Solid-state 13C nuclear magnetic resonance spectroscopy of simultaneously metabolized acetate and phenol in a soil Pseudomonas sp

Abstract

We investigated concentration-dependent primary and secondary substrate relationships in the simultaneous metabolism of the ubiquitous pollutant phenol and the naturally occurring substrate acetate by a Pseudomonas sp. soil isolate capable of utilizing either substance as a sole source of carbon and energy. In addition to conventional analytical techniques, solid-state 13C nuclear magnetic resonance spectroscopy was used to follow the cellular distribution of [1-_3C]acetate in the presence of unlabeled phenol. With 5 mM acetate as the primary substrate, Pseudomonas sp. 9S8D2 removed 1 mM phenol (secondary substrate) at a rate of 2 nmol/mg of total cell protein. Although extensive acetate metabolism was indicated by a significant redistribution of the carboxyl label, this redistribution was not affected by the presence of phenol as a secondary substrate. When the primary and secondary substrate roles were reversed, however, the presence of 1 mM phenol altered the metabolism of 0.1 mM acetate, as evidenced by both the two-to fourfold increases in carboxyl label that appeared in terminal methyl and acyl chain methylene carbon resonances and the decrease in label that occurred in the carbohydrate spectral region. These results suggest that, when phenol is present as the primary substrate, acetate is preferentially shuttled into fatty acyl chain synthesis, whereas phenol carbon is funnelled into the tricarboxylic acid cycle. Thus, simultaneous use of a xenobiotic compound and a natural substrate apparently does occur, and the relative concentrations of the two substrates do influence the rate and manner in which the compounds are utilized. These results also demonstrate the unique advantage of using solid-state nuclear magnetic resonance techniques combined with 13C labeling of specific sites in substrates when doing microbial degradation studies. In this work, the entire cellular biomass was examined directly without extensive extraction, fractionation, or isolation of subcellular units; thus, there is no uncertainty about chemical alteration of substrate metabolites as a result of these often harsh treatments.

Characterization of a Pseudomonas sp. Capable of Aniline Degradation in the Presence of Secondary Carbon Sources

“…In several other aniline-degrading microbes, catabolism of aniline is repressed by the presence of other organic substrates (12, 37). Similar phenomena have been observed with other aromatic substrates (11,27). However, strain Kl was able to synthesize catechol dioxygenase and to metabolize aniline in the presence of high concentrations of lactate.…”

Kinetics of Growth and Aniline Degradation by Stenotrophomonas maltophilia

“…In several anilinedegrading microorganisms, aniline catabolism is repressed by the presence of other organic substrates (Aoki et aI., 1984;Helm and Reber] 979;Nakanishi et aI., 1991;and Zeyer et aI., 1985). Similar phenomena have been observed with other aromatic substrates (Heiman andCooper, 1987, andSchmidt andAlexander, 1985). However, there are some microorganisms that metabolize aniline in the presence of a high concentration of organic substrates (Aoki et aI., 1983, andKonopca et aI., 1989).…”

“…When more than one substrate is used for growth, the microorganism may use the compounds simultaneously or sequentially (diauxic growth). Helm and Reber (1979) reported that when Pseudomonas multivorans strain AnI cultivated with a mixture of aniline and a particular additional carbon source (glucose, 4-hydroxybenzoate, and succinate), diauxic growth was observed be- The left side of equation 7 versus 5, was plotted and Y X1S1 was determined as 0.894 mg cells/mg aniline from the slope of the line ( Figure 5).…”

Effects of co-occurring aromatic hydrocarbons on degradation of individual polycyclic aromatic hydrocarbons in marine sediment slurries

“…Previous studies have examined both naturally occurring and xenobiotic compound utilization as co-occurring primary and secondary substrates in pure cultures. When phenol was the primary (i.e., present at the higher concentration) substrate, acetate metabolism was altered so that it was shuttled to fatty acyl synthesis by a soil pseudomonad (18). This result was not obtained when phenol was the secondary substrate.…”