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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

Distribution of the ‘heavy’ and ‘light’ DNA in separated SIP fractions. The top of the panel shows the denaturing gradient gel electrophoresis (DGGE) profiles of bacterial PCR products from separated [13C]-PHE fractions with decreasing densities from left to right. The position of unlabeled Escherichia coli DNA, which was used as an internal control in all three isopycnic centrifugations, is shown on the right. The distribution of qPCR-quantified 16S rRNA gene sequences in fractions from [13C]-PHE incubations is shown below the DGGE image for Cycloclasticus (●) and for E. coli (○). Fractions 6–10 (shaded area) were determined to represent 13C heavy DNA and were combined for further analysis. Gene abundance in a fraction are presented as a percentage of the total bacterial 16S rRNA genes quantified in the displayed range of fractions. DGGE banding patterns for a given fraction are aligned with the corresponding gene abundance data below.
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Figure 4: Distribution of the ‘heavy’ and ‘light’ DNA in separated SIP fractions. The top of the panel shows the denaturing gradient gel electrophoresis (DGGE) profiles of bacterial PCR products from separated [13C]-PHE fractions with decreasing densities from left to right. The position of unlabeled Escherichia coli DNA, which was used as an internal control in all three isopycnic centrifugations, is shown on the right. The distribution of qPCR-quantified 16S rRNA gene sequences in fractions from [13C]-PHE incubations is shown below the DGGE image for Cycloclasticus (●) and for E. coli (○). Fractions 6–10 (shaded area) were determined to represent 13C heavy DNA and were combined for further analysis. Gene abundance in a fraction are presented as a percentage of the total bacterial 16S rRNA genes quantified in the displayed range of fractions. DGGE banding patterns for a given fraction are aligned with the corresponding gene abundance data below.

Mentions: Denaturing gradient gel electrophoresis analysis of the fractions derived from the labeled and unlabeled incubations showed clear evidence of isotopic enrichment of DNA in 13C-PHE incubations, separation of 13C-labeled and unlabeled DNA, and different banding patterns between the 13C-enriched and unenriched DNA fractions (Figure 4). One band in particular was especially dominant in fractions containing 13C-enriched DNA. For the 13C-incubation shown in Figure 4, fractions 6–10 were combined and used in the generation of the 16S rRNA gene clone library. Fractions 4–8 of the duplicate gradient were combined in a similar fashion (data not shown). After excluding vector sequences, poor sequence reads, chimeras, and singleton sequences, the clone library constructed from pooled 13C-enriched DNA comprised 68 sequences. Of these 68 sequences, 3 OTUs were identified of which two were singleton sequences affiliated to Marinobacterium and Propionibacterium and not further analyzed. OTU-1, designated SIP clone PHE1, which comprised the majority (95%) of the 68 sequences (>99% sequence identity), was found affiliated to the genus Cycloclasticus.


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)

Distribution of the ‘heavy’ and ‘light’ DNA in separated SIP fractions. The top of the panel shows the denaturing gradient gel electrophoresis (DGGE) profiles of bacterial PCR products from separated [13C]-PHE fractions with decreasing densities from left to right. The position of unlabeled Escherichia coli DNA, which was used as an internal control in all three isopycnic centrifugations, is shown on the right. The distribution of qPCR-quantified 16S rRNA gene sequences in fractions from [13C]-PHE incubations is shown below the DGGE image for Cycloclasticus (●) and for E. coli (○). Fractions 6–10 (shaded area) were determined to represent 13C heavy DNA and were combined for further analysis. Gene abundance in a fraction are presented as a percentage of the total bacterial 16S rRNA genes quantified in the displayed range of fractions. DGGE banding patterns for a given fraction are aligned with the corresponding gene abundance data below.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 4: Distribution of the ‘heavy’ and ‘light’ DNA in separated SIP fractions. The top of the panel shows the denaturing gradient gel electrophoresis (DGGE) profiles of bacterial PCR products from separated [13C]-PHE fractions with decreasing densities from left to right. The position of unlabeled Escherichia coli DNA, which was used as an internal control in all three isopycnic centrifugations, is shown on the right. The distribution of qPCR-quantified 16S rRNA gene sequences in fractions from [13C]-PHE incubations is shown below the DGGE image for Cycloclasticus (●) and for E. coli (○). Fractions 6–10 (shaded area) were determined to represent 13C heavy DNA and were combined for further analysis. Gene abundance in a fraction are presented as a percentage of the total bacterial 16S rRNA genes quantified in the displayed range of fractions. DGGE banding patterns for a given fraction are aligned with the corresponding gene abundance data below.
Mentions: Denaturing gradient gel electrophoresis analysis of the fractions derived from the labeled and unlabeled incubations showed clear evidence of isotopic enrichment of DNA in 13C-PHE incubations, separation of 13C-labeled and unlabeled DNA, and different banding patterns between the 13C-enriched and unenriched DNA fractions (Figure 4). One band in particular was especially dominant in fractions containing 13C-enriched DNA. For the 13C-incubation shown in Figure 4, fractions 6–10 were combined and used in the generation of the 16S rRNA gene clone library. Fractions 4–8 of the duplicate gradient were combined in a similar fashion (data not shown). After excluding vector sequences, poor sequence reads, chimeras, and singleton sequences, the clone library constructed from pooled 13C-enriched DNA comprised 68 sequences. Of these 68 sequences, 3 OTUs were identified of which two were singleton sequences affiliated to Marinobacterium and Propionibacterium and not further analyzed. OTU-1, designated SIP clone PHE1, which comprised the majority (95%) of the 68 sequences (>99% sequence identity), was found affiliated to the genus Cycloclasticus.

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