Identification of a novel metabolite in the degradation of pyrene by Mycobacterium sp. strain AP1: actions of the isolate on two- and three-ring polycyclic aromatic hydrocarbons

Abstract

Mycobacterium sp. strain AP1 grew with pyrene as a sole source of carbon and energy. The identification of metabolites accumulating during growth suggests that this strain initiates its attack on pyrene by either monooxygenation or dioxygenation at its C-4, C-5 positions to give trans- or cis-4,5-dihydroxy-4,5-dihydropyrene, respectively. Dehydrogenation of the latter, ortho cleavage of the resulting diol to form phenanthrene 4,5-dicarboxylic acid, and subsequent decarboxylation to phenanthrene 4-carboxylic acid lead to degradation of the phenanthrene 4-carboxylic acid via phthalate. A novel metabolite identified as 6,6'-dihydroxy-2,2'-biphenyl dicarboxylic acid demonstrates a new branch in the pathway that involves the cleavage of both central rings of pyrene. In addition to pyrene, strain AP1 utilized hexadecane, phenanthrene, and fluoranthene for growth. Pyrene-grown cells oxidized the methylenic groups of fluorene and acenaphthene and catalyzed the dihydroxylation and ortho cleavage of one of the rings of naphthalene and phenanthrene to give 2-carboxycinnamic and diphenic acids, respectively. The catabolic versatility of strain AP1 and its use of ortho cleavage mechanisms during the degradation of polycyclic aromatic hydrocarbons (PAHs) give new insight into the role that pyrene-degrading bacterial strains may play in the environmental fate of PAH mixtures.

FIG. 1

Utilization of pyrene by Mycobacterium sp. strain AP1 in liquid mineral medium with pyrene as the sole source of carbon and energy at 25°C and 200 rpm. Growth is shown as an increase in the level of cell protein in cultures (▴) and in controls without a carbon source (▵). Pyrene concentrations were determined by HPLC analyses of organic extracts from cultures (●) and uninoculated controls (○).

FIG. 2

HPLC elution profiles of neutral (A) and acidic (B) extracts from cultures of strain AP1 in liquid mineral medium containing pyrene (1 g/liter). The UV-visible spectra of identified metabolites are displayed as insets. These metabolites were identified as follows: II, phenanthrene 4-carboxylic acid; III, 4,5-dihydroxy-4,5-dihydropyrene; IV, 6,6′-dihydroxy-2,2′-biphenyl dicarboxylic acid; VI, phenanthrene 4,5-dicarboxylic acid; and VIII, phthalic acid. mAU, milliabsorbance unit.

FIG. 3

¹H (A) and ¹³C (B) NMR spectra and chemical structure of metabolite IV.

FIG. 4

Schematic pathway proposed for the degradation of pyrene by Mycobacterium sp. strain AP1. The product in brackets has not been isolated. Dotted arrows indicate two or more successive reactions.

FIG. 5

Metabolites identified in washed-cell suspensions of Mycobacterium sp. strain AP1 incubated with PAHs. Reactions are grouped by type. Products in brackets have not been isolated.