Analysis of a heme-dependent signal transduction system in Corynebacterium diphtheriae: deletion of the chrAS genes results in heme sensitivity and diminished heme-dependent activation of the hmuO promoter

Abstract

The Corynebacterium diphtheriae hmuO gene encodes a heme oxygenase that is involved in the utilization of heme as an iron source. Transcription of hmuO is activated by heme or hemoglobin and repressed by iron and DtxR. Previous studies with Escherichia coli showed that heme-dependent transcriptional activation of an hmuO promoter-lacZ fusion was dependent on the cloned C. diphtheriae chrA and chrS genes (chrAS), which encode the response regulator and sensor kinase, respectively, of a two-component signal transduction system. In this study, nonpolar deletions in the chrAS genes were constructed on the chromosome of C. diphtheriae. Mutations in chrAS resulted in marked reduction in heme-dependent transcription of hmuO, which indicates that the ChrA/S system is a key regulator at the hmuO promoter. However, low but significant levels of heme-specific transcriptional activity were observed at the hmuO promoter in the chrAS mutants, suggesting that an additional heme-dependent activator is involved in hmuO expression. The chrAS mutants were also sensitive to heme, which was observed only in stationary-phase cultures and correlated with reduced cell viability. The heme sensitivity of the mutants was not due to reduced expression of hmuO, and these results suggest that additional factors controlled by the ChrA/S system may be involved in protection against heme toxicity. Transcriptional analysis of the chrAS operon revealed that it was not autoregulated or affected by iron or heme levels.

FIG. 1.

Genetic map of the chrAS region. The regions deleted in the C. diphtheriae C7 chrSΔ2 and chrAΔ mutants are indicated below the map. P indicates putative promoter, and arrows indicate direction of transcription. Sizes of predicted gene products are indicated in kilodaltons.

FIG. 2.

The chrA deletion mutant is sensitive to heme. Wild-type C7(-) (solid symbols with solid lines) and the chrA deletion mutant chrAΔ (open symbols with dashed lines) were grown in HIBTW medium with no added heme (diamond), 5 μM heme (triangle), or 10 μM heme (circle). Data shown are the mean of three experiments ± standard deviations.

FIG. 3.

The viability of chrAΔ is diminished in the presence of heme. Wild type C7(-) (black) and chrAΔ (gray) were grown in HIBTW medium with 10 μM heme. Dilutions of the cultures were spread onto HIBTW agar plates at the indicated time points, and viable bacteria were determined. Data shown are the means of three experiments ± standard deviations.

FIG. 4.

Heme sensitivity of chrAΔ is reduced in log-phase cultures. Growth curves of wild-type C7(-) (solid symbols with solid lines) and chrAΔ (open symbols with dashed lines) are shown. Strains were grown in HIBTW, and heme was added to both C7(-) and chrAΔ at a final concentration of 10 μM at various times (T) after inoculation of the medium: T = 0 h (square), T = 1 h (triangle), T = 2 h (circle), and T = 3 h (×). Results shown are from a representative experiment. Wild-type C7 without heme (solid diamond) is shown as a control.