Rugosibacter aromaticivorans gen. nov., sp. nov., a bacterium within the family Rhodocyclaceae, isolated from contaminated soil, capable of degrading aromatic compounds

A bacterial strain designated Ca6T was isolated from polycyclic aromatic hydrocarbon (PAH)-contaminated soil from the site of a former manufactured gas plant in Charlotte, NC, USA, and linked phylogenetically to the family Rhodocyclaceae of the class Betaproteobacteria. Its 16S rRNA gene sequence was highly similar to globally distributed environmental sequences, including those previously designated ‘Pyrene Group 1’ demonstrated to grow on the PAHs phenanthrene and pyrene by stable-isotope probing. The most closely related described relative was Sulfuritalea hydrogenivorans strain sk43HT (93.6 % 16S rRNA gene sequence identity). In addition to a limited number of organic acids, Ca6T was capable of growth on the monoaromatic compounds benzene and toluene, and the azaarene carbazole, as sole sources of carbon and energy. Growth on the PAHs phenanthrene and pyrene was also confirmed. Optimal growth was observed aerobically under mesophilic temperature, neutral pH and low salinity conditions. Major fatty acids present included summed feature 3 (C16 : 1ω7c or C16 : 1ω6c) and C16 : 0. The DNA G+C content of the single chromosome was 55.14 mol% as determined by complete genome sequencing. Due to its distinct genetic and physiological properties, strain Ca6T is proposed as a member of a novel genus and species within the family Rhodocyclaceae, for which the name Rugosibacter aromaticivorans gen. nov., sp. nov. is proposed. The type strain of the species is Ca6T (=ATCC TSD-59T=DSM 103039T)

Description of Immundisolibacter cernigliae gen. nov., sp. nov., a high-molecular-weight polycyclic aromatic hydrocarbon-degrading bacterium within the class Gammaproteobacteria, and proposal of Immundisolibacterales ord. nov. and Immundisolibacteraceae fam. nov.

The bacterial strain TR3.2T was isolated from aerobic bioreactor-treated soil from a polycyclic aromatic hydrocarbon (PAH)-contaminated site in Salisbury, NC, USA. Strain TR3.2T was identified as a member of ‘Pyrene Group 2’ or ‘PG2’, a previously uncultivated cluster of organisms associated with the degradation of high-molecular-weight PAHs by stable-isotope probing. Based on its 16S rRNA gene sequence, the strain was classified as a member of the class Gammaproteobacteria but possessed only 90.5 % gene identity to its closest described relative, Methylococcus capsulatus strain Bath. Strain TR3.2T grew on the PAHs pyrene, phenanthrene, anthracene, benz[a]anthracene and fluorene, as well as the azaarene carbazole, and could additionally metabolize a limited number of organic acids. Optimal growth occurred aerobically under mesophilic temperature, neutral pH and low salinity conditions. Strain TR3.2T was catalase and oxidase positive. Predominant fatty acids were C17 : 0 cyclo and C16 : 0. Genomic G+C content of the single chromosome was 67.79 mol% as determined by complete genome sequencing. Due to the high sequence divergence from any cultivated species and its unique physiological properties compared to its closest relatives, strain TR3.2T is proposed as a representative of a novel order, family, genus and species within the class Gammaproteobacteria, for which the name Immundisolibacter cernigliae gen. nov., sp. nov. is proposed. The associated order and family are therefore proposed as Immundisolibacteralesord. nov. and Immundisolibacteraceaefam. nov. The type strain of the species is TR3.2T (=ATCC TSD-58T=DSM 103040T)

Structural and functional fine mapping of cysteines in mammalian glutaredoxin reveal their differential oxidation susceptibility

Abstract Protein-S-glutathionylation is a post-translational modification involving the conjugation of glutathione to protein thiols, which can modulate the activity and structure of key cellular proteins. Glutaredoxins (GLRX) are oxidoreductases that regulate this process by performing deglutathionylation. However, GLRX has five cysteines that are potentially vulnerable to oxidative modification, which is associated with GLRX aggregation and loss of activity. To date, GLRX cysteines that are oxidatively modified and their relative susceptibilities remain unknown. We utilized molecular modeling approaches, activity assays using recombinant GLRX, coupled with site-directed mutagenesis of each cysteine both individually and in combination to address the oxidizibility of GLRX cysteines. These approaches reveal that C8 and C83 are targets for S-glutathionylation and oxidation by hydrogen peroxide in vitro. In silico modeling and experimental validation confirm a prominent role of C8 for dimer formation and aggregation. Lastly, combinatorial mutation of C8, C26, and C83 results in increased activity of GLRX and resistance to oxidative inactivation and aggregation. Results from these integrated computational and experimental studies provide insights into the relative oxidizability of GLRX’s cysteines and have implications for the use of GLRX as a therapeutic in settings of dysregulated protein glutathionylation

Protein Sulfenylation: A Novel Readout of Environmental Oxidant Stress

Oxidative stress is a commonly cited mechanism of toxicity of environmental agents. Ubiquitous environmental chemicals such as the diesel exhaust component 1,2-naphthoquinone (1,2-NQ) induce oxidative stress by redox cycling, which generates hydrogen peroxide (H₂O₂). Cysteinyl thiolate residues on regulatory proteins are subjected to oxidative modification by H₂O₂ in physiological contexts and are also toxicological targets of oxidant stress induced by environmental contaminants. We investigated whether exposure to environmentally relevant concentrations of 1,2-NQ can induce H₂O₂-dependent oxidation of cysteinyl thiols in regulatory proteins as a readout of oxidant stress in human airway epithelial cells. BEAS-2B cells were exposed to 0–1000 μM 1,2-NQ for 0–30 min, and levels of H₂O₂ were measured by ratiometric spectrofluorometry of HyPer. H₂O₂-dependent protein sulfenylation was measured using immunohistochemistry, immunoblotting, and isotopic mass spectrometry. Catalase overexpression was used to investigate the relationship between H₂O₂ generation and protein sulfenylation in cells exposed to 1,2-NQ. Multiple experimental approaches showed that exposure to 1,2-NQ at concentrations as low as 3 μM induces H₂O₂-dependent protein sulfenylation in BEAS-2B cells. Moreover, the time of onset and duration of 1,2-NQ-induced sulfenylation of the regulatory proteins GAPDH and PTP1B showed significant differences. Oxidative modification of regulatory cysteinyl thiols in human lung cells exposed to relevant concentrations of an ambient air contaminant represents a novel marker of oxidative environmental stress