kind of attenuation that is directed by the LEU element in the upstream region of the gene (45, 56). In case of the leuB gene, negative transcriptional regulation has been proposed, since the activity of the leuB promoter was significantly reduced when leucine was present in the growth medium, but an attenuator is absent in front of the coding region (46).

Production of the aromatic amino acid tryptophan by fermentation has made tremendous progress in recent years by applying more rational approaches for the improvement of C. glutamicum strains (26, 28). Biosynthesis of aromatic amino acids begins with the condensation of phosphoenolpyruvate and erythrose 4-phosphate and proceeds to chorismate, from which the specific pathways to tryptophan, phenylalanine and tyrosine branch. The genes of the tryptophan biosynthesis pathway in C. glutamicum are organized as an operon, consisting of six structural genes (trpE, trpG, trpD, trpCF, trpB, and trpA) that encode all enzymatic activities required for the conversion of chorismate to tryptophan (39, 17). In addition to feedback inhibition control of three enzymes within this pathway (anthranilate synthase, anthranilate phosphoribosyltransferase, and tryptophan synthase), expression of the trp operon is repressed in the presence of tryptophan (57, 28). Therefore, the structure and regulatory function of the trp operon control region have been studied extensively (53, 17). A small leader peptide, encoded by the trpL gene, contains three consecutive tryptophan codons within the coding region, suggesting regulation of the trp operon expression by classical transcriptional attenuation (53, 56). Mutations in the predicted attenuator region were apparently responsible for high levels of readthrough transcription and a constitutive antitermination response of C. glutamicum (38, 21). Moreover, a putative operator sequence with similarity to the DNA binding site of the tryptophan operon repressor TrpR from Escherichia coli was detected upstream of the −10 promoter region of the trp operon (53, 17), although a typical TrpR protein is not encoded in the C. glutamicum genome sequence (5). However, site-directed mutagenesis of the palindromic nucleotide sequence revealed a regulatory role in expression of the C. glutamicum trp operon (17).

DNA-binding transcriptional regulators represent key components in the control of bacterial gene expression. They possess a DNA binding domain to recognize cognate operator sequences in front of their target genes and trigger a specific transcriptional response of the cell to changing environmental conditions (37). To understand transcriptional regulation of amino acid biosynthesis pathways in the wild-type strain C. glutamicum ATCC 13032, we recently screened the complete genome sequence by different bioinformatics tools and characterized the repertoire of potential regulators in this species (5). In this study, the cg1486 gene, encoding a transcriptional regulator of the IclR protein family, was analyzed by comparative transcriptomics, using the C. glutamicum wild type and a defined cg1486 mutant strain. Therefore, a genome-wide approach with oligonucleotide microarray hybridizations in conjunction with bioinformatics predictions revealed the target genes of Cg1486 protein and potential DNA binding sites in the C. glutamicum genome. Verification of DNA binding was accomplished by DNA band shift assays with purified Cg1486 protein. The resulting data provided clear evidence that the Cg1486 protein is involved in transcriptional regulation of the leucine and tryptophan biosynthesis pathways of C. glutamicum and that orthologs of this regulatory protein are highly conserved in almost all sequenced bacterial species belonging to the taxonomic class Actinobacteria.

MATERIALS AND METHODS

Bacterial strains, plasmids, and growth conditions.

E. coli DH5αMCR (15) was routinely grown at 37°C in Luria-Bertani medium (52) supplemented with 2 g liter−1 glucose and used for standard cloning procedures. Selection for the presence of plasmids was performed with kanamycin (50 μg ml−1 for E. coli and 25 μg ml−1 for C. glutamicum) or ampicillin (100 μg ml−1 for E. coli). Isopropyl-β-d-thiogalactopyranoside (IPTG) was used to induce gene expression by the Ptrc promoter of pEC-XK99E (31). Induction of gene expression on pASK-IBA3 (IBA, Göttingen, Germany) was carried out by using 200 μg ml−1 anhydrotetracycline. The wild-type strain C. glutamicum ATCC 13032 (American Type Culture Collection, Manassas, VA) and the mutant strain C. glutamicum IB1486 were grown at 30°C in Luria-Bertani medium (52) or in CGXII minimal medium containing 30 mg liter−1 protocatechuic acid (30). Growth of shake-flask cultures was monitored by measuring the optical density at 600 nm with an Eppendorf BioPhotometer. Plasmids used and constructed in this study are listed in Table Table11.

TABLE 1.
Plasmids used and constructed in this study

DNA isolation, manipulation, and transfer.

Plasmid DNA was prepared from E. coli cells by an alkaline lysis technique using a QIAprep Spin Miniprep Kit (QIAGEN, Hilden, Germany). Chromosomal DNAs were prepared from small aliquots of cells that were resuspended in H2O and boiled for 5 min. The supernatants were cleared by centrifugation at 16,000 × g for 10 min and finally stored at −20°C. Modification of DNA, analysis by agarose gel electrophoresis, and ligation were performed by standard procedures (52). Transformation of E. coli and C. glutamicum cells was performed by electroporation (58, 59).

PCR techniques.

PCR experiments were carried out with a PTC-100 thermocycler (MJ Research, Watertown, MA), Pwo DNA polymerase (Roche Diagnostics, Mannheim, Germany), and chromosomal DNA as template. PCR products were purified by using a PCR Purification Spin Kit (QIAGEN). Oligonucleotides used for PCR amplification were purchased from Operon Biotechnologies (Cologne, Germany).

Generation of a defined cg1486 mutant and genetic complementation.

The gene SOEing method (25) was applied to construct a pK19mobsacB derivative that is suitable to perform an allelic exchange of the cg1486 gene in the chromosome of C. glutamicum ATCC 13032 (54). The amplified DNA fragment was digested with EcoRI and cloned into the vector pK19mobsacB (Table (Table1).1). The resulting plasmid pK19Δ cg1486 carries a modified cg1486 gene that is specified by a defined deletion of 134 nucleotides. Gene replacement in the chromosome of C. glutamicum ATCC 13032 resulted in the mutant strain C. glutamicum IB1486. For genetic complementation of C. glutamicum IB1486, the complete cg1486 coding region along with the native promoter region was amplified by PCR with the primer pair compl_cg1486_a and compl_cg1486_b (Table (Table2).2). The PCR product was digested with EcoRI and SalI and cloned into the compatible sites of the vector pEC-K18mob2 (Table (Table1).1). The resulting plasmid pNJ010 was transferred to C. glutamicum IB1486 by electrotransformation. For heterologous complementation of the cg1486 deletion in C. glutamicum IB1486, the orthologous genes of Corynebacterium jeikeium K411 (National Collection of Type Cultures, London, United Kingdom), Corynebacterium diphtheriae DSM44123, Bifidobacterium longum DSM20219 (DSMZ, Braunschweig, Germany), and Mycobacterium bovis BCG were amplified by PCR and cloned either into the shuttle vector pEC-K18mob2 (in the case of the jk1222 and dip1126 genes) or into the IPTG-inducible expression vector pEC-XK99E (in the case of the Mb3013 and BL1261 genes). The respective primer sequences are listed in Table Table2.2. The resulting plasmids pNJ011 to pNJ014 (Table (Table1)1) were transferred to C. glutamicum IB1486 by electrotransformation.

TABLE 2.
Primers used for gene amplification in this study

Total RNA preparation from C. glutamicum cultures.

For the preparation of total RNA, C. glutamicum cultures were grown in Luria-Bertani medium (52). Approximately 5 × 108 cells were harvested from exponentially growing cultures (optical density at 600 nm of 2.5) by centrifugation with 11,000 × g for 15 s. Subsequently, the supernatant was decanted, and the pellet was transferred into liquid nitrogen. RNA isolation was performed with an RNeasy Mini Kit (QIAGEN) in such a way that the frozen cells were resuspendend in 800 μl of RLT buffer and immediately disrupted by means of the RiboLyser instrument (Hybaid, Heidelberg, Germany), using Fast Protein Tubes (Qbiogene, Heidelberg, Germany) and two time intervals of 30 s at speed level 6.5 with intermediary cooling of the probe. The RNase-free DNase set (QIAGEN) was used for on-column digestion of chromosomal DNA. A second DNase I digestion was performed with DNase I (RNase free) from MBI Fermentas (St. Leon-Rot, Germany) to completely remove DNA from the sample. After the second DNase I treatment, the RNA was purified according to the RNeasy Mini Kit clean-up protocol and finally stored at −80°C.

Detection of differential gene expression by real-time RT-PCR.

Purified total RNA of C. glutamicum cultures was used for real-time reverse transcription-PCR (RT-PCR) experiments with the LightCycler instrument (Roche Diagnostics) and a Quanti-Tect SYBR Green RT-PCR Kit (QIAGEN). Oligonucleotides used to measure relative gene expression were purchased from Operon Biotechnologies. Verification of RT-PCR products was performed by melting curve analysis. Differences in gene expression were determined by comparing the crossing points of two samples measured in duplicate. Crossing points were calculated by the LightCycler software (Roche Diagnostics).

Identification of transcriptional start sites by the RACE-PCR method.

For the identification of transcriptional start sites, total RNA was isolated from C. glutamicum cultures grown in Luria-Bertani medium (52). Rapid amplification of cDNA ends (RACE)-PCR primers (18-mer oligonucleotides), binding 200 to 300 nucleotides downstream of the annotated translational start codon of leuC and cg1486, and 1 μg of total RNA were used for cDNA synthesis. The resulting cDNA was modified and subsequently amplified by two additional PCRs using a 5′/3′ RACE Kit, second generation (Roche Diagnostics). PCR products were cloned into the pCR2.1-TOPO vector and transferred into chemically competent E. coli TOP10 cells (Invitrogen, Karlsruhe, Germany). DNA sequencing of cloned RACE-PCR products was performed by IIT Biotech (Bielefeld, Germany).

Hybridization of whole-genome DNA microarrays.

Eight micrograms of total RNA from C. glutamicum cultures was used for cDNA synthesis. Labeling of probes and hybridization of the C. glutamicum whole-genome DNA microarray were performed as described previously (6). Hybridization experiments were carried out in duplicate using label swapping. Since each DNA microarray contains four replicates per C. glutamicum gene, a total number of eight spots per gene were available for calculating differential gene expression. During the image acquisition and data analysis process, mean signal and mean local background intensities were determined for each spot of the microarray images by using the ImaGene 6.0 software for spot detection, image segmentation, and signal quantification (BioDiscovery, Los Angeles, CA). After subtraction of the local background intensities from the signal intensities, the log2 value of the ratio of intensities was calculated for each spot according to the formula Mi = log2(Ri/Gi). In particular, Ri = Ich1i − Bgch1i and Gi = Ich2i − Bgch2i, where Ich1i or Ich2i is the intensity of a spot in channel 1 or channel 2 and Bgch1i or Bgch2i is the background intensity of a spot in channel 1 or channel 2, respectively. The average intensity in both channels was calculated for each spot according to the formula Ai = log2(RiGi) (13). Normalization and evaluation of the hybridization data were accomplished by the EMMA 2.2 microarray data analysis software (11) (http://www.cebitec.uni-bielefeld.de/groups/brf/software/emma_info/), using a signal intensity (A value) cutoff of ≥10.0 and a signal intensity ratio (M value) cutoff of ±1, which corresponds to relative expression changes equal to or greater than twofold.

Bioinformatics tools for Cg1486 protein binding site detection.

The annotated version of the C. glutamicum ATCC 13032 genome sequence (29) was used to perform a genome-wide screening for putative Cg1486 protein binding sites. The search was accomplished by profile hidden Markov model (HMM) analysis using the HMMER version 2.3.2 software package (http://hmmer.janelia.org/). An alignment of predicted Cg1486 binding sites compiled with the CLUSTAL X program (62) was used to create a profile HMM by using the HMMBUILD module. The calculated profile HMM, in conjunction with the HMMSEARCH module, was applied to screen the C. glutamicum genome sequence for the presence of Cg1486 protein binding sites. The genomic positions of the resulting hits were correlated with coding sequences that revealed differential expression in C. glutamicum IB1486 compared with the wild-type strain by DNA microarray hybridization. Furthermore, the EMBOSS motif search program fuzznuc (51) and the Predict Regulon server (72) were applied to search for Cg1486 binding sites. The identified Cg1486 binding sites of C. glutamicum were aligned with the CLUSTAL X program and used to generate a profile HMM for screening of the genome sequences of Corynebacterium efficiens (42), C. diphtheriae (8), C. jeikeium (60), and Corynebacterium urealyticum (61).

Construction and purification of a streptavidin-tagged Cg1486 protein.

The cg1486 coding region was amplified by PCR with the primer pair cg1486-strep1 and cg1486-strep2 (Table (Table2)2) to generate a fusion protein of Cg1486 and a carboxy-terminal streptavidin (Strep) tag. The PCR product was digested with BsaI and cloned into the vector pASK-IBA3 (Table (Table1).1). The resulting plasmid pNJ015 was transferred to E. coli DH5αMCR by electroporation. To isolate the Strep-tagged Cg1486 protein, E. coli DH5αMCR (pNJ015) was grown for 16 h at 25°C in selective Luria-Bertani medium containing 200 μg ml−1 anhydrotetracyline to induce the protein expression. Approximately 5 × 1010 E. coli cells were harvested by centrifugation, resuspended in buffer W (1 M Tris-HCl, 1.5 M NaCl, 10 mM EDTA, one tablet of Roche Complete Mini protease inhibitors, pH 8.0) and transferred into a RiboLyser tube (Hybaid). Cell disruption by means of the RiboLyser instrument was carried out with a speed rate of 6.5 and two time intervals of 30 s. The Strep-tagged Cg1486 protein was purified from the protein crude extract with a Strep-Tactin Sepharose-packed column (IBA, Göttingen, Germany) according to the manufacturer's instructions. After the protein crude extract was loaded, the column was washed five times with 1 ml of buffer W. The Strep-tagged Cg1486 protein was eluted with 0.5 ml of buffer E (buffer W containing 2.5 mM desthiobiotin). The resulting eluate was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (52), further concentrated by using 5,000-molecular-weight-cutoff Amicon Ultra-4 centrifugal filter units (Millipore, Schwalbach, Germany), and finally stored at −20°C. The protein concentration was determined with a Bio-Rad protein assay kit (Bio-Rad Laboratories, Munich, Germany). To verify the purification of the Strep-tagged Cg1486 protein, an in-gel digestion with modified trypsin (Promega, Mannheim, Germany) was carried out, and the resulting peptide masses were determined by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry, applying an Ultraflex mass spectrometer (Bruker Daltonics, Bremen, Germany) and the MASCOT software (48) as described previously (18).

DNA band shift assays with purified Cg1486 protein.

DNA band shift assays were performed with Cy3-labeled 40-mer oligonucleotides (Operon Biotechnologies) that were annealed with the corresponding complementary oligonucleotides to double-stranded DNA fragments by heating at 94°C for 5 min and annealing on ice for 15 min. During DNA band shift assays, 50 pmol of Strep-tagged Cg1486 protein was mixed with 0.05 pmol of DNA, 15% (vol/vol) glycerol, and binding buffer (20 mM Na2HPO4, 50 mM NaCl, 5 mM MgCl2, 2 nM dithiothreitol, 10% [vol/vol] glycerol, 100 μg ml−1 bovine serum albumin, pH 7.0) to get a total volume of 20 μl. The binding buffer was modified where appropriate by adding either 5 mM leucine or 5 mM tryptophan. The assay was incubated at room temperature for 15 min and then separated at 4°C with a 2% agarose gel prepared in gel buffer (20 mM Na2HPO4, pH 7.0). A voltage of 80 V was supplied for 1.5 h. The agarose gel was scanned with a Typhoon 8600 Variable Mode Imager (Amersham Biosciences Europe, Freiburg, Germany).

RESULTS

Molecular features of the transcriptional regulator Cg1486 and genetic characterization of the mutant strain C. glutamicum IB1486.

The leuCD operon (cg1487/cg1488) of C. glutamicum ATCC 13032 encodes the subunits of the IPM dehydratase that catalyzes the second step of the leucine biosynthesis pathway (29). The regulatory gene cg1486 was localized directly upstream of the leuCD operon on the opposite DNA strand of the C. glutamicum genome sequence (Fig. (Fig.1A).1A). The predicted regulatory protein Cg1486 has a length of 235 amino acids and a theoretical molecular mass of 24.8 kDa. According to protein structure predictions performed with the SUPERFAMILY server (34), the Cg1486 protein contains a helix-turn-helix (HTH) motif of the winged-helix type within its amino-terminal region. Moreover, Cg1486 contains a large domain with significant structural similarity to GAF-like effector binding domains that were initially identified in cyclic GMP (cGMP)-specific phosphodiesterases, in several adenylyl cyclases, and in the E. coli transcription factor Fhl (36). Due to these protein structure predictions and additional amino acid sequence similarities of the HTH domain with the DNA binding domain of IclR-type regulators (14), the Cg1486 protein can be classified into the IclR family of transcriptional regulators (5). The IclR family comprises repressors, activators, and regulatory proteins with dual function that control a multiplicity of cellular processes, such as the glyoxylate bypass reactions or the degradation of aromatic compounds (41). No member of the IclR protein family was hitherto described to be involved in transcriptional regulation of amino acid biosynthesis pathways (41).

FIG. 1.
Detailed genetic maps of the leuCD, leuB, and trpE regulatory regions of C. glutamicum. (A) Genetic organization of the leuCD upstream region. The mapped transcriptional start sites (+1) and the deduced −35 and −10 hexamers of ...

To elucidate a potential role of the regulatory protein Cg1486 in expression of the leucine biosynthesis pathway of C. glutamicum, the mutant strain C. glutamicum IB1486, carrying a defined deletion in the cg1486 coding region, was constructed by an allelic exchange procedure (54). Inactivation of the cg1486 coding region should in principle affect the expression of potential target genes of the transcriptional regulator Cg1486 in the mutant strain and eventually expression of genes involved in leucine biosynthesis. One experimental method to detect regulatory interactions is to measure differences in gene expression of potential target genes (23). Therefore, differential expression of the leuA, leuB, leuCD, and ilvE genes was examined by real-time RT-PCR assays, comparing the gene expression in the C. glutamicum IB1486 mutant with that of the wild-type strain. For this purpose, total RNA was prepared from two independently grown C. glutamicum cultures of each strain. Subsequent real-time RT-PCR assays demonstrated that the expression of the leuC and leuD genes was enhanced approximately 100-fold in C. glutamicum IB1486, whereas expression of the leuB gene was enhanced 11.8-fold in the mutant strain (see Fig. Fig.3).3). On the other hand, expression of the leuA gene was moderately changed and enhanced only 3.3-fold in C. glutamicum IB1486, whereas no significant difference in gene expression was measured in the case of the ilvE gene.

FIG. 3.
Relevant genes and gene clusters showing differential expression in the cg1486 mutant C. glutamicum IB1486. The respective values of differential gene expression shown above the arrows were deduced from real-time RT-PCR assays. The values are means of ...

The cg1486 deletion mutant C. glutamicum IB1486 was subsequently complemented by transformation with plasmid pNJ010 carrying the cloned cg1486 gene of the wild-type strain. To demonstrate this complementation, the relative expression of the leuA, leuB, and leuCD genes was measured by real-time RT-PCR. C. glutamicum ATCC 13032 and C. glutamicum IB1486, both carrying the empty cloning vector pEC-K18mob2 (Table (Table1),1), served as controls. As expected, expression of the leuA, leuB, and leuCD genes was enhanced also in the vector-carrying mutant strain C. glutamicum IB1486 (pEC-K18mob2) compared with the respective wild-type control, C. glutamicum ATCC 13032 (pEC-K18mob2). On the other hand, enhanced expression of the leuA, leuB, and leuCD genes was no longer detected in C. glutamicum IB1486 (pNJ010), compared with C. glutamicum ATCC 13032 (pEC-K18mob2), demonstrating that the cg1486 gene on pNJ10 complemented the cg1486 mutant (data not shown). The observed deregulation of leu gene expression in C. glutamicum IB1486 and C. glutamicum IB1486 (pEC-K18mob2) can therefore be attributed to the deletion of the cg1486 gene.

To elucidate the role of leucine in differential expression of the leu genes of C. glutamicum, both the wild-type strain C. glutamicum ATCC 13032 and the cg1486 mutant C. glutamicum IB1486 were grown in minimal medium CGXII, and relative gene expression was measured by real-time RT-PCR. No differential expression of the leu genes was detected when C. glutamicum IB1486 was grown in minimal medium containing 5 mM leucine compared with growth in nonsupplemented minimal medium (data not shown). On the other hand, expression of the leu genes decreased in the wild-type strain when it was cultivated in leucine-containing minimal medium, indicating that the LtbR mutant lacks a leucine-dependent regulation of leu gene expression. These data furthermore suggested that the Cg1486 protein is involved in negative transcriptional regulation of the leu genes of C. glutamicum, either directly or indirectly.

Detection of potential operator sequences by promoter mapping and bioinformatics analysis.

The real-time RT-PCR assays demonstrated that the expression of the leu genes is enhanced in the absence of the regulatory protein Cg1486. To localize potential regulator binding sites in the upstream regions of the differentially expressed genes, a DNA sequence alignment was performed with the CLUSTAL X tool (62). The DNA alignment revealed the identical 12-bp sequence CATATAGTGAGA that is located in different distances to the translational start codons of the leuB and leuC genes (Fig. (Fig.1).1). Since the transcriptional start site and the promoter of the leuB gene were determined previously (Fig. (Fig.1B)1B) (46), we additionally mapped the promoters of the leuCD operon and the divergently oriented cg1486 gene for a more precise functional analysis of the respective intergenic region (Fig. (Fig.1A).1A). Total RNA was isolated from exponentially grown C. glutamicum IB1486 cells and used to amplify and sequence the 5′ ends of the leuCD and cg1486 transcripts by the RACE-PCR method. The resulting DNA sequences allowed the identification of the transcriptional start site in front of the leuC gene that is located 60 nucleotides upstream of the translational start codon (Fig. (Fig.1A).1A). In the case of the cg1486 gene, the mapped transcriptional start site was identical with the adenine residue of the annotated translational start codon (Fig. (Fig.1A),1A), indicating the expression of a leaderless transcript (47).

The mapped transcriptional start sites were subsequently used to determine appropriate −35 and −10 promoter regions according to the characteristic features of corynebacterial promoters (47). The deduced −35 (GTGACA) and −10 (TATACT) regions of the leuCD promoter are separated by a spacer of 18 nucleotides (Fig. (Fig.1A).1A). Both hexamers revealed considerable similarity with the −35 (TTGCCA) and −10 [TA(C/T)AAT] consensus sequences of corynebacterial promoters (47). In the case of the cg1486 promoter, only the deduced −10 region (TAAATT), which is located 7 bp upstream of the transcriptional and translational start site, revealed similarity to the corynebacterial consensus promoter, whereas the −35 region (ACCACG) is less well conserved (Fig. (Fig.1A).1A). According to these mapping data, the common 12-bp sequence CATATAGTGAGA is located between the −10 promoter region and the translational start codon of the leuB and leuC genes (Fig. 1A and B). This location of the common 12-bp sequence in front of differentially expressed genes suggests a potential role as a DNA binding site of a repressor protein (33), which is consistent with the enhanced expression of the leuB and leuCD genes in the cg1486 mutant C. glutamicum IB1486.

To detect identical 12-bp motifs in the C. glutamicum ATCC 13032 genome sequence, bioinformatics analyses were performed with the pattern recognition tool fuzznuc (51) and the Predict Regulon server (72). Furthermore, a profile HMM was built by means of an alignment of the identified 12-bp sequences and used in a motif search against the C. glutamicum genome sequence. These bioinformatics searches revealed that the 12-bp sequence is solely present in front of the leuB and leuC genes. However, a multitude of similar sequence stretches was detected in the C. glutamicum genome sequence when one to four mismatches within the motif sequence were included (data not shown). For instance, a similar 12-bp sequence with four mismatches in the motif sequence was localized upstream of the mapped cg1486 promoter (Fig. (Fig.1A),1A), but no similar 12-bp sequences were detected by the different computational methods in the known promoter region of leuA (47) and in the upstream region of the ilvE gene. This result prompted us to examine the global gene expression of the cg1486 mutant C. glutamicum IB1486 by whole-genome transcriptional profiling with DNA microarray hybridizations to search for differentially expressed genes that are specified by the presence of imperfectly conserved 12-bp sequences.

Detection of differentially expressed genes in C. glutamicum IB1486 by whole-genome DNA microarray hybridization.

To detect genes with differential expression in the cg1486 mutant strain, whole-genome DNA microarray hybridizations were carried out, thereby comparing the gene expression of C. glutamicum IB1486 with that of the wild-type strain C. glutamicum ATCC 13032. Cell samples for RNA purification were harvested from two independently grown C. glutamicum cultures of each strain, and purified total RNAs were used for two DNA microarray hybridization assays, including label swapping. The resulting hybridization data were evaluated and visualized with the Imagene software. Normalization of the data by the LOWESS function and t test statistics were conducted with the EMMA 2.2 software package. To minimize the number of false-positive signals, the data were stringently filtered to obtain genes with at least six statistically significant values out of the eight technical replicates present on the two hybridized microarrays, along with an error probability of less than 5% for the t test. By applying the experimental threshold values of ≥10 for the signal intensity A and ±1 for the log2 transformed signal intensity ratio M (corresponding to relative expression changes of at least twofold), a sum total of 50 genes revealed differential expression in C. glutamicum IB1486, including 42 genes with enhanced expression (M value equal to or greater than +1) and eight genes with decreased expression (M value equal to or less than −1), compared with the wild-type strain. The resulting data are depicted graphically in a ratio/intensity (M/A) plot (Fig. (Fig.2).2). The respective genes showing differential expression in C. glutamicum IB1486 are listed in Table Table33.

FIG. 2.
Ratio/intensity (M/A) plot deduced from DNA microarray hybridizations comparing the gene expression of the cg1486 mutant C. glutamicum IB1486 with that of the wild-type strain C. glutamicum ATCC 13032. The analyzed strains were cultivated in Luria-Bertani ...
TABLE 3.
Genes differentially expressed in the cg1486 mutant C. glutamicum IB1486

Among the genes with significantly enhanced expression in C. glutamicum IB1486, the leuABCD genes involved in the leucine biosynthesis pathway were detected. The genes with the highest changes in transcription were leuC and leuD, whereas leuB and leuA showed smaller changes, which is consistent with the initial measurements of differential gene expression in the cg1486 mutant by real-time RT-PCR assays. In addition, almost all genes of the tryptophan biosynthesis operon (trpEGDCFBA) revealed an enhanced expression in C. glutamicum IB1486, although differential expression of the trpB and trpA genes showed smaller changes, which may be caused by the presence of an internal promoter in the trp operon of C. glutamicum (43). Moreover, the genes of the putative rbs operon (rbsRACBD) that is apparently involved in the uptake of ribose (68) were differentially expressed in C. glutamicum IB1486, with the exception of rbsD, which is again the last gene of the putative polycistronic transcript.

Identification of differentially expressed genes of C. glutamicum IB1486 specified by the presence of a 12-bp sequence.

The results of the DNA microarray hybridization were subsequently combined with data from bioinformatics pattern searches to determine the fraction of differentially expressed genes of C. glutamicum IB1486 that are specified by the presence of 12-bp sequences. Three computational methods were applied to search for 12-bp sequences in the genome sequence of C. glutamicum, revealing the presence of seven motifs in the upstream region of genes or within coding regions that were detected as differentially expressed in C. glutamicum IB1486 (Table (Table4).4). This set of 12-bp sequences included the identical motifs initially localized in front of the leuB and leuCD genes. Furthermore, an imperfectly conserved 12-bp sequence, showing two mismatches, was detected upstream of the trpE gene (Table (Table4).4). A closer inspection of the respective gene region of C. glutamicum revealed that the 12-bp sequence is located 16 nucleotides in front of the trpL gene, thereby spanning the already mapped transcriptional start site of the tryptophan operon (Fig. (Fig.1C)1C) (17). The trpL locus codes for a small leader peptide and is involved in transcriptional attenuation of the trp operon expression (38, 21). The other 12-bp sequences are less well conserved and contain three to four mismatches (Table (Table4).4). These sequence motifs are located (i) in front of the aroG gene, encoding 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase, which is the first enzyme of the biosynthesis pathway for aromatic amino acids (9); (ii) within the rbsC gene, encoding a permease subunit of a putative ABC-type ribose transporter (68); (iii) upstream of the cg1617 gene; and (iv) within the cg2545 coding region that revealed a lower expression in C. glutamicum IB1486 (Table (Table33).

TABLE 4.
The 12-bp sequences localized in front of or within differentially expressed genes of C. glutamicum IB1486

Considering potential operon structures of the C. glutamicum genome (49), the detected 12-bp sequences are related to seven transcription units comprising a sum total of 20 genes (Fig. (Fig.3).3). Differential expression of these genes in C. glutamicum IB1486 was verified by real-time RT-PCR assays, using the same experimental setup as applied for whole-genome DNA microarray hybridization. The resulting data are summarized in Fig. Fig.3,3, confirming the enhanced or decreased expression of the selected genes in C. glutamicum IB1486. The combination of DNA microarray hybridization with bioinformatics pattern searches therefore defined a set of genes that are not only differentially expressed in the cg1486 mutant strain C. glutamicum IB1486 but are also specified by the presence of 12-bp sequences. The respective DNA regions of the C. glutamicum genome represent appropriate candidate sequences to prove a direct regulatory interaction by the transcriptional regulator Cg1486.

Direct interaction of the Cg1486 protein with 40-mer DNA sequences analyzed in vitro by DNA band shift assays.

To demonstrate a direct interaction of the transcriptional regulator Cg1486 with the 12-bp regions in front of the differentially expressed genes of C. glutamicum IB1486, DNA band shift assays were performed. For this purpose, a modified Cg1486 protein was constructed by adding a carboxy-terminal Strep tag. The complete coding region of cg1486 was amplified by PCR and cloned in E. coli into the anhydrotetracyline-inducible expression vector pASK-IBA3 (Table (Table1).1). The Strep-tagged Cg1486 protein was subsequently expressed in an induced E. coli culture and purified to homogeneity by Strep-Tactin affinity chromatography. Purification of the Cg1486 protein was verified by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (data not shown).

DNA band shift assays were performed with the Strep-tagged Cg1486 protein and double-stranded DNA fragments containing the 12-bp sequences (Table (Table4).4). Moreover, the 12-bp sequence located upstream of the cg1486 gene (Fig. (Fig.1A)1A) was included in this assay. The selected 12-mers were equally extended on both sides with the respective genome-specific sequences to yield a total length of 40 nucleotides. Appropriate synthetic oligonucleotides were annealed to produce Cy3-labeled, double-stranded 40-mer fragments. Each DNA band shift assay contained 0.05 pmol of the labeled 40-mer fragment and 50 pmol of the purified Cg1486 protein. As shown in Fig. Fig.4A,4A, three 40-mer fragments revealed a different electrophoretic mobility when previously incubated with the Strep-tagged Cg1486 protein, comprising the probes of the leuC, leuB, and trpE genes. No differences in electrophoretic mobility were observed when 40-mer sequences present upstream of the cg1412 (rbsC), cg1486, cg1617, cg2391 (aroG), and cg2545 genes were used (Fig. (Fig.4B).4B). The specificity of the DNA band shift with the Strep-tagged Cg1486 protein was furthermore examined by competition assays. When an excess of nonlabeled 40-mer was added to the assay, the DNA band shift of the leuC probe was prevented more and more (Fig. (Fig.4C),4C), apparently by competition between labeled and increasing amounts of nonlabeled 40-mers. Moreover, the DNA band shift assay was performed with a modified binding buffer containing either leucine or tryptophan as potential effector substances of the regulatory protein Cg1486 (Fig. (Fig.4D).4D). DNA band shifts were detected in the absence and presence of leucine and tryptophan, indicating that both amino acids are dispensable for the in vitro interaction of Cg1486 with the 40-mer fragments and not involved in alleviating the DNA binding properties of the Cg1486 protein. Nevertheless, the DNA band shift assays confirmed in vitro the direct binding of the Strep-tagged Cg1486 protein to three specific 40-mer DNA fragments containing the 12-bp sequences that were localized in the upstream regions of the leuC, leuB, and trpE genes, thereby demonstrating that the Cg1486 protein is involved in transcriptional regulation of the leucine and tryptophan biosynthesis pathways of C. glutamicum. Due to the apparent direct regulatory role of the Cg1486 protein in leucine and tryptophan biosynthesis of C. glutamicum, the cg1486 gene was named ltbR (leucine and tryptophan biosynthesis regulator).

FIG. 4.
Agarose gels of DNA band shift assays performed with the streptavidin-tagged Cg1486 protein. (A) DNA band shift assays with Cy3-labeled, double-stranded 40-mers covering the 12-bp sequences in front of the leuC, leuB, and trpE genes. DNA band shifts were ...

Detection of ltbR orthologs in genome sequences of Actinobacteria.

A recent classification of genes encoding DNA-binding transcriptional regulators in six sequenced corynebacterial genomes revealed that the LtbR (Cg1486) protein belongs to the common set of regulators in these species (4, 5). The homologous genes of C. efficiens (ce1426), C. diphtheriae (dip1126), C. jeikeium (jk1222), and C. urealyticum (cu09_0562 and cu11_1897) are located upstream of the leuCD genes in a divergent orientation as well. In addition, DNA motifs with similarity to the 12-bp sequence of C. glutamicum were identified in front of the leuB and leuCD genes of these species (data not shown). A similar 12-bp sequence was, furthermore, detected in front of the trpL locus of the C. efficiens trp operon (data not shown). The presence of ltbR-like regulatory genes in the leuCD gene region was therefore examined in 42 sequenced genomes of gram-positive bacteria belonging to the taxonomic class Actinobacteria. The analysis was accomplished by means of the SEED software environment (44) and additional protein similarity searches that were performed with the BLASTP program (1). These computational methods revealed that the genomic organization of the ltbR and leuCD gene region is conserved in 38 of the hitherto sequenced actinobacterial genomes, including all sequenced species of the genera Acidothermus, Arthrobacter, Bifidobacterium, Brevibacterium, Corynebacterium, Frankia, Janibacter, Kineococcus, Mycobacterium, Nocardia, Nocardioides, Propionibacterium, Rhodococcus, Rubrobacter, Salinispora, Streptomyces, and Thermobifida (data not shown). Exceptions are Leifsonia xyli subsp. xyli, the sequenced strains of Tropheryma whipplei, and the unclassified actinobacterial species Symbiobacterium thermophilum. The conserved genomic organization suggests that genes homologous with ltbR of C. glutamicum may have a similar regulatory function in gene expression of other species belonging to the taxonomic class Actinobacteria.

Heterologous complementation of C. glutamicum IB1486 with ltbR-like genes from Corynebacterium, Mycobacterium, and Bifidobacterium species.

To assess whether the actinobacterial ltbR-like genes are functionally equivalent in transcriptional regulation of gene expression, heterologous complementation assays were conducted with the ltbR mutant C. glutamicum IB1486 and the ltbR-like genes of C. diphtheriae DSM44123 (dip1126), C. jeikeium K411 (jk1222), M. bovis BCG (Mb3013), and B. longum DSM20219 (BL1261). The selected ltbR-like genes were amplified by PCR and cloned in E. coli into either the shuttle vector pEC-K18mob2 (dip1126 and jk1222) or the shuttle expression vector pEC-XK99E (Mb3013 and BL1261). The resulting plasmids pNJ011 to pNJ014 (Table (Table1)1) were finally transferred into the ltbR mutant C. glutamicum IB1486 by electrotransformation. Relative expression of the leuCD, leuB, and trpE genes in the plasmid-carrying mutant C. glutamicum IB1486 was subsequently measured by real-time RT-PCR assays and compared with that of the wild-type strain C. glutamicum ATCC 13032 carrying the empty cloning vectors. Therefore, C. glutamicum IB1486 was also transformed with pEC-K18mob2 or pEC-XK99E and served as control. As expected, the expression of leuCD, leuB, and trpE was significantly enhanced in the control strains C. glutamicum IB1486 (pEC-K18mob2) and C. glutamicum IB1486 (pEC-XK99E) compared to the respective gene expression levels of the plasmid-carrying wild-type controls (Fig. (Fig.5).5). On the other hand, expression of the leuCD, leuB, and trpE genes significantly decreased in C. glutamicum IB1486 harboring plasmids pNJ011 to pNJ014, indicating that the cloned ltbR-like genes from C. diphtheriae, C. jeikeium, M. bovis, and B. longum complemented the deregulation of the leuCD, leuB, and trpE gene expression in the mutant strain (Fig. (Fig.5).5). An exception was the ltbR-like gene BL1261 from B. longum that reduced the expression of the leuCD genes in C. glutamicum IB1486 (pNJ014) to only 30% of the values measured in the control strain C. glutamicum IB1486 (pEC-XK99E), indicating that only partial complementation by the BL1261 gene occurred in this assay (Fig. (Fig.5).5). Nevertheless, the heterologous complementation assays with the ltbR mutant C. glutamicum IB1486 demonstrated that the ltbR-like genes of C. diphtheriae, C. jeikeium, M. bovis, and B. longum not only are located in a conserved position in the actinobacterial genomes but also can functionally replace the transcriptional regulator LtbR of C. glutamicum.

FIG. 5.
Heterologous complementation assays of C. glutamicum IB1486 with ltbR-like genes from C. diphtheriae (CD), C. jeikeium (CJ), M. bovis (MB), and B. longum (BL). Relative expression of the leuCD, leuB, and trpE genes was measured by real-time RT-PCR and ...

DISCUSSION

The leucine biosynthesis genes leuB and leuCD are subject to transcriptional control by the IclR-type repressor LtbR.

In the present study, the regulon of the transcriptional regulator LtbR of C. glutamicum was examined, and the topology of its gene regulatory network was deduced from DNA microarray hybridizations, bioinformatics analysis, and DNA band shift assays. The ltbR gene is located on the opposite DNA strand upstream of the leuCD genes, encoding IPM dehydratase (29). IPM dehydratase as well as IPM synthase and IPM dehydrogenase, encoded by the leuA and leuB genes, represent the enzymes unique to the leucine biosynthesis pathway of C. glutamicum (45, 46). Expression of the three enzymes is coordinately down-regulated when appropriate amounts of leucine are present in the growth medium, suggesting negative transcriptional regulation of the respective genes (45). However, a previous comparison of the regulatory DNA regions of the leuA and leuB genes revealed no nucleotide sequence similarities, already indicating different mechanisms for transcriptional regulation of leu gene expression in C. glutamicum (46). A small gene coding for a potential leader peptide with four consecutive leucine residues was detected upstream of the leuA coding region (45, 56). Additional structural features of the regulatory DNA region in front of the leuA gene, collectively named LEU element, provided hints for an alternative type of transcriptional attenuation to control leuA gene expression (56, 57). It has been proposed that the actinobacterial LEU element is a binding site for any regulatory protein that probably exerts an RNA-binding regulatory role (56). As shown in this study, the leuA gene is not part of the LtbR regulon, since the LEU element is apparently the key feature in controlling leuA gene expression in C. glutamicum. The moderate differential expression of the leuA gene in the ltbR mutant C. glutamicum IB1486 is most likely caused by an indirect effect of the ltbR mutation. Given the common enzymatic function of the branched-chain amino acid aminotransferase IlvE in the biosynthesis pathways of isoleucine, valine, and phenylalanine in C. glutamicum (35, 40), it is apparent that the ilvE gene expression is also not controlled by the LtbR protein.

The LtbR protein belongs to the IclR family of transcriptional regulators that are involved in regulation of a multiplicity of cellular processes, although no member of the IclR family has been implicated in controlling amino acid biosynthesis pathways so far (41). It contains an amino-terminal HTH motif of the winged-helix type and a large domain with similarity to GAF-like effector binding domains. GAF-like protein domains represent one of the largest and most widespread families of small-molecule binding units in bacteria (36). Although initially identified as a cGMP binding domain, bioinformatics methods revealed that GAF domains are present in many proteins, of which several do not sense cGMP. Therefore, the domain architecture of LtbR provides no indication on the small molecule that is sensed by this protein. Nevertheless, binding of the purified LtbR protein to 40-mer fragments containing the 12-bp sequence CATATAGTGAGA was demonstrated in vitro by DNA band shift assays. Thus, the LtbR protein of C. glutamicum represents a new type of regulatory proteins that directly controls the biosynthesis of the branched-chain amino acid leucine.

Integrating the transcriptional regulation of the leuCD genes into the regulatory network of C. glutamicum.

The current knowledge about the gene regulatory network of C. glutamicum was summarized and processed very recently to implement the publicly available database CoryneRegNet (3, 4). Integration of the data obtained in the present study into the transcriptional regulatory network of C. glutamicum displayed a complex regulation of leucine biosynthesis due to coregulation of the leuCD genes by LtbR and the RipA protein. Expression of the leuCD genes of C. glutamicum is apparently repressed under iron limitation by the AraC-type regulator RipA (69). The regulatory role of the RipA protein is reflected by the observation that the reaction center of IPM dehydratase contains an iron-sulfur cluster (67). Moreover, the regulatory gene ripA is subject to regulation by the iron-dependent transcriptional regulator DtxR and is repressed under iron excess conditions (7, 70). The DtxR protein is an iron-dependent dual regulator that is involved in global regulation of gene expression in C. glutamicum, thereby providing a hierarchical control level of leuCD gene expression (7, 70). Under iron limitation, repression of the DtxR target genes is alleviated, and the DtxR regulon is thus derepressed, resulting in expression of RipA and concomitant repression of genes belonging to the RipA regulon (69). The mapped promoter sequence in front of the leuC gene of C. glutamicum revealed that the formerly described RipA binding site overlaps with the −35 region, whereas the newly identified operator of LtbR is located downstream of the −10 region (Fig. (Fig.1).1). Expression of the leuCD genes therefore depends on at least two environmental stimuli, one of which is linked to the availability of iron for cellular metabolism. The second environmental stimulus is represented by the currently unknown effector molecule probably interacting with the GAF domain of the LtbR protein. Since in vitro binding of the LtbR protein to specific 40-mer DNA fragments was also observed in the presence of leucine or tryptophan, a regulatory role of these amino acids as effector substances is unlikely. The physiological function of the LtbR repressor seems to be more intriguing, since the regulatory network of LtbR connects the transcriptional control of leucine biosynthesis with that of the biosynthesis pathway for the aromatic amino acid tryptophan.

The tryptophan operon of C. glutamicum is subject to transcriptional control by the IclR-type repressor LtbR.

DNA band shift assays confirmed in vitro the direct regulatory interaction of the LtbR protein with the proposed operator site in the regulatory region of the tryptophan (trp) operon. Former examination of the trp regulatory region revealed the presence of a leader peptide that contains three consecutive tryptophan codons, suggesting regulation of gene expression by classical attenuation (53, 56). The 12-bp motif of the DNA binding site of the LtbR protein is located between the −10 promoter region and the ribosome-binding site of the trpL gene, encoding the leader peptide of the trp operon. An additional 14-bp palindromic operator with similarity to TrpR binding sites of the E. coli genome was previously identified upstream of the −10 promoter region (53, 17), although no typical TrpR regulator is encoded in C. glutamicum (5). Directed mutagenesis of the palindromic DNA sequence revealed a regulatory role in expression of the C. glutamicum trp operon that was dependent on the presence of tryptophan in the growth medium (53, 17). Due to the different positions of the potential operator sequences, it is unlikely that the LtbR regulator is involved in the observed regulatory effect. On the other hand, an additional growth rate-dependent regulation of trp operon expression in C. glutamicum has been proposed (53, 17). Whether this type of trp gene regulation is mediated by the LtbR protein remains to be elucidated. Nevertheless, integration of the trp operon genes into the regulon of the LtbR repressor revealed a completely new regulatory connection between leucine and tryptophan biosynthesis pathways in bacteria.

The very recent work related to a genome-based approach to create a minimally mutated C. glutamicum strain for l-lysine production revealed interesting insights into differential expression of the leu and trp genes (19, 20). It is well known that some industrial C. glutamicum strains used for l-lysine production are characterized by leucine auxotrophy (63, 55). Mutation of the leuC gene in a rationally designed l-lysine producer resulted in significant up-regulation of many amino acid biosynthesis genes, including leuCD and the trp operon genes (19). It has been proposed that leucine limitation caused by the leuC mutation resulted in rel-independent global expression changes of amino acid biosynthesis genes through a certain regulatory mechanism (19). The newly identified transcriptional regulator LtbR offers new perspectives for the elucidation of these complex regulatory connections between amino acid biosynthesis pathways. Based on the results presented in this study it might also be interesting to examine whether deletion of the regulatory gene ltbR can replace the defined leuC mutation and result in higher l-lysine production. The next step in characterizing the regulatory linkage between the leucine and tryptophan biosynthesis pathways of C. glutamicum is to elucidate the common regulatory molecule that acts as effector for the LtbR protein. In principle, the transcriptional regulator LtbR can be regarded as a regulatory gateway of different amino acid biosynthesis pathways in C. glutamicum.

Genes orthologous with ltbR from C. glutamicum are a common feature of bacterial genomes within the taxonomic class Actinobacteria.

Comparative content analysis of corynebacterial genome sequences revealed that ltbR-like genes are conserved in all sequenced species (5, 4). In addition, orthologous genes of ltbR from C. glutamicum were also detected in almost all genome sequences of bacterial species belonging to the Actinobacteria. These orthologous candidate genes were found upstream of the leuCD genes in 38 out of 42 genome sequences publicly available at the time of writing the manuscript. A remnant of an ltbR-like gene was detected in the genome of Mycobacterium leprae that is generally characterized by massive gene loss events (10). Likewise, the reduced genome of T. whipplei lacks an ltbR-like gene, but this species is also devoid of a leucine biosynthesis pathway (50). The absence of an ltbR-like gene in S. thermophilum is apparent, given the uncertain taxonomic position of this bacterium (66). Although the genome data revealed a high G+C content of the genomic DNA, the genetic organization of the coding regions was more similar to genomes of Firmicutes that lack an ltbR-like gene. Thus, the ltbR gene might represent some kind of signature protein for bacterial species belonging to the class Actinobacteria. The only exception is the genome sequence of L. xyli subsp. xyli that revealed the presence of a pseudogene upstream of the leuCD genes, implying that structural rearrangements might have occurred in this region of the genome.

In the present study, we also provided first experimental data for a functional conservation of the orthologous regulatory proteins by heterologous complementation of the ltbR mutant C. glutamicum IB1486 with selected candidates genes. The cloned ltbR-like genes of C. diphtheriae, C. jeikeium, B. longum, and M. bovis complemented the deregulation of leuB, leuCD, and trpE gene expression in the ltbR mutant C. glutamicum IB1486. This result demonstrated that the respective proteins of other actinobacterial species can functionally replace the LtbR repressor of C. glutamicum. In M. bovis BCG, the LeuCD proteins are apparently part of the thiol-specific oxidative stress response (12). The role of IPM dehydratase in relation to oxidative stress has not been elucidated, whereas leucine auxotrophy is known to restrict the growth of M. bovis BCG in macrophages (2). Likewise, targeted mutation of the leuD gene rendered Mycobacterium tuberculosis incapable of replicating in macrophages in vitro (24). The LtbR protein might therefore represent an important regulatory switch to provide optimal expression of the leuCD genes in mycobacteria.

Footnotes

[down-pointing small open triangle]Published ahead of print on 26 January 2007.

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