Identification and functional analysis of the genes encoding dibenzothiophene-desulfurizing enzymes from thermophilic bacteria.
Thermophilic bacteria Bacillus subtilis WU-S2B and Mycobacterium phlei WU-F1 desulfurize dibenzothiophene (DBT) and alkylated DBTs through specific cleavage of the carbon-sulfur bonds over a temperature range up to 52 degrees C. In order to identify and functionally analyze the DBT-desulfurization genes, the gene cluster containing bdsA, bdsB, and bdsC was cloned from B. subtilis WU-S2B. The nucleotide and amino acid sequences of bdsABC show homologies to those of the other known DBT-desulfurization genes and enzymes; e.g. a nucleotide sequence homology of 61.0% to dszABC of the mesophilic bacterium Rhodococcus sp. IGTS8 and 57.8% to tdsABC of the thermophilic bacterium Paenibacillus sp. A11-2. Deletion and subcloning analysis of bdsABC revealed that the gene products of bdsC, bdsA and bdsB oxidized DBT to DBT sulfone (DBTO(2)), converted DBTO(2) to 2'-hydroxybiphenyl-2-sulfinate (HBPSi), and desulfurized HBPSi to 2-hydroxybiphenyl (2-HBP), respectively. Resting cells of a recombinant Escherichia coli JM109 harboring bdsABC converted DBT to 2-HBP over a temperature range of 30-52 degrees C, indicating that the gene products of bdsABC were functional in the recombinant. The activities of DBT degradation at 50 degrees C and DBT desulfurization (2-HBP production) at 40 degrees C in resting cells of the recombinant were approximately five times and twice, respectively, as high as those in B. subtilis WU-S2B. The recombinant E. coli cells also degraded alkylated DBTs, such as 2,8-dimethylDBT and 4,6-dimethylDBT. The nucleotide sequences of B. subtilis WU-S2B bdsABC and the corresponding genes from M. phlei WU-F1 were found to be completely identical to each other although the strains are genetically different.
Kirimura
Kohtaro
K
Department of Applied Chemistry, School of Science and Engineering, Waseda University, 3-4-1 Ohkubo, Shinjuku-ku, Tokyo 169-8555, Japan. kkohtaro@waseda.jp
Harada
Koji
K
Iwasawa
Hidekazu
H
Tanaka
Takeomi
T
Iwasaki
Yuichiro
Y
Furuya
Toshiki
T
Ishii
Yoshitaka
Y
Kino
Kuniki
K
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001713:Biphenyl Compounds / Q000378:metabolism; D013437:Carbon-Sulfur Lyases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D017124:Conserved Sequence; D004269:DNA, Bacterial / Q000737:chemistry / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005798:Genes, Bacterial; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009168:Mycobacterium phlei / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D010105:Oxygenases / Q000235:genetics; D010802:Phylogeny; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012240:Rhodococcus / Q000235:genetics; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D012689:Sequence Homology, Nucleic Acid; D013696:Temperature; D013876:Thiophenes / Q000378:metabolism*
D001426:Bacterial Proteins; D001713:Biphenyl Compounds; D004269:DNA, Bacterial; D011994:Recombinant Proteins; D013876:Thiophenes; C058517:dibenzothiophene 5-oxide; C004369:2-phenylphenol; D006899:Mixed Function Oxygenases; D010105:Oxygenases; C421210:dibenzothiophene sulfone monooxygenase; D013437:Carbon-Sulfur Lyases; C016366:dibenzothiophene