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. 2007 Sep;75(9):4506-13.
doi: 10.1128/IAI.00487-07. Epub 2007 Jun 25.

Glutathione-dependent alcohol dehydrogenase AdhC is required for defense against nitrosative stress in Haemophilus influenzae

Stephen P Kidd  1 , Donald Jiang, Michael P Jennings, Alastair G McEwan
Affiliations

Glutathione-dependent alcohol dehydrogenase AdhC is required for defense against nitrosative stress in Haemophilus influenzae

Stephen P Kidd et al. Infect Immun. 2007 Sep.

Abstract

In Haemophilus influenzae Rd KW20, we identified a gene, adhC, which encodes a class III alcohol dehydrogenase (AdhC) and has S-nitrosoglutathione reductase activity. adhC exists on an operon with estD, which encodes an esterase. Divergent to the adhC-estD operon is the Haemophilus influenzae nmlR gene (nmlR(HI)), which encodes a MerR family regulator that is homologous to the Neisseria MerR-like regulator (NmlR). Analysis of an nmlR(HI) mutant indicated that expression of the adhC-estD operon is regulated by NmlR(HI) in strain Rd KW20. Chromosomal inactivation of either adhC or nmlR(HI) resulted in sensitivity to S-nitrosoglutathione and decreased S-nitrosoglutathione reductase activity. Examination of the NmlR(HI)-AdhC system in the genome sequences of nontypeable H. influenzae strains R2846, R2866, and 86-028NP identified significant variations. The adhC gene of 86-028NP was predicted to be nonfunctional due to a premature stop codon. Polymorphisms in the operator/promoter region of R2866 resulted in reduced enzyme activity. This correlated with an increased sensitivity to S-nitrosoglutathione. The adhC-nmlR(HI) system was examined in thirty-three clinical isolates (both capsular and nontypeable strains). Nucleic acid sequence data showed that only strain 86-028NP contained a premature stop codon. There were some variations in the DNA sequence of the operator/promoter region which altered the nmlR(HI) promoter. However, the clinical isolates still possessed S-nitrosoglutathione reductase activity and showed at least the equivalent ability to grow in the presence of S-nitrosoglutathione as Rd KW20. These data suggest that the nmlR(HI)-adhC system has a role in the defense against nitrosative stress in Haemophilus influenzae.

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Figures

FIG. 1.
FIG. 1.
Organization of the nmlRHI and adhC gene cluster of H. influenzae. The DNA sequence for the intergenic spacer region of adhC-estD (HI0185 and HI0184) to nmlRHI (HI0186) is shown. The −10 and −35 promoter elements for PnmlRHI are boxed and annotated −10 nmlRHI and −35 nmlRHI; for PadhC, they are underlined and annotated −10 adhC and − 35 adhC. The dyad symmetry is indicated with arrows. The start of translation is in bold type.
FIG. 2.
FIG. 2.
Regulation of the adhC-estD operon. (A) Analysis of transcription of adhC-estD using RT-PCR. Template RNA from H. influenzae Rd KW20 (lane 2) and H. influenzae nmlRHI (lane 1). RNA size marker (lane 3) and the PCR product using genomic DNA (lane 4). (B) Consensus NmlR binding site. The binding sites as determined by in silico analysis for H. influenzae strains are as follows: Rd KW20 (Hi RdKW20) (GenBank accession number L42023; nucleotide coordinates 200673-200692), R2846 (accession number AADO0000000; coordinates 86235-86258), R2866 (accession number AADP0000000; coordinates 46407-46387), 86-028NP (accession number CP000057; coordinates 261657-261638), Lactobacillus plantarum (Lac.plant) (accession number CR377164; coordinates 2681213-2681231), Enterococcus faecium (En.faec) (accession number AAAK00000000; coordinates 9495-9515), Clostridium acetobutylicum (Cl.aceto) (accession number AE001437.1; coordinates 113616-113636), Oceanobacillus iheyensis (Oc.ihey) (accession number BA000028; coordinates 841653-841673), Streptococcus pneumoniae (Str.pn) (accession number AE008532; coordinates 9226-9245), Streptococcus agalactiae (Str.aga) (accession number AE0014211; coordinates 10838-10861), and Neisseria meningitidis (NMA) (accession number AL162756.2; coordinates 80451-80471), as well as the two proven binding sites by genetic studies for NmlR in N. gonorrhoeae PadhC (Ng PadhC) and PcopA (Ng PcopA) (18). The dyad symmetries are underlined. (C) WebLogo consensus sequence for the binding site for NmlR.
FIG. 3.
FIG. 3.
Roles for H. influenzae, H. influenzae adhC, and H. influenzae nmlRHI in GSNO metabolism. (A) S-Nitrosoglutathione reductase activities of H. influenzae strains Rd KW20 wild type (Rd), Rd KW20 adhC (Rd adhC), and Rd KW20 nmlRHI (Rd nmlR). Y-axis error bars indicate ±1 standard deviation of the mean. Each assay experiment was conducted in triplicate and repeated with separated bacterial cultures for both strains. (B) Effect of GSNO on H. influenzae, H. influenzae adhC, and H. influenzae nmlRHI. Wild-type strain Rd KW20 was grown in CDM (⧫) as were Rd KW20 adhC (X) and Rd KW20 nmlRHI (▴). Results are shown alongside results for each strain grown in the presence of 0.5 mM GSNO, shown as dotted lines (Rd KW20 [▪] Rd KW20 adhC [•] and Rd KW20 nmlRHI [X]. Y-error bars indicate ±1 standard deviation of the mean. Experiments were conducted in triplicate.
FIG. 4.
FIG. 4.
Effect of GSNO on clinical isolates of H. influenzae. S-Nitrosoglutathione reductase activities of H. influenzae strains are shown with filled bars. Y-error bars indicate ±1 standard deviation of the mean. Each enzyme assay experiment was conducted in triplicate and repeated with separated bacterial cultures for both strains. The sensitivities of clinical isolates of H. influenzae to GSNO are shown with open bars. Separate cultures were grown in CDM and CDM with 0.5 mM GSNO. The growth was followed over 18 h, and the results are presented as the percentages of CFU/ml in 0.5 mM GSNO relative to that of CDM alone. Y-error bars indicate ±1 standard deviation of the mean. Each assay experiment was conducted in triplicate.
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