Article pubs.acs.org/ac
Monitoring Trehalose Uptake and Conversion by Single Bacteria using Laser Tweezers Raman Spectroscopy Anna Avetisyan,† John Beck Jensen,† and Thomas Huser*,‡,§ †
Department for Arctic and Marine Biology, University of Tromsø, N-9037 Tromsø, Norway NSF Center for Biophotonics Science and Technology, University of California, Davis, Sacramento, California, United States § Biomolecular Photonics, Department of Physics, University of Bielefeld, 33501 Bielefeld, Germany ‡
ABSTRACT: Having the ability to monitor metabolic activity at the scale of single bacterial cells noninvasively would enable us to follow changes in the distribution of activity in bacterial systems which is of major importance for topics such as integration of metabolism and development, metabolic engineering, microbial activity and drug resistance, cell−cell interactions, and quorum sensing. Here, we used laser tweezers Raman spectroscopy to monitor the in vivo real-time uptake and conversion of trehalose by single bacterial cells. This approach can be used for the quantitative determination of sugar uptake by a single bacterium and its metabolic response to the sugar application with time. We show that uptake of trehalose can be quantified in single living bacterial cells held in place by an optical trap while simultaneously collecting Raman spectra upon application of sugar to the medium. This technique yields real-time chemical information in a label-free manner, thus eliminating the limitations of toxicity of the isotopic probes common in studying transport processes. It can substitute the laborious and time-consuming analytical evaluation. Although the single-cell Raman spectroscopy method demonstrated here is focused on the study of trehalose uptake by Sinorhizobium meliloti, the demonstrated approach is applicable to many different organisms and carbohydrates in general.
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cell extracts, which might not accurately reveal the intracellular sugar amounts due to incomplete extraction and quenching of the metabolism and may result in loss of the link of cellular dynamics and functionality, which leads to a gap in understanding and controlling fundamental biological processes. At the same time, methods used to monitor real-time changes in single cells normally require the use of exogenous fluorophores or stains,3 possessing potential risks of fluorophore photobleaching or inhomogeneous expression and distribution of the FRET sensors. These limitations stimulate the development of an entirely new approach permitting the real-time monitoring of sugar uptake and metabolism in single living cells. Raman spectroscopy has been shown to be a powerful spectroscopic technique for studying biological processes down to the single cell level. It is based on the inelastic scattering of light by chemical bond vibrations within a molecule resulting in a “molecular fingerprint” which can be used for biochemical analysis. Raman spectroscopy is an attractive alternative for collecting direct quantitative chemical information in a labelfree, nondestructive, and real-time manner at the single cell level without any exogenous modification of samples. When combined with laser (optical) tweezers, also called lasertweezers Raman spectroscopy (LTRS), it permits the simultaneous immobilization of single cells in the laser beam and the excitation and collection of data from chemical bond
ugars are fundamental biomolecules for the growth and development of bacteria. They serve as a source of carbon and energy, structural components, and signaling molecules. Therefore, detailed information on carbohydrate uptake and metabolism are of great interest for a general understanding of biological processes occurring in bacterial cells and for optimizing the metabolic state of bacteria in bioengineering processes. Trehalose [α-D-glucopyranosyl-(1,1)-α-D-glucopyranoside] is a nonreducing disaccharide present in a wide variety of living organisms, including bacteria, fungi, yeast, insects, invertebrates and plants. Besides being a carbon and energy source, it plays an important role in stress tolerance, providing cells with osmoprotection, protection against heat, cold and oxygen radicals. 1 In the Gram-negative soil bacterium Sinorhizobium meliloti, which exists in nature as a free-living saprophyte and a nitrogen-fixing symbiont inside root nodules, trehalose has been found in both living forms, emphasizing its importance and possible association with virulence-attributed properties of bacteria. Thus, studying trehalose uptake and the metabolic flux will provide deeper insights not only into the mechanisms of cellular processes in single cells but also in better understanding of host−microbe interactions. Uptake and metabolic flux of carbohydrates is commonly analyzed using isotope probing, followed by chromatography, mass spectrometry or NMR.2 These methods provide a compositional identification of the carbohydrates but are relatively time-consuming and effort-intensive. They are performed on bulk cell populations, which lack information of extra- or intracellular variations. Moreover, they are based on © 2013 American Chemical Society
Received: April 19, 2013 Accepted: June 24, 2013 Published: June 24, 2013 7264
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vibrations. LTRS readily enables the detailed chemical analysis of cells in their natural state. It produces in vivo spectra from single living cells with excellent signal-to-noise ratios and enables a new level of quantitative analysis. Due to these advantages, LTRS has become an analytical method of choice in a wide range of biological applications. LTRS has been applied to eukaryotic cells, permitting a variety of experiments on mammalian cells.4 In microbiology, LTRS has been used for identification of bacteria and assessment of bacterial viability,5 as well as in studying bacterial metabolic activity, such as induction of protein expression,6 heat denaturation of microorganisms,7 kinetic germination of bacterial spores8 and cell growth and differentiation.9 LTRS can also be used for the direct quantitative measurement of metabolites in single living cells.10 Studies on carbohydrate uptake and metabolism using Raman spectroscopy, however, are very limited, and so far are only presented on bulk dried bacterial cultures.11 Up to now, no experiments relating to the real-time uptake and metabolism of carbohydrates by single cells have been reported. Direct real-time monitoring of glucose transport in yeast using CARS, however, was recently shown.12 Here, we have utilized LTRS to directly monitor the in vivo uptake and conversion of trehalose by continuous acquisition of Raman spectra in the soil bacterium S. meliloti. We showed that this approach can be used for both the qualitative and quantitative determination of the sugar uptake within single bacterial cells. Although we have focused our study on trehalose uptake by S. meliloti, the approach demonstrated here is applicable to many different organisms and carbohydrates in general.
Figure 1. (A) Schematic of our confocal LTRS setup, showing the 785-nm laser focused into a sample with a high NA objective. Raman signals are epi-detected, spatially filtered by a confocal pinhole and dispersed onto a liquid nitrogen cooled CCD camera. (B) Experimental setup: application of trehalose solution to the cell chamber with the minimal medium with (2) and without (1) bacterial cell.
Cell suspensions were diluted to the point where very few cells were visible within the microscope’s field of view. An aliquot (500 μL) of the cell suspension was placed in a 35 mm diameter culture dish formed by a stainless steel cell chamber (AttoChamber, Invitrogen, Eugene, OR) and a MgF2 coverslip. Cells were trapped manually by manipulating the xy translation stage of the microscope and adjusting the focus. They were then moved to 30 μm height above the coverslip prior to acquiring Raman spectra. A spectral integration time of as little as 10 s was found to be sufficient to obtain Raman spectra with excellent signal-to-noise ratio to identify the characteristic Raman bands of the bacteria. Trehalose was added to a final concentration of 0.4% (w/v). The dropwise addition of trehalose to the solution, although applied as carefully as possible by hand, typically caused some turbulence that often led to the removal of the initial bacterial cell from the optical trap. In all cases where this happened, the optical trap was reoccupied within seconds by another bacterium, which always resulted in the same initial (t0) spectra that we observed before the addition of trehalose. We always confirmed that truly just a single cell was held in the optical trap by visual inspection of the microscope image after data acquisition. Bacterial cells were held in the optical trap for 5 min in total and continuous spectra were collected through this time frame. For timeresolved measurements, the initial spectrum was subtracted from each subsequent spectrum to obtain difference spectra that will represent dynamic changes upon trehalose uptake inside the cell with time. The dimensions of these rod-like S. meliloti bacterial cells are about 1.7 μm in length and 0.5 μm in diameter, which means that the entire bacterial cell was captured within the focal volume of the tightly focused laser beam. When a cell is caught in the optical trap, the long side of the cell is realigned to be parallel to the direction of the laser beam since the longitudinal trapping force is weaker than the transverse trapping force.15 Background spectra were collected by application of trehalose using the same experimental conditions without trapped cell (Figure 1B). The experiments were initially repeated to obtain spectral measurements of the trehalose diffusion gradient (the background gradient), which was found to be similar if the amount of applied sugar and the distance to the trapped cell were kept constant.
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MATERIALS AND METHODS Bacterial Cultures. The inoculum of S. meliloti strain Rm1021,13 grown overnight in TY medium containing streptomycin (500 μg/mL) at 28 °C, was washed twice with minimal medium (MM)14 and transferred into streptomycinfree MM with 0.2% (w/v) mannitol and 0.2% (w/v) trehalose for continued growth. The cultures were incubated in a rotary shaker at 28 °C to an optical density at 600 (OD600) value of ∼0.3. Cells were washed and incubated in the MM without carbon source for 30 min prior to conducting Raman measurements. All chemicals were purchased from SigmaAldrich (St. Louis, MO). Laser Tweezers Raman Spectroscopy of Single Bacterial Cells. Laser-tweezers Raman spectroscopy of a single bacterial cell in suspension was achieved based on a custom-built, inverted optical microscope. The main microscope platform consists of an Olympus IX-71 inverted optical microscope equipped with a 60×, NA 1.2, water immersion objective optimized for near-IR operation (MO - Olympus America, Center Valley, PA). The laser source is an 80 mW, 785 nm diode-pumped solid-state laser (Crystalaser, Reno, NV). The Raman signal from the probed cells was detected by a spectrometer (Acton 2300i; Princeton Instruments) equipped with 1-μm blaze wavelength 300 grooves/mm grating. The microscope was also equipped for imaging to identify and visualize individual cells. Cells could be viewed through an analog CCD camera (Edmunds Optics), which was digitized and displayed on a personal computer using a frame grabber board (Data Translation, Inc.) (Figure 1A). The spectral resolution of the setup is 4 cm−1. Further details can be found in Chan et al.6 7265
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Figure 2. (A) Raman spectra of a single S. meliloti cell in minimal medium. (I) Raman spectrum of the solution background (minimal medium); (II) Raman spectrum of a single optically trapped S. meliloti cell in minimal medium; (III) Raman spectrum of the single cell of S. meliloti after background subtraction (I subtracted from II). (B) Intensity variations of select Raman peaks of the bacterial cells in minimal medium with standard deviation at the start and the end of the acquisition (n = 4). Single bacterial cells were trapped by laser tweezers and spectra acquired every 10 s for 5 min with an excitation power of 30 mW at 785 nm. The data are normalized to the total area.
The measurements were conducted at room temperature. All experiments were repeated at least 3 times in order to ensure reproducibility. Acquisition of Raman Spectra from Trehalose (Calibration curve). The calibration curve was built from measurement of 2.5, 5, 7.5 and 10% (w/v) of aqueous solutions of trehalose. Five-hundred microliters of the solutions were examined in the cell chamber for 10 s integration time under the same experimental conditions. In total, 5 spectra of each solution were collected. Data Analysis/Data Processing. All the data were acquired, smoothed and background spectra removed using WinSpec32 software (Roper Scientific, Tucson, AZ) and processed using Excel 2010. Spectra were normalized to the total area under the curve. Quantification was based on the values of peak amplitudes by fitting the 529 cm−1 peak with a Lorentzian fit function. Data fitting was done with Origin 8.6 (OriginLab Corporation). Trehalose Uptake Assay. Cells were grown to exponential phase in M9 minimal medium in the presence of 0.2% (w/v) mannitol and 0.2% (w/v) trehalose. The cells were harvested by centrifugation (4000 rpm for 10 min) at room temperature and washed once with M9 salts without any carbon source and adjusted to an OD600 value of ∼0.3 with the same medium. [14C] Trehalose (1.1 GBq/mmol) was added at a final concentration of 1 μM and the suspension was incubated at 25 °C. At various time points after the addition of trehalose, 110 μL aliquot samples were removed and filtered through 0.45 μm pore-size filter (Millipore) under vacuum. The filters were washed with 5 mL M9 and transferred to scintillation vials containing 4 mL of Ultima Gold scintillation liquid (Packard Instruments). The radioactivity trapped in the cells was
measured with a scintillation counter (2300TR; Packard Instruments). [14C] Trehalose was prepared according to the method of Horlacher et al.16 The amount of trehalose was calculated based on the cell number of the exponentially growing cultures and the averaged volume of the S. meliloti cells.
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RESULTS AND DISCUSSION Raman Spectra of Single S. meliloti Cells. The spectral signature of a trapped single S. meliloti cell from an exponentially grown culture before trehalose addition is shown in Figure 2A. Several characteristic Raman bands were observed, corresponding to specific molecular vibrations of typical cellular biopolymers, such as proteins, carbohydrates, lipids and nucleic acids. The summary of the spectral peak assignments is provided in Table 1. Nucleic acids can be identified by characteristic peaks of sugar−phosphate backbone vibrations and nucleotide vibrations. It is worth noting that since prokaryotic cells (E. coli) contain about 7 times more RNA than DNA by dry weight the major contributions to these Raman peaks to vibrational modes are due to RNA (see Chen, et al.17 and references therein). The main peaks related to nucleic acid contributions appear at 830 cm−1 for the O−P−O asymmetric stretching of DNA, 906 cm−1 for the DNA backbone as well as 730, 1207, 1349 and 1455 cm−1 for adenine, guanine and thymine. The peak intensities of the DNA/RNA bands change during the bacteria growth. Higher values of peaks associated with nucleic acids could be explained by the growth phase of bacteria. The studied bacteria were in exponential phase, which is characterized with active metabolic activity involving DNA and RNA synthesis. Actively growing bacterial cells prepare for and then double the 7266
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glucose, indicating that this monosaccharide could be one of the components.26 It is important to note that some peaks, assigned to proteins, carbohydrates, lipids, and DNA/RNA have overlapping vibrational modes, which makes the proper interpretation of the spectra difficult. For example, the Raman peak around 1455 cm−1 attributed to a deformation mode of CH2 groups of aliphatic carbon chains is a results of both, lipids and long-chain amino acids. The peak at 830 cm−1 assigned to a ribosephosphate vibrations of B-form DNA overlaps with a tyrosine band at 830 cm−1 (see Table 1). For comparison we also characterized the pure forms of trehalose and 3-ketotrehalose in aqueous suspension at high concentration, and focused on Raman modes that we could clearly assign to vibrations from these molecules. Cell Viability. To evaluate potential photodamage that might occur to a trapped cell by LTRS,27 single S. meliloti cells were monitored in the optical trap for 5 min. No degradation of the Raman signal intensity was found after a 5-min exposure in the laser trap (Figure 2B), which suggests that there is no cellular photodamage and/or any drastic negative impact on the state of health of the cells at current experimental conditions. This is consistent with other studies that have used 785 nm laser light to probe red blood cells and bacteria.28 The results indicate also that S. meliloti cells do not change their activity under these experimental conditions. Consequently, the Raman spectra acquired from the cells are signatures that accurately represent the biochemical composition and state of live, viable bacterial cells. Time Dependence. The Raman spectrum of trehalose in aqueous solution (Figure 3A) shows a strong peak in the 529
Table 1. Assignments for Raman Bands wavenumber (cm−1) 424 529 621 730 784 830 843 850 906 917 1057 800−1100 1100 1207 1300 1349 1455 1462 1578 ∼1661 1734 2932
assignments18,19,21−26,35 a
c : skeletal endocyclic def. c: skeletal exocyclic def. p: C−C twist/phenylalanine na: A (ring breathing) na: U,C,T ring breathing/bk. O−P−O str. na: O−P−O asym. str.; p: tyrosine (ring breath) c: C−H in α-anomers in the 4C1 conformation p: tyrosine (ring breath); c: hemiacetal str./methylene def. na: DNA bk.; c: 1-o-methyl-α-D-glucose c: C−OH def. in α-D-glucose l: C−O str./alkyl C−C trans str. c: C−C, C−O, (C−O−H) def.; C−O−C glycos. ring na: bk. O−P−O sym. str. na: A, T; p: phenylalanine C−C6H5 str/tyrosine/amid III p: amid III, CH def.; l: alkyl CH2 twist na: A,G; c: CH2 def. and C−O−H bending na: A, G; p/l: CH2 def. c: CH2 def./O−C−H, C−C−H and C−OH def. na: G, A p: amid I; l: alkyl CC str.-cis unsaturated l: ester, c: CO str. CH2 and CH3 asym. and sym. str.
a
Abbreviations: na-nucleic acids, A-adenine, U-uracil, G-guanine, Tthymine, p-proteins, l-lipids, c-carbohydrates, def.-deformation, str.stretching, bk.-backbone, sym./asym.-symmetric/asymmetric.
DNA amount before binary fission. Increase of nucleic acid bands in E. coli entering the log phase has been reported.18 Similar results were shown by Moritz et al., where the 784 cm−1 peak intensity was reduced by a factor of 2 during transition from log to stationary phase in E. coli.19 The spectra of proteins are dominated by peaks corresponding to amide I (1660−1700 cm−1)20 and amide III (1200−1300 cm−1) vibrations. The amide I band centered at 1660 cm−1 suggests predominantly α-helical confirmation in the protein secondary structure, compared to β-sheets, which are present as weak peaks at around 1680 cm−1.20 Peaks corresponding mainly to amino acids, containing phenyl groups, are observed at 830 and 850 cm−1 and are assigned to tyrosine.21 The strongest Raman peaks of lipids are observed at 1057 and 1734 cm−1 for ester CO stretching vibrations, as well as 1455 and 2932 cm−1 for CH2 and CH3 asymmetric and symmetric stretching modes. Unsaturated chains of lipids are represented by CC stretching modes at 1655 cm−1.22 The presence of C−C trans stretching vibrations at 1057 cm−1 could represent the phase state of the constituent lipids since fatty acid tails are packed in orderly trans conformation in the solid phase.23 Saturated fatty acids can be traced by the vibrations of saturated CH2 bonds such as CH2 twist at 1300 cm−1 and CH2 symmetric and asymmetric stretching vibrations at 2800−3000 cm−1.23 Carbohydrates can be identified due to some of the characteristic Raman peaks of sugars observed around 1030− 1130 cm−1, such as C−C, C−O, C−O−H deformation modes24 and 800−1100 cm−1 assigned to vibrations of the glycosidic bonds and sugar rings.25 The strong band at 906 cm−1 is found to be a derivative of glucose, 1-o-methyl-α-D-
Figure 3. Chemical structure and Raman spectra of a 5% (w/v) aqueous solution of trehalose (A) and 3-ketotrehalose (B). In the keto-sugar molecule the hydroxyl group is replaced with a carbonyl group at the 3rd carbon atom providing a characteristic peak at 1734 cm−1.
cm−1 region, which does not have a significant counterpart in the cell spectrum (see Figure 2AIII). This peak was chosen as a suitable probe for the selective mapping of trehalose in such a complex biochemical setting as a single bacterial cell. Tracking this peak within a single cell provides information about trehalose uptake process, its rate and undergoing metabolism. Cells were trapped and trehalose was added to the cell suspension to reach a final concentration of 0.4% (w/v). The t0 spectrum was obtained as described in the experimental setup 7267
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transporter system.29 This can explain the high intensity of protein bands in the initial spectrum (Figure 2 and Table 1). Figure 4B shows a plot of the intensity of the 529 cm−1 band as a function of time. When S. meliloti cells are exposed to trehalose solution, it appears that they begin to immediately take up the sugar based on the strong onset of the 529 cm−1 peak. High uptake of trehalose within the first minute could be a result of the preceding cell starvation. It appears that the amount of trehalose is equilibrated within the cell during the second minute and undergoes metabolic conversion/transformation resulting in temporal increase and decrease of sugar content in the cell, detected at 3−4 min. Since the cells were incubated in the presence of trehalose, the synthesis of sugar transporters was likely triggered in order to facilitate the uptake. We suggest that short-term starvation could result in depletion in sugar content, without affecting the activity of the channels, which could be compensated by rapid sugar uptake upon trehalose addition. Quantitative Analysis of Trehalose Uptake. In addition to the qualitative data acquired during Raman scattering-based cell monitoring, it is also possible to quantify the sugar uptake by a single bacterium. As the spontaneous Raman scattering signal is linearly dependent on the analyte concentration, by measuring relevant experimentally acquired Raman bands assigned to specific sugars, concentrations of the corresponding molecular species can be estimated. For construction of the calibration curve several measurements of the trehalose solution were carried out. Figure 5A shows the calibration curve of the peak amplitudes at 529 cm−1 used for quantification of intracellular trehalose content. According to these calculations the intracellular trehalose concentration ranges from 65 mM at its maximum through 18 mM at its minimum, with an average
section and was subtracted from each subsequent spectrum to result in difference spectra that represent changes inside the cell upon trehalose uptake with time. Subtraction of the initial spectrum of the cell from each subsequent spectrum also allows us to monitor the uptake and the metabolism of trehalose itself inside the single bacterial cell upon trehalose uptake with time. As we mentioned earlier, the addition of trehalose into the medium typically resulted in some turbulence which caused the trapped cells to move out of the laser trap. The system stabilized shortly thereafter, resulting in the acquisition of reproducible spectra within 30 s or less following trehalose application. Figure 4A illustrates selected time-point Raman spectra acquired during continued laser exposure. The overall spectral
Figure 4. (A) Time dependence of trehalose uptake and metabolism in single S. meliloti cells. Spectral average with standard error represents three different cells. The acquisition time for each spectrum was 10 s with an excitation power of 30 mW at 785 nm. Each spectrum is background-subtracted and normalized to the total area. Data presented as continuous acquisitions of a single cell for 4 min 30 s where the initial cell spectrum, i.e., t0, was subtracted. Dashed gray and red lines show the peaks present and absent in the trehalose standard, respectively. The light-blue stripes mark the peaks at 529 and 1734 cm−1, representing trehalose and a cell, respectively. Their intensity changes of peaks from 30 s to 4 min and 30 s with standard errors (n = 3) are presented in (B) and (C).
signatures reveal changes in the intensities of bands attributed to trehalose, such as the ones at 424, 529, and 1125 cm−1 with time, confirming that we were able to trace the uptake of the sugar by LTRS. Uptake of carbohydrates is facilitated by proteins which could be expressed for that purpose during the incubation with the sugar source. Trehalose uptake in S. meliloti has been reported to occur via the ATP-binding cassette (ABC)
Figure 5. (A) Calibration curve of a 2.5−10% (w/v) aqueous solution of trehalose. Five spectra of the peak at 529 cm−1 were backgroundsubtracted and fitted to Lorentzian functions. Data represent absolute peak values with standard errors. (B) Trehalose uptake per single S. meliloti cell measured by [14C] labeling. 7268
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CONCLUSION Single-cell laser-trapping Raman spectroscopy was used to follow the biochemical process of sugar-metabolism in individual bacterial cells in vivo. We have demonstrated the ability of the laser trapping Raman spectroscopy technique to monitor sugar uptake in a trapped single bacterium in real time by continuous acquisition of Raman spectra. LTRS provided label-free chemical information from individual living bacterial cells and allowed monitoring the temporal evolution of a biological process upon trehalose uptake. This work further illustrates the promise of LTRS for the direct, rapid, nondestructive, and quantitative characterization of carbohydrate uptake and metabolism at the single cell level under native conditions. This demonstration of a simple quantitative in vivo analysis of bacterial cells is suitable for a better understanding of the metabolic system of organisms/bacteria, where knowledge of fast dynamics of metabolites and pathways is desired. We have shown that LTRS is suitable for the study of transport kinetics, which opens new opportunities for studies on the regulation mechanisms of carbohydrates transport. Such information is important for a fundamental understanding of the mechanisms behind metabolic diseases, monitoring of intracellular metabolites during fermentation, and for the optimization of metabolic engineering processes.
of 22 mM intracellular trehalose after 2 min of trehalose exposure (Figure 4B). Since no experimental data are available on sugar concentration in single S. meliloti cells, for comparison the conventional method of uptake quantification was performed using radioactively labeled [14C] and bulk bacterial cultures using the same culture conditions, which was then extrapolated to the single cell level to obtain an approximate intracellular trehalose content. Figure 5B shows the uptake of trehalose within 1 to 4 min. It should be noted that both the methods are not quite compatible, since the radioactive tracer will also represent the metabolized sugar. This estimation is very rough and tends to show the approximate range of intracellular trehalose. The linear increase in the amount of trehalose confirms rapid uptake within the first minute of trehalose addition. The amount of sugar measured is in the mM range and roughly similar to the LRTS measurements. The radioactivity assay results are approximately 1 order of magnitude less than reported by the single cell Raman experiments. We believe this is due to the bulk averaging of the radioactivity readings. It should also be noted that uptake of sugars differs in different microorganisms and is highly dependent on growth conditions, nutritional state of the cells and stage of growth. Metabolism of Trehalose. The application of trehalose does not only involve activation of the trehalose transport mechanisms, but also induces metabolic processes within the cells, which can be followed by LTRS. Changes of trehalose peak intensities are accompanied with changes of other peaks.30 The most profound intensity variations with time are marked by light-blue stripes, indicating trehalose marker peak at 529 cm−1 and the peak at 1734 cm−1, suggesting that the active metabolic process is directly related to trehalose application. We attribute the occurrence of the 1734 cm−1 peak to the conversion of trehalose into ketotrehalose (Figure 3B), which adds a carbonyl group to the sugar. The 1734 cm−1 peak is the typical Raman band for carbonyl CO vibrations. The time-dependent peak intensities of the chosen peaks (Figure 4B and C) show some complementing trend, i.e., when one increases, the other in the contrary decreases, suggesting that they might be metabolically connected. We assume that the change in time-dependent trehalose content within the cells is most likely caused by the sugar metabolism. There are three known pathways of trehalose catabolism in microorganisms. First is trehalose hydrolization into two glucose moieties using trehalase.30 Second, trehalose is transported across the membranes to a cell either by permease or by a phosphotransferase system, leaving trehalose unmodified or as a phospholiated trehalose 6-phosphate form (T6P).31 Degradation of trehalose or T6P follows involving trehalase, T6P-hydrolase, phospho-(1−1)-glucosidase, or phosphotrehalase.32 Third, trehalose is taken up via the PTS system as T6P, and the enzyme trehalose-6-phosphate phosphorylase phosphorylates the T6P and splits the molecule to yield βglucose-1-phosphate and glucose-6-phosphate. Phosphorylated glucose residues, rather than glucose, are the products of trehalose metabolism in this pathway found in Lactococcus lactis.31,33 However, none of these pathways has been found in S. meliloti, suggesting that there is an alternative mechanism of trehalose utilization.34
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Phone: +49 521 106 5451. Fax: + 49 521 106 2958. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank Dr. John C. (Jack) Meeks and Dr. Brett Chromy, both UC Davis, for kindly providing us with the opportunity to work in their laboratories at the CBST and UC Davis. This project was supported by the Research Council of Norway under project number 183242/S20 “Functional Genomics Research in Northern Norway”.
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Analytical Chemistry
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NOTE ADDED AFTER ASAP PUBLICATION This paper was published on the Web on July 10, 2013. Additional text corrections were made throughout the manuscript and Figures 4 and 5 were revised. The corrected version was reposted on July 19, 2013.
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dx.doi.org/10.1021/ac4011638 | Anal. Chem. 2013, 85, 7264−7270
