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...

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SUMMARY The DF signal molecule regulates the production of both yellow pigments (xanthomonadins) and extracellular polysaccharide (EPS) in Xanthomonas campestris pv. campestris. These two bacterial products are crucial to the epiphytic survival and pathogenicity of this pathogen on its plant hosts. Previous work suggested that DF is a butyrolactone, which the Streptomyces bacteria are known to utilize as signals. pigB is one of seven transcriptional units in the X. c. pv. campestris xanthomonadin gene cluster, and its inactivation results in the loss of DF signal, xanthomonadin and EPS production. Here, determination and analysis of the pigB DNA sequence reveals the presence of two open reading frames, the first (xanB1) encoding a putative reductase/halogenase, and the second (xanB2) showing the highest level of identity to Streptomyces genes encoding putative pteridine-dependent dioxygenase-like proteins. We show that xanB2 (but not xanB1) is needed for production of the DF signal, and that some Streptomyces strains produce functional analogues of DF. A role for xanB2 in the biosynthesis of DF is proposed.