Analysis of the Manganese and MntR Regulon in Corynebacterium diphtheriae

Abstract

Corynebacterium diphtheriae is the causative agent of a severe respiratory disease in humans. The bacterial systems required for infection are poorly understood, but the acquisition of metals such as manganese (Mn) is likely critical for host colonization. MntR is an Mn-dependent transcriptional regulator in C. diphtheriae that represses the expression of the mntABCD genes, which encode a putative ABC metal transporter. However, other targets of Mn and MntR regulation in C. diphtheriae have not been identified. In this study, we use comparisons between the gene expression profiles of wild-type C. diphtheriae strain 1737 grown without or with Mn supplementation and comparisons of gene expression between the wild type and an mntR deletion mutant to characterize the C. diphtheriae Mn and MntR regulon. MntR was observed to both repress and induce various target genes in an Mn-dependent manner. Genes induced by MntR include the Mn-superoxide dismutase, sodA, and the putative ABC transporter locus, iutABCD. DNA binding studies showed that MntR interacts with the promoter regions for several genes identified in the expression study, and a 17-bp consensus MntR DNA binding site was identified. We found that an mntR mutant displayed increased sensitivity to Mn and cadmium that could be alleviated by the additional deletion of the mntABCD transport locus, providing evidence that the MntABCD transporter functions as an Mn uptake system in C. diphtheriae. The findings in this study further our understanding of metal uptake systems and global metal regulatory networks in this important human pathogen. IMPORTANCE Mechanisms for metal scavenging are critical to the survival and success of bacterial pathogens, including Corynebacterium diphtheriae. Metal import systems in pathogenic bacteria have been studied as possible vaccine components due to high conservation, critical functionality, and surface localization. In this study, we expand our understanding of the genes controlled by the global manganese regulator, MntR. We determined a role for the MntABCD transporter in manganese import using evidence from manganese and cadmium toxicity assays. Understanding the nutritional requirements of C. diphtheriae and the tools used to acquire essential metals will aid in the development of future vaccines.

Keywords: Corynebacterium; diphtheria; manganese.

FIG 1

Gene expression array and qPCR correlation. Expression of the indicated genes was probed by qPCR for comparison to the microarray. The comparison of relative expression of genes from wild-type C. diphtheriae strain 1737 grown without and with 5 μM MnCl₂ supplementation is shown. Data are the means and standard deviations from the three biological replicates used in the microarray analysis (R² = 0.9691).

FIG 2

Further analysis of the mntABCD-R locus. (A) Genetic map of the mntABCD-mntR gene locus with predicted gene products described. The mntR gene is located 14 bp downstream of mntD. The location of transcription start sites (TSS) and direction of transcription are indicated. (B) qPCR was used to measure the relative expression of mntA, mntB, and mntD in the wild type (WT) and ΔmntR mutant in response to Mn supplementation; mntR expression was examined only in the wild type. Data were analyzed using the ΔΔC_q method using gyrB for normalization compared against the wild type with empty vector in the absence of Mn supplementation. *, P < 0.0001 by 2-way ANOVA Holm-Sidak’s multiple comparisons test comparing the wild type with added Mn to that with no added Mn (n = 3).

FIG 3

MntR binds promoters of target genes. (A) Electrophoretic mobility shift assays were performed with recombinant His-tagged MntR and PCR-amplified putative promoter regions for the indicated genes. MntR was either excluded (−) or added (+) to the binding reaction prior to separation by electrophoresis. Binding reactions shown were performed with MnCl₂ (top) or EDTA (bottom) as noted. (B) Binding reactions were performed using complementary annealed nucleotides encompassing predicted binding sites identified using MEME (29), indicated below, with the most highly conserved bases in boldface. The black arrow head indicates migration of the double-stranded annealed fragment. The gray arrow head indicates migration of biotinylated single-stranded oligonucleotides. Two potential overlapping binding sites were found in the iutA promoter region; key bases for one are in boldface and the others are underlined. (C) The MntR binding consensus sequence based upon sequences that showed binding to MntR. *, iutA sequence was not used to establish the binding consensus.

FIG 4

Promoter fusions for iutA, sodA, and ciuE. (A) Notable elements in the iutA promoter region are indicated, including the DtxR and Zur binding sites that were identified previously (25) and promoter elements (−35, −10, and +1). The region containing two independent, overlapping motifs with similarity to the MntR binding consensus is noted below; MntR binding to this region could not be confirmed using DNA oligonucleotides (see Fig. 5B). (B) Promoter fusions for iutA, sodA, and ciuE were tested for activity in mPGT without or with MnCl₂ supplementation in wild-type C. diphtheriae 1737 and the ΔmntR mutant. The data are the means and standard deviations from four biological replicates. Statistical significance between the corresponding with and without Mn samples determined by multiple paired t tests: *, P < 0.05; **, P < 0.01.

FIG 5

Growth of the C. diphtheriae wild type and mutant derivatives in Mn. (A) Line diagram indicating the deletions introduced into C. diphtheriae for the mntA-R locus. Dashed lines indicate the region of DNA removed in each respective mutant. (B) Overnight growth of strains in mPGT with MnCl₂ supplementation as indicated, measured by OD₆₀₀ (n = 4). (C) Growth of the wild-type strain carrying empty vector (pKN2.6Z) and the ΔmntR strain with either empty vector or mntR clone (pKNmntR) in mPGT with MnCl₂ supplementation as indicated. Data are the means and standard deviations from biological replicates (n = 3). Statistical significance comparing the corresponding wild-type growth and indicated strain (by color) determined by Holm-Sidak’s multiple-comparison test: *, P < 0.05; **, P < 0.01.

FIG 6

MntA-D is responsible for C. diphtheriae CdCl₂ sensitivity. (A) Wild-type or mutant strains as indicated were seeded in HIA and filter discs with 10 μl of 1 mM CdCl₂ were placed over the agar. Plates were incubated overnight at 37°C, and the diameters (mm) of the zone of clearance were measured. Data are the means and standard deviations from biological replicates (n = 4); only nonsignificant comparisons are noted. (B) Strains were grown in HIBTW with CdCl₂ added at the final concentrations indicated, and the optical density (OD₆₀₀) was measured following overnight incubation; data are the means and standard deviations from 3 biological replicates. (C) Wild-type or mutant strains with plasmids as indicated were seeded in HIA, and agar assays were performed as described for panel A. (D) Strains with the indicated plasmids were grown in HIBTW with CdCl₂ as for panel B; data are the means and standard deviations from 3 biological replicates. (A and C) Significance for data was assessed by Holm-Sidak’s multiple-comparison test. NS (not significant), P > 0.05; all other comparisons were significant. (B and D) Statistical significance comparing the corresponding wild-type growth and indicated strain (by color) determined by Holm-Sidak’s multiple-comparison test: *, P < 0.05; **, P < 0.01; ****, P < 0.0001.