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 ␤-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 ␤-ketoadipate 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...

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.It is now widely accepted that the diversity of microorganisms extends far beyond the few thousand species in culture collections (15). This diversity of microbes and their metabolism constitutes a vast source of enzymes and genes for biotechnology applications. The identification of useful metabolic genes has traditionally proceeded either through a direct genetic approach or by the reverse genetics approach, starting with the purification of the enzyme of interest followed by identification of its gene through the use of antibodies or amino acid sequence information obtained from the pure protein.Although both strategies are routinely used, they are often limited by technical problems. The direct genetic approach can be used only for organisms that have a developed genetic system or whose genes can be expressed in heterologous hosts. The reverse genetics approach requires purification of the protein of interest, which often takes a long time, and the successful amplification of a DNA probe from degenerate primers, a technique that sometimes fails. Recently mRNA techniques have made it possible to access regulated genes directly without the purification of their gene products and in the absence of a genetic system. These approaches are based on comparison of the mRNA population between two cultures or tissues and identification of the subset of genes whose mRNA is more abundant under conditions of induction. These techniques rely on the hybridization of labeled mRNAs onto arrays of DNA on membranes (4) or DNA microarrays (9), large-scale sample sequencing of expressed sequence tag libraries (28), or the sampling of mRNA by the production of randomly amplified DNA fragments by reverse transcription (RT) followed by PCR (RT-PCR) (19,20,35). Because it can easily be done by individual scientists at low cost, the latter approach has been used extensively since it was first described.Two variations of this RT-PCR method have been published. The first, called differential display (DD) (19,20), begins with the synthesis of cDNAs by RT of mRNA using a poly(dT) primer that hybridizes to the poly(A) tail o...