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 ¹⁵N stable isotope probing (SERS–¹⁵N 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 ¹⁵N content, because of the substitution of “light” nitrogen by “heavier” ¹⁵N stable isotope. These shifts, especially in 730 cm^–1 band, were highly distinguishable and universal in different bacteria, providing a robust indicator for nitrogen assimilation in bacteria. SERS–¹⁵N SIP was also demonstrated in important N₂-fixing bacteria via ¹⁵N₂ incubations. The same prominent shifts as that induced by ¹⁵NH₄Cl were observed, indicating the applicability of SERS–¹⁵N SIP to different nitrogen sources. SERS–¹⁵N SIP was further applied to environmental microbial community via ¹⁵NH₄Cl, ¹⁵NO₃^–, and ¹⁵N₂ 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–¹⁵N SIP was obtained. The nondestructive SERS–¹⁵N 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 ¹³C content, which were grown in a mixture of ¹²C- and fully carbon-labeled ¹³C-glucose (99%) as a sole carbon source, were fed to the nematode. The ¹³C signal in <i>Caenorhabditis elegans</i> was proportional to the ¹³C content in <i>E. coli</i>. Two Raman spectral biomarkers (Raman bands for phenylalanine at 1001 cm^–1 and thymine at 747 cm^–1 Raman bands), were used to quantify the ¹³C 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 ¹³C-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 ¹³C content, which were grown in a mixture of ¹²C- and fully carbon-labeled ¹³C-glucose (99%) as a sole carbon source, were fed to the nematode. The ¹³C signal in <i>Caenorhabditis elegans</i> was proportional to the ¹³C content in <i>E. coli</i>. Two Raman spectral biomarkers (Raman bands for phenylalanine at 1001 cm^–1 and thymine at 747 cm^–1 Raman bands), were used to quantify the ¹³C 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 ¹³C-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 - ¹³C-DNA; 3, 4 - ¹²C-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

Primers used in this study.

<p>Primers used in this study.</p

Strains and plasmids used in this study.

<p>Strains and plasmids used in this study.</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 ¹²C- and ¹³C-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