Limits...
DNA-based stable isotope probing coupled with cultivation methods implicates Methylophaga in hydrocarbon degradation.

Mishamandani S, Gutierrez T, Aitken MD - Front Microbiol (2014)

Bottom Line: Those with an almost exclusive ability to utilize hydrocarbons as a sole carbon and energy source have been found confined to just a few genera.This is a contentious group since, though some of its members have been proposed to degrade hydrocarbons, substantive evidence has not previously confirmed this.Both showed substantial increases in gene copy number during the experiments.

View Article: PubMed Central - PubMed

Affiliation: Department of Environmental Sciences and Engineering, Gillings School of Global Public Health, University of North Carolina Chapel Hill, NC, USA.

ABSTRACT
Marine hydrocarbon-degrading bacteria perform a fundamental role in the oxidation and ultimate removal of crude oil and its petrochemical derivatives in coastal and open ocean environments. Those with an almost exclusive ability to utilize hydrocarbons as a sole carbon and energy source have been found confined to just a few genera. Here we used stable isotope probing (SIP), a valuable tool to link the phylogeny and function of targeted microbial groups, to investigate hydrocarbon-degrading bacteria in coastal North Carolina sea water (Beaufort Inlet, USA) with uniformly labeled [(13)C]n-hexadecane. The dominant sequences in clone libraries constructed from (13)C-enriched bacterial DNA (from n-hexadecane enrichments) were identified to belong to the genus Alcanivorax, with ≤98% sequence identity to the closest type strain-thus representing a putative novel phylogenetic taxon within this genus. Unexpectedly, we also identified (13)C-enriched sequences in heavy DNA fractions that were affiliated to the genus Methylophaga. This is a contentious group since, though some of its members have been proposed to degrade hydrocarbons, substantive evidence has not previously confirmed this. We used quantitative PCR primers targeting the 16S rRNA gene of the SIP-identified Alcanivorax and Methylophaga to determine their abundance in incubations amended with unlabeled n-hexadecane. Both showed substantial increases in gene copy number during the experiments. Subsequently, we isolated a strain representing the SIP-identified Methylophaga sequences (99.9% 16S rRNA gene sequence identity) and used it to show, for the first time, direct evidence of hydrocarbon degradation by a cultured Methylophaga sp. This study demonstrates the value of coupling SIP with cultivation methods to identify and expand on the known diversity of hydrocarbon-degrading bacteria in the marine environment.

No MeSH data available.


Related in: MedlinePlus

Growth of Methylophaga sp. strain SM14 on n-hexadecane (0.002% v/v) as the sole carbon and energy source and disappearance of the n-hexadecane as measured by GC. Each data point is the mean ± standard deviation from triplicate incubations. Open symbols represent live cultures (non-acid treated); filled symbols represent acid-inhibited controls. Circles and squares are cell density and GC measurements, respectively. Some error bars are smaller than the symbol.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3936186&req=5

Figure 4: Growth of Methylophaga sp. strain SM14 on n-hexadecane (0.002% v/v) as the sole carbon and energy source and disappearance of the n-hexadecane as measured by GC. Each data point is the mean ± standard deviation from triplicate incubations. Open symbols represent live cultures (non-acid treated); filled symbols represent acid-inhibited controls. Circles and squares are cell density and GC measurements, respectively. Some error bars are smaller than the symbol.

Mentions: Enrichment experiments using the North Carolina field sample as inoculum and with methanol as the sole carbon and energy source yielded two isolates, designated strain SM13 and strain SM14. Both strains were found affiliated to the genus Methylophaga based on sequencing of their 16S rRNA gene—they shared 94% sequence identity between them. Strain SM13 proved difficult to maintain in laboratory culture as it eventually ceased to grow upon subsequent subculturing and was therefore no longer used for further experimentation. On the other hand, strain SM14 yielded small (0.05–0.15 mm) off-white colonies after 2 weeks incubation on ONR7a agar amended with Na-pyruvate as the sole carbon and energy source (not shown). The strain also grew well in ONR7a broth amended with Na-pyruvate or methanol. Evidence of the strain's ability to grow on n-hexadecane as a sole carbon and energy source is shown in Figure 4. At an initial n-hexadecane concentration of 0.002% (v/v), the strain reached a low cell density of ca. 0.015 at 600 nm. However, growth coincided with the disappearance of the n-hexadecane, which was indicative that the hydrocarbon was being degraded by the strain and utilized as a carbon source for growth. No growth was measured in uninoculated controls, or in inoculated incubations in the absence of any added n-hexadecane. Cultures amended with higher concentrations of n-hexadecane yielded higher cell densities (results not shown). Evidence that this was a pure culture of Methylophaga strain SM14 was confirmed by 16S rRNA gene sequencing of DNA isolated from cell pellets collected toward the end of these growth experiments.


DNA-based stable isotope probing coupled with cultivation methods implicates Methylophaga in hydrocarbon degradation.

Mishamandani S, Gutierrez T, Aitken MD - Front Microbiol (2014)

Growth of Methylophaga sp. strain SM14 on n-hexadecane (0.002% v/v) as the sole carbon and energy source and disappearance of the n-hexadecane as measured by GC. Each data point is the mean ± standard deviation from triplicate incubations. Open symbols represent live cultures (non-acid treated); filled symbols represent acid-inhibited controls. Circles and squares are cell density and GC measurements, respectively. Some error bars are smaller than the symbol.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3936186&req=5

Figure 4: Growth of Methylophaga sp. strain SM14 on n-hexadecane (0.002% v/v) as the sole carbon and energy source and disappearance of the n-hexadecane as measured by GC. Each data point is the mean ± standard deviation from triplicate incubations. Open symbols represent live cultures (non-acid treated); filled symbols represent acid-inhibited controls. Circles and squares are cell density and GC measurements, respectively. Some error bars are smaller than the symbol.
Mentions: Enrichment experiments using the North Carolina field sample as inoculum and with methanol as the sole carbon and energy source yielded two isolates, designated strain SM13 and strain SM14. Both strains were found affiliated to the genus Methylophaga based on sequencing of their 16S rRNA gene—they shared 94% sequence identity between them. Strain SM13 proved difficult to maintain in laboratory culture as it eventually ceased to grow upon subsequent subculturing and was therefore no longer used for further experimentation. On the other hand, strain SM14 yielded small (0.05–0.15 mm) off-white colonies after 2 weeks incubation on ONR7a agar amended with Na-pyruvate as the sole carbon and energy source (not shown). The strain also grew well in ONR7a broth amended with Na-pyruvate or methanol. Evidence of the strain's ability to grow on n-hexadecane as a sole carbon and energy source is shown in Figure 4. At an initial n-hexadecane concentration of 0.002% (v/v), the strain reached a low cell density of ca. 0.015 at 600 nm. However, growth coincided with the disappearance of the n-hexadecane, which was indicative that the hydrocarbon was being degraded by the strain and utilized as a carbon source for growth. No growth was measured in uninoculated controls, or in inoculated incubations in the absence of any added n-hexadecane. Cultures amended with higher concentrations of n-hexadecane yielded higher cell densities (results not shown). Evidence that this was a pure culture of Methylophaga strain SM14 was confirmed by 16S rRNA gene sequencing of DNA isolated from cell pellets collected toward the end of these growth experiments.

Bottom Line: Those with an almost exclusive ability to utilize hydrocarbons as a sole carbon and energy source have been found confined to just a few genera.This is a contentious group since, though some of its members have been proposed to degrade hydrocarbons, substantive evidence has not previously confirmed this.Both showed substantial increases in gene copy number during the experiments.

View Article: PubMed Central - PubMed

Affiliation: Department of Environmental Sciences and Engineering, Gillings School of Global Public Health, University of North Carolina Chapel Hill, NC, USA.

ABSTRACT
Marine hydrocarbon-degrading bacteria perform a fundamental role in the oxidation and ultimate removal of crude oil and its petrochemical derivatives in coastal and open ocean environments. Those with an almost exclusive ability to utilize hydrocarbons as a sole carbon and energy source have been found confined to just a few genera. Here we used stable isotope probing (SIP), a valuable tool to link the phylogeny and function of targeted microbial groups, to investigate hydrocarbon-degrading bacteria in coastal North Carolina sea water (Beaufort Inlet, USA) with uniformly labeled [(13)C]n-hexadecane. The dominant sequences in clone libraries constructed from (13)C-enriched bacterial DNA (from n-hexadecane enrichments) were identified to belong to the genus Alcanivorax, with ≤98% sequence identity to the closest type strain-thus representing a putative novel phylogenetic taxon within this genus. Unexpectedly, we also identified (13)C-enriched sequences in heavy DNA fractions that were affiliated to the genus Methylophaga. This is a contentious group since, though some of its members have been proposed to degrade hydrocarbons, substantive evidence has not previously confirmed this. We used quantitative PCR primers targeting the 16S rRNA gene of the SIP-identified Alcanivorax and Methylophaga to determine their abundance in incubations amended with unlabeled n-hexadecane. Both showed substantial increases in gene copy number during the experiments. Subsequently, we isolated a strain representing the SIP-identified Methylophaga sequences (99.9% 16S rRNA gene sequence identity) and used it to show, for the first time, direct evidence of hydrocarbon degradation by a cultured Methylophaga sp. This study demonstrates the value of coupling SIP with cultivation methods to identify and expand on the known diversity of hydrocarbon-degrading bacteria in the marine environment.

No MeSH data available.


Related in: MedlinePlus