Phospholipids of Five Pseudomonad Archetypes for Different Toluene Degradation Pathways

Liquid chromatography/electrospray ionization/mass spectrometry (LC/ESI/MS) was used to determine intact phospholipid profiles for five reference pseudomonad strains harboring different (aerobic) toluene catabolic pathways: Pseudomonas putida mt-2, Pseudomonas putida F1, Burkholderia cepacia G4, Burkholderia pickettii PKO1, and Pseudomonas mendocina KR1. These five strains contained a predominant pool of phosphatidylethanolamines. Other phospholipids identified include phosphatidylglycerol, phosphatidylserine, phosphatidylmethylethanolamine, and phosphatidyldimethylethanolamine. There was a clear separation in phospholipid profiles that allows for the differentiation between the Pseudomonas and Burkholderia genera. Factor analysis of the phospholipid profiles showed that B. cepacia G4, P. putida mt-2, and B. pickettii PKO1 were clearly separated, while P. putida F1 and P. mendocina KR1 were clustered as a group. These results suggest that intact phospholipid profiling could be used to evaluate the relative abundance of specific degraders in bioreactors or in aquifer material. Nevertheless, the usefulness of this technique for taxonomic characterization of such complex samples remains to be demonstrated because of potential confounding effects of overlapping profiles and potential changes in phospholipid composition due to different growth conditions. Corresponding author: Tel: (734) 936-3177; Fax: (734) 763-6513; E-mail: [email protected] Introduction The monoaromatic hydrocarbons, benzene, toluene, ethylbenzene, and xylenes (BTEX), are common environmental contaminants that represent a serious threat to groundwater resources and to public health (Tursman and Cork, 1992). Microbial degradation of these contaminants is widely regarded as a cost-effective approach to clean up BTEX-contaminated aquifers (National Research Council, 1994). Indeed, considerable progress has been made toward understanding and managing hydrogeochemical factors that influence the success of BTEX bioremediation. Nevertheless, process optimization is limited by our incomplete understanding of the diversity and prevalence of specific biochemical processes. Microbial catabolic diversity is reflected in the fact that different pseudomonad strains can degrade toluene by five different oxygen-dependent pathways, which are named after the operons that code them (Zylstra, 1994). The TOL pathway, which was first discovered in Pseudomonas putida (arvilla) mt-2, is coded in the tol pWW0 plasmid. The TOD pathway is expressed by P. putida F1, which uses toluene dioxygenase to convert toluene into cis-toluene dihydrodiol (Gibson et al., 1968), which is subsequently dehydrogenated to 3-methylcatechol. The TOM pathway occurs in B. cepacia G4 (formerly known as Pseudomonas cepacia G4), which uses toluene omonooxygenase in the initial attack to form o-cresol (Shields and Montgomery, 1989). The TBU pathway occurs in B. pickettii PK01 (formerly known as Pseudomonas pickettii PK01), which hydroxylates the ring using toluene m-monooxygenase to yield m-cresol (Kukor and Olson, 1996). Finally, the TMO pathway is expressed by P. mendocina KR1, which uses toluene p-monooxygenase to yield p-cresol (Whited and Gibson, 1991). These five reference strains exhibit different substrate ranges and kinetic properties. For example, the