Limits...
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

Abundance of 16S rRNA genes of Cycloclasticus (SIP clone PHE1) in samples collected from incubations with unlabeled PHE that were run in parallel during SIP. Bars are the averages and SD of results from triplicate qPCRs measuring the abundance of group-specific 16S rRNA genes. Circles are the means and standard deviations of triplicate measurements of the total mass of DNA per sample. Bars or data points with asterisks represent values with one or more readings below the quantification limit of the assay and are presented as the largest possible value for that point.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 5: Abundance of 16S rRNA genes of Cycloclasticus (SIP clone PHE1) in samples collected from incubations with unlabeled PHE that were run in parallel during SIP. Bars are the averages and SD of results from triplicate qPCRs measuring the abundance of group-specific 16S rRNA genes. Circles are the means and standard deviations of triplicate measurements of the total mass of DNA per sample. Bars or data points with asterisks represent values with one or more readings below the quantification limit of the assay and are presented as the largest possible value for that point.

Mentions: During incubations of the 4567-24 sediment sample with unlabeled PHE in parallel with the SIP incubation, the abundance of 16S rRNA genes for SIP clone PHE1 (OTU-1) increased by several orders of magnitude (Figure 5), thus providing further confirmation of its enrichment on PHE as a growth substrate. By day 3 of the PHE enrichment, the gene abundance increased by over three orders of magnitude, coinciding with the time-frame for the initial stages of disappearance, and mineralization of this compound (Figure 3). By day 9, the gene abundance had increased by ca. six orders of magnitude, coinciding with the almost complete disappearance and high mineralization rate of the PHE. The increase in gene abundance coincided with an increase in the total concentration of DNA, an indicator of cell growth. Collectively, the low bacterial diversity identified in the heavy DNA fractions, which is almost exclusively represented by the Cycloclasticus OTU (SIP clone PHE1), and the dramatic increase in the abundance of these organisms in the incubations with unlabeled PHE, strongly supports that this OTU was solely responsible for degradation of the PAH. In addition, since growth of these organisms coincided with PAH disappearance and the appearance of their 16S rRNA genes only in the most heavily enriched 13C-DNA fractions of incubations containing the labeled substrate, their presence in clone libraries was unlikely due to cross-feeding on a PAH metabolite. We cannot, however, disregard the possibility that other bacterial taxa in the Guaymas 4567-24 sediment sample also possessed the capacity to degrade PHE or its metabolites – they were merely not strongly represented in the most highly 13C-enriched fractions analyzed. However, as discussed below, we also isolated PHE-degrading strains from this sediment sample that are affiliated to other genera. In previous pyrosequencing analyses of the bacterial diversity of Guaymas Basin sediments, Cycloclasticus related sequences composed 0.12% of the average bacterial community at nearby sites, suggesting that representatives of the clade may be poised to act in this oily habitat (Biddle et al., 2012).


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)

Abundance of 16S rRNA genes of Cycloclasticus (SIP clone PHE1) in samples collected from incubations with unlabeled PHE that were run in parallel during SIP. Bars are the averages and SD of results from triplicate qPCRs measuring the abundance of group-specific 16S rRNA genes. Circles are the means and standard deviations of triplicate measurements of the total mass of DNA per sample. Bars or data points with asterisks represent values with one or more readings below the quantification limit of the assay and are presented as the largest possible value for that point.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4493657&req=5

Figure 5: Abundance of 16S rRNA genes of Cycloclasticus (SIP clone PHE1) in samples collected from incubations with unlabeled PHE that were run in parallel during SIP. Bars are the averages and SD of results from triplicate qPCRs measuring the abundance of group-specific 16S rRNA genes. Circles are the means and standard deviations of triplicate measurements of the total mass of DNA per sample. Bars or data points with asterisks represent values with one or more readings below the quantification limit of the assay and are presented as the largest possible value for that point.
Mentions: During incubations of the 4567-24 sediment sample with unlabeled PHE in parallel with the SIP incubation, the abundance of 16S rRNA genes for SIP clone PHE1 (OTU-1) increased by several orders of magnitude (Figure 5), thus providing further confirmation of its enrichment on PHE as a growth substrate. By day 3 of the PHE enrichment, the gene abundance increased by over three orders of magnitude, coinciding with the time-frame for the initial stages of disappearance, and mineralization of this compound (Figure 3). By day 9, the gene abundance had increased by ca. six orders of magnitude, coinciding with the almost complete disappearance and high mineralization rate of the PHE. The increase in gene abundance coincided with an increase in the total concentration of DNA, an indicator of cell growth. Collectively, the low bacterial diversity identified in the heavy DNA fractions, which is almost exclusively represented by the Cycloclasticus OTU (SIP clone PHE1), and the dramatic increase in the abundance of these organisms in the incubations with unlabeled PHE, strongly supports that this OTU was solely responsible for degradation of the PAH. In addition, since growth of these organisms coincided with PAH disappearance and the appearance of their 16S rRNA genes only in the most heavily enriched 13C-DNA fractions of incubations containing the labeled substrate, their presence in clone libraries was unlikely due to cross-feeding on a PAH metabolite. We cannot, however, disregard the possibility that other bacterial taxa in the Guaymas 4567-24 sediment sample also possessed the capacity to degrade PHE or its metabolites – they were merely not strongly represented in the most highly 13C-enriched fractions analyzed. However, as discussed below, we also isolated PHE-degrading strains from this sediment sample that are affiliated to other genera. In previous pyrosequencing analyses of the bacterial diversity of Guaymas Basin sediments, Cycloclasticus related sequences composed 0.12% of the average bacterial community at nearby sites, suggesting that representatives of the clade may be poised to act in this oily habitat (Biddle et al., 2012).

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