Article Figures & Data
Figures
- FIG. 1.
Proposed Dsz pathway for BDS of DBT by R. erythropolis IGTS8. DBTO, DBT sulfoxide; DBTO2, DBT sulfone; HPBS, 2-(2"-hydroxyphenyl)-benzene sulfinate; HBP, 2-hydroxybiphenyl; DBT-MO, DBT monooxygenase; DBTO2-MO, DBTO2 monooxygenase; FMN, flavin mononucleotide.
- FIG. 2.
A synthetic oligonucleotide (upper strand) was 5" phosphorylated to allow ligation repair from upstream sequence. The oligonucleotide was randomized at codon 261, and the MunI site was removed with two silent mutations.
Tables
- TABLE 1.
Bacterial strains and plasmids used in this study
Strain or plasmid Genotype or description Source or reference Strains R. erythropolis IGTS8 BDS type strain 16 R. erythropolis I-19 Derivative of R. erythropolis IGTS8 with multiple copies of dszACD 9 R. erythropolis BKO53 DszB derivative of R. erythropolis I-19 9; B. R. Folsom, presented at the First International Conference on Petroleum Biotechnology, Mexico City, Mexico, 21-23 February 2000 R. erythropolis JB55 ΔdszABC derivative of IGTS8; dszD still present 4 R. erythropolis C4-104 Chemostat mutant with octyl sulfide gain of function; contains mutant plasmid pJA101 This study R. erythropolis C4-224 Chemostat mutant with 5-MBT gain of function; contains mutant plasmid pJA102 This study E. coli DH10B Electrocompetent cells used to amplify plasmid libraries before transformation into Rhodococcus Gibco BRL Plasmids pEBC388 dszABC, chloramphenicol resistance 21 pEBC1100 dszABCD, kanamycin resistance 4 pJA101 pEBC388 derivative isolated from chemostat mutant R. erythropolis C4-104; contains dszA1 allele This study pJA102 pEBC388 derivative isolated from chemostat mutant R. erythropolis C4-224; contains dszA2 and dszC1 alleles This study pJA103 pEBC1100 derivative with dszC allele replaced by dszC1 allele from pJA102 This study Codon 261 mutant plasmids Series of plasmids derived from pEBC1100, each with one of all possible codons at position 261 of DszC (see Results) This study - TABLE 2.
Summary of organosulfur model compound biotransformations by R. erythropolis BKO53a
Total sulfur in oil (μM) Compound Stock 24 h % Remaining DBT 940 0 0 Benzothiophenes Benzothiophene 1,200 920 77 2-MBT 670 230 34 3-MBT 970 250 26 5-MBT 850 850 100 3,5-Dimethylbenzothiophene 810 0 0 Thiophenes 2,3,5-Trimethylthiophene 760 530 100b 2,5-Di(tert-butyl)thiophene 250 280 112 3-n-Octylthiophene 830 940 113 3-Phenylthiophene 780 760 97 Dialkyl sulfides Hexyl sulfide 8,400 1,080 13 Octyl sulfide 760 740 97c Decyl sulfide 600 690 115 Dodecyl sulfide 940 940 100 ↵ a Assay results are corrected for the presence of the internal standard (dodecyl sulfide).
↵ b For 2,3,5-trimethylthiophene, a similar loss was observed for the cell-free control, indicating volatilization.
↵ c GC-SCD indicated the appearance of a product peak that was confirmed by GC-MS to be octyl sulfone.
- TABLE 3.
Summary of model compound assaysa
μmol removed Rhodococcus strain Genotype 5-MBTb Benzothiophenec Octyl sulfided DBT sp acte (μmol g [dry wt] of cells−1 min −1) JB55 Δdsz −0.7 −0.3 1.2 0.06 JB55(pEBC388) dszA+B+C+ 0 −1.3 0.3 0.65 JB55(pEBC1100) dszA+B+C+D+ 1.2 NTf NT 1.14 C4-104 dszA1B+C+ −0.1 NT 8.9g 0.54 C4-224 dszA2B+C1 3.9 3.9 9.2 0.45 JB55(pJA103) dszA+B+C1D+ 7.5 6.9 3.5 1.02 ↵ a Assays ended at 24 h unless otherwise stated.
↵ b The initial amount of 5-MBT was 8.6 μmol.
↵ c The initial amount of benzothiophene was 8.6 μmol.
↵ d The initial amount of octyl sulfide was 58 μmol.
↵ e The initial amount of DBT was 115 μmol; the assay ended at 30 min.
↵ f NT, not tested.
↵ g The assay ended at 16 h.
- TABLE 4.
Summary of DBT activities of codon 261 substitution mutants
Amino acid at DszC codon 261 Transformation of DBTa Nonpolar Ala + Val +++ Lcu +++ Ile +++ Met +++ Phe ++ Trp − Pro − Polar uncharged Asn − Gln − Gly − Ser − Thr +++ Cys ++ Tyr + Charged Lys − Arg − His − Asp − Glu − ↵ a Assayed as described in Materials and Methods. Results indicate percent activity of the wild type: +++, >75%; ++, 25 to 75%; +, 2 to 25%; −, <2%.
