Metaproteomic and metagenomic analyses of de¢ned oceanic
microbial populations using microwave cell ¢xation and £ow
cytometric sorting
Isabelle Mary1,2, Anna Oliver3, Paul Skipp4, Ross Holland1, Juliette Topping1, Glen Tarran5,
David J. Scanlan6, C. David O’Connor4, Andrew S. Whiteley3, Peter H. Burkill7,8& Mikhail V. Zubkov1
1
National Oceanography Centre, Southampton, UK;2LMGE, Laboratoire Microorganismes: G ´enome & Environnement, UMR CNRS 6023, Universit ´e
Blaise Pascal (Clermont-Ferrand II), Aubie`re, France;3Biodiversity and Ecosystem Function Group, Molecular Microbial Ecology Section,
CEH-Oxford, Oxford, UK;4Centre for Proteomic Research, School of Biological Sciences, University of Southampton, Southampton,
UK;5Plymouth Marine Laboratory, Plymouth, UK;6Department of Biological Sciences, University of Warwick, Coventry, UK;7Sir Alister Hardy
Foundation for Ocean Science, Plymouth, UK; and8Marine Institute, University of Plymouth, Plymouth, UK
Correspondence: Isabelle Mary, LMGE, Laboratoire Microorganismes: G ´enome & Environnement, UMR CNRS 6023, Universit ´e Blaise Pascal (Clermont-Ferrand II), 24 avenue des Landais, Aubie`re 63177, France. Tel.: 133 4 73 40 74 70; fax: 133 4 73 40 76 70; e-mail: isabelle.mary@univ-bpclermont.fr Received 9 December 2009; revised 13 May 2010; accepted 22 May 2010.
Final version published online 5 July 2010. DOI:10.1111/j.1574-6941.2010.00927.x Editor: Kornelia Smalla
Keywords
high-speed cell sorting; microwave irradiation; protein sequencing; genome sequencing; oceanic cyanobacteria; microbial ecology.
Abstract
A major obstacle in the molecular investigation of natural, especially oceanic, microbial cells is their adequate preservation for further land-based molecular analyses. Here, we examined the use of microwaves for cell fixation before high-speed flow cytometric sorting to define the metaproteomes and metagenomes of key microbial populations. The microwave fixation procedure was established using cultures of Synechococcus cyanobacteria, the photosynthetic eukaryote Micromonas pusilla and the gammaproteobacterium Halomonas variabilis. Shot-gun proteomic analyses showed that the profile of microwave-fixed and -unfixed Synechococcus sp. WH8102 cells was the same, and hence proteome identification of microwave-fixed sorted cells by nanoLC-MS/MS is possible. Microwave-fixed flow-sorted Synechococcus cells can also be successfully used for whole-genome amplification and fosmid library construction. We then carried out successful metaproteomic and metagenomic analyses of microwave-fixed Synechococcus cells flow sorted from concentrates of microbial cells, collected in the North Atlantic Ocean. Thus, the microwave fixation procedure developed appears to be useful for molecular studies of microbial populations in aquatic ecosystems.
Introduction
One of the main aims of aquatic microbiology is to identify key organisms and to characterize their physiology in situ. However, most bacterioplankton groups either defy cultiva-tion (Giovannoni et al., 1990; Amann, 1995) or grow poorly in laboratory culture (Rapp´e et al., 2002). Among culture-independent approaches, developed in recent years, the large-scale environmental shotgun sequencing of total DNA extracted from natural bacterioplankton communities re-vealed a complex microbial diversity in the ocean (Venter et al., 2004; Rusch et al., 2007). However, the very same high diversity has restricted the genomic characterization of microbial communities to the partial genome assembly of only the most numerically dominant taxa. Although
meta-genomic sequences provide valuable information on the potential functions of the components identified, the func-tions ought to be confirmed by information about proteins, synthesized under specific conditions (Petersohn et al., 2001; Eymann et al., 2002). Metaproteomic analyses of natural microbial communities have been successfully used to evaluate in situ functioning of a low-complexity biofilm (Ram et al., 2005) or more complex estuarine communities (Kan et al., 2005). Nevertheless, the detection of proteins from rarer microbial populations could be missed if a complex microbial community is analysed in its totality unless a cell-targeted approach is introduced to overcome this limitation.
Flow cytometry (FCM) provides an efficient and fast characterization of natural microbial populations (Fuchs
MICR
OBIOLOGY ECOLOGY
et al., 2000; Zubkov et al., 2001), whereas cell flow sorting offers a tool to reduce cell diversity and to access the in situ physiology of cells. For example, metagenomic analysis of flow-sorted individual cells or of limited numbers of cells has allowed the assessment of the metabolic potential of taxonomically diverse uncultured marine bacterioplankton (Podar et al., 2007; Stepanauskas & Sieracki, 2007).
Traditionally, proteomic studies are performed on un-fixed cells; however, working with natural samples, collected during fieldwork, for example oceanic cruises, either immediate analysis or efficient sample fixation and storage are required, the latter to preserve cells for further proces-sing in specialized laboratories. Samples for FCM are gen-erally flash-frozen in liquid nitrogen and stored at 80 1C until analysis after fixation (Marie et al., 1996; Troussellier et al., 1999; Gasol & del Giorgio, 2000). However, when a nucleic acid-specific stain is used to discriminate, count and sort bacterioplankton clusters or viruses, fixation of bacter-ioplankton cells is required (Marie et al., 1999; Fuchs et al., 2000). Moreover, it becomes essential for preserving intact proteome profiles during long sorting runs. The latter is necessary to collect enough protein material from a natural sample for proteomic analysis, in contrast to genomic analyses, where whole-genome amplification technology is possible. Thus, fixation conditions are critical for preserving cell integrity with minimal alteration of cellular macromolecules.
Paraformaldehyde is the reference fixative generally used, for example FCM and in situ hybridization analyses. How-ever, a major concern with choosing paraformaldehyde is its potential to cause nucleic acid alterations via the formation of methylene bridges between functional groups in nucleic acids (Clevenger & Shankey, 1993), making the amplifica-tion of target DNA sequences in paraformaldehyde-fixed cells more difficult (Hodson et al., 1995). The use of aldehydes is also incompatible with proteomic approaches because of protein cross-linking, which limits protein re-covery and complicates analysis (Hayat, 1981). In addition, paraformaldehyde fixation can affect flow cytometric discrimination of marine bacterial populations (Troussellier et al., 1999; Kamiya et al., 2007). Hence, there is an urgent need to develop a suitable fixation procedure for subsequent flow sorting and molecular analyses of microbial cells.
An alternative, artefact-free fixation of microbial cells could be fixation with microwaves, which has been proven to be successful in immunocytochemistry, for example for the flow cytometric detection of intracellular anti-gens (Millard et al., 1998), for the fixation of cells grown in tissue culture before fluorescent antibody staining (Medina et al., 1994; Ler´ıa et al., 2004) and for preserving cell morphology (Lan et al., 1996). The main advantage of microwave fixation is that no chemicals are involved,
which would otherwise alter the macromolecules in the cells.
Thus, the aim of the present study was to develop an approach for separating environmental microbial popula-tions for subsequent genomic and proteomic analyses using efficient cell concentrating, microwave fixation and high-speed flow cytometric sorting. First, we tested the feasibility of this approach and optimized the protocol by isolating specific populations from nonaxenic cultures of marine microorganisms. Then we applied the protocol to separate Synechococcus populations from a complex microbial com-munity of the euphotic layer of the North Atlantic Ocean.
Materials and methods
Strain cultivation and sample preparation Synechococcus sp. WH8102 and WH8016 were grown in ASW medium (Wilson et al., 1996) at 24 1C under a continuous white light at 25 mmol photons m2s1. Micromonas pusilla was grown in a K medium (Keller et al., 1987) under the same light and temperature conditions. Halomonas variabilis was grown on marine agar (Difco) at 10 1C and resuspended in 0.1-mm-filtered seawater for experimentation.
Environmental sampling
Samples were collected from a depth of 5 m at a station, 29.531N, 36.271W, in the North Atlantic Ocean in October 2005. Seawater samples, 4–10 L, supplemented with Pluro-nic F68 surfactant for effective cell recovery (Biegala et al., 2003) (Sigma, Poole, UK; 0.05% v/v final concentration), were prefiltered through 10-mm polycarbonate filters and concentrated into 0.2-mm CellTrapTM filter cartridges (Mem-Teq, Orrell, UK) to a final volume of 1.8 mL using a peristaltic pump. This system allowed rapid concentration of a large seawater sample volume with minimal cell damage and clumping. Samples were transferred into autoclaved 2-mL polypropylene tubes with screw caps and then micro-wave was fixed for 25 s at 1.06 J as described below, flash-frozen in liquid nitrogen and stored at 80 1C for several months until further processing.
Microwaving fixation
Microwave fixation was tested on Synechococcus sp. WH8102, H. variabilis and M. pusilla cultures in order to fix the cells with minimal protein and DNA degradation. Pluronic solution at a final concentration of 0.05% v/v was added to the samples to prevent cell aggregation. Polypro-pylene tubes (2 mL) with screw caps were filled with 1.6 mL of cell suspensions. Tubes were placed in 120 mL of cold (4 1C) water and irradiated using a domestic microwave oven (Panasonic NNT573). The energy of microwave
emitted was calibrated by measuring the water temperature change at various time points and different powers of irradiation and by converting these values using the specific capacity constant of 4.186 J kg1K1 for liquid water. For calibration, the same water volume (120 mL) was used under similar experimental conditions and water was mixed before performing temperature measurements using an electronic thermometer with an accuracy of 0.1 1C.
Cell integrity was checked using a FACSort flow cytometer (Becton Dickinson, Oxford, UK) and the protein concentration was measured using the BCA (bicinchoninic acid) assay (Sigma). For heterotrophic bacteria and cyano-bacteria, the metabolic activity of cells after fixation was checked using an amino acid uptake assay (Zubkov et al., 2001). Samples at a concentration of 106cells mL1 were incubated with 0.5 nM [4,5-3H]leucine (specific activity
6 TBq mmol1) or 0.1 nML-[35S]methionine (specific
activ-ity 4 37 TBq mmol1) and fixed at 10, 20, 30 and 60 min, respectively, by adding 20% paraformaldehyde to 1% w/v final concentration. The sample particulate material was harvested onto polycarbonate filters with a pore size of 0.2 mm. Radioactivity retained on filters was measured using an ultra-low level liquid scintillation counter (1220 Quan-tulus, Wallac, Finland).
The photosynthetic activity of cultured Synechococcus and M. pusilla after microwave fixation was measured using H14CO3radiotracer incubation. Samples at a concentration
of 106cells mL1 were incubated with 31.2 nM [14 C]NaH-CO3(specific activity 0.5 GBq mmol1) and the uptake was
stopped at 2, 4 and 6 h, respectively, by adding H3PO4(0.3%
final concentration) to fix cells and to change the pH in order to remove the unfixed 14CO2 (Steemann Nielsen,
1952). The fixed samples were transferred into scintillation vials, amended with a scintillation cocktail (Gold-Star, Meridian, UK) and radioassayed.
Flow cytometric sorting
Flow cytometric sorting of target cells for proteomic analysis was carried out using a MoFloTM flow cytometer (Dako Cytomation, UK), equipped with a UV tunable Argon-Ion laser (Innova 90c, Coherent, UK) and a Green (510nm) Argon laser (Compass 315nm-150, Coherent). Synechococcus cyanobacteria were chosen as a model organism for devel-oping the methodology, because their cells can be easily identified by FCM based on their fluorescent pigment signature. Phycoerythrin excitation by the green laser enabled clear visualization of Synechococcus populations on a bivariate dot plot (SUMMIT software version 3.1, Dako
Cytomation) of forward light scatter and orange fluores-cence (580 30-nm bandpass filter). Chlorophyll excitation by the green laser enabled clear visualization of picoeukar-yote groups on a bivariate dot plot of forward light scatter
against red fluorescence (670 30-nm bandpass filter). Excitation of the DNA stain Hoechst 33342 (Sigma) by the UV laser enabled the visualization of DNA-stained cells on a plot of blue fluorescence (450 60-nm bandpass filter) against forward light scatter. This avoided contamination with heterotrophic bacteria during the sort. Logical gating facilitated the omission of orange fluorescent (Synechococ-cus) populations from the red fluorescence/forward light scatter plot, and the subsequent definition of sort logic. The MoFlo was operated in the two-way, sort-purify ‘single-drop’ sort mode, with a nozzle amplitude of 12.74 V and a frequency of 96 210 Hz. The resultant drop delay was 33114/16, calibrated manually by sorting a specified num-ber of 3 mm yellow-green microspheres (Polysciences, Ger-many) onto a glass slide and counting the beads under an epifluorescence microscope. Performance was validated by sorting 0.5 mm yellow-green microspheres (Polysciences) of different fluorescence from a mixed suspension and reana-lysing the sorted material flow cytometrically to confirm sort purity and recovery of the beads with different fluores-cence. A saline solution of 0.1% w/v in MilliQ water was used as the sheath fluid.
For convenience, flow cytometric sorting of target cells for metagenomic analysis was carried out using a FACSAria flow cytometer (BD Biosciences, Oxford, UK) equipped with a blue (488 nm) solid-state laser (CoherentsSapphireTM, Coherent). The blue laser excited SYBR Green I dye, which was used to stain cell DNA (Marie et al., 1997; Zubkov et al., 2001). Bivariate plots of green fluorescence (530 15-nm bandpass filter) and red fluorescence (695 20 nm) (BD FACSDIVA software version 4.1) were used to discriminate
between Synechococcus and other bacteria (Supporting In-formation, Fig. S1). The FACSAria was operated using a 100 mm nozzle, with a nozzle amplitude of 12.5 V and a frequency of 30 000 Hz. BD FACSTMAccudrop beads were used to provide a drop delay of 27.96 drops, a Drop 1 position of 337 pixels and a gap between the stream and the first drop of 8–9 pixels. Synechococcus cells were sorted in the single cell mode (Yield Mask = 0, Purity Mask = 32, Phase Mask = 16), with the sweet spot on. Performance was validated by sorting a mixture of 0.5 mm yellow-green microspheres (Polysciences) of different fluorescence, and reanalysing the sorted material flow cytometrically to con-firm sort purity and recovery of the beads with different fluorescence. Autoclaved 0.2-mm-filtered seawater was used as the sheath fluid at a pressure of 20 p.s.i.
GeLC-MS/MS
To determine the amount of cells required for protein analysis, protein extracts from a range from 106 to 109 Synechococcus sp. WH8102 cells were analysed on a one-dimensional sodium dodecyl sulphate-polyacrylamide gel
electrophoresis (SDS-PAGE) gel stained with Coomassie Blue (data not shown). Based on the detection of at least 10 intense bands, a minimum of 108–2 108cells were sorted and concentrated by centrifugation. Cell extracts were pre-pared by sonicating three times for 10 s on ice in 0.5 M triethylammonium bicarbonate (Sigma). Cell debris was sedimented at 10 000 g for 10 min and the proteins were separated on a NuPAGE 4–12% gradient SDS-polyacryla-mide gel (Invitrogen). After visualization with colloidal Coomassie Blue, each gel lane (length: 7 cm) was excised, cut into 23 equal-sized pieces and subjected to in situ trypsin digestion (Shevchenko et al., 1996). Using a Multiprobe II robotics system (Perkin Elmer, MA), gel slices were washed/ dehydrated three times in 100 mM ammonium bicarbonate buffer and 100 mM ammonium bicarbonate and acetoni-trile. Cysteine bonds were reduced with 10 mM dithiothrei-tol for 1 h at 40 1C and alkylated with 50 mM iodoacetamide for 45 min at room temperature. Gel slices were subse-quently washed/dehydrated twice, rehydrated with 100 mM ammonium bicarbonate and incubated with 140 ng of trypsin in 25 mL of 100 mM ammonium bicarbonate for 4.5 h at 37 1C. Peptides were extracted initially using 30 mL of 2% acetonitrile containing 1% formic acid and then finally using 24 mL of 50% acetonitrile containing 1% formic acid. Peptides extracted from each gel piece were subsequently analysed using nanoLC-MS/MS (Schirle et al., 2003; Skipp et al., 2005).
NanoLC-MS/MS was performed using a CapLC system (Waters, Manchester, UK) online to a Q-tof Global Ultima (Waters) mass spectrometer fitted with a nanoLockSprayTM source. Peptides were loaded to a trap column (PepMap C18
RP, 0.3 mm i.d. 50 mm, LC-Packings, Sunnyvale, CA) and washed with 5% v/v acetonitrile containing 0.1% v/v formic acid (buffer A) at a rate of 20 mL min1for 10 min. Peptides were eluted from the trap column onto a PepMap C18 RP
analytical column (75 mm i.d. 15 mm, LC-Packings) and separation of peptides was performed using a gradient of 0–85% buffer B [95% acetonitrile (v/v) containing 0.1% v/v formic acid] over 60 min, at a flow rate of 200 nL min1. MS/ MS data were acquired from 300 to 1700 m/z with the switching criteria for MS to MS/MS including ion intensity and charge state.
MS/MS data were processed usingMASCOT DISTILLER
ver-sion 2.1.1 (Matrix Science, London, UK) and searched against a protein translation of the NCBI nonredundant database (September 2006) in aFASTAformat usingMASCOT
(Matrix Science). The following parameters were used: the parent mass tolerance was 150 p.p.m., the fragment mass tolerance was 0.25 Da, carbamidomethylation was set as a fixed modification, the oxidation of methionine as a variable modification and a maximum of one missed cleavage was allowed. The significance threshold for the search results was set at Po 0.05 (indicates identity or extensive homology).
Whole -genome amplification, fosmid library construction and sequencing
The integrity of DNA of microwave-fixed Synechococcus sp. WH8016 cells was initially checked using an Agilent 2100 Bioanalyzer (Agilent Technologies) (data not shown). Whole-genome amplification was performed on either 300 cells from Synechococcus sp. WH8016 or 10 000 cells from natural samples using GenomiPhi V2 DNA amplification (GE Healthcare) according to the manufacturer’s instruc-tions. For the latter, DNA was extracted as described previously (Neufeld et al., 2007). Fosmid libraries were constructed using the CopyControlTMFosmid Library Pro-duction Kit (Epicentre) as recommended by the manufac-turer. The quality of the library was checked by end sequencing of purified fosmid clones (Strataprep Plasmid Miniprep Kit, Agilent Technologies) that had been induced to a high copy number (according to the Epicentre manual). End sequencing was carried out by Geneservice Ltd, Cam-bridge (now based in Nottingham).
Results and discussion
Microbial cell fixation
Our objective was to fix microbial cells in the shortest period of time so as to maintain a low sample temperature and hence to reduce alterations to proteins and DNA (Hayat, 1981; Moriguchi et al., 2002). Cells of Synechococcus, H. variabilis and M. pusilla were irradiated with microwave of different energies. Cell integrity, protein content and metabolic activity were determined using FCM, the BCA protein assay and radiotracer cell uptake, respectively (Fig. 1). The results of these experiments showed that the optimal microwave fixation conditions for the three cultures tested were achieved with 0.66 J microwave mL1 of sus-pended cells in a tube immersed in 120 mL of water, which equated to 600 W microwave irradiation in the microwave oven for 25 s. Cells fixed under these conditions had flow cytometric signatures in terms of light scatter, DNA and pigment contents similar to the unfixed control cells (Fig. 1). The cell protein content measured using a BCA assay was not affected by microwave fixation, while CO2fixation by
Synechococcus and M. pusilla cells or leucine uptake by H. variabilis cells was halted after the optimal or higher microwave irradiation (Fig. 1).
It is noteworthy that the phycoerythrin fluorescence of Synechococcus cells was negatively affected (57% cell recovery after 0.7 J mL1) by microwave irradiation higher than the chosen optimal one. Therefore, considerable atten-tion was paid to maintain the chosen irradiaatten-tion condiatten-tions. Microwave fixation of a natural sample showed cell recovery ranging from 88% for picoeukaryotes to 100% for Prochloro-coccus and a loss of bacterial activity for fixed cells (Table S1).
A decline in the cell abundance of Synechococcus and to a lesser extent M. pusilla and picoeukaryotes, observed when the microwave dose exceeded 0.66 J mL1, reflected an micro-wave-induced reduction in the phycoerythrin autofluores-cence of Synechococcus cells and to a lesser degree in the chlorophyll autofluorescence of picoeukaryotic cells rather than real cell loss. In other words, cells remained intact, but the autofluorescence of some cells decreased below the detection threshold of the flow cytometers used for cell enumeration.
Analysis of theSynechococcusproteome from flow cytometrically sorted cells
Proteome studies, performed using two-dimensional gel electrophoresis, allow the relative comparison of proteins between samples, but have a poor resolution, and hence limit the identification of proteins of medium to low abundance (Gygi et al., 2000). To overcome this limitation, a shotgun proteomics approach can be used to identify proteins in complex mixtures using a combination of HPLC and MS (see e.g. Banfield et al., 2005; Denef et al., 2007; Sowell et al., 2008). To determine the optimal number of cells required for proteomic analysis, i.e. the minimum number required to obtain a detectable profile of proteins by Coomassie staining, samples prepared from 107 to
5 108
cells were examined by SDS-PAGE. Synechococcus sp. WH8102 was used for this study due to the availability of its genome sequence, which facilitates protein identification. Coomassie staining indicated that 108–2 108 cells
pro-duced a detectable profile while allowing flow sorting from a natural seawater sample in a reasonable time considering the proportion of targeted cells in the sample and flow sorting efficiency. Nevertheless, 14–28 h were required to flow sort enough cells from concentrated samples.
The protein profile obtained from a fixed sample was similar to the unfixed control sample and the major proteins identified were the same (Fig. S2). For the relatively small number of cells used, 10 intense bands and several others of lower intensity were detected on the gel. GeLC-MS/MS of the protein extracts from live and fixed cells allowed the identification of the same proteins mainly involved in photosynthesis, DNA or RNA metabolism, membrane transport and stress response (Table S2). A total of 125 peptide assignments were made from the sorted Synechococ-cus culture, resulting in the identification of 39 proteins. Approximately 73% of the proteins were identified on the basis of more than two peptides, of which nearly half had a function in electron transport or photosynthesis (Table 1).
We then used the established protocol for metaproteomic analysis of Synechococcus cells flow sorted from the micro-wave-fixed concentrated samples, collected in the North
(a) 0.0 0.3 0.6 0.9 Abundance (%) 0 50 100 (b) 0.0 0.3 0.6 0.9 0 50 100 (d) 0.0 0.3 0.6 0.9 Protein content (%) 0 50 100 (g) MW, J mL–1 0.0 0.3 0.6 0.9 Metabolic activity (%) 0 50 100 (c) 0.0 0.3 0.6 0.9 0 50 100
Synechococcus Micromonas Halomonas
(e) 0.0 0.3 0.6 0.9 0 50 100 (f) 0.0 0.3 0.6 0.9 0 50 100 (h) MW, J mL–1 0.0 0.3 0.6 0.9 0 50 100 (i) MW, J mL–1 0.0 0.3 0.6 0.9 0 50 100
Fig. 1. Microwave irradiation of Synechococcus WH8102, Micromonas pusilla and Halomonas variabilis cells. Cell abundance (a, b, c) was measured by FCM, protein content (d, e, f) was measured by the BCA assay and the metabolic activity (g, h, i) of cells was assayed by measuring
the [14C]NaHCO
3uptake rates of Synechococcus
and Micromonas or by measuring the [3H]leucine
uptake rate of Halomonas. Microwave thermal energy (MW) was calibrated by measuring water temperature increase after irradiation. Data are presented as the percentage change compared with MW untreated control measurements. Error bars show single coefficients of variance of repli-cated measurements. Thin dotted lines connect consecutive measurements to aid visualization of trends. Vertical dashed lines indicate the selected MW energy used for routine sample fixation. Synechococcus sp. cells were enumerated as cells with characteristic phycoerythrin orange fluorescence, which was adversely affected by MW irradiation.
Atlantic Ocean. Microwave fixation of bacterioplankton was checked using [35S]methionine tracer uptake and the integ-rity of the cells was checked by FCM (Table S1). A total of 86 peptide assignments were made from the sorted Synechococcus natural sample, resulting in the identification of 22 proteins. Approximately 65% of the proteins were identified on the basis of more than two peptides (Tables 1 and S3).
Construction of fosmid libraries from flow cytometrically sortedSynechococcuscells Synechococcus sp. WH8016 was used as a test organism to develop a protocol for fosmid library construction from microwave-fixed, flow-sorted cells. Three hundred micro-wave-fixed Synechococcus cells were flow sorted and used as a template for whole-genome amplification. The purity of cell sorting was checked by sequencing of the 16S rRNA gene amplified using general primers. A single Synechococcus sequence identical to the test organism was identified (data not shown). The amplified DNA was then cloned using the CopyControlTMFosmid Library Production Kit (Epicentre). The fosmid library comprised 12 000 clones ranging from 15
to 45 kb, with an average insert size of 30 kb (data not shown).
The protocol established in laboratory experiments was then applied to flow sort Synechococcus cells from the microwave-fixed concentrated microbial samples collected in the North Atlantic Ocean. The whole genome of 300 sorted Synechococcus cells was amplified and used for fosmid library construction. The fosmid library comprised 9000 clones, with an average insert size of 15 kb, ranging from 4 to 50 kb. Sequencing of both 50and 30ends of a subsample of 38 individual clones generated sequences identified as Synechococcus genes for approximately 80% of these reads, usingBLASTNsearches of the nr database, while the remainder
matched with Prochlorococcus or other marine bacterial genes. Thus, the main functional categories represented in this sequencing included genes for various metabolic path-ways, DNA or RNA metabolism and energy production (Tables 2 and S4). A number of cells, required for genomic analysis, could be even lower for example 100 cells (Stepa-nauskas & Sieracki, 2007). Generally, the number of sorted cells should be proportional to the degree of genetic diversity of the targeted group.
During the development of the microwave fixation ap-proach, including concentrating oceanic microorganisms, an important technical limitation emerged: the number of cells required for proteomic analysis is relatively high due to the limit of sensitivity of the nanoLC-MS/MS machine, restricting analyses to microbial groups relatively abundant in natural communities. Consequently, this method allows the detection of only the most abundant cellular proteins after sorting. However, future increases in the speed of flow cytometric sorting of target populations from complex natural microbial communities and the sensitivity of mass spectrometric detection of peptides will alleviate such blems, facilitating better characterization of microbial pro-tein expression profiles in the ocean. Further, recent advances in cell manipulation, electrophoretic protein se-paration and single-molecule fluorescence detection have allowed the measurement of protein expression within a single cell of Synechococcus under conditions of nitrogen starvation (Huang et al., 2007). The ability to stabilize cells using microwave fixation should be useful for subsequent cell manipulations using the emerging molecular technolo-gies. In case additional tests can prove the microwave stability of RNA, microwave-fixed cells may also be used for transcriptome analyses.
In conclusion, this study demonstrates that microwave treatment is a potentially powerful technique for in situ proteomic and genomic studies that require sample preser-vation before analysis. Moreover, combined with flow cyto-metric sorting, it has wide-ranging applications in environmental microbiology, and should improve our un-derstanding of natural microbial communities and their Table 1. Functional category distribution of peptides recovered from
sorted Synechococcus sp. WH8102 cells and sorted Synechococcus cells collected in the North Atlantic Ocean
Category
No. of unique peptides Synechococcus WH8102 North Atlantic Synechococcus Electron transport, photosynthesis 43
Cell redox homeostasis 3
Calvin cycle 4 ATP synthesis 3 3 Transport 13 Glutamin biosynthetic process 3 Oxidation reduction 9 Cobalamin biosynthetic process 3 Glutamate biosynthetic process 8 DNA recombination 6 Transcription 4 Translation 5 Translation elongation 5 Protein biosynthesis 3 5 Protein folding 5 4 Metabolic process 3 Other 6 None 4 Hypothetical protein 3
functioning. So far, this method has only been applied on pigmented cells. For microbial cells, which lack fluorescent pigments, it could be combined with in situ hybridization with specific rRNA probes (Sekar et al., 2004; Podar et al., 2007). However, one should also be aware that microwave energy changes with time and the model of instrumentation and a calibration should always be performed before an experiment to standardize the fixation protocol, and the efficiency of microwave fixation should always be verified experimentally.
Acknowledgements
This work was supported by the Natural Environment Research Council (NERC) as a research grant awarded to M.V.Z., D.J.S., C.D.O’C., P.H.B. within a Marine Microbial Metagenomics consortium (NE/C50800X/1) and through the Oceans 2025 core programmes of the National Oceano-graphy Centre, Southampton and Plymouth Marine La-boratory. The research of M.V.Z. was partly supported by the NERC advanced research fellowship (NER/I/S/2000/ 01426). CEH Oxford was supported by NERC grant NE/ C507937/1. We acknowledge the NERC Atlantic Meridional Transect (AMT) programme for the cruise opportunity (AMT contribution number 187).
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Table 2. Summary of sequencing of a fosmid library created from the amplified genome of sorted Synechococcus cells collected in the North Atlantic Ocean
Functional category of fosmid 50-end sequences
No. of sequences
Hypothetical protein 10
Energy production and conservation 4
DNA or RNA replication, restriction, modification, recombination and repair
3
Other 3
Pentose phosphate pathway 2
Metabolic enzyme 2
DNA or RNA metabolism 2
Translation, ribosomal structure and biogenesis 1
Transcription 1
Surface polysaccharides, lipopolysaccharides and antigens 1
Pyruvate dehydrogenase 1
Pyrimidine ribonucleotide biosynthesis 1
Protein and peptide secretion 1
Inorganic ion transport and metabolism 1
Glycolate pathway 1
Cell wall/membrane biogenesis 1
Aromatic amino acid family 1
Amino acid transport and metabolism 1
Functional category of fosmid 30-end sequences
No. of sequences
Aminoacyl tRNA synthetase and tRNA modification 1
Hypothetical protein 8
Other 5
Protein modification and translation factors 2
Dehydrogenase 2
Enzyme of unknown specificity 1
Glutamate family/nitrogen assimilation 1
CO2fixation 1
Regulatory functions 1
Sugars 1
Amino acids and amines 1
Transport and binding proteins 1
Aromatic amino acid family 1
Function unknown 1
Transport and binding proteins 1
Chaperones 1
Purine ribonucleotide biosynthesis 1
NADH dehydrogenase 1
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Supporting Information
Additional Supporting Information may be found in the online version of this article:
Fig. S1. FCM output and gating of Synechococcus sp. cells in samples from the North Atlantic Ocean.
Fig. S2. One-dimensional PAGE profiles of total proteins obtained from Synechococcus sp. WH8102.
Table S1. The effect of microwave fixation on bacterioplank-ton and picoplankbacterioplank-ton collected in the euphotic layer of the tropical North Atlantic Ocean.
Table S2. Proteins identified in microwave-fixed, flow-sorted cells of Synechococcus sp. WH8102.
Table S3. Proteins identified in microwave-fixed Synecho-coccus cells flow sorted from a CellTrapTM concentrated microbial sample collected in the North Atlantic Ocean. Table S4. GenBank ID numbers andBLASTannotation for the
sequences obtained by end-sequencing of the fosmid library constructed after whole-genome amplification of micro-wave-fixed Synechococcus cells flow sorted from a Cell-TrapTM concentrated microbial sample collected in the North Atlantic Ocean.
Please note: Wiley-Blackwell is not responsible for the content or functionality of any supporting materials sup-plied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article.
