Biodegradation of dimethylsilanediol in soils.

The biodegradation potential of [14C]dimethylsilanediol, the monomer unit of polydimethylsiloxane, in soils was investigated. Dimethylsilanediol was found to be biodegraded in all of the tested soils, as monitored by the production of 14CO2. When 2-propanol was added to the soil as a carbon source in addition to [14C]dimethylsilanediol, the production of 14CO2 increased. A method for the selection of primary substrates that support cometabolic degradation of a target compound was developed. By this method, the activity observed in the soils was successfully transferred to liquid culture. A fungus, Fusarium oxysporum Schlechtendahl, and a bacterium, an Arthrobacter species, were isolated from two different soils, and both microorganisms were able to cometabolize [14C]dimethylsilanediol to 14CO2 in liquid culture. In addition, the Arthrobacter sp. that was isolated grew on dimethylsulfone, and we believe that this is the first reported instance of a microorganism using dimethylsulfone as its primary carbon source. Previous evidence has shown that polydimethylsiloxane is hydrolyzed in soil to the monomer, dimethylsilanediol. Now, biodegradation of dimethylsilanediol in soil has been demonstrated.

Novel pathway for bacterial metabolism of bisphenol A. Rearrangements and stilbene cleavage in bisphenol A metabolism.

Bisphenol A (BPA) is metabolized by a Gram-negative aerobic bacterium via a novel pathway involving oxidative skeletal rearrangement of the BPA. Oxidation of the aliphatic methyl group of BPA leads to coproduction of the methyl-hyroxylated 2,2-bis(4-hydroxyphenyl)-1-propanol and a skeletally rearranged triol 1,2-bis(4-hydroxyphenyl)-2-propanol. The major route of metabolism (> 80%) is through the rearrangement. The 1,2-bis(4-hydroxyphenyl)-2-propanol is dehydrated to 4,4'-dihydroxy-alpha-methylstilbene, which is rapidly cleaved by oxidation to 4-hydroxybenzaldehyde and 4-hydroxyacetophenone. 4-Hydroxybenzaldehyde is oxidized to 4-hydroxybenzoic acid. Both 4-hydroxybenzoic acid and 4-hydroxyacetophenone are mineralized. The minor product of BPA hydroxylation, 2,2-bis(4-hydroxyphenyl)-1-propanol, is further oxidized to form both 2,2-bis(4-hydroxyphenyl)propanoic acid and a skeletally rearranged tetraol, 2,3-bis(4-hydroxyphenyl)-1,2-propanediol. As is the case in the hydroxylation of BPA, the major product is skeletally rearranged. 2,3-Bis(4-hydroxyphenyl)-1,2-propanediol is slowly transformed to 4-hydroxyphenacyl alcohol.

Biodegradation of bisphenol A and other bisphenols by a gram-negative aerobic bacterium.

A novel bacterium designated strain MV1 was isolated from a sludge enrichment taken from the wastewater treatment plant at a plastics manufacturing facility and shown to degrade 2,2-bis(4-hydroxyphenyl)propane (4,4'-isopropylidenediphenol or bisphenol A). Strain MV1 is a gram-negative, aerobic bacillus that grows on bisphenol A as a sole source of carbon and energy. Total carbon analysis for bisphenol A degradation demonstrated that 60% of the carbon was mineralized to CO2, 20% was associated with the bacterial cells, and 20% was converted to soluble organic compounds. Metabolic intermediates detected in the culture medium during growth on bisphenol A were identified as 4-hydroxybenzoic acid, 4-hydroxyacetophenone, 2,2-bis(4-hydroxyphenyl)-1-propanol, and 2,3-bis(4-hydroxyphenyl)-1,2-propanediol. Most of the bisphenol A degraded by strain MV1 is cleaved in some way to form 4-hydroxybenzoic acid and 4-hydroxyacetophenone, which are subsequently mineralized or assimilated into cell carbon. In addition, about 20% of the bisphenol A is hydroxylated to form 2,2-bis(4-hydroxyphenyl)-1-propanol, which is slowly biotransformed to 2,3-bis(4-hydroxyphenyl)-1,2-propanediol. Cells that were grown on bisphenol A degraded a variety of bisphenol alkanes, hydroxylated benzoic acids, and hydroxylated acetophenones during resting-cell assays. Transmission electron microscopy of cells grown on bisphenol A revealed lipid storage granules and intracytoplasmic membranes.

Isolation and characterization of a small catalytic domain released from the adenylate cyclase from Escherichia coli by digestion with trypsin.

An expression plasmid containing a hybrid gene encoding a protein having the primary amino acid sequence of the adenylate cyclase from Escherichia coli was constructed. When the gene was induced, the adenylate cyclase could be expressed at high levels in a cya- strain of E. coli. The majority of the enzymatic activity and protein (having a molecular weight of 95,000) induced was insoluble. However, treatment of the insoluble fraction of cell lysates with trypsin resulted in both an increase in and solubilization of the total amount of adenylate cyclase activity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the soluble protein produced by treatment with trypsin revealed a polypeptide having a molecular weight of 30,000. This soluble, catalytically active fragment of adenylate cyclase was purified and subjected to amino-terminal sequence analyses; two amino-terminal sequences were identified beginning at residue 82 and at residue 342 of the intact enzyme. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the purified fragment followed by either silver or Coomassie Blue staining revealed the presence of only a single polypeptide having a molecular weight of 30,000; a short oligopeptide associated with the amino terminus at residue 342 could not be detected. Site-directed mutagenesis was used to place a stop codon at residue 341; the truncated enzyme was catalytically active, so the short oligopeptide is not necessary for catalysis. The Km for ATP, the Ka for Mg2+, and the Vmax determined for the product containing the 30,000-dalton fragment were similar to the values reported for the intact enzyme from E. coli.

Evidence for a small catalytic domain in the adenylate cyclase from Salmonella typhimurium.

Deletions of large portions of the carboxyl-terminal end of the adenylate cyclase (ATP pyrophosphate lyase (cyclizing), EC 4.6.1.1) from Salmonella typhimurium do not significantly affect the enzymatic activity exhibited by the shortened polypeptide. The deletion mutations were generated by nuclease Bal31 digestion from the 3'-end of the cya gene fragment cloned by Wang et al. (Wang, J. Y.-J., Clegg, D. O., and Koshland, D.E. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 4684-4688); the shortened cya genes were inserted in pBR322 and used to transform a cya- strain of Escherichia coli. The original gene fragment encodes for an enzymatically active polypeptide having an apparent molecular weight of 77,000. Mutant polypeptides as small as 46,000 Da were found to retain significant enzymatic activity and to confer several cya+ phenotypes on the E. coli host. More extensive deletions resulting in polypeptides as small as 33,000 Da did not have assayable amounts of adenylate cyclase activity, but the biochemical properties of the transformed cya- host implicate the presence of low levels of enzymatic activity. These data suggest that the structure of the intact enzyme is composed of discrete functional domains. Such a structure for this adenylate cyclase should both facilitate investigations of the chemical mechanism of the reaction and allow structure-function relationships in this physiologically important enzyme to be investigated on a molecular level.