13 research outputs found
Surface-Enhanced Raman Spectroscopy Combined with Stable Isotope Probing to Monitor Nitrogen Assimilation at Both Bulk and Single-Cell Level
Microbe-mediated
biogeochemical cycle of nitrogen is a critical
process in the environment. In this study, surface-enhanced Raman
spectroscopy combined with <sup>15</sup>N stable isotope probing (SERS–<sup>15</sup>N SIP) was developed as a new, nondestructive, and robust
approach to probe nitrogen assimilation by bacteria at both bulk and
single-cell level, and from pure culture to environmental microbial
community. Multiple distinguishable SERS band shifts were observed
and displayed a linear relationship with <sup>15</sup>N content, because
of the substitution of “light” nitrogen by “heavier” <sup>15</sup>N stable isotope. These shifts, especially in 730 cm<sup>–1</sup> band, were highly distinguishable and universal in
different bacteria, providing a robust indicator for nitrogen assimilation
in bacteria. SERS–<sup>15</sup>N SIP was also demonstrated
in important N<sub>2</sub>-fixing bacteria via <sup>15</sup>N<sub>2</sub> incubations. The same prominent shifts as that induced by <sup>15</sup>NH<sub>4</sub>Cl were observed, indicating the applicability
of SERS–<sup>15</sup>N SIP to different nitrogen sources. SERS–<sup>15</sup>N SIP was further applied to environmental microbial community
via <sup>15</sup>NH<sub>4</sub>Cl, <sup>15</sup>NO<sub>3</sub><sup>–</sup>, and <sup>15</sup>N<sub>2</sub> incubation. Bacteria-
and nitrogen source-dependent activity in nitrogen assimilation were
revealed in environmental microbial community, pointing to the bacterial
diversity and necessity of single-cell level investigation. Finally,
by mixing optimized ratio of bacteria with Ag NPs, explicit single-cell
SERS–<sup>15</sup>N SIP was obtained. The nondestructive SERS–<sup>15</sup>N SIP approach will be useful not only to identify active
nitrogen-assimilating cells, but also enable Raman activated cell
sorting and downstream genomic analysis, which will bring in deep
insights into nitrogen metabolism of environmental microorganisms
Stable Isotope Probing and Raman Spectroscopy for Monitoring Carbon Flow in a Food Chain and Revealing Metabolic Pathway
Accurately measuring carbon flows is a challenge for
understanding
processes such as diverse intracellular metabolic pathways and predator-prey
interactions. Combined with stable isotope probing (SIP), single-cell
Raman spectroscopy was demonstrated for the first time to link the
food chain from carbon substrate to bacterial prey up to predators
at the single-cell level in a quantitative and nondestructive manner. <i>Escherichia coli</i> OP50 with different <sup>13</sup>C content,
which were grown in a mixture of <sup>12</sup>C- and fully carbon-labeled <sup>13</sup>C-glucose (99%) as a sole carbon source, were fed to the
nematode. The <sup>13</sup>C signal in <i>Caenorhabditis elegans</i> was proportional to the <sup>13</sup>C content in <i>E. coli</i>. Two Raman spectral biomarkers (Raman bands for phenylalanine at
1001 cm<sup>–1</sup> and thymine at 747 cm<sup>–1</sup> Raman bands), were used to quantify the <sup>13</sup>C content in <i>E. coli</i> and <i>C. elegans</i> over a range of
1.1–99%. The phenylalanine Raman band was a suitable biomarker
for prokaryotic cells and thymine Raman band for eukaryotic cells.
A biochemical mechanism accounting for the Raman red shifts of phenylalanine
and thymine in response to <sup>13</sup>C-labeling is proposed in
this study and is supported by quantum chemical calculation. This
study offers new insights of carbon flow via the food chain and provides
a research tool for microbial ecology and investigation of biochemical
pathways
Unidentified specimen sp.
Accurately measuring carbon flows is a challenge for
understanding
processes such as diverse intracellular metabolic pathways and predator-prey
interactions. Combined with stable isotope probing (SIP), single-cell
Raman spectroscopy was demonstrated for the first time to link the
food chain from carbon substrate to bacterial prey up to predators
at the single-cell level in a quantitative and nondestructive manner. <i>Escherichia coli</i> OP50 with different <sup>13</sup>C content,
which were grown in a mixture of <sup>12</sup>C- and fully carbon-labeled <sup>13</sup>C-glucose (99%) as a sole carbon source, were fed to the
nematode. The <sup>13</sup>C signal in <i>Caenorhabditis elegans</i> was proportional to the <sup>13</sup>C content in <i>E. coli</i>. Two Raman spectral biomarkers (Raman bands for phenylalanine at
1001 cm<sup>–1</sup> and thymine at 747 cm<sup>–1</sup> Raman bands), were used to quantify the <sup>13</sup>C content in <i>E. coli</i> and <i>C. elegans</i> over a range of
1.1–99%. The phenylalanine Raman band was a suitable biomarker
for prokaryotic cells and thymine Raman band for eukaryotic cells.
A biochemical mechanism accounting for the Raman red shifts of phenylalanine
and thymine in response to <sup>13</sup>C-labeling is proposed in
this study and is supported by quantum chemical calculation. This
study offers new insights of carbon flow via the food chain and provides
a research tool for microbial ecology and investigation of biochemical
pathways
Naphthalene dioxygenase (NDO) PCR products.
<p>Two pairs of degenerate primers, COM1_F&COM1_R (<i>Comamonas</i>-type), and PSE1_F&PSE1_R (<i>Pseudomonas</i>-type) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047530#pone-0047530-t001" target="_blank">Table 1</a>) and different DNA templates: 1, 2 - <sup>13</sup>C-DNA; 3, 4 - <sup>12</sup>C-DNA; 5– blank control were used. M - DNA molecular weight Ladder.</p
Structure of the <i>nag</i> operons revealed by BGT and pyrosequencing.
<p>The <i>nag2</i> operon was of a mosaic-type pattern. The gray scale of individual genes within the <i>nag2</i> operon indicates % similarity to <i>nag</i>-U2 (black) or <i>nag</i>-CJ2 (white). The numbers beneath the <i>nag</i>2 operon indicates the % similarity to <i>nag</i>-U2/<i>nag</i>-CJ2/neither.</p
A. Schematic of a toolbox to dissect microbial community structure and their functional genes in a complex community.
<p>The culture-independent SMB toolbox comprises stable isotope probing, metagenomic sequencing and biosensor-based gene transducer. <b>B. </b><b>Schematic of the biosensor-based gene transducer.</b> This approach can be used to screen for gene encoded functions for the metabolism of molecules with no distinguishable activity or phenotypes.</p
Taxonomic assignments of the microbial communities in the <sup>12</sup>C- and <sup>13</sup>C-DNA fractions revealed by pyrosequencing.
<p>Relative abundance of the species as a % of total 16S-rRNA reads is shown.</p
Bioluminescence of biosensor transducers incorporating DHN degradation genes.
<p>ADPWH_Nag containing a <i>nag</i>2 gene cluster was rapidly activated to show bioluminescence after the addition of 50 µM DHN, confirming the function of the gene cluster. No bioluminescence was detected in the absence DHN. The negative control ADPWH_1274 did not respond to DHN. ADPWH_Nah containing a <i>nah</i> gene cluster was used as a positive control.</p