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...
In this study, the enzymes involved in polycyclic aromatic hydrocarbon (PAH) degradation in the chrysenedegrading organism Sphingomonas sp. strain CHY-1 were investigated. [14 C]chrysene mineralization experiments showed that PAH-grown bacteria produced high levels of chrysene-catabolic activity. One PAH-induced protein displayed similarity with a ring-hydroxylating dioxygenase beta subunit, and a second PAH-induced protein displayed similarity with an extradiol dioxygenase. The genes encoding these proteins were cloned, and sequence analysis revealed two distinct loci containing clustered catabolic genes with strong similarities to corresponding genes found in Novosphingobium aromaticivorans F199. In the first locus, two genes potentially encoding a terminal dioxygenase component, designated PhnI, were followed by a gene coding for an aryl alcohol dehydrogenase (phnB). The second locus contained five genes encoding an extradiol dioxygenase (phnC), a ferredoxin (phnA3), another oxygenase component (PhnII), and an isomerase (phnD). PhnI was found to be capable of converting several PAHs, including chrysene, to the corresponding dihydrodiols. The activity of PhnI was greatly enhanced upon coexpression of genes encoding a ferredoxin (phnA3) and a reductase (phnA4). Disruption of the phnA1 a gene encoding the PhnI alpha subunit resulted in a mutant strain that had lost the ability to grow on PAHs. The recombinant PhnII enzyme overproduced in Escherichia coli functioned as a salicylate 1-hydroxylase. PhnII also used methylsalicylates and anthranilate as substrates. Our results indicated that a single enzyme (PhnI) was responsible for the initial attack of a range of PAHs, including chrysene, in strain CHY-1. Furthermore, the conversion of salicylate to catechol was catalyzed by a three-component oxygenase unrelated to known salicylate hydroxylases.
Ribulose 1,5-bisphosphate carboxylase-oxygenase (RuBPC-O) activity was greatly enhanced when Rhodopseudomonas sphaeroides was grown in a mineral salts medium supplied with 1.5% CO2 in hydrogen. Analysis of cell extracts by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that cells growing on 1.5% CO2 in H2 specifically accumulated RuBPC-O polypeptides. Quantitative immunological determinations revealed that accumulation of form I and form II RuBPC-O closely correlates with the increase of specific activity. However, the two enzymes appeared to be derepressed at different levels. Upon transfer from heterotrophic to autotrophic (1.5% CO2) growth conditions, the intracellular form I RuBPC-O concentration was augmented 17-fold, whereas the form II RuBPC-O content increased only fourfold. As a result, the form I-form II ratio changed from 0.5 to about 2.0. Since this change in the RuBPC-O ratio occurred in the early stage of growth, it suggests that form I RuBPC-O is required for growth under drastic CO2 limitation. The difference in the extent of derepression of form I and form II RuBPC-O also indicates that the synthesis of each enzyme is regulated somewhat independently of the other.Ribulose 1,5-bisphosphate carboxylase-oxygenase (RuBPC-O) is a bifunctional enzyme which catalyzes the initial reaction of the two competing metabolic pathways of photosynthetic carbon assimilation and photorespiratory glycolate metabolism. Because this enzyme is such a poor catalyst, organisms which depend on CO2 for carbon must synthesize enormous quantities of . This is particularly evident in Rhodospirillum rubrum, where up to 50% of the soluble protein is found to be RuBPC-O when cells are grown photolithotrophically under an atmosphere of hydrogen and C02, with CO2 supplied at levels of 2% or less of the total gas mixture (18,19).Rhodopseudomonas sphaeroides, another member of the family Rhodospirillaceae, presents an interesting situation. Two different forms of RuBPC-O are found in R. sphaeroides (4) and in the closely related R. capsulata (5,20). Form I RuBPC-O resembles the enzyme found in eucaryotes and most procaryotes in that it is a very large molecule with a native molecular weight of about 550,000 and is composed of eight large (catalytic) and eight small subunits. The form II enzyme, on the other hand, resembles the Rhodospirillum rubrum enzyme in that it is an aggregate of large subunits only and has a native molecular weight of about 290,000 (6a). Antiserum to the form I or form II enzyme does not cross-react with the heterologous enzyme (4, 6). The large and small subunits of the form I enzyme have been separated, and detailed peptide mapping experiments have shown that there are significant differences in the primary structure of the catalytic subunits of the form I and form II RuBPC-O from R. sphaeroides, suggesting that these proteins are products of different genes (6a). In addition, form II RuBPC-O has been cloned (3,11,16) and expressed in Escherichia coli (11, 16;
The rnf genes of Rhodobacter capsulatus, essential for nitrogen fixation, are thought to encode a system for electron transport to nitrogenase. In the present study, we have attempted to overexpress the rnf genes in Escherichia coli to investigate the molecular properties of the corresponding proteins. Corrections were made to the published DNA sequence of the rnf operon, resulting in the identification of two genes, rnfG and rnfH. The rnfABCDGEH operon thus comprises seven genes and shows similarities in gene arrangement and deduced protein sequences to homologous regions in the genomes of Haemophilus influenzae and E. coli. Four of the rnf gene products were found to be similar in sequence to components of an Na ϩ -dependent NADH:ubiquinone oxidoreductase from Vibrio alginolyticus. Three of the rnf genes were successfully overexpressed in E. coli as His-tagged polypeptides, whereas the products of rnfA, rnfD and rnfE, predicted to be transmembrane proteins, could not be stably maintained in E. coli. The rnfB and rnfC gene products were isolated as two brown proteins with apparent molecular-mass values of 25 kDa and 55 kDa, respectively. RnfB was shown to contain one [2Fe-2S] cluster, based on absorption spectrophotometry, EPR spectroscopy and iron content. Recombinant RnfC contained at least one ironsulfur cluster, most likely of the [4Fe-4S] type. Unambiguous identification of the prosthetic groups was, however, precluded by the extreme instability of this protein. In R. capsulatus, RnfB and RnfC were found by immunoblot analysis to be tightly bound to the membrane, despite their hydrophilic character. The RnfB and RnfC proteins were absent in mutant strains bearing insertions at various positions within the rnfABCDGEH operon, suggesting that their stability depends on the cosynthesis of the other rnf gene products. We observed that iron limitation during growth resulted in a decrease both in the cellular content of RnfB and in the level of transcription of the rnfABCDGEH operon, indicating that the expression of this operon is regulated as a function of iron availability
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