Glutathione-Dependent Alcohol Dehydrogenase AdhC Is Required for Defense against Nitrosative Stress in Haemophilus influenzae

Abstract

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 .