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

Abstract

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 genes hcnABC encoding HCN synthase were expressed from the T7 promoter in Escherichia coli, resulting in HCN production in this bacterium. Analysis of the nucleotide sequence of the hcnABC 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. The hcnApromoter 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 from P. fluorescens CHA0 and sequenced. ANR of strain CHA0 was most similar to ANR of P. aeruginosa and CydR of Azotobacter vinelandii. An anr mutant of P. fluorescens (CHA21) produced little HCN and was unable to express an hcnA-lacZ translational fusion, whereas in wild-type strain CHA0, microaerophilic conditions strongly favored the expression of the hcnA-lacZ fusion. Mutant CHA21 as well as an hcn deletion mutant were impaired in their capacity to suppress black root rot of tobacco, a disease caused by Thielaviopsis basicola, under gnotobiotic conditions. This effect was most pronounced in water-saturated artificial soil, where the anr 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.

Bacterial cyanogenesis: impact on biotic interactions

“…A similar result was obtained with Pseudomonas protegens CHA0 (formerly known as Pseudomonas fluorescens CHA0, Ramette et al 2011) and its cyanide-negative mutant CHA77. Under low oxygen conditions in vitro, conditions favourable to expression of the hcnABC operon (Laville et al 1998), egg hatching rates of the plant pathogenic nematode Meloidogyne javanica exposed to Ps. protegens CHA0 were 40% as compared to 85% hatching in nematodes exposed to Ps.…”

“…This genetic locus was first characterized in Ps. protegens CHA0 (Laville et al 1998). Due to the impact of cyanide production on soil biota, the hcnABC gene cluster has been analysed in a number of different ways.…”

Multiple transcription factors of the FNR family in denitrifying Pseudomonas stutzeri : characterization of four fnr‐like genes, regulatory responses and cognate metabolic processes

“…Genes targeted subsequently by the DNR regulators encoded from dnrS and dnr are indicated by the arrows. The locus controlling cyanogenesis in P. aeruginosa, which has not been identified as such, is termed hcn, analogous to the Pseudomonas fluorescens locus (Laville et al, 1998); qox is the operon for a quinol oxidase. two-component system, and not by FixK 2 , which lacks the iron-sulphur cluster (Nellen-Anthamatten et al, 1998).…”

Multicopy suppression of a gacA mutation by the infC operon in Pseudomonas fluorescens CHA0: competition with the global translational regulator RsmA

“…Standard recombinant techniques were used [16] or have been referenced elsewhere [20,24]. For subcloning experiments with cosmid pVK100 (23 kb), DNA fragments were ligated, after electrophoretic separation, in low melting point agarose gels.…”

“…The gacA deletion mutant CHA500, which does not produce HCN, could be functionally complemented for HCN production by recombinant cosmids which contained either the gacA gene [3], the hcnABC structural genes [22,24], or a common 7.5 kb HindIII fragment unrelated to gacA or hcnABC. HCN production was assessed by a qualitative test [25].…”