Cloning and characterization of genes from Agrobacterium sp. IP I-671 involved in hydantoin degradation.
Cloning and sequencing of a 7.1 kb DNA fragment from Agrobacterium sp IP I-671 revealed seven open reading frames (ORFs) encoding D-hydantoinase, D-carbamoylase and putative hydantoin racemase, D-amino acid oxidase and NAD(P)H-flavin oxidoreductase. Two incomplete ORFs flanking the hydantoin utilization genes showed similarities to genes involved in transposition. Expression of the D-hydantoinase and D-carbamoylase gene in Escherichia coli gave mainly inactive protein concentrated in inclusion bodies, whereas homologous expression on an RSF1010 derivative increased hydantoinase and D-carbamoylase activity 2.5-fold and 10-fold, respectively, in this strain. Inactivation of the D-carbamoylase gene in Agrobacterium sp IP I-671 led to a complete loss of detectable carbamoylase activity whereas the low hydantoinase activity remaining after inactivation of the D-hydantoinase gene indicated the presence of a second hydantoinase-encoding gene. Two plasmids of 80 kb and 190 kb in size were identified by pulsed-field gel electrophoresis and the cloned hydantoin utilization genes were found to be localized on the 190 kb plasmid.
Hils
M
M
Institute of Industrial Genetics, University of Stuttgart, Germany.
Münch
P
P
Altenbuchner
J
J
Syldatk
C
C
Mattes
R
R
D000581:Amidohydrolases / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D018390:Gene Targeting; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D006827:Hydantoins / Q000378:metabolism*; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010957:Plasmids / Q000235:genetics; D015183:Restriction Mapping; D012231:Rhizobium / Q000235:genetics* / Q000378:metabolism*
D006827:Hydantoins; D000581:Amidohydrolases; C080477:N-carbamoyl-D-amino acid amidohydrolase; C020047:dihydropyrimidinase