The redox-sensitive transcriptional activator OxyR regulates the peroxide response regulon in the obligate anaerobe Bacteroides fragilis

Abstract

The peroxide response-inducible genes ahpCF, dps, and katB in the obligate anaerobe Bacteroides fragilis are controlled by the redox-sensitive transcriptional activator OxyR. This is the first functional oxidative stress regulator identified and characterized in anaerobic bacteria. oxyR and dps were found to be divergently transcribed, with an overlap in their respective promoter regulatory regions. B. fragilis OxyR and Dps proteins showed high identity to homologues from a closely related anaerobe, Porphyromonas gingivalis. Northern blot analysis revealed that oxyR was expressed as a monocistronic 1-kb mRNA and that dps mRNA was approximately 500 bases in length. dps mRNA was induced over 500-fold by oxidative stress in the parent strain and was constitutively induced in the peroxide-resistant mutant IB263. The constitutive peroxide response in strain IB263 was shown to have resulted from a missense mutation at codon 202 (GAT to GGT) of the oxyR gene [oxyR(Con)] with a predicted D202G substitution in the OxyR protein. Transcriptional fusion analysis revealed that deletion of oxyR abolished the induction of ahpC and katB following treatment with hydrogen peroxide or oxygen exposure. However, dps expression was induced approximately fourfold by oxygen exposure in DeltaoxyR strains but not by hydrogen peroxide. This indicates that dps expression is also under the control of an oxygen-dependent OxyR-independent mechanism. Complementation of DeltaoxyR mutant strains with wild-type oxyR and oxyR(Con) restored the inducible peroxide response and the constitutive response of the ahpCF, katB, and dps genes, respectively. However, overexpression of OxyR abolished the catalase activity but not katB expression, suggesting that higher levels of intracellular OxyR may be involved in other physiological processes. Analysis of oxyR expression in the parents and in DeltaoxyR and overexpressing oxyR strains by Northern blotting and oxyR'::xylB fusions revealed that B. fragilis OxyR does not control its own expression.

FIG. 1

Multiple alignment of the B. fragilis (Bf) deduced amino acid sequences for OxyR (A) and Dps (B) with other bacterial OxyR and Dps homologue amino acid sequences, respectively. E. coli (Ec), B. burgdorferi (Bb), B. pertussis (Bp), B. subtilis (Bs), E. chrysanthemi (Ech), H. influenzae (Hi), H. pylori (Hp), L. innocua (Li), M. leprae (Ml), N. miningitis (Nm), P. gingivalis (Pg), P. aeruginosa (Pa), R. prowazekii (Rp), S. pneumoniae (Sp), Synechocystis sp. (Sy), and Xanthomonas campestris (Xc) sequences were used. Lines drawn above and below the amino acid sequences indicate the LyR helix-turn-helix DNA-binding nucleotide sequence motif and functional redox-active cysteine residues of E. coli OxyR C199 and C208 (29, 45) in panel A and the Dps protein signature consensus pattern associated with DNA binding properties (6, 43) in panel B. Consensus of at least 50% identical amino acid residues are labeled with black boxes. Conserved amino acid substitutions are depicted by grey boxes. The respective protein descriptions and GenBank accession numbers for the sequences are listed in Materials and Methods.

FIG. 1

Multiple alignment of the B. fragilis (Bf) deduced amino acid sequences for OxyR (A) and Dps (B) with other bacterial OxyR and Dps homologue amino acid sequences, respectively. E. coli (Ec), B. burgdorferi (Bb), B. pertussis (Bp), B. subtilis (Bs), E. chrysanthemi (Ech), H. influenzae (Hi), H. pylori (Hp), L. innocua (Li), M. leprae (Ml), N. miningitis (Nm), P. gingivalis (Pg), P. aeruginosa (Pa), R. prowazekii (Rp), S. pneumoniae (Sp), Synechocystis sp. (Sy), and Xanthomonas campestris (Xc) sequences were used. Lines drawn above and below the amino acid sequences indicate the LyR helix-turn-helix DNA-binding nucleotide sequence motif and functional redox-active cysteine residues of E. coli OxyR C199 and C208 (29, 45) in panel A and the Dps protein signature consensus pattern associated with DNA binding properties (6, 43) in panel B. Consensus of at least 50% identical amino acid residues are labeled with black boxes. Conserved amino acid substitutions are depicted by grey boxes. The respective protein descriptions and GenBank accession numbers for the sequences are listed in Materials and Methods.

FIG. 2

Diagram of oxyR and dps genetic organization and structure of the promoter regions. The open and grey long arrows indicate the open reading frames and their respective direction of transcription. The dps-oxyR intergenic nucleotide sequence region and the first 5 codons of dps and 13 codons of oxyR also are shown. A partial restriction endonuclease map of the sequenced genes is indicated. The dark arrowheads indicate the transcription initiation nucleotide for dps and oxyR mRNAs. Based on a B. fragilis consensus (D. P. Bayley and C. J. Smith, unpublished data), the predicted −10 and −35 promoter region for each gene is underlined. The bottom panels show the primer extension autoradiographs used to determine the transcription start sites for dps and oxyR mRNAs. To the left of each panel is a DNA sequencing ladder generated with the same primer used for primer extension reactions. The following treatments were used as described in the Materials and Methods: anaerobic growth (lane 1), hydrogen peroxide treatment (lane 2), and oxygen exposure (lane 3).

FIG. 3

(A and C) Autoradiographs of Northern hybridization membranes of total RNA from mid-log-phase B. fragilis 638R and IB263 following exposure to different oxidative stress conditions. The probe was a dps (A) or oxyR (C) internal gene fragment. Lanes: 1, anaerobic growth; 2, cultures treated with hydrogen peroxide; 3, cultures exposed to oxygen. The approximate sizes of the transcripts are indicated. (B and D) Respective ethidium bromide-stained agarose gels loaded with approximately 30 μg total RNA in each lane. The 23S and 16S rRNAs are also indicated.

FIG. 4

Expression of peroxide-inducible genes in oxyR mutants. Determination of β-xylosidase (A, B, and D) and catalase (C) activities in crude extracts of mid-log-phase cells of B. fragilis parent and ΔoxyR mutant strains is shown. Bacteria were grown in BHIS and exposed to different oxidative stress conditions as indicated.

FIG. 5

Complementation of oxyR mutations. Determination of β-xylosidase (A, B, and D) and catalase (C) activities in crude extracts of mid-log-phase cells of B. fragilis ΔoxyR mutant strains completed with constitutive OxyR(Con), pFD770[oxyR(Con)], or wild-type OxyR, pFd772(oxyR) is shown. oxyR^c is equivalent to oxyR(Con). Bacteria were grown in BHIS and exposed to different oxidative stress conditions as indicated.

FIG. 6

Comparison of KatB enzyme activity to the expression of katB transcriptional fusion in OxyR-overproducing strains. Determination of catalase (A) and β-xylosidase (B) activities in crude extracts of mid-log-phase cells of B. fragilis 638R katB′::xylB transformed with pFd772(oxyR) is shown. Bacteria were grown to mid-log phase in BHIS and exposed to different oxidative stress conditions as indicated.