Role of the alkyl hydroperoxide reductase (ahpCF) gene in oxidative stress defense of the obligate Anaerobe bacteroides fragilis

Abstract

In this study we report the identification and role of the alkyl hydroperoxide reductase (ahp) gene in Bacteroides fragilis. The two components of ahp, ahpC, and ahpF, are organized in an operon, and the deduced amino acid sequences revealed that B. fragilis AhpCF shares approximately 60% identity to orthologues in other gram-positive and gram-negative bacteria. Northern blot hybridization analysis of total RNA showed that the ahpCF genes were transcribed as a polycistronic 2.4-kb mRNA and that ahpC also was present as a 0.6-kb monocistronic mRNA. ahpC and ahpCF mRNAs were induced approximately 60-fold following H(2)O(2) treatment or oxygen exposure of the parent strain but were constitutive in a peroxide-resistant strain. Further investigation using an ahpCF'::beta-xylosidase gene transcriptional fusion confirmed that ahpCF had lost normal regulation in the peroxide-resistant strain compared to the parent. The ahpCF mutant was more sensitive to growth inhibition and mutagenesis by organic peroxides than the parent strain, as determined by disk inhibition assays and the frequency of mutation to fusidic acid resistance. This finding suggests that the ahp genes play an important role in peroxide resistance in B. fragilis. Under anaerobic conditions, we observed increases in the number of spontaneous fusidic acid-resistant mutants of five- and sevenfold in ahpCF and ahpF strain backgrounds, respectively, and eightfold in the ahpCF katB double mutant strain compared to the parent and katB strains. In addition, ahpCF, ahpF, and ahpCF katB mutants were slightly more sensitive to oxygen exposure than the parent strain. Moreover, the isolation of a strain with enhanced aerotolerance and high-level resistance to alkyl hydroperoxides from an ahpCF katB parent suggests that the physiological responses to peroxide toxicity and to the toxic effects of molecular oxygen are overlapping and complex in this obligate anaerobe.

FIG. 1

Functional and genetic maps of the ahpCF locus in B. fragilis strains. (A) Wild-type gene in strains 638R (wild type) and IB263 (hpr). The striped and grey arrows represent the ahpC and ahpF genes, respectively; The letter P and a dark arrowhead mark the promoter region and the transcription start site. Arrows under the map represent the length and orientation of the transcripts. Also shown are the ahpC and ahpF double-stranded dsDNA fragments used as probes for Northern blot hybridizations. (B to D) Schematic representations of the single-crossover insertional disruptions of the ahpC and ahpF genes and the ahpC′::XA construct in the chromosome. A partial restriction map is shown above the diagrams. The twisted line is not drawn to scale and represents the suicide vector pFD516 (7.7 kb). The bifunctional XA reporter gene is depicted as the hatched arrow.

FIG. 2

Alignment of the B. fragilis (Bf) deduced amino acid sequences for AhpC (A) and AhpF (B) with sequences of proteins from E. coli (Ec), S. typhimurium (St), P. putida (Pp), P. gingivalis (Pg), E. faecalis (Ef), and T. pallidum (Tp), and X. campestris (Xc). Asterisks above the sequences indicate functional redox-active cysteine residues (4, 11). Predicted adenine dinucleotide binding sites in AhpF are underline according to the putative assignment of bacterial AhpF functional domains (29). Consensus of at least 50% identical amino acid residues is denoted by black boxes; conserved amino acid substitutions are depicted by grey boxes. Only a partial alignment of the most conserved AhpF regions is shown. For comparison, amino acid residue positions are shown for E. coli AhpF and B. fragilis AhpF.

FIG. 3

Autoradiograph of Northern hybridization filter of total RNA from mid-log-phase B. fragilis 638R and IB263 (hpr) following exposure to different oxidative stress conditions. The probe was an ahpC (A) or ahpF (B) internal gene fragment; samples consisted of anaerobic cultures (lane 1), cultures treated with 50 μM H₂O₂ (lane 2), cultures treated with 100 μM potassium ferricyanide (lane 3 in panel A), cultures treated with 1 mM potassium ferricyanide (lanes 4 in panel A and lane 3 in panel B), and cultures exposed to aeration for 1 h (lane 5 in panel A and lane 4 in panel B). Approximate sizes of the transcripts are indicated on the left. The arrow in panel B points to an ahpF mRNA component of approximately 1.7 kb. Bottom panels are corresponding ethidium bromide-stained agarose gels loaded with 30 μg of total RNA in each lane. Positions of 23S and 16S rRNA are also indicated.

FIG. 4

(A) Autoradiograph following electrophoresis of ahpCF primer extension reactions. Total RNA was obtained from mid-log-phase cells of B. fragilis 638R grown anaerobically in BHI medium and then subjected to oxidative stress conditions. Lane 1, anaerobic culture; lane 2, culture exposed to 50 μM H₂O₂; lane 3, culture exposed to oxygen for 1 h. The nucleotide sequence of ahpCF, using the same primer, was run in parallel. (B) Nucleotide sequence of the ahpCF regulatory region. Putative −35 and −10 regions of the promoter and the putative ribosome binding site (rbs) are underlined. The arrow indicates the initial +1 guanine nucleotide 37 bp upstream of the ahpC translation start codon. The start codon of the ahpC gene is indicated in boldface, and only the first 13 codons are shown.

FIG. 5

Analysis of ahpC′::XA transcriptional fusions in strains grown under different oxidative stress conditions. β-Xylosidase activity in crude extracts was determined for wild-type (wt) strain IB277 and constitutive hpr mutant strain IB278 grown to mid-log phase and then either shaken in air for 1 h (culture exposed to oxygen), challenged with two successive 50 μM H₂O₂ treatments for 15 min each (addition of hydrogen peroxide), or incubated anaerobically (anaerobic culture).

FIG. 6

Survival of mid-log-phase anaerobic cells of B. fragilis strains shifted to aerobic conditions. Cultures of mid-log-phase cells at an optical density at 550 nm of 0.3 were divided at time zero; one half was shaken at 250 rpm in air at 37°C, and the other half was maintained anaerobically. Viable cell counts were determined at the times shown. For clarity, data for the anaerobic control cultures are not shown.