Substrate-regulated cyanide hydratase (chy) gene expression in Fusarium solani: the potential of a transcription-based assay for monitoring the biotransformation of cyanide complexes.
The fungus Fusarium solani detoxifies cyanide through induction of the cyanide hydratase gene activity (chy) in the presence of either KCN or the metal-complexed cyanides, K2Ni(CN)4 or K4Fe(CN)6, at pH 7.0 and 4.0 respectively. Sequence analysis of the chy gene identified primers for reverse transcriptase-polymerase chain reaction (RT-PCR)-directed analysis of mRNA transcripts, which demonstrated that activity correlated to the substrate-specific induction of gene expression. chy transcription was initiated 30-60 min after exposure of F. solani cultures to cyanide complexes. Maximum expression was detected within 4.5 h, after which chy mRNA synthesis declined below the limits of detection within 26 h. A lag period of approximately 2 h, following initial transcription, was recorded before cyanide complexes were converted to formamide. mRNA transcripts of chy were not detected in the absence of cyanide or cyanide complexes. The presence of introns within the gene resulted in a difference in size of 100 bp for DNA compared with mRNA of the corresponding 5' region. This size difference facilitated PCR detection of gene and transcript respectively. Comparisons of the predicted amino acid sequence of the F. solani chy gene and those of Gloeocerospora sorghi, Fusarium lateritium and Leptosphaeria maculans demonstrate that cyanide hydratase genes are highly conserved and of a similar evolutionary origin. These data predict that the functional assay described here to monitor the induction of chy gene expression and, potentially, cyanide degradation would be applicable to a variety of polluted environments.
Barclay
M
M
Oxford Centre for Environmental Biotechnology at Natural Environment Research Council, Molecular Microbial Ecology Laboratory, Institute of Virology and Environmental Microbiology, CEH-Oxford, Mansfield Road, OX1 3SR, UK.
Day
J C
JC
Thompson
I P
IP
Knowles
C J
CJ
Bailey
M J
MJ
D000595:Amino Acid Sequence; D001483:Base Sequence; D001673:Biodegradation, Environmental; D003486:Cyanides / Q000378:metabolism*; D018076:DNA, Complementary / Q000737:chemistry; D021901:DNA, Intergenic / Q000737:chemistry / Q000235:genetics; D005559:Formamides / Q000032:analysis / Q000378:metabolism; D005670:Fusarium / Q000201:enzymology* / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D006836:Hydro-Lyases / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D012331:RNA, Fungal / Q000737:chemistry / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D003486:Cyanides; D018076:DNA, Complementary; D021901:DNA, Intergenic; D005559:Formamides; D012331:RNA, Fungal; C031066:formamide; D006836:Hydro-Lyases; C076848:cyanide hydratase