Soil Type-Dependent Responses to Phenanthrene as Revealed by Determining the Diversity and Abundance of Polycyclic Aromatic Hydrocarbon Ring-Hydroxylating Dioxygenase Genes by Using a Novel PCR Detection System

A protein fraction exhibiting 1-hydroxy-2-naphthoic acid (1-H2NA) dioxygenase activity was purified via ion exchange, hydrophobic interactions, and gel filtration chromatography from Arthrobacter phenanthrenivorans sp. nov. strain Sphe3 isolated from a Greek creosote-oil-polluted site. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and tandem MS (MS-MS) analysis revealed that the amino acid sequences of oligopeptides of the major 45-kDa protein species, as analyzed by SDS-PAGE and silver staining, comprising 29% of the whole sequence, exhibited strong homology with 1-H2NA dioxygenase of Nocardioides sp. strain KP7. A BLAST search of the recently sequenced Sphe3 genome revealed two putative open reading frames, named diox1 and diox2, showing 90% nucleotide identity to each other and 85% identity at the amino acid level with the Nocardia sp. homologue. diox1 was found on an indigenous Sphe3 plasmid, whereas diox2 was located on the chromosome. Both genes were induced by the presence of phenanthrene used as a sole carbon and energy source, and as expected, both were subject to carbon catabolite repression. The relative RNA transcription level of the chromosomal (diox2) gene was significantly higher than that of its plasmid (diox1) homologue. Both diox1 and diox2 putative genes were PCR amplified, cloned, and overexpressed in Escherichia coli. Recombinant E. coli cells expressed 1-H2NA dioxygenase activity. Recombinant enzymes exhibited Michaelis-Menten kinetics with an apparent K m of 35 M for Diox1 and 29 M for Diox2, whereas they showed similar kinetic turnover characteristics with K cat /K m values of 11 ؋ 10 6 M ؊1 s ؊1 and 12 ؋ 10 6 M ؊1 s ؊1 , respectively. Occurrence of two diox1 and diox2 homologues in the Sphe3 genome implies that a replicative transposition event has contributed to the evolution of 1-H2NA dioxygenase in A. phenanthrenivorans.

Section: Discussionmentioning

confidence: 59%

Heterologous Expression and Characterization of Two 1-Hydroxy-2-Naphthoic Acid Dioxygenases from Arthrobacter phenanthrenivorans

Vandera

Kavakiotis

Kallimanis

et al. 2012

“…The persistence over time of antagonistic effects in this study and others (18,21,23) is important to the field of bioremediation because it may explain the recalcitrance of PAHs in soil where bacteria that have the ability to degrade them are present (12,18). Often, bioremediation potential has been measured by monitoring the presence of relevant microbes or catabolic genes (51,52), but if in situ organisms experience antagonistic interference with degradation, the predictive power of these techniques may depend on which organisms and PAH mixtures are present.…”

Section: Discussionmentioning

confidence: 81%

Effects of Polycyclic Aromatic Hydrocarbon Mixtures on Degradation, Gene Expression, and Metabolite Production in Four Mycobacterium Species

Hennessee

2016

Polycyclic aromatic hydrocarbons (PAHs) are widespread environmental contaminants that are hazardous to human health. It has been demonstrated that members of the Mycobacterium genus are among the most effective degraders of PAHs, but few studies have focused on the degradation of PAH mixtures. In this study, single and mixed PAH metabolism was investigated in four phylogenetically distinct Mycobacterium species with respect to (i) parent compound degradation, (ii) bacterial growth, (iii) catabolic gene expression, and (iv) metabolite production. Synergistic and antagonistic effects on four model PAH compounds (benzo[a]pyrene, pyrene, fluoranthene, and phenanthrene) characterized degradation of mixtures in a strain-and mixture-dependent manner. The mixture of pyrene and phenanthrene, in particular, resulted in antagonized degradation by three out of four bacterial species, and further studies were narrowed to investigate the degradation of this mixture. Antagonistic effects persisted over time and were correlated with reduced bacterial growth. Antagonized degradation of PAH was not caused by preferential degradation of secondary PAHs, nor were mixture compounds or concentrations toxic to cells growing on sugars. Reverse transcription-PCR (RT-PCR) studies of the characterized catabolic pathway of phenanthrene showed that in one organism, antagonism of mixture degradation was associated with downregulated gene expression. Metabolite profiling revealed that antagonism in mixture degradation was associated with the shunting of substrate through alternative pathways not used during the degradation of single PAHs. The results of this study demonstrate metabolic differences between single and mixed PAH degradation with consequences for risk assessment and bioremediation of PAH-contaminated sites. IMPORTANCEMycobacterium species are promising organisms for environmental bioremediation because of their ubiquitous presence in soils and their ability to catabolize aromatic compounds. PAHs can be degraded effectively as single compounds, but mixed substrates often are subject to degradative inhibition, which may explain the persistence of these pollutants in soils. Single and mixed PAH degradation by diverse Mycobacterium species was compared, with associated bacterial growth, gene expression, and metabolite production. The results demonstrate that antagonism characterized degradation in a strain-and mixture-dependent manner. One strain that was versatile in its pathway use of single chemicals also efficiently degraded the mixture, whereas antagonism in other the strains was associated with altered metabolic profiles, indicating unusual pathway use. The impacts of this work on risk assessment and bioremediation modeling studies indicate the need to account for mixture-generated intermediates and to recognize mixture degradation as a property distinct from that of PAH substrate range. P olycyclic aromatic hydrocarbons (PAHs) are a class of chemical pollutants that are widespread and persistent in the environment (1-3...

“…Soil microbial community dynamics in response to hydrocarbon pollution is more diverse and niche specific than microbial dynamics in seawater, probably due to the limited dispersal compared with that in marine environments. Different soil types may respond differentially following pollution with the same PAH (i.e., phenanthrene), whereas the same soil may develop different community structures in response to related PAHs (e.g., naphthalene, phenanthrene, and pyrene) (53,54). The organic matter content is probably a major physicochemical soil feature inducing differential responses (55).…”

Section: Fig 2 Effect Of Diesel Pollution and Bioremediation Treatmenmentioning

confidence: 99%

From Rare to Dominant: a Fine-Tuned Soil Bacterial Bloom during Petroleum Hydrocarbon Bioremediation

Fuentes

Barra

Caporaso

et al. 2016

113

Hydrocarbons are worldwide-distributed pollutants that disturb various ecosystems. The aim of this study was to characterize the short-lapse dynamics of soil microbial communities in response to hydrocarbon pollution and different bioremediation treatments. Replicate diesel-spiked soil microcosms were inoculated with either a defined bacterial consortium or a hydrocarbonoclastic bacterial enrichment and incubated for 12 weeks. The microbial community dynamics was followed weekly in microcosms using Illumina 16S rRNA gene sequencing. Both the bacterial consortium and enrichment enhanced hydrocarbon degradation in diesel-polluted soils. A pronounced and rapid bloom of a native gammaproteobacterium was observed in all dieselpolluted soils. A unique operational taxonomic unit (OTU) related to the Alkanindiges genus represented ϳ0.1% of the sequences in the original community but surprisingly reached >60% after 6 weeks. Despite this Alkanindiges-related bloom, inoculated strains were maintained in the community and may explain the differences in hydrocarbon degradation. This study shows the detailed dynamics of a soil bacterial bloom in response to hydrocarbon pollution, resembling microbial blooms observed in marine environments. Rare community members presumably act as a reservoir of ecological functions in high-diversity environments, such as soils. This rare-to-dominant bacterial shift illustrates the potential role of a rare biosphere facing drastic environmental disturbances. Additionally, it supports the concept of "conditionally rare taxa," in which rareness is a temporary state conditioned by environmental constraints.