The Zur regulon of Corynebacterium glutamicum ATCC 13032

Abstract

Background: Zinc is considered as an essential element for all living organisms, but it can be toxic at large concentrations. Bacteria therefore tightly regulate zinc metabolism. The Cg2502 protein of Corynebacterium glutamicum was a candidate to control zinc metabolism in this species, since it was classified as metalloregulator of the zinc uptake regulator (Zur) subgroup of the ferric uptake regulator (Fur) family of DNA-binding transcription regulators.

Results: The cg2502 (zur) gene was deleted in the chromosome of C. glutamicum ATCC 13032 by an allelic exchange procedure to generate the zur-deficient mutant C. glutamicum JS2502. Whole-genome DNA microarray hybridizations and real-time RT-PCR assays comparing the gene expression in C. glutamicum JS2502 with that of the wild-type strain detected 18 genes with enhanced expression in the zur mutant. The expression data were combined with results from cross-genome comparisons of shared regulatory sites, revealing the presence of candidate Zur-binding sites in the mapped promoter regions of five transcription units encoding components of potential zinc ABC-type transporters (cg0041-cg0042/cg0043; cg2911-cg2912-cg2913), a putative secreted protein (cg0040), a putative oxidoreductase (cg0795), and a putative P-loop GTPase of the COG0523 protein family (cg0794). Enhanced transcript levels of the respective genes in C. glutamicum JS2502 were verified by real-time RT-PCR, and complementation of the mutant with a wild-type zur gene reversed the effect of differential gene expression. The zinc-dependent expression of the putative cg0042 and cg2911 operons was detected in vivo with a gfp reporter system. Moreover, the zinc-dependent binding of purified Zur protein to double-stranded 40-mer oligonucleotides containing candidate Zur-binding sites was demonstrated in vitro by DNA band shift assays.

Conclusion: Whole-genome expression profiling and DNA band shift assays demonstrated that Zur directly represses in a zinc-dependent manner the expression of nine genes organized in five transcription units. Accordingly, the Zur (Cg2502) protein is the key transcription regulator for genes involved in zinc homeostasis in C. glutamicum.

Figure 1

Comparative analysis of Zur proteins from actinobacteria. (A), Multiple amino acid sequence alignment of actinobacterial Zur proteins, including FurB from M. tuberculosis H37Rv. The winged-helix DNA binding domain is highlighted in grey. Three zinc binding sites (Zn 1 to Zn 3) deduced from the crystal structure of the mycobacterial FurB protein [23] are specifically coloured. Zn 1 (yellow): Asp-71, Cys-85, His-91, and His-93; Zn 2 (red): Cys-96, Cys-99, Cys-136, and Cys-139; Zn 3 (blue): His-90, His-92, Glu-111, and His-128 (according to the C. glutamicum protein positions). (B), Maximum likelihood phylogenetic tree of Zur protein orthologues from actinobacteria. The source of the abbreviated Zur-like proteins is indicated by the respective GenBank identifiers.

Figure 2

Genomic organization of the znr-zur gene region in corynebacterial genomes and M. tuberculosis H37Rv. (A), Comparison of the znr-zur genome region. The respective gene regions were obtained from C. glutamicum ATCC 13032 (NC_006958), C. efficiens YS-314 (NC_004369), C. diphtheriae NCTC 13239 (NC_002935), C. aurimucosum DSM44827 (NC_012590), C. accolens ATCC 49725 (NZ_ACGD00000000), C. urealyticum DSM7109 (NC_010545), C. jeikeium K411 (NC_007164), C. kroppenstedtii DSM44385 (NC_012704), and M. tuberculosis H37Rv (NC_000962). Orthologous genes are specifically labeled. Please note that the gene regions of C. jeikeium and C. accolens are shown in reversed orientation. (B), The znr upstream region of C. glutamicum ATCC 13032. The mapped transcription start site (+1) and the deduced core promoter regions (- 35 and - 10) are marked in bold. A stretch of six thymine residues representing a potential up-element is boxed. A putative ribosome-binding site (RBS) is indicated, the GTG start codon of znr is underlined.

Figure 3

Fluorescence microscopy of E. coli DH5α MCR and C. glutamicum ATCC 13032. The cells are carrying either the empty pEPR1 vector, pEPR1 containing the znr upstream region, or pEPR1 containing the znr-zur intergenic region. Images at a 400-fold magnification were taken with transmitted light or UV light at 395 nm to detect GFP fluorescence.

Figure 4

Predicted actinobacterial Zur regulons. (A), Chromosomal clusters of predicted Zur-regulated genes and their orthologues in members of the class Actinobacteria. The locations of candidate Zur-binding sites are shown by red circles. The Zur-binding sites confirmed in this study are marked with 'c'; the previously known Zur-binding sites are marked with 'k'. Homologous genes are marked by matching colour, including zinc ABC-type transporter znuABC (shades of blue), yciC for zinc allocation protein (pink), cmrA and sapD genes for surface-anchored proteins (shades of green), alcohol dehydrogenase adhA (orange), genes encoding ribosomal proteins (yellow), zur (black), znr (dark grey). (B), Consensus sequence logo for the predicted Zur-binding sites.

Figure 5

Ratio/intensity (m/a) plot deduced from DNA microarray hybridizations comparing the transcriptome of the zur mutant C. glutamicum JS2502 with that of the wild-type strain C. glutamicum ATCC 13032. Two biological replicates including label swapping were used for DNA microarray hybridizations. Genes showing significantly enhanced expression in C. glutamicum JS2502 are marked by black dots, decreased transcript levels are indicated by triangles, and genes without differential expression pattern are shown by grey diamonds. Genes were regarded as differentially expressed using the following cut-offs: m-value ≥ 1.0, upregulation; m-value ≤- 1.0, downregulation. The cut-offs correspond to relative changes in gene expression of at least two-fold.

Figure 6

Promoter organization of the Zur regulon members in C. glutamicum ATCC 13032. A schematic presentation of relevant DNA regions from the C. glutamicum ATCC 13032 genome with detected promoters and candidate Zur-binding sites is presented. The 21-bp motifs are shown as grey boxes. A stretch of ten nucleotides (boxed), located upstream of the cg2911 promoter region, revealed similarity to the right half site of the 21-bp motif. The transcription start sites (+1) were mapped by 5' RACE-PCR and are marked in bold letters. Underlined nucleotides show the deduced - 10 and - 35 regions belonging to the corynebacterial promoters. Putative ribosome-binding sites (RBS) are indicated, start codons are underlined. The transcription start site and the - 10 region of cg0043 were deduced from bioinformatic predictions.

Figure 7

Zinc-dependent activity of the cg0042 and cg2911 operon promoters. The promoter activities of the Zur-regulated operons cg0042 and cg2911 was measured in the wild-type strain C. glutamicum ATCC 13032 (WT) and in the zur mutant C. glutamicum JS2502 (zur) under low, high and zinc-chelated (TPEN) conditions. The relative expression of the gfp reporter gene was determined by real-time RT-PCR. The values are means of four measurements. The relative expression was calculated by using a C. glutamicum control carrying the empty expression vector pEPR1.

Figure 8

Agarose gels of DNA band shift assays with purified Zur protein. (A), The DNA band shift assays with fluorescein-labeled 40-mers covering the candidate Zur-binding sites in the cg0042-cg0043 intergenic region and in front of cg0794, cg0795, cg2911, and cg3107. DNA band shift assays were performed with 40 pmol of streptavidin-tagged Zur protein incubated with 0.05 pmol of fluorescein-labeled, double-stranded 40-mer DNA fragments. The assays were performed in the absence of zinc ions and in the presence of 50 μM ZnCl₂. Lanes 1: control assays without Zur protein; lanes 2: DNA band shift assays with added Zur protein. The negative control assay was performed with a 40-mer deduced from the upstream region of cg0841. (B), DNA band shift assays with mutated versions of the 40-mers. Mutated versions were generated by introducing transitions into the candidate Zur-binding sites or into the genomic flanking regions. The EMSAs were carried out in the presence of 50 μM ZnCl₂. (C), DNA band shift assays with binding buffers containing varying metal ions. The EMSAs were performed in the presence of 50 μM ZnCl₂, MgSO₄, NiCl₂, CuSO₄, MnSO₄, or FeSO₄ with the 40-mer region representing cg2911.