Expression and transfer of engineered catabolic pathways harbored by Pseudomonas spp. introduced into activated sludge microcosms

Abstract

Two genetically engineered microorganisms (GEMs), Pseudomonas sp. strain B13 FR1(pFRC20P) (FR120) and Pseudomonas putida KT2440(pWWO-EB62) (EB62), were introduced into activated sludge microcosms that had the level of aeration, nutrient makeup, and microbial community structure of activated sludge reactors. FR120 contains an experimentally assembled ortho cleavage route for simultaneous degradation of 3-chlorobenzoate (3CB) and 4-methyl benzoate (4MB); EB62 contains a derivative TOL plasmid-encoded degradative pathway for toluene experimentally evolved so that it additionally processes 4-ethyl benzoate (4EB). Experiments assessed survival of the GEMs, their ability to degrade target substrates, and lateral transfer of plasmid-encoded recombinant DNA. GEMs added at initial densities of 106 to 107 bacteria per ml of activated sludge declined to stable population densities of 104 to 105 bacteria per ml. FR120 degraded combinations of 3CB and 4MB (1 mM each) following 3 days of adaptation in the microcosms. Indigenous microorganisms required an 8-day adaptation period before degradation of 4MB was observed; 3CB was degraded only after the concentration of 4MB was much reduced. The indigenous microbial community was killed when both compounds were present at concentrations of 4.0 mM. However, in parallel microcosms containing FR120, the microbial community maintained a normal density of viable cells. Indigenous microbes readily degraded 4EB (2 mM), and EB62 did not significantly increase the observed rate of degradation. In filter matings, transfer of pFRC20P, which specifies mobilization but not transfer functions, from FR120 to P. putida UWC1 was not detectable (<10-7 transconjugants per donor cell). In contrast, pWWO-EB62, a TOL plasmid derivative which contains all functions necessary for conjugation, transferred to P. putida UWC1 at a frequency of 10-1 transconjugant per donor cell. Within the microcosms, pWWO-EB62 transferred readily to P. putida UWC1; transconjugants reached a density of approximately 103 bacteria per ml, appearing twice as fast with 4EB present as in its absence. Transconjugants arising from transfer of pFRC20P to P. putida UWC1 were rarely observed.