The IclR-type transcriptional repressor LtbR regulates the expression of leucine and tryptophan biosynthesis genes in the amino acid producer Corynebacterium glutamicum

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.

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 corynebacterial promoters are specifically marked. Potential ribosome-binding sites in front of the coding regions are indicated; translational start codons are underlined. Detected 12-bp sequences are shown as gray boxes. A RipA binding site in front of the leuC gene is boxed (69). (B) Genetic organization of the leuB promoter region. The leuB promoter was mapped in an earlier study (46). The 12-bp sequence is shown as a gray box. (C) Genetic organization of the regulatory region of the tryptophan operon (17). The 12-bp sequence detected in this study is marked. The trpL locus codes for a small leader peptide in front of the trpE gene and is apparently involved in transcriptional attenuation. A predicted operator sequence in the regulatory region of the tryptophan operon is boxed (17). RBS, ribosome-binding site.

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 medium to mid-exponential growth phase, revealing no apparent difference in growth behavior. Total RNA was subsequently isolated from two biological replicates and applied for DNA microarray hybridizations, including label swapping. Genes showing enhanced expression in C. glutamicum IB1486 are marked by black diamonds, genes with decreased expression are indicated by black triangles, and genes without differential expression pattern are shown by gray spots. Selected differentially expressed genes are named by their identifiers. The A value represents the signal intensity, and the M value represents the signal intensity ratio, which corresponds to relative expression changes. The M value cutoff of ±1 corresponds to relative expression changes equal to or greater than twofold.

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 four measurements. Predicted Rho-independent transcriptional terminators are indicated by hairpin structures; 12-bp sequences defined by bioinformatics analyses are shown as white boxes. The differentially expressed genes are involved in leucine and tryptophan biosynthesis, in ribose uptake, and in other physiological processes.

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 performed with 0.05 pmol of the Cy3-labeled 40-mer fragment and 50 pmol of the Strep-tagged Cg1486 protein. Assays were separated in a 2% agarose gel and visualized by fluorescence imaging. Gene identifiers are shown below the agarose gels. Lane 1, control assay without Cg1486 protein; lane 2, DNA band shift assay with Cg1486 protein. (B) DNA band shift assays with Cy3-labeled, double-stranded 40-mers covering the 12-bp sequences of the cg1412 (rbsC), cg1486, cg1617, cg2391 (aroG), and cg2545 genes. (C) DNA competition experiment with a 40-mer fragment covering the 12-bp sequence in front of the leuC gene. A total of 50 pmol of the Strep-tagged Cg1486 protein and 0.05 pmol of a Cy3-labeled 40-mer along with increasing concentrations of the same nonlabeled 40-mer fragment (40mer) were added to the assay. (D) DNA band shift assays with the Cy3-labeled 40-mer fragment representing the upstream region of the leuC gene and modified binding buffer containing either 5 mM leucine (Leu) or 5 mM tryptophan (Trp).

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 normalized by using the values determined in the plasmid-carrying control strains C. glutamicum ATCC 13032 (pEC-K18mob2) and C. glutamicum ATCC 13032 (pEC-XK99E), abbreviated C1 and C2, respectively. The values are means of four measurements; standard deviations are indicated.