Signature-tagged mutagenesis (STM) was used to identify genetic determinants of fitness associated with two key ecological processes mediated by bacteria. Burkholderia vietnamiensis strain G4 was used as a model bacterium to investigate: phenol degradation as a model of bioremediation, and pea rhizosphere colonization as a prerequisite to biological control and phytoremediation. A total of 1900 mutants were screened and 196 putative fitness mutants identified; the genetic basis of 137 of these mutations was determined by correlation to the G4 genome. The phenol-STM screen was more successful at identifying phenol degradation mutations (83 mutants; 4.4% hit rate) than a conventional agar-based phenol screen (49 mutants, 5319 screened, 0.92% hit rate). The combination of both screens completely defined the components of the TOM pathway in strain G4 and also identified novel accessory genes not previously implicated in phenol utilization. The rhizosphere-STM screen identified 113 mutants (5.9% hit rate); 107 had reduced tag signals indicative of poor rhizosphere colonization (Rhiz-), while six mutants produced high hybridization signals suggesting increased rhizosphere competence (Rhiz+). Competition assays confirmed that 69% of Rhiz- mutants tested (24/35) were severely compromised in their rhizosphere fitness. Seventy Rhiz- mutations mapped to genes with the following putative functions: amino acid biosynthesis (25; 36%), general metabolism (18; 26%), hypothetical (9; 13%), regulatory genes (4; 5.7%), transport and stress (2 each; 2.8% respectively). One of the most interesting discoveries mediated by the rhizosphere-STM screen was the identification of three Rhiz+ mutants inactivated within a single virulence-associated autotransporter adhesin gene; this mutation consistently produced a hyper-colonization phenotype suggesting a highly novel role for this surface adhesin during plant interactions. Our study has shown that STM can be successfully applied to ecologically important microbial interactions, defining the underlying genetic systems important for biotechnological fitness of environmental bacteria such those from the Burkholderia cepacia complex.