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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.

mRNA differential display has been used to identify cyclohexanone oxidation genes in a mixed microbial community derived from a wastewater bioreactor. Thirteen DNA fragments randomly amplified from the total RNA of an enrichment subculture exposed to cyclohexanone corresponded to genes predicted to be involved in the degradation of cyclohexanone. Nine of these DNA fragments are part of genes encoding three distinct Baeyer-Villiger cyclohexanone monooxygenases from three different bacterial species present in the enrichment culture. In Arthrobacter sp. strain BP2 and Rhodococcus sp. strain Phi2, the monooxygenase is part of a gene cluster that includes all the genes required for the degradation of cyclohexanone, while in Rhodococcus sp. strain Phi1 the genes surrounding the monooxygenase are not predicted to be involved in this degradation pathway but rather seem to belong to a biosynthetic pathway. It is now well recognized that the diversity of microbial species and their metabolic capabilities constitute a tremendous source of biocatalysts (6,10,39). Only a small fraction of microorganisms in most environments can be readily isolated (1, 58); therefore, gene discovery techniques which overcome the need for strain isolation provide access to the diversity of microbial chemistry. Direct cloning approaches can be very successful (21,27,28,48), but they require a genetic selection or an easy screen as well as the efficient expression of the cloned DNA in an appropriate host (15). Other approaches, based on PCR amplification from environmental DNA, target only highly conserved gene families (50). While these techniques are powerful, they often are not applicable. Differential display (DD) is an alternate technique that can be used for the discovery of bacterial genes, requiring neither a genetic selection or screen nor the presence of highly conserved genes. This technique of DD involves the reproducible amplification of DNA fragments from the mRNA population at arbitrary sites by reverse transcription (RT) followed by PCR (RT-PCR) (36,37,57). DD is used to compare the mRNA pools from cells grown under different physiological conditions. Genes expressed at the same level in all cultures will be amplified equally from all cultures, while genes expressed only under a specific condition will give rise to RT-PCR bands only under that condition. DD is a gene discovery technique that can be applied to identify differentially expressed genes. It does not rely on prior knowledge of the genes targeted or on a genomic sequence but only on the fact that the activity that these genes encode is inducible.DD has been applied extensively to eukaryotic systems and takes advantage of the poly(A) tails of eukaryotic mRNA by using poly(dT) primers to synthesize cDNAs by RT (36,37,57). This approach of DD cannot be applied to prokaryotes, which lack stable poly(A) tails. A second variation of DD uses arbitrary oligonucleotide primers to initiate RT of the message at random sites (57) and thus can be applied to archaeal and bacterial s...

A series of alterations in the Bacillus amyloliquefaciens levansucrase signal peptide were made by in vitro mutagenesis, and their effect on the secretion of levansucrase in Bacillus subtilis was studied. Some of the alterations resulted in a completely defective signal peptide. These included the removal of positively charged residues from the N-terminus and disruption of the hydrophobic core of the signal peptide either by introducing a charged residue or by deleting five or more amino acids. Analysis of the signal peptide processing-site alterations revealed that small residues are preferred at the -1 and -3 positions. However, a wide variety of amino acids are tolerated at the + 1 position.Most of our current knowledge on protein secretion in bacteria is based on studies on Escherichia coli. Signal sequence mutants of E. coli have aided both in understanding the role of signal sequences in the secretion process and in identifying E. coli genes involved in protein secretion (1,3,5,25). The mechanism of protein secretion in Bacillus subtilis has not been extensively studied, and few signal sequence mutants of B. subtilis have been reported (18,21). We were interested in determining whether signal sequence mutants of B. subtilis will have the same phenotype as analogous mutants of E. coli. The Bacillus signal peptides have some of the common features observed in signal peptides from both procaryotes and eucaryotes (22,28,30). The signal peptides of Bacillus secreted proteins are 28 to 34 residues long; the number of positively charged residues at the N-terminus varies from two to five, and the hydrophobic core consists of 12 to 18 residues. The signal peptide processing site is between two alanine residues in the majority of cases.Bacillus amyloliquefaciens levansucrase signal peptide was chosen as a model Bacillus signal peptide for structurefunction studies for several reasons. (i) The levansucrase gene is inducible in B. subtilis, and thus studies of signal sequence mutations that may be lethal to B. subtilis should be feasible (27). (ii) The conversion of levansucrase precursor to mature protein involves a single processing event (signal peptide cleavage); this is in contrast to the multiple processing events involved in the conversion of precursor to mature protein for several Bacillus secreted proteins (protease and RNase) due to the presence of a propeptide between the signal peptide and mature protein (19,29). Thus, construction of levansucrase signal peptide processing site mutants is feasible. (iii) A genetic system to isolate suppressors of signal sequence mutants based on the secretion of levansucrase can be developed.We earlier reported the isolation of the levansucrase gene from B. amyloliquefaciens (sacB [BamP]) and its sucroseinducible expression in B. subtilis (27). The levansucrase signal peptide is 29 amino acids long and has three positively * Corresponding author. t Present address: Department of Microbiology, The Technical University of Denmark, Bygning 221, DK2800 Lyngby, Denmark. charged...

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Industrial wastewater bioreactors are potentially important sources of novel biocatalysts. However, the microbial populations in these bioreactors are not well characterized. The microbial community in an industrial wastewater bioreactor was surveyed by extracting DNA from a sample of activated sludge, followed by PCR amplification and sequencing of cloned 16S rRNA genes. A total of 407 cloned 16S rRNA gene sequences were compared with 88 bacterial isolates cultured from the same sample of sludge using a variety of standard media. Most of the bacteria detected by the PCR-based approach were beta-subdivision Proteobacteria, whereas most of the cultured bacteria were gamma-subdivision Proteobacteria. Only a few types of bacteria were detected by both approaches. These observations indicate that multiple techniques are necessary to characterize the microbial diversity in any complex ecosystem.

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.