Molecular Characterization of Aromatic Hydrocarbon Degradation in Extreme Halophiles

Many hypersaline environments such as natural saline lakes, salt marshes, saline industrial effluents, oil fields and coastal areas are often contaminated with high levels of petroleum hydrocarbons. Since non-halophiles do not operate efficiently at high salinity, halophilic and halotolerant hydrocarbon degraders are considered as potential candidates for bioremediation of hydrocarbon-impacted hypersaline sites. Several studies have reported the ability of microbial consortia and pure cultures of halophiles and halotolerants to degrade petroleum compounds. However, information regarding the genes and mechanisms of hydrocarbon degradation pathways at high salinity is scarce. This work describes the metabolic potential of halophilic bacteria and archaea to degrade aromatic hydrocarbons at high salinity. A combination of omics-based approaches was used to study the molecular basis of hydrocarbon degradation inArhodomonas sp. strain Seminole and Arhodomonassp. strain Rozel. Genomic analysis of strain Seminole predicted clusters of genes encoding enzymes involved in the metabolism of benzene, toluene, 4-hydroxybenzoic acid and phenylacetic acid to Krebs cycle intermediates. Many key enzymes of the predicted steps were identified in the cytosolic proteomes of hydrocarbon-grown cells by liquid chromatography-mass spectrometry, thus confirming the genomic data. Although these proteins have been described in non-halophiles, they differed from their non-halophilic homologs by exhibiting low-pI values, a unique feature known to maintain the stability and activity of halophilic proteins at high salinities. In addition, another aim of this project was to study the degradation potential of archaea in the presence of high salt. A highly enriched culture developed using sediment samples from Rozel Point; Great Salt Lake utilized benzoate as the growth substrate at salinity ranging from 2 to 5 M NaCl with highest rate of degradation at 4 M. Pyrosequencing of 16S rRNA gene sequences revealed that the enrichment composed solely of Halobacteriaceae members. Of these, Halopenitus was the most dominant member comprising of 91% of the enrichment. PCR amplification with degenerate primers revealed the presence of 4-hydroxybenzoate 3-monooxygenase and protocatechuate 3,4-dioxygenase genes, suggesting that the enrichment might degrade benzoate via protocatechuate. These results suggest the potential role of Halopenitus members in degrading oxygenated aromatic compounds at high salinity.Microbiology, Cell, & Molecular Biolog

Electrical Resistivity Imaging for Long-Term Autonomous Monitoring of Hydrocarbon Degradation: Lessons from the Deepwater Horizon Oil Spill

Conceptual models for the geophysical responses associated with hydrocarbon degradation suggest that the long-term evolution of an oil plume will result in a more conductive anomaly than the initial contamination. In response to the Deepwater Horizon (DH) oil spill into the Gulf of Mexico in 2010, an autonomous resistivity monitoring system was deployed on Grand Terre, Louisiana, in an attempt to monitor natural degradation processes in hydrocarbon-impacted beach sediments of this island. A 48-electrode surface array with a 0.5-m spacing was installed to obtain twice-daily images of the resistivity structure of the shallow subsurface impacted by oil. Over the course of approximately 18 months, we observed a progressive decrease in the resistivity of the DH spill-impacted region. Detailed analysis of pixel/point resistivity variation within the imaged area showed that long-term decreases in resistivity were largely associated with the DH-impacted sediments. A microbial diversity survey revealed the presence of hydrocarbon-degrading organisms throughout the test site. However, hydrocarbon degradation activity was much higher in the DH-impacted locations compared to nonimpacted locations, suggesting the presence of active hydrocarbon degraders, supporting biodegradation processes. The results of this long-term monitoring experiment suggested that resistivity might be used to noninvasively monitor the long-term degradation of crude oil spills

Arhodomonas sp. strain Seminole and its genetic potential to degrade aromatic compounds under high-salinity conditions

Arhodomonas sp. strain Seminole was isolated from a crude oil-impacted brine soil and shown to degrade benzene, toluene, phenol, 4-hydroxybenzoic acid (4-HBA), protocatechuic acid (PCA), and phenylacetic acid (PAA) as the sole sources of carbon at high salinity. Seminole is a member of the genus Arhodomonas in the class Gammaproteobacteria, sharing 96% 16S rRNA gene sequence similarity with Arhodomonas aquaeolei HA-1. Analysis of the genome predicted a number of catabolic genes for the metabolism of benzene, toluene, 4-HBA, and PAA. The predicted pathways were corroborated by identification of enzymes present in the cytosolic proteomes of cells grown on aromatic compounds using liquid chromatography-mass spectrometry. Genome analysis predicted a cluster of 19 genes necessary for the breakdown of benzene or toluene to acetyl coenzyme A (acetyl-CoA) and pyruvate. Of these, 12 enzymes were identified in the proteome of toluene-grown cells compared to lactate-grown cells. Genomic analysis predicted 11 genes required for 4-HBA degradation to form the tricarboxylic acid (TCA) cycle intermediates. Of these, proteomic analysis of 4-HBA-grown cells identified 6 key enzymes involved in the 4-HBA degradation pathway. Similarly, 15 genes needed for the degradation of PAA to the TCA cycle intermediates were predicted. Of these, 9 enzymes of the PAA degradation pathway were identified only in PAA-grown cells and not in lactate-grown cells. Overall, we were able to reconstruct catabolic steps for the breakdown of a variety of aromatic compounds in an extreme halophile, strain Seminole. Such knowledge is important for understanding the role of Arhodomonas spp. in the natural attenuation of hydrocarbon-impacted hypersaline environments.Peer reviewedMicrobiology and Molecular GeneticsBiochemistry and Molecular Biolog

Proteogenomic elucidation of the initial steps in the benzene degradation pathway of a novel halophile, Arhodomonas sp. strain Rozel, isolated from a hypersaline environment

Lately, there has been a special interest in understanding the role of halophilic and halotolerant organisms for their ability to degrade hydrocarbons. The focus of this study was to investigate the genes and enzymes involved in the initial steps of the benzene degradation pathway in halophiles. The extremely halophilic bacteria Arhodomonas sp. strain Seminole and Arhodomonas sp. strain Rozel, which degrade benzene and toluene as the sole carbon source at high salinity (0.5 to 4 M NaCl), were isolated from enrichments developed from contaminated hypersaline environments. To obtain insights into the physiology of this novel group of organisms, a draft genome sequence of the Seminole strain was obtained. A cluster of 13 genes predicted to be functional in the hydrocarbon degradation pathway was identified from the sequence. Two-dimensional (2D) gel electrophoresis and liquid chromatography-mass spectrometry were used to corroborate the role of the predicted open reading frames (ORFs). ORFs 1080 and 1082 were identified as components of a multicomponent phenol hydroxylase complex, and ORF 1086 was identified as catechol 2,3-dioxygenase (2,3-CAT). Based on this analysis, it was hypothesized that benzene is converted to phenol and then to catechol by phenol hydroxylase components. The resulting catechol undergoes ring cleavage via the meta pathway by 2,3-CAT to form 2-hydroxymuconic semialdehyde, which enters the tricarboxylic acid cycle. To substantiate these findings, the Rozel strain was grown on deuterated benzene, and gas chromatography-mass spectrometry detected deuterated phenol as the initial intermediate of benzene degradation. These studies establish the initial steps of the benzene degradation pathway in halophiles.Peer reviewedMicrobiology and Molecular Genetic

A human model of Batten disease shows role of CLN3 in phagocytosis at the photoreceptor–RPE interface

CLN3 disease is characterised by childhood-onset vision loss and premature death. Using patient-derived retinal cells, the authors show that CLN3 is required for retinal pigment epithelium (RPE) cell structure, microvilli and phagocytosis of photoreceptor outer segments that are essential for vision. They further suggest that gene-therapy targeting RPE cells can be effective for CLN3 disease