Sphingomonas wittichii RW1 gene reporters interrogating the dibenzofuran metabolic network highlight conditions for early successful development in contaminated microcosms.

In order to better understand the fate and activity of bacteria introduced into contaminated material for the purpose of enhancing biodegradation rates, we constructed Sphingomonas wittichii RW1 variants with gene reporters interrogating dibenzofuran metabolic activity. Three potential promoters from the dibenzofuran metabolic network were selected and fused to the gene for enhanced green fluorescent protein (EGFP). The stability of the resulting genetic constructions in RW1 was examined, with plasmids based on the broad-host range vector pME6012 being the most reliable. One of the selected promoters, upstream of the gene Swit_4925 for a putative 2-hydroxy-2,4-pentadienoate hydratase, was inducible by growth on dibenzofuran. Sphingomonas wittichii RW1 equipped with the Swit_4925 promoter egfp fusion grew in a variety of non-sterile sandy microcosms contaminated with dibenzofuran and material from a former gasification site. The strain also grew in microcosms without added dibenzofuran but to a very limited extent, and EGFP expression indicated the formation of consistent small subpopulations of cells with an active inferred dibenzofuran metabolic network. Evidence was obtained for competition for dibenzofuran metabolites scavenged by resident bacteria in the gasification site material, which resulted in a more rapid decline of the RW1 population. Our results show the importance of low inoculation densities in order to observe the population development of the introduced bacteria and further illustrate that the limited availability of unique carbon substrate may be the most important factor impinging growth.