A new family of transcriptional regulators of tungstoenzymes and molybdate/tungstate transport.

Bacterial genes for molybdenum-containing and tungsten-containing enzymes are often differentially regulated depending on the metal availability in the environment. Here, we describe a new family of transcription factors with an unusual DNA-binding domain related to excisionases of bacteriophages. These transcription factors are associated with genes for various molybdate and tungstate-specific transporting systems as well as molybdo/tungsto-enzymes in a wide range of bacterial genomes. We used a combination of computational and experimental techniques to study a member of the TF family, named TaoR (for tungsten-containing aldehyde oxidoreductase regulator). In Desulfovibrio vulgaris Hildenborough, a model bacterium for sulfate reduction studies, TaoR activates expression of aldehyde oxidoreductase aor and represses tungsten-specific ABC-type transporter tupABC genes under tungsten-replete conditions. TaoR binding sites at aor promoter were identified by electrophoretic mobility shift assay and DNase I footprinting. We also reconstructed TaoR regulons in 45 Deltaproteobacteria by comparative genomics approach and predicted target genes for TaoR family members in other Proteobacteria and Firmicutes.

Generalized schemes for high-throughput manipulation of the Desulfovibrio vulgaris genome.

The ability to conduct advanced functional genomic studies of the thousands of sequenced bacteria has been hampered by the lack of available tools for making high-throughput chromosomal manipulations in a systematic manner that can be applied across diverse species. In this work, we highlight the use of synthetic biological tools to assemble custom suicide vectors with reusable and interchangeable DNA "parts" to facilitate chromosomal modification at designated loci. These constructs enable an array of downstream applications, including gene replacement and the creation of gene fusions with affinity purification or localization tags. We employed this approach to engineer chromosomal modifications in a bacterium that has previously proven difficult to manipulate genetically, Desulfovibrio vulgaris Hildenborough, to generate a library of over 700 strains. Furthermore, we demonstrate how these modifications can be used for examining metabolic pathways, protein-protein interactions, and protein localization. The ubiquity of suicide constructs in gene replacement throughout biology suggests that this approach can be applied to engineer a broad range of species for a diverse array of systems biological applications and is amenable to high-throughput implementation.