The small-molecule biosynthetic diversity encoded within the genomes of uncultured bacteria is an attractive target for the discovery of natural products using functional metagenomics. Phenotypes commonly associated with the production of small molecules, such as antibiosis, altered pigmentation, or altered colony morphology, are easily identified from screens of arrayed metagenomic library clones. However, functional metagenomic screening methods are limited by their intrinsic dependence on a heterologous expression host. Toward the goal of increasing the small-molecule biosynthetic diversity found in functional metagenomic studies, we report the phenotypic screening of broad-host-range environmental DNA libraries in six different proteobacteria: Agrobacterium tumefaciens, Burkholderia graminis, Caulobacter vibrioides, Escherichia coli, Pseudomonas putida, and Ralstonia metallidurans. Clone-specific small molecules found in culture broth extracts from pigmented and antibacterially active clones, as well as the genetic elements responsible for the biosynthesis of these metabolites, are described. The host strains used in this investigation provided access to unique sets of clones showing minimal overlap, thus demonstrating the potential advantage conferred on functional metagenomics through the use of multiple diverse host species.Uncultured bacteria are predicted to be a significant reservoir of novel small-molecule biosynthetic machinery (19,34). One means by which to access the biosynthetic potential contained within the genomes of uncultured bacteria is functional metagenomics (19). This approach involves cloning DNA directly from naturally occurring microbial populations (environmental DNA [eDNA]) and screening the resulting clone libraries for phenotypes traditionally associated with the production of secondary metabolites. A major limitation of functional metagenomics is its reliance on a foreign host to facilitate the expression of eDNA-derived genes and gene clusters (17). Codon bias, missing substrates, and the inability to recognize foreign regulatory elements, including promoters and ribosomal binding sites, are just some of the obstacles that are likely to limit the success of expression-dependent studies with any single host organism. Circumventing these obstacles through an expansion of the collection of hosts available for functional metagenomic studies should increase the efficacy of this approach.Soil ecosystems are rich in bacterial diversity, and the majority of soil-dwelling bacteria remain recalcitrant to standard microbial culture methods (33, 39). Large-scale metagenomic sequencing studies indicate that soil microbiomes are often dominated by five bacterial phyla: Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Acidobacteria, and Actinobacteria (14). Bacteria from these common phyla are therefore appealing hosts for use in functional metagenomic studies of soil-derived eDNA libraries.In this study, six unique bacterial hosts [Agrobacterium tumefaciens (Alphaproteobacteria), ...