ABSTRACT
Exploration of metabolically diverse rhodococci is generally hampered by the lack of genetic tools. A small cryptic plasmid (pAN12) isolated from Rhodococcus erythropolis strain AN12 was sequenced. Plasmid pAN12 encodes proteins that share homology to replication proteins and putative cell division proteins. Based on in vitro transposon mutagenesis, we determined that the Rep protein of pAN12 is essential for plasmid replication in Rhodococcus spp., and the putative cell division protein Div is important for plasmid stability. The pAN12 replicon is able to replicate in R. erythropolis strains AN12 and CW23 (ATCC 47072) and is compatible with the nocardiophage Q4 replicon present on a Rhodococcus shuttle plasmid pDA71. pAN12 appears to belong to the pIJ101/pJV1 family of rolling circle replication plasmids. Expression of an isoprenoid pathway gene ( dxs) on the pAN12-derived multicopy shuttle vector increased production of carotenoid pigments in R. erythropolis ATCC 47072.
Subject(s)
Genetic Vectors , Plasmids/genetics , Rhodococcus/genetics , Adenosine Triphosphatases/genetics , Amino Acid Sequence , Base Sequence , Cloning, Molecular , DNA Helicases/genetics , Gene Expression Regulation, Bacterial , Models, Genetic , Molecular Sequence Data , Plasmids/analysis , Plasmids/isolation & purification , Sequence AlignmentABSTRACT
Biological oxidation of cyclic ketones normally results in formation of the corresponding dicarboxylic acids, which are further metabolized in the cell. Rhodococcus ruber strain SC1 was isolated from an industrial wastewater bioreactor that was able to utilize cyclododecanone as the sole carbon source. A reverse genetic approach was used to isolate a 10-kb gene cluster containing all genes required for oxidative conversion of cyclododecanone to 1,12-dodecanedioic acid (DDDA). The genes required for cyclododecanone oxidation were only marginally similar to the analogous genes for cyclohexanone oxidation. The biochemical function of the enzymes encoded on the 10-kb gene cluster, the flavin monooxygenase, the lactone hydrolase, the alcohol dehydrogenase, and the aldehyde dehydrogenase, was determined in Escherichia coli based on the ability to convert cyclododecanone. Recombinant E. coli strains grown in the presence of cyclododecanone accumulated lauryl lactone, 12-hydroxylauric acid, and/or DDDA depending on the genes cloned. The cyclododecanone monooxygenase is a type 1 Baeyer-Villiger flavin monooxygenase (FAD as cofactor) and exhibited substrate specificity towards long-chain cyclic ketones (C11 to C15), which is different from the specificity of cyclohexanone monooxygenase favoring short-chain cyclic compounds (C5 to C7).
Subject(s)
Genes, Bacterial , Hydrocarbons, Alicyclic/metabolism , Multigene Family , Oxygenases/genetics , Oxygenases/physiology , Rhodococcus/metabolism , Amino Acid Sequence , Chromosome Mapping , Cloning, Molecular , Cosmids/genetics , Escherichia coli/genetics , Flavin-Adenine Dinucleotide/analysis , Models, Chemical , Molecular Sequence Data , Oxidation-Reduction , Oxygenases/metabolism , Rhodococcus/genetics , Rhodococcus/growth & development , Sequence Homology, Amino Acid , Substrate SpecificityABSTRACT
Biological oxidation of cyclic alcohols normally results in formation of the corresponding dicarboxylic acids, which are further metabolized and enter the central carbon metabolism in the cell. We isolated an Acinetobacter sp. from an industrial wastewater bioreactor that utilized cyclohexanol as a sole carbon source. A cosmid library was constructed from Acinetobacter sp. strain SE19, and oxidation of cyclohexanol to adipic acid was demonstrated in recombinant Escherichia coli carrying a SE19 DNA segment. A region that was essential for cyclohexanol oxidation was localized to a 14-kb fragment on the cosmid DNA. Several putative open reading frames (ORFs) that were expected to encode enzymes catalyzing the conversion of cyclohexanol to adipic acid were identified. Whereas one ORF showed high homology to cyclohexanone monooxygenase from Acinetobacter sp. strain NCIB 9871, most of the ORFs showed only moderate homology to proteins in GenBank. In order to assign functions of the various ORFs, in vitro transposon mutagenesis was performed using the cosmid DNA as a target. A set of transposon mutants with a single insertion in each of the ORFs was screened for cyclohexanol oxidation in E. coli. Several of the transposon mutants accumulated a variety of cyclohexanol oxidation intermediates. The in vitro transposon mutagenesis technique was shown to be a powerful tool for rapidly assigning gene functions to all ORFs in the pathway.
Subject(s)
Acinetobacter/genetics , Adipates/metabolism , Bacterial Proteins , Cyclohexanols/metabolism , Genes, Bacterial , Multigene Family , Acinetobacter/growth & development , Acinetobacter/isolation & purification , Aldehyde Oxidoreductases/genetics , Base Sequence , Cloning, Molecular , Cosmids , DNA, Bacterial , Molecular Sequence Data , Oxidation-Reduction , Trans-Activators/geneticsABSTRACT
A CryV-type protein (CGCryV) has been isolated from supernatant fluids of Bacillus thuringiensis AB88 cultures. Previous reports have suggested the cryptic nature of the cryV-type genes on the basis of the absence of CryV-type proteins in parasporal crystals. The CryV-type protein reported here is expressed early in stationary phase, and evidence indicates that it is an exported protein. Analysis of the deduced protein sequence from this gene reveals the presence of an N-terminal domain that likely acts as a signal peptide. The CGCryV protein is the first reported case of a delta-endotoxin being a secreted protein, which may influence the biological relevance of these proteins.