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The secondary metabolite hydrogen cyanide (HCN) is produced byPseudomonas fluorescens from glycine, essentially under microaerophilic conditions. The genetic basis of HCN synthesis inP. fluorescens CHA0 was investigated. The contiguous structural geneshcnABC encoding HCN synthase were expressed from the T7 promoter inEscherichia coli , resulting in HCN production in this bacterium. Analysis of the nucleotide sequence of thehcnABC genes showed that each HCN synthase subunit was similar to known enzymes involved in hydrogen transfer, i.e., to formate dehydrogenase (for HcnA) or amino acid oxidases (for HcnB and HcnC). These similarities and the presence of flavin adenine dinucleotide- or NAD(P)-binding motifs in HcnB and HcnC suggest that HCN synthase may act as a dehydrogenase in the reaction leading from glycine to HCN and CO2 . ThehcnA promoter was mapped by primer extension; the −40 sequence (TTGGC … .ATCAA) resembled the consensus FNR (fumarate and nitrate reductase regulator) binding sequence (TTGAT … .ATCAA). The gene encoding the FNR-like protein ANR (anaerobic regulator) was cloned fromP. fluorescens CHA0 and sequenced. ANR of strain CHA0 was most similar to ANR ofP. aeruginosa and CydR ofAzotobacter vinelandii . Ananr mutant ofP. fluorescens (CHA21) produced little HCN and was unable to express anhcnA-lacZ translational fusion, whereas in wild-type strain CHA0, microaerophilic conditions strongly favored the expression of thehcnA-lacZ fusion. Mutant CHA21 as well as anhcn deletion mutant were impaired in their capacity to suppress black root rot of tobacco, a disease caused byThielaviopsis basicola , under gnotobiotic conditions. This effect was most pronounced in water-saturated artificial soil, where theanr mutant had lost about 30% of disease suppression ability, compared with wild-type strain CHA0. These results show that the anaerobic regulator ANR is required for cyanide synthesis in the strictly aerobic strain CHA0 and suggest that ANR-mediated cyanogenesis contributes to the suppression of black root rot.

journal of bacteriology

Characterization of the<i>hcnABC</i>Gene Cluster Encoding Hydrogen Cyanide Synthase and Anaerobic Regulation by ANR in the Strictly Aerobic Biocontrol Agent<i>Pseudomonas fluorescens</i>CHA0

Characterization of thehcnABCGene Cluster Encoding Hydrogen Cyanide Synthase and Anaerobic Regulation by ANR in the Strictly Aerobic Biocontrol AgentPseudomonas fluorescensCHA0