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 (C 11 to C 15 ), which is different from the specificity of cyclohexanone monooxygenase favoring short-chain cyclic compounds (C 5 to C 7 ).

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.

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 ␦-endotoxin being a secreted protein, which may influence the biological relevance of these proteins.The insecticidal properties of Bacillus thuringiensis are associated with parasporal inclusions that are formed during sporulation. These inclusions are composed of proteins which exhibit highly specific insecticidal activities against larvae of lepidoptera, coleoptera, and/or diptera. These proteins are known as ␦-endotoxins because of their intracellular location and as Cry proteins because of the crystalline nature of their inclusions. The Cry proteins have been classified as CryI, -II, -III, or -IV depending on the host specificity as well as the degree of amino acid homology (9). Various authors have proposed additions to the Höfte and Whiteley nomenclature (9) as new endotoxins have been discovered. Thus, Chambers et al. (5) identified a partial sequence of a new insecticidal protein that was designated CryIX because it had 62% sequence homology with CryIB yet was significantly distinct. Tailor et al. (15) reported a new class of crystal protein gene whose encoded protein could not be found in preparations of solubilized crystal proteins from B. thuringiensis. This gene was designated cryV on the basis of the following criteria: (i) while it had 62% sequence homology with CryIB, the new protein was significantly distinct from other characterized crystal proteins; and (ii) when expressed in Escherichia coli, the encoded 81-kDa protein showed dual specificity, with insecticidal activity against lepidopterans (Ostrinia nubilalis) and coleopterans (Leptinotarsa decimlineata). A comparison of the cryIX and cryV genes showed them to be very similar.Recently, other groups (6, 13) have reported the cloning and characterization of cryV-type genes in different strains of B. thuringiensis. Although DNA dot blot analysis and/or PCR analysis has confirmed the presence of cryV-type genes in a large proportion of B. thuringiensis strains (13), cryV-encoded proteins have not been found in crystal preparations. cryV genes are generally located approximately 500 bp downstream of cryI-type genes, but cryV gene expression is thought to be absent because these genes lack an upstream promoter-like sequence (13,15). Thus, known cryV-type genes are reported to be silent in their host, B. thuringiensis.In this paper we present data demonstrating that B. thuringiensis AB88 expresses a CryV-type protein in early stationary phase and that this protein is secreted i...

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.