Affiliation:
1. Mikrobiologisches Institut1 and
2. Laboratoire de Biologie Microbienne, Universitéde Lausanne, CH-1015 Lausanne,2 Switzerland
3. Institut für Pflanzenwissenschaften/Phytopathologie, Eidgenössische Technische Hochschule, CH-8092 Zürich,3and
Abstract
ABSTRACT
The secondary metabolite hydrogen cyanide (HCN) is produced by
Pseudomonas fluorescens
from glycine, essentially under microaerophilic conditions. The genetic basis of HCN synthesis in
P. 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 CO
2
. The
hcnA
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 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.
Publisher
American Society for Microbiology
Subject
Molecular Biology,Microbiology
Cited by
197 articles.
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