Patricia C. Brzostowicz

mRNA differential display in a microbial enrichment culture: simultaneous identification of three cyclohexanone monooxygenases from three species.

ABSTRACT 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. Furthermore, in the case of Arthrobacter strain BP2, three other genes flanking the monooxygenase were identified by differential display, demonstrating that the repeated sampling of bacterial operons shown earlier for a pure culture (D. M. Walters, R. Russ, H. Knackmuss, and P. E. Rouvière, Gene 273:305-315, 2001) is also possible for microbial communities. The activity of the three cyclohexanone monooxygenases was confirmed and characterized following their expression in Escherichia coli.

Simultaneous Identification of Two Cyclohexanone Oxidation Genes from an Environmental Brevibacterium Isolate Using mRNA Differential Display

The technique of mRNA differential display was used to identify simultaneously two metabolic genes involved in the degradation of cyclohexanone in a new halotolerant Brevibacterium environmental isolate. In a strategy based only on the knowledge that cyclohexanone oxidation was inducible in this strain, the mRNA population of cells exposed to cyclohexanone was compared to that of control cells using reverse transcription-PCR reactions primed with a collection of 81 arbitrary oligonucleotides. Three DNA fragments encoding segments of flavin monooxygenases were isolated with this technique, leading to the identification of the genes of two distinct cyclohexanone monooxygenases, the enzymes responsible for the oxidation of cyclohexanone. Each monooxygenase was expressed in Escherichia coli and characterized. This work validates the application of mRNA differential display for the discovery of new microbial metabolic genes.

Identification of two gene clusters involved in cyclohexanone oxidation in Brevibacterium epidermidis strain HCU

Abstract.Brevibacteriumepidermidis HCU can grow on cyclic ketones and alcohols as a sole carbon source. We have previously reported the identification of two cyclohexanone-induced Bayer-Villiger monooxygenase genes by mRNA differential display. Using the related technique of Out-PCR, we have amplified large DNA fragments flanking the two monooxygenase genes. Two large gene clusters were sequenced. Several ORFs in each gene cluster encoded proteins homologous to cyclohexanol and cyclohexanone oxidation enzymes from Acinetobacter. However, the structure of these two gene clusters differs significantly from that of Acinetobacter, where the complete pathway has been described. To assess activity of these genes, they were cloned and expressed in Escherichia coli. In vivo and in vitro assays enabled us to assign functions to the expressed ORFs. These ORFs included a cyclohexanol dehydrogenase, two different ε-caprolactone hydrolases and two 6-hydroxyhexanoate dehydrogenases belonging to different enzyme families. Because this environmental isolate is difficult to manipulate, we cannot determine at this time which cluster is involved in the degradation of cyclohexanone under physiological conditions. However, the original differential display experiments and some of the experiments reported here suggest the involvement of both gene clusters in the oxidation of cyclic ketones.

Transcriptional Cross-Regulation of the Catechol and Protocatechuate Branches of the β-Ketoadipate Pathway Contributes to Carbon Source-Dependent Expression of the Acinetobacter sp. Strain ADP1 pobA Gene

ABSTRACT Transcriptional control of carbon source preferences by Acinetobacter sp. strain ADP1 was assessed with a pobA::lacZ fusion during growth on alternative substrates. The pobA-encoded enzyme catalyzes the first step in the degradation of 4-hydroxybenzoate, a compound consumed rapidly as a sole carbon source. If additional aromatic carbon sources are available, 4-hydroxybenzoate consumption is inhibited by unknown mechanisms. As reported here, during growth on aromatic substrates, pobA was not expressed despite the presence of 4-hydroxybenzoate, an inducer that normally causes the PobR regulator to activate pobA transcription. Growth on organic acids such as succinate, fumarate, and acetate allowed higher levels of pobA expression. In each case, pobA expression increased at the end of the exponential growth phase. Complex transcriptional regulation controlled 4-hydroxybenzoate catabolism in multisubstrate environments. Additional studies focused on the wild-type preference for benzoate consumption prior to 4-hydroxybenzoate consumption. These compounds are degraded via the catechol and protocatechuate branches of the β-ketoadipate pathway, respectively. Here, mutants were characterized that degraded benzoate and 4-hydroxybenzoate concurrently. These mutants lacked the BenM and CatM transcriptional regulators that normally activate genes for benzoate catabolism. A model is presented in which BenM and CatM prevent pobA expression indirectly during growth on benzoate. These regulators may affect pobA expression by lowering the PcaK-mediated uptake of 4-hydroxybenzoate. Consistent with this model, BenM and CatM bound in vitro to an operator-promoter fragment controlling the expression of several pca genes, including pcaK. These studies provide the first direct evidence of transcriptional cross-regulation between the distinct but analogous branches of the β-ketoadipate pathway.