Rugosibacter aromaticivorans gen. nov., sp. nov., a bacterium within the family Rhodocyclaceae, isolated from contaminated soil, capable of degrading aromatic compounds.
ABSTRACT: 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?:?1?7c or C16?:?1?6c) 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).
Project description: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).
Project description:Four bacterial strains identified as members of the Acidovorax genus were isolated from two geographically distinct but similarly contaminated soils in North Carolina, USA, characterized, and their genomes sequenced. Their 16S rRNA genes were highly similar to those previously recovered during stable-isotope probing (SIP) of one of the soils with the polycyclic aromatic hydrocarbon (PAH) phenanthrene. Heterotrophic growth of all strains occurred with a number of organic acids, as well as phenanthrene, but no other tested PAHs. Optimal growth occurred aerobically under mesophilic temperature, neutral pH, and low salinity conditions. Predominant fatty acids were C16:1?7c/C16:1?6c, C16:0, and C18:1?7c, and were consistent with the genus. Genomic G+C contents ranged from 63.6 to 64.2%. A combination of whole genome comparisons and physiological analyses indicated that these four strains likely represent a single species within the Acidovorax genus. Chromosomal genes for phenanthrene degradation to phthalate were nearly identical to highly conserved regions in phenanthrene-degrading Delftia, Burkholderia, Alcaligenes, and Massilia species in regions flanked by transposable or extrachromosomal elements. The lower degradation pathway for phenanthrene metabolism was inferred by comparisons to described genes and proteins. The novel species Acidovorax carolinensis sp. nov. is proposed, comprising the four strains described in this study with strain NA3T as the type strain (=LMG 30136, =DSM 105008).
Project description:The polycyclic aromatic hydrocarbon (PAH)-degrading strain Q8 was isolated from oilfield produced water. According to the analysis of a biochemical test, 16S rRNA gene, house-keeping genes and DNA-DNA hybridization, strain Q8 was assigned to a novel species of the genus Gordonia. The strain could not only grow in mineral salt medium (MM) and utilize naphthalene and pyrene as its sole carbon source, but also degraded mixed naphthalene, phenanthrene, anthracene and pyrene. The degradation ratio of these four PAHs reached 100%, 95.4%, 73.8% and 53.4% respectively after being degraded by Q8 for seven days. A comparative experiment found that the PAHs degradation efficiency of Q8 is higher than that of Gordonia alkaliphila and Gordonia paraffinivorans, which have the capacities to remove PAHs. Fourier transform infrared spectra, saturate, aromatic, resin and asphaltene (SARA) and gas chromatography-mass spectrometry (GC-MS) analysis of crude oil degraded by Q8 were also studied. The results showed that Q8 could utilize n-alkanes and PAHs in crude oil. The relative proportions of the naphthalene series, phenanthrene series, thiophene series, fluorene series, chrysene series, C21-triaromatic steroid, pyrene, and benz(a)pyrene were reduced after being degraded by Q8. Gordonia sp. nov. Q8 had the capacity to remediate water and soil environments contaminated by PAHs or crude oil, and provided a feasible way for the bioremediation of PAHs and oil pollution.
Project description:Bacteria play an important role in the removal of polycyclic aromatic hydrocarbons (PAHs) from polluted environments. In marine environments, Cycloclasticus is one of the most prevalent PAH-degrading bacterial genera. However, little is known regarding the degradation mechanisms for multiple PAHs by Cycloclasticus Cycloclasticus sp. strain P1 was isolated from deep-sea sediments and is known to degrade naphthalene, phenanthrene, pyrene, and other aromatic hydrocarbons. Here, six ring-hydroxylating dioxygenases (RHDs) were identified in the complete genome of Cycloclasticus sp. P1 and were confirmed to be involved in PAH degradation by enzymatic assays. Further, five gene clusters in its genome were identified to be responsible for PAH degradation. Degradation pathways for naphthalene, phenanthrene, and pyrene were elucidated in Cycloclasticus sp. P1 based on genomic and transcriptomic analysis and characterization of an interconnected metabolic network. The metabolic pathway overlaps in many steps in the degradation of pyrene, phenanthrene, and naphthalene, which were validated by the detection of metabolic intermediates in cultures. This study describes a pyrene degradation pathway for Cycloclasticus. Moreover, the study represents the integration of a PAH metabolic network that comprises pyrene, phenanthrene, and naphthalene degradation pathways. Taken together, these results provide a comprehensive investigation of PAH metabolism in Cycloclasticus IMPORTANCE PAHs are ubiquitous in the environment and are carcinogenic compounds and tend to accumulate in food chains due to their low bioavailability and poor biodegradability. Cycloclasticus is an obligate marine PAH degrader and is widespread in marine environments, while the PAH degradation pathways remain unclear. In this report, the degradation pathways for naphthalene, phenanthrene, and pyrene were revealed, and an integrated PAH metabolic network covering pyrene, phenanthrene, and naphthalene was constructed in Cycloclasticus This overlapping network provides streamlined processing of PAHs to intermediates and ultimately to complete mineralization. Furthermore, these results provide an additional context for the prevalence of Cycloclasticus in oil-polluted marine environments and pelagic settings. In conclusion, these analyses provide a useful framework for understanding the cellular processes involved in PAH metabolism in an ecologically important marine bacterium.
Project description:The pyrene-degrading Mycobacterium sp. strain AP1 grew in nutrient-supplemented artificial seawater with a heavy fuel oil as the sole carbon source, causing the complete removal of all linear (C(12) to C(40)) and branched alkanes from the aliphatic fraction, as well as an extensive degradation of the three- and four-ring polycyclic aromatic hydrocarbons (PAHs) phenanthrene (95%), anthracene (80%), fluoranthene (80%), pyrene (75%), and benzo(a)anthracene (30%). Alkylated PAHs, which are more abundant in crude oils than the nonsubstituted compounds, were selectively attacked at extents that varied from more than 90% for dimethylnaphthalenes, methylphenanthrenes, methylfluorenes, and methyldibenzothiophenes to about 30% for monomethylated fluoranthenes/pyrenes and trimethylated phenanthrenes and dibenzothiophenes. Identification of key metabolites indicated the utilization of phenanthrene, pyrene, and fluoranthene by known assimilatory metabolic routes, while other components were cooxidized. Detection of mono- and dimethylated phthalic acids demonstrated ring cleavage and further oxidation of alkyl PAHs. The extensive degradation of the alkanes, the two-, three-, and four-ring PAHs, and their 1-, 2-, and 3-methyl derivatives from a complex mixture of hydrocarbons by Mycobacterium sp. strain AP1 illustrates the great substrate versatility of alkane- and PAH-degrading mycobacteria.
Project description:Two new polyaromatic hydrocarbon-degrading marine bacteria have been isolated from burrow wall sediments of benthic macrofauna by using enrichments on phenanthrene. Strain LC8 (from a polychaete) and strain M4-6 (from a mollusc) are aerobic and gram negative and require sodium chloride (>1%) for growth. Both strains can use 2- and 3-ring polycyclic aromatic hydrocarbons as their sole carbon and energy sources, but they are nutritionally versatile. Physiological and phylogenetic analyses based on 16S ribosomal DNA sequences suggest that strain M4-6 belongs to the genus Cycloclasticus and represents a new species, Cycloclasticus spirillensus sp. nov. Strain LC8 appears to represent a new genus and species, Lutibacterium anuloederans gen. nov., sp. nov., within the Sphingomonadaceae. However, when inoculated into sediment slurries with or without exogenous phenanthrene, only L. anuloederans appeared to sustain a significant phenanthrene uptake potential throughout a 35-day incubation. In addition, only L. anuloederans appeared to enhance phenanthrene degradation in heavily contaminated sediment from Little Mystic Cove, Boston Harbor, Boston, Mass.
Project description:Mycobacterium sp. strain CH1 was isolated from polycyclic aromatic hydrocarbon (PAH)-contaminated freshwater sediments and identified by analysis of 16S rDNA sequences. Strain CH1 was capable of mineralizing three- and four-ring PAHs including phenanthrene, pyrene, and fluoranthene. In addition, strain CH1 could utilize phenanthrene or pyrene as a sole carbon and energy source. A lag phase of at least 3 days was observed during pyrene mineralization. This lag phase decreased to less than 1 day when strain CH1 was grown in the presence of phenanthrene or fluoranthene. Strain CH1 also was capable of using a wide range of alkanes as sole carbon and energy sources. No DNA hybridization was detected with the nahAc gene probe, indicating that enzymes involved in PAH metabolism are not related to the well-characterized naphthalene dioxygenase gene. DNA hybridization was not detected when the alkB gene from Pseudomonas oleovorans was used under high-stringency conditions. However, there was slight but detectable hybridization under low-stringency conditions. This suggests a distant relationship between genes involved in alkane oxidation.
Project description:Acidovorax sp. strain NA3 was isolated from polycyclic aromatic hydrocarbon (PAH)-contaminated soil that had been treated in a bioreactor and enriched with phenanthrene. The 16S rRNA gene of the isolate possessed 99.8 to 99.9% similarity to the dominant sequences recovered during a previous stable-isotope probing experiment with [U-(13)C]phenanthrene on the same soil (D. R. Singleton, S. N. Powell, R. Sangaiah, A. Gold, L. M. Ball, and M. D. Aitken, Appl. Environ. Microbiol. 71:1202-1209, 2005). The strain grew on phenanthrene as a sole carbon and energy source and could mineralize (14)C from a number of partially labeled PAHs, including naphthalene, phenanthrene, chrysene, benz[a]anthracene, and benzo[a]pyrene, but not pyrene or fluoranthene. Southern hybridizations of a genomic fosmid library with a fragment of the large subunit of the ring-hydroxylating dioxygenase gene from a naphthalene-degrading Pseudomonas strain detected the presence of PAH degradation genes subsequently determined to be highly similar in both nucleotide sequence and gene organization to an uncharacterized Alcaligenes faecalis gene cluster. The genes were localized to the chromosome of strain NA3. To test for gene induction by selected compounds, RNA was extracted from amended cultures and reverse transcribed, and cDNA associated with the enzymes involved in the first three steps of phenanthrene degradation was quantified by quantitative real-time PCR. Expression of each of the genes was induced most strongly by phenanthene and to a lesser extent by naphthalene, but other tested PAHs and PAH metabolites had negligible effects on gene transcript levels.
Project description:A phenanthrene-degrading endophytic bacterium, Pn2, was isolated from Alopecurus aequalis Sobol grown in soils contaminated with polycyclic aromatic hydrocarbons (PAHs). Based on morphology, physiological characteristics and the 16S rRNA gene sequence, it was identified as Massilia sp. Strain Pn2 could degrade more than 95% of the phenanthrene (150 mg · L(-1)) in a minimal salts medium (MSM) within 48 hours at an initial pH of 7.0 and a temperature of 30 °C. Pn2 could grow well on the MSM plates with a series of other PAHs, including naphthalene, acenaphthene, anthracene and pyrene, and degrade them to different degrees. Pn2 could also colonize the root surface of ryegrass (Lolium multiflorum Lam), invade its internal root tissues and translocate into the plant shoot. When treated with the endophyte Pn2 under hydroponic growth conditions with 2 mg · L(-1) of phenanthrene in the Hoagland solution, the phenanthrene concentrations in ryegrass roots and shoots were reduced by 54% and 57%, respectively, compared with the endophyte-free treatment. Strain Pn2 could be a novel and useful bacterial resource for eliminating plant PAH contamination in polluted environments by degrading the PAHs inside plants. Furthermore, we provide new perspectives on the control of the plant uptake of PAHs via endophytic bacteria.
Project description:A betaproteobacterium within the family Rhodocyclaceae previously identified as a pyrene degrader via stable-isotope probing (SIP) of contaminated soil (designated pyrene group 1 or PG1) was cultivated as the dominant member of a mixed bacterial culture. A metagenomic library was constructed, and the largest contigs were analyzed for genes associated with polycyclic aromatic hydrocarbon (PAH) metabolism. Eight pairs of genes with similarity to the ?- and ?-subunits of ring-hydroxylating dioxygenases (RHDs) associated with aerobic bacterial PAH degradation were identified and linked to PG1 through PCR analyses of a simplified enrichment culture. In tandem with a ferredoxin and reductase found in close proximity to one pair of RHD genes, six of the RHDs were cloned and expressed in Escherichia coli. Each cloned RHD was tested for activity against nine PAHs ranging in size from two to five rings. Despite differences in their predicted protein sequences, each of the six RHDs was capable of transforming phenanthrene and pyrene. Three RHDs could additionally transform naphthalene and fluorene, and these genotypes were also associated with the ability of the E. coli constructs to convert indole to indigo. Only one of the six cloned RHDs was capable of transforming anthracene and benz[a]anthracene. None of the tested RHDs were capable of significantly transforming fluoranthene, chrysene, or benzo[a]pyrene.