Flow cytometric analysis of bacteria- and virus-like particles in lake sediments

Abstract

Flow cytometry (FCM) was successfully used to analyze freshwater bacteria and viruses in lake sediments after relatively simple sample treatment and optimization of dilution/fixation/staining procedures. Biological particles from Lakes Geneva and Bourget were first separated from the sediments by using both Sodium Pyrophosphate (0.01 M final concentration) and Polyoxyethylene-Sorbitan Monooleate (10% final concentration) and sonicating for 3 min in a water bath. The best results (based on FCM signature and the highest virus and bacterial yields from the sediments) were obtained by formaldehyde fixation carried out within less than one hour (2% final concentration, vs. no fixation or using glutaraldehyde at different concentrations), SYBR-Green II staining (×1 / 20,000 stock solution concentration, vs. use of SYBR-Gold and SYBR-Green I dyes at different concentrations). There was a considerable loss of particles after only a few days of storage at either 4 or − 22 °C. For FCM analysis, the samples were diluted in Tris–EDTA buffer (pH 8) and heated for 10 min at 75 °C after incubating for 5 min in the dark. The bacterial and viral counts paralleled those obtained using epifluorescence microscopy (EFM), but EFM always gave lower counts than FCM. Analysis of the distribution of the viruses in the water column and in the sediments of Lakes Bourget revealed a marked gradient, with larger quantities in the top layer of the sediment than in the water above it. These results are discussed, as well as the possible novel application of flow cytometry in the study of aquatic viral ecology.

Introduction

Flow cytometry provides a method for high-speed multi-parametric data acquisition and analysis. During the last two decades, it has been successfully used to analyze and count pelagic microbial communities of organisms such as protists, small algae, bacteria and viruses (Chisholm et al., 1988, Button and Robertson, 1989, Courties et al., 1994, Marie et al., 1999a, Marie et al., 1999b, Lindström et al., 2002, Rose et al., 2004), to identify and quantify population DNA content and/or to investigate the cell cycle (Boucher et al., 1991, Marie et al., 1996, Marie et al., 1997, Gasol et al., 1999), to identify populations of interest using molecular probes (Simon et al., 1995, Wallner et al., 1996, Lange et al., 1996), to assess cellular physiology (Jochem, 2000, Lebaron et al., 2001), etc. Reviews have been published of the accuracy of this technique applied to the field of aquatic sciences and ecology in particular, and to the modification/optimization of the apparatus and procedures (Olson et al., 1991, Yentsch et al., 1983, Davey and Kell, 1996, Porter et al., 1996, Dubelaar et al., 1999, Veldhuis and Kraay, 2000, Vives-Rigo et al., 2000, Collier and Campbell, 2000, Gruden et al., 2004).

Since Marie et al., 1999a, Marie et al., 1999b, viruses in the water column have been counted using benchtop flow cytometers on several occasions (Marie et al., 1999a, Marie et al., 1999b, Brussaard et al., 2000, Chen et al., 2001, Jacquet et al., 2002a, Jacquet et al., 2002b). Previously, viruses in aquatic environments were investigated using either transmission electron microscopy (TEM) or epifluorescence microscopy (EFM). Estimates of viral levels were first obtained using TEM after ultrafiltration or ultracentrifugation procedures (Bergh et al., 1989, Borsheim et al., 1990, Sime-Ngando et al., 1996). Since 1959, TEM has also been used to visualize phages, and characterize their morphology (Field, 1982). The use of EFM combined with the development of a variety of highly fluorescent nucleic acid specific dyes soon became the accepted method, because it involved a faster and less expensive technology. Nowadays, viruses (especially bacteriophages) are still usually counted by EFM using fluorochromes, such as SYBR Green I, SYBR Green II, SYBR Gold or Yo-Pro I (Xenopoulos and Bird, 1997, Marie et al., 1999a, Marie et al., 1999b, Shopov et al., 2000, Hewson et al., 2001a, Hewson et al., 2001b, Hewson et al., 2001c, Chen et al., 2001, Middelboe et al., 2003, Wen et al., 2004).

There have only been a few studies comparing the efficiency of the different techniques (such as EFM, FCM and TEM) for direct total counts of viruses in aquatic pelagic ecosystems. However, these comparisons make it possible to conclude that all these methods are fairly suitable for counting viruses (Hara et al., 1991, Hennes and Suttle, 1995, Weinbauer and Suttle, 1997, Marie et al., 1999a, Marie et al., 1999b, Bettarel et al., 2000, Chen et al., 2001), even though it seems that FCM has been reported to be as efficient as EFM or between 1 and 2 times more efficient (Marie et al., 1999a, Marie et al., 1999b, Brussaard et al., 2000, Chen et al., 2001, Dorigo et al., in revision), which in turn is reported to be up to seven times more efficient than TEM (Hara et al., 1991, Hennes and Suttle, 1995, Weinbauer and Suttle, 1997, Noble and Fuhrman, 1998, Bettarel et al., 2000). To the best of our knowledge, only Noble (2001) found TEM and EFM to have exactly the same efficiency levels for counting bacteriophages. The advantages and disadvantages of the three methods mentioned above, and a comparison of their efficiency can also be found in Weinbauer's excellent review on prokaryotic viruses (Weinbaueur, 2004).

Viruses are now considered to constitute an important component of aquatic microbial communities. They have been shown to be the most abundant biological compartment, and to play a crucial role in bacterial mortality, diversity and diversification in the pelagos (Wommack and Colwell, 2000, Weinbauer, 2004). Typically, viral infections are responsible for 20–50% of daily prokaryotic mortality, and they are a major source of dissolved organic matter. There has been little investigation of their importance in the sediment domain, where even basic information, such as their temporal dynamics and spatial distribution, is almost non-existent. However, sediments play a key role in the aquatic carbon cycle, and a high proportion of carbon degradation may be mediated by benthic processes (Glud and Middelboe, 2004). Since the work of Paul et al. (1993) and Maranger and Bird (1996), it has been known that high concentrations of viruses can occur in lake and marine surface sediments, typically reaching concentrations 10 to 1000 fold higher than those in the water column above, these densities being generally related to the trophic status of the ecosystem. In theory, aquatic sediments could provide an optimal environment (and hence constitute a reservoir) for virus development, since potential hosts (typically bacteria) are found in higher numbers than in the water column above, concentrations of organic matter are relatively high, and the distances between cells are very small (Wiggins and Alexander, 1985). Although, Danovaro and Serresi (2000) found large quantities of viruses and bacteria in a variety of sediments in the Eastern Mediterranean Sea, the low virus-to-bacterium ratios and their inverse relationship with trophic status suggest that the role played by viruses in controlling deep-sea benthic bacterial assemblages and biogeochemical cycles may be less relevant than in the pelagic systems. In another study, Danovaro et al. (2002) found that the lowest viral counts were obtained at stations where the largest cell sizes and the lowest bacterial growth and turnover rates were reported. These authors have suggested that the bacterial doubling time may play an important role in limiting virus development in sediments, and may influence the life strategies of benthic viruses. Recently, Middelboe and colleagues (Middelboe et al., 2003, Glud and Middelboe, 2004) clearly showed that the benthic viral community can be very dynamic, morphologically diverse and coupled with benthic bacterial activity. In freshwater ecosystems, high viral abundance but low virus-to-bacterium ratios have also been reported in lake sediments, suggesting that these particles are again only loosely related (Maranger and Bird, 1996, Lemke et al., 1997; Gessner et al. personal communication). At least, it seems that viral activity has only limited impact on benthic biogeochemical cycling (Middelboe, 2005). Clearly, only scant information about benthic viral ecology is available, and there are still no obvious conclusions about the importance and role of the viriobenthos.

To date, and to the best of our knowledge, viral abundance in aquatic sediments has always been determined using EFM or TEM. In this paper, we set out i) to propose an alternative way of counting viruses using flow cytometry combined with optimization of the extraction/fixation/dilution/staining procedure and ii) to report for the first time some typical concentrations of viruses and bacteria in the lake sediments of the two largest natural French lakes: Lakes Bourget and Geneva.