A Salmonella enterica serovar typhimurium hemA mutant is highly susceptible to oxidative DNA damage

Abstract

The first committed step in the biosynthesis of heme, an important cofactor of two catalases and a number of cytochromes, is catalyzed by the hemA gene product. Salmonella enterica serovar Typhimurium hemA26::Tn10d (hemA26) was identified in a genetic screen of insertion mutants that were sensitive to hydrogen peroxide. Here we show that the hemA26 mutant respires at half the rate of wild-type cells and is highly susceptible to the effects of oxygen species. Exposure of the hemA26 strain to hydrogen peroxide results in extensive DNA damage and cell death. The chelation of intracellular free iron fully abrogates the sensitivity of this mutant, indicating that the DNA damage results from the iron-catalyzed formation of hydroxyl radicals. The inactivation of heme synthesis does not change the amount of intracellular iron, but by diminishing the rate of respiration, it apparently increases the amount of reducing equivalents available to drive the Fenton reaction. We also report that hydrogen peroxide has opposite effects on the expression of hemA and hemH, the first and last genes of heme biosynthesis pathway, respectively. hemA mRNA levels decrease, while the transcription of hemH is induced by hydrogen peroxide, in an oxyR-dependent manner. The oxyR-dependent induction is suppressed under conditions that accelerate the Fenton reaction by a mechanism that is not yet understood.

FIG. 1.

Mapping of the hemA promoter and the insertion mutant. (A) Primer extension analysis of chromosomal (30 μg, total RNA) and plasmid-encoded (3 μg, total RNA) hemA promoter region. Total RNA was extracted from exponential-phase (A₆₀₀ = 0.3) SL1344 and SL1344 cells carrying the plasmid encoding the hemA promoter region (pGEM-5′hemA). The sequencing reaction was carried out with the same primer. (B) Sequence of the hemA promoter region. The horizontal arrows indicate the start sites observed by primer extension. Brackets indicate the −10 regions of P1 and P2. The bases matching the −10 hexamers of the σ⁷⁰ consensus are underlined. Both P1 and P2 promoters have no obvious −35 sequence. The vertical arrow indicates the site of insertion of the transposon Tn10d. (C) S1 mapping of the hemA transcripts. Total RNA was extracted from exponential cultures (A₆₀₀ = 0.3) of SL1344 wild type and SL1344 hemA26 mutant prior to and after exposure to 1 mM hydrogen peroxide (15 min). To analyze hemA transcription from the tetR promoter, hemA26 strain was exposed to 10 μg of tetracycline/ml for 15 min. S1 mapping was carried out with an end-labeled single-stranded DNA fragment (612 bases) complementary to hemA. The fragments protected by hemA mRNA in wild-type and hemA26 cells were 93 (indicated as “+1”) and 119 (indicated as “+26”) nucleotides long, respectively. +1, Transcription start site of P1; +26, insertion site (i.e., the position at which transcription originating at the tetR promoter enters into hemA).

FIG. 2.

Hydrogen peroxide killing assay. Bacterial cultures (SL1344 wild-type, SL1344 katG katE, and SL1344 hemA26 strains) grown to A₆₀₀ = 0.2 to 0.4 in LB medium were treated with 2.5 mM hydrogen peroxide. Viability was assayed at the indicated time points by plating the cells on LB or LB-Tet plates.

FIG. 3.

ALA supplementation in hydrogen peroxide killing. Bacterial cultures (i.e., SL1344 wild type [A], SL1344 hemA26 mutant [B], SL1344 hemA26 mutant carrying pTE367 [prfA] [C], and SL1344 hemA26 mutant carrying pSA35 [Ptac-dorf1 lacI] [D]) grown to A₆₀₀ = 0.2 to 0.4 in NB in the absence or in the presence of ALA (at 50 μg/ml) were treated with 1 mM hydrogen peroxide. Viability was assayed at the indicated time points by plating the cells on LB or LB-Tet plates. To induce expression of dorf1 from the Ptac-dorf1 plasmid, the cells were dilute in NB medium supplemented with IPTG (isopropyl-β-d-thiogalactopyranoside; 0.1 μg/ml).

FIG. 4.

Effect of iron chelator. (A) SNG survival assay. Bacterial cultures of each strain carrying the prfA plasmid were grown to an A₆₀₀ of 0.18 in LB medium, and then half were treated with 1 mM 2,2′-dipyridyl (dpy) for 20 min. Thereafter, the iron chelator-treated cultures and the control untreated cultures were treated with 1 μg of SNG/ml. Viability was assayed at 0, 10, 20, and 40 min after the addition of SNG by plating the bacterial cells onto LB or LB-Tet plates. The average of five independent experiments is shown. (B) Hydrogen peroxide killing. Wild-type and hemA26 strains carrying the prfA plasmid were examined for hydrogen peroxide sensitivity (2.5 mM) with or without prior treatment with 1 mM 2,2′-dipyridyl for 15 min. In both experiments, the cells were introduced with the prfA plasmid (pTE367) to adjust the total number of hemA26 CFU.

FIG. 5.

Analysis of plasmid DNA topology. (A) Detection of single-strand breaks. Cultures of wild-type SL1344 and hemA26 mutant carrying pKK177-3 were grown to an A₆₀₀ of 0.25 in LB mutant and were then exposed to 0, 0.5, and 1 mM hydrogen peroxide for 15 min. Where indicated, the iron chelator 2,2′-dipyridyl (1 mM) was added to the cells 15 min prior to the treatment with hydrogen peroxide. Plasmid DNA samples were separated on a 1% agarose gel. DNA was visualized by ethidium bromide staining. uncut, pKK177-3 DNA; cut, pKK177-3 DNA digested with EcoRI. (B) Detection of changes in the negative supercoiling of the DNA. The plasmid samples from above were analyzed on 1.4% agarose gels containing 10 μg of chloroquine/ml. At this chloroquine concentration, the most relaxed molecules migrate most rapidly through the gel (41). DNA was visualized by ethidium bromide staining.

FIG. 6.

EPR analysis of intracellular free iron. Samples for EPR analysis were taken from exponential-phase cultures of SL1344 wild-type, hemA26, and fur-1 strains. The iron concentration was calculated based on the following ferric sulfate standards: wild type, 36 μM intracellular chelatable iron; hemA26, 45 μM iron; fur-1, 190 μM iron.

FIG. 7.

Respiratory blocks sensitize Salmonella to killing by hydrogen peroxide. (A) Oxygen consumption was measured in exponential phase cultures grown in LB medium. Tetracycline (10 μg/ml), or ALA (50 μg/ml) were added where indicated. (B) Killing by hydrogen peroxide in respiration deficient cells. Where indicated, 3 mM potassium cyanide was added to cells 5 min before challenge with 2.5 mM hydrogen peroxide. Viability was assayed at the indicated time points. The addition of cyanide alone did not diminish cell viability.

FIG. 8.

Primer extension assays. (A) Cultures of SL1344 wild-type, ΔoxyR, and fur1 strains were grown to an A₆₀₀ of 0.3 to 0.4 in LB medium, and then half of each culture was treated with 1 mM hydrogen peroxide for 15 min. P1 of hemA mutant is shown. No expression could be detected from P2. (B) A wild-type culture was split into four subcultures, and two were treated with 1 mM hydrogen peroxide. Rifampin (0.2 mg/ml) was added after 1 min to one treated and one untreated culture. Samples were taken from control untreated and treated cultures 1, 3, 6, 11, and 16 min after the addition of hydrogen peroxide. Approximately 80% of the hemA mRNA in the cells exposed to hydrogen peroxide and 95% of the nonexposed mRNA were degraded within the first 2 min of rifampin treatment, as measured with a BioImaging Analyzer.

FIG. 9.

Primer extension assays. (A) Cultures at exponential phase were exposed to 0, 0.2, and 1 mM of hydrogen peroxide for 5 min. (B) The cultures shown in panel A were exposed to the iron chelator (1 mM) for 15 min prior to the treatment with hydrogen peroxide. (C) Cultures of wild-type and hemA26 strains grown to an A₆₀₀ of 0.25 in LB medium were split. One part of each culture was treated with the iron chelator 2,2′-dipyridyl (1 mM) for 15 min. Thereafter, the dipyridyl treated and the untreated cultures were exposed to 1 mM hydrogen peroxide for 5 min. In addition, two parts of hemA26 culture were exposed to 10 μg of tetracycline/ml for 10 min, and then one was further exposed to hydrogen peroxide (1 mM) for 5 min.