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Cultivation-dependent and cultivation-independent characterization of hydrocarbon-degrading bacteria in Guaymas Basin sediments.

Gutierrez T, Biddle JF, Teske A, Aitken MD - Front Microbiol (2015)

Bottom Line: We used quantitative PCR primers targeting the 16S rRNA gene of the SIP-identified Cycloclasticus to determine their abundance in sediment incubations amended with unlabeled PHE and showed substantial increases in gene abundance during the experiments.We also isolated a strain, BG-2, representing the SIP-identified Cycloclasticus sequence (99.9% 16S rRNA gene sequence identity), and used this strain to provide direct evidence of PHE degradation and mineralization.In addition, we isolated Halomonas, Thalassospira, and Lutibacterium sp. with demonstrable PHE-degrading capacity from Guaymas Basin sediment.

View Article: PubMed Central - PubMed

Affiliation: Department of Environmental Sciences and Engineering, Gillings School of Global Public Health, University of North Carolina at Chapel Hill, Chapel Hill, NC USA ; School of Life Sciences, Heriot-Watt University, Edinburgh UK.

ABSTRACT
Marine hydrocarbon-degrading bacteria perform a fundamental role in the biodegradation of crude oil and its petrochemical derivatives in coastal and open ocean environments. However, there is a paucity of knowledge on the diversity and function of these organisms in deep-sea sediment. Here we used stable-isotope probing (SIP), a valuable tool to link the phylogeny and function of targeted microbial groups, to investigate polycyclic aromatic hydrocarbon (PAH)-degrading bacteria under aerobic conditions in sediments from Guaymas Basin with uniformly labeled [(13)C]-phenanthrene (PHE). The dominant sequences in clone libraries constructed from (13)C-enriched bacterial DNA (from PHE enrichments) were identified to belong to the genus Cycloclasticus. We used quantitative PCR primers targeting the 16S rRNA gene of the SIP-identified Cycloclasticus to determine their abundance in sediment incubations amended with unlabeled PHE and showed substantial increases in gene abundance during the experiments. We also isolated a strain, BG-2, representing the SIP-identified Cycloclasticus sequence (99.9% 16S rRNA gene sequence identity), and used this strain to provide direct evidence of PHE degradation and mineralization. In addition, we isolated Halomonas, Thalassospira, and Lutibacterium sp. with demonstrable PHE-degrading capacity from Guaymas Basin sediment. This study demonstrates the value of coupling SIP with cultivation methods to identify and expand on the known diversity of PAH-degrading bacteria in the deep-sea.

No MeSH data available.


Related in: MedlinePlus

Phylogenetic tree of SIP clone PHE1 and of the isolated strains from Guaymas Basin surface (0–4 cm) sediment shown alongside closely related sequences and type strains from GenBank. Guaymas strain EbS7 and Guaymas clones SB-9 and SB-21 are included as representatives of sulfate-reducing bacteria identified at Guaymas Basin that are capable of oxidizing aromatic hydrocarbons. Aerobic PAH-degrading Cycloclasticus P1, isolated from deep-sea sediments of the West Pacific, and other strains of this genus isolated from shallower sediment locations (strains M4-6 and PS-1T), are also included. The tree was constructed using the neighbor-joining algorithm. Nodes with bootstrap support (1000 replications) of at least 65% (○) and 90% (●) are marked. Accession numbers of all sequences are given in parentheses. The scale bar indicates the difference of number of substitutions per site.
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Figure 6: Phylogenetic tree of SIP clone PHE1 and of the isolated strains from Guaymas Basin surface (0–4 cm) sediment shown alongside closely related sequences and type strains from GenBank. Guaymas strain EbS7 and Guaymas clones SB-9 and SB-21 are included as representatives of sulfate-reducing bacteria identified at Guaymas Basin that are capable of oxidizing aromatic hydrocarbons. Aerobic PAH-degrading Cycloclasticus P1, isolated from deep-sea sediments of the West Pacific, and other strains of this genus isolated from shallower sediment locations (strains M4-6 and PS-1T), are also included. The tree was constructed using the neighbor-joining algorithm. Nodes with bootstrap support (1000 replications) of at least 65% (○) and 90% (●) are marked. Accession numbers of all sequences are given in parentheses. The scale bar indicates the difference of number of substitutions per site.

Mentions: The near-complete 16S rRNA gene sequence (>1400 bp) of each of these PHE-degrading strains were compared with related GenBank sequences, including sequences from studies investigating hydrocarbon-degrading bacteria in deep-sea sediments (Figure 6). From a BLAST analysis, the highest level (99.9%) of sequence identity for strain BG-2 was to Cycloclasticus sp. strain P1 isolated from deep-sea sediment of the West Pacific at 2682 m water depth (Wang et al., 2008), Cycloclasticus spirillensus strain M4-6 isolated from marine macrofaunal burrow sediments of Lowes Cove in Maine, USA (Chung and King, 2001), and to Cycloclasticus sp. clone SWNAP12 which was identified in 13C-enriched DNA of a SIP enrichment of a surface oil slick sample collected during the Deepwater Horizon oil spill (Gutierrez et al., 2013b). The next closest cultivated relative to BG-2 was C. pugetii strain PS-1T (99.7% sequence identity) isolated from marine sediment of Puget Sound (Dyksterhouse et al., 1995). Notably, isolated strain BG-2 shared 99.7% 16S rRNA gene sequence identity with SIP clone PHE1. Strain BG-3a was most closely related to the type strains Halomonas alkaliantarctica strain CRSST (Poli et al., 2007; 99.4% sequence identity), H. neptunia strain Eplume1T (Kaye et al., 2004; 99.3% sequence identity), and the exopolysaccharide (EPS)-producer H. variabilis strain ANT-3b (Pepi et al., 2005; 99.3% sequence identity). The next closest cultivated relatives to BG-3a were H. titanicae strain BH1T (Sanchez-Porro et al., 2010) and H. variabilis strain DSM 3051T (Arahal et al., 2002; 98.8% sequence identity). Strain BG-3b shared 99.4% sequence identity to Thalassospira lucentensis strain VBW014 (Rajasabapathy et al., 2014), and to the type strains T. alkalitolerans strain MBE#61T (Tsubouchi et al., 2014) and T. profundimaris strain WP0211T (Liu et al., 2007) with 99.1% and 99.3% sequence identity, respectively. Strain BG-4 was most closely related to the type strains Lutibacterium anuloederans strain LC8T (Chung and King, 2001) and Erythrobacter marinus strain HWDM-33T (Jung et al., 2012) with 98.7 and 98.6% sequence identity, respectively. In a previous deep sequencing survey of bacterial diversity at Guaymas basin, Halomonas sp. accounted for 0.1% of sequences, whereas Thalassospira and Lutibacterium were not detected (Biddle et al., 2012). This highlights the importance of using enrichment [cultivation-independent (DNA-SIP) and/or cultivation-dependent] methods to uncover minority taxa that may not be identified by solely sequencing surveys, and to linking these organisms to a particular function – in this case the degradation of hydrocarbons.


Cultivation-dependent and cultivation-independent characterization of hydrocarbon-degrading bacteria in Guaymas Basin sediments.

Gutierrez T, Biddle JF, Teske A, Aitken MD - Front Microbiol (2015)

Phylogenetic tree of SIP clone PHE1 and of the isolated strains from Guaymas Basin surface (0–4 cm) sediment shown alongside closely related sequences and type strains from GenBank. Guaymas strain EbS7 and Guaymas clones SB-9 and SB-21 are included as representatives of sulfate-reducing bacteria identified at Guaymas Basin that are capable of oxidizing aromatic hydrocarbons. Aerobic PAH-degrading Cycloclasticus P1, isolated from deep-sea sediments of the West Pacific, and other strains of this genus isolated from shallower sediment locations (strains M4-6 and PS-1T), are also included. The tree was constructed using the neighbor-joining algorithm. Nodes with bootstrap support (1000 replications) of at least 65% (○) and 90% (●) are marked. Accession numbers of all sequences are given in parentheses. The scale bar indicates the difference of number of substitutions per site.
© Copyright Policy
Related In: Results  -  Collection

License
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getmorefigures.php?uid=PMC4493657&req=5

Figure 6: Phylogenetic tree of SIP clone PHE1 and of the isolated strains from Guaymas Basin surface (0–4 cm) sediment shown alongside closely related sequences and type strains from GenBank. Guaymas strain EbS7 and Guaymas clones SB-9 and SB-21 are included as representatives of sulfate-reducing bacteria identified at Guaymas Basin that are capable of oxidizing aromatic hydrocarbons. Aerobic PAH-degrading Cycloclasticus P1, isolated from deep-sea sediments of the West Pacific, and other strains of this genus isolated from shallower sediment locations (strains M4-6 and PS-1T), are also included. The tree was constructed using the neighbor-joining algorithm. Nodes with bootstrap support (1000 replications) of at least 65% (○) and 90% (●) are marked. Accession numbers of all sequences are given in parentheses. The scale bar indicates the difference of number of substitutions per site.
Mentions: The near-complete 16S rRNA gene sequence (>1400 bp) of each of these PHE-degrading strains were compared with related GenBank sequences, including sequences from studies investigating hydrocarbon-degrading bacteria in deep-sea sediments (Figure 6). From a BLAST analysis, the highest level (99.9%) of sequence identity for strain BG-2 was to Cycloclasticus sp. strain P1 isolated from deep-sea sediment of the West Pacific at 2682 m water depth (Wang et al., 2008), Cycloclasticus spirillensus strain M4-6 isolated from marine macrofaunal burrow sediments of Lowes Cove in Maine, USA (Chung and King, 2001), and to Cycloclasticus sp. clone SWNAP12 which was identified in 13C-enriched DNA of a SIP enrichment of a surface oil slick sample collected during the Deepwater Horizon oil spill (Gutierrez et al., 2013b). The next closest cultivated relative to BG-2 was C. pugetii strain PS-1T (99.7% sequence identity) isolated from marine sediment of Puget Sound (Dyksterhouse et al., 1995). Notably, isolated strain BG-2 shared 99.7% 16S rRNA gene sequence identity with SIP clone PHE1. Strain BG-3a was most closely related to the type strains Halomonas alkaliantarctica strain CRSST (Poli et al., 2007; 99.4% sequence identity), H. neptunia strain Eplume1T (Kaye et al., 2004; 99.3% sequence identity), and the exopolysaccharide (EPS)-producer H. variabilis strain ANT-3b (Pepi et al., 2005; 99.3% sequence identity). The next closest cultivated relatives to BG-3a were H. titanicae strain BH1T (Sanchez-Porro et al., 2010) and H. variabilis strain DSM 3051T (Arahal et al., 2002; 98.8% sequence identity). Strain BG-3b shared 99.4% sequence identity to Thalassospira lucentensis strain VBW014 (Rajasabapathy et al., 2014), and to the type strains T. alkalitolerans strain MBE#61T (Tsubouchi et al., 2014) and T. profundimaris strain WP0211T (Liu et al., 2007) with 99.1% and 99.3% sequence identity, respectively. Strain BG-4 was most closely related to the type strains Lutibacterium anuloederans strain LC8T (Chung and King, 2001) and Erythrobacter marinus strain HWDM-33T (Jung et al., 2012) with 98.7 and 98.6% sequence identity, respectively. In a previous deep sequencing survey of bacterial diversity at Guaymas basin, Halomonas sp. accounted for 0.1% of sequences, whereas Thalassospira and Lutibacterium were not detected (Biddle et al., 2012). This highlights the importance of using enrichment [cultivation-independent (DNA-SIP) and/or cultivation-dependent] methods to uncover minority taxa that may not be identified by solely sequencing surveys, and to linking these organisms to a particular function – in this case the degradation of hydrocarbons.

Bottom Line: We used quantitative PCR primers targeting the 16S rRNA gene of the SIP-identified Cycloclasticus to determine their abundance in sediment incubations amended with unlabeled PHE and showed substantial increases in gene abundance during the experiments.We also isolated a strain, BG-2, representing the SIP-identified Cycloclasticus sequence (99.9% 16S rRNA gene sequence identity), and used this strain to provide direct evidence of PHE degradation and mineralization.In addition, we isolated Halomonas, Thalassospira, and Lutibacterium sp. with demonstrable PHE-degrading capacity from Guaymas Basin sediment.

View Article: PubMed Central - PubMed

Affiliation: Department of Environmental Sciences and Engineering, Gillings School of Global Public Health, University of North Carolina at Chapel Hill, Chapel Hill, NC USA ; School of Life Sciences, Heriot-Watt University, Edinburgh UK.

ABSTRACT
Marine hydrocarbon-degrading bacteria perform a fundamental role in the biodegradation of crude oil and its petrochemical derivatives in coastal and open ocean environments. However, there is a paucity of knowledge on the diversity and function of these organisms in deep-sea sediment. Here we used stable-isotope probing (SIP), a valuable tool to link the phylogeny and function of targeted microbial groups, to investigate polycyclic aromatic hydrocarbon (PAH)-degrading bacteria under aerobic conditions in sediments from Guaymas Basin with uniformly labeled [(13)C]-phenanthrene (PHE). The dominant sequences in clone libraries constructed from (13)C-enriched bacterial DNA (from PHE enrichments) were identified to belong to the genus Cycloclasticus. We used quantitative PCR primers targeting the 16S rRNA gene of the SIP-identified Cycloclasticus to determine their abundance in sediment incubations amended with unlabeled PHE and showed substantial increases in gene abundance during the experiments. We also isolated a strain, BG-2, representing the SIP-identified Cycloclasticus sequence (99.9% 16S rRNA gene sequence identity), and used this strain to provide direct evidence of PHE degradation and mineralization. In addition, we isolated Halomonas, Thalassospira, and Lutibacterium sp. with demonstrable PHE-degrading capacity from Guaymas Basin sediment. This study demonstrates the value of coupling SIP with cultivation methods to identify and expand on the known diversity of PAH-degrading bacteria in the deep-sea.

No MeSH data available.


Related in: MedlinePlus