Identification of Pyrene-Induced Proteins in Mycobacterium sp. Strain 6PY1: Evidence for Two Ring-Hydroxylating Dioxygenases

Abstract

In this study, the enzymes involved in polycyclic aromatic hydrocarbon (PAH) degradation were investigated in the pyrene-degrading Mycobacterium sp. strain 6PY1. [14 C]pyrene mineralization experiments showed that bacteria grown with either pyrene or phenanthrene produced high levels of pyrene-catabolic activity but that acetate-grown cells had no activity. As a means of identifying specific catabolic enzymes, protein extracts from bacteria grown on pyrene or on other carbon sources were analyzed by two-dimensional gel electrophoresis. Pyrene-induced proteins were tentatively identified by peptide sequence analysis. Half of them resembled enzymes known to be involved in phenanthrene degradation, with closest similarity to the corresponding enzymes from Nocardioides sp. strain KP7. The genes encoding the terminal components of two distinct ring-hydroxylating dioxygenases were cloned. Sequence analysis revealed that the two enzymes, designated Pdo1 and Pdo2, belong to a subfamily of dioxygenases found exclusively in gram-positive bacteria. When overproduced in Escherichia coli, Pdo1 and Pdo2 showed distinctive selectivities towards PAH substrates, with the former enzyme catalyzing the dihydroxylation of both pyrene and phenanthrene and the latter preferentially oxidizing phenanthrene. The catalytic activity of the Pdo2 enzyme was dramatically enhanced when electron carrier proteins of the phenanthrene dioxygenase from strain KP7 were coexpressed in recombinant cells. The Pdo2 enzyme was purified as a brown protein consisting of two types of subunits with M r s of about 52,000 and 20,000. Immunoblot analysis of cell extracts from strain 6PY1 revealed that Pdo1 was present in cells grown on benzoate, phenanthrene, or pyrene and absent in acetate-grown cells. In contrast, Pdo2 could be detected only in PAH-grown cells. These results indicated that the two enzymes were differentially regulated depending on the carbon source used for growth.Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous contaminants in soils and sediments and are of environmental concern because of their mutagenic and/or carcinogenic effects. While low-molecular-weight PAHs (composed of two or three rings) are readily degraded by bacteria, PAHs consisting of four rings or more are recalcitrant to biodegradation and persist in the environment (6, 41). The biodegradation of lowmolecular-weight PAHs, especially naphthalene, has been extensively studied with pseudomonads, leading to a good understanding of the bacterial catabolic pathway (42). On the other hand, relatively little information is available on the metabolism of high-molecular-weight PAHs (20). A number of bacterial isolates capable of pyrene mineralization have been described. Most of them are actinomycetes and belong to the genus Mycobacterium (2, 7, 37), Rhodococcus (4, 40), or Gordonia (21). A few pyrene-degrading strains have been identified as gram-negative species, including Stenotrophomonas maltophilia, Pseudomonas fluorescens (3), Sphingomonas paucimobilis (22), and Burkholder...

Response of bacterial pdo1, nah, and C12O genes to aged soil PAH pollution in a coke factory area

“…In this study, the clone library of the pdo1 gene showed that all pdo1 sequences were grouped into Mycobacterium, which was unsurprising as the primer used was designed based on the pdo1 gene from Mycobacterium sp. strain 6PY1 (Krivobok et al 2003;Johnsen et al 2006). Johnsen et al (2007) also found that Mycobacteria were dominant members of phenanthrene, pyrene, and fluoranthene degraders in soils originating from an asphalt and tar production plant.…”

A New Classification System for Bacterial Rieske Non-Heme Iron Aromatic Ring-Hydroxylating Oxygenases

“…Phylogenetic analysis of these oxygenase components, NidA, NidA3 and PhtAa, showed that they clustered with a new group of α subunits found in Nocardioides, Rhodococcus, Terrabacter, Arthrobacter and other Mycobacterium spp. [20][21][22][23][24][25]. Interestingly, RHO genes found in this group of bacteria appear to have features in common; genes for ETC components are not always closely positioned with oxygenase genes (genetic discreteness) and limited numbers of ferredoxin and reductase components are shared by multiple oxygenases (numerical imbalance).…”

The rhodanese RhdA helps Azotobacter vinelandii in maintaining cellular redox balance

“…Starting from the finding that bovine rhodanese (Nandi and Westley, 1998), Escherichia coli GlpE (Ray et al, 2000), and the 3-mercaptopyruvate sulfurtransferase from Leishmania (Williams et al, 2003) have higher affinity for reduced thioredoxins than for cyanide, the concept that cyanide detoxification was not the only physiological role of the rhodanese-like proteins has emerged. The relevance of specific rhodanese-like proteins in the maintenance of redox homeostasis has been suggested by in vitro studies with rat mercaptopyruvate sulfurtransferase (Nagahara and Katayama, 2005;Nagahara et al, 2007), and proteomic analyses (Krivobok et al, 2003;Santos et al, 2004) indicated possible roles of bacterial rhodanese-domain proteins in physiological processes related to xenobiotic-induced oxidative-stress and detoxification. This experimental evidence suggests that rhodanese domains might function as regulatory devices in specific physiological situations, but the redundancy of these proteins in the same organism make it difficult to define their different cellular roles.…”