How endo- is endo-? Surface sterilization of delicate samples: a Bryopsis (Bryopsidales, Chlorophyta) case study
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- Hollants, J., Leliaert, F., De Clerck, O. et al. Symbiosis (2010) 51: 131. doi:10.1007/s13199-010-0068-0
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In the search for endosymbiotic bacteria, elimination of ectosymbionts is a key point of attention. Commonly, the surface of the host itself or the symbiotic structures are sterilized with aggressive substances such as chlorine or mercury derivatives. Although these disinfectants are adequate to treat many species, they are not suitable for surface sterilization of delicate samples. In order to study the bacterial endosymbionts in the marine green alga Bryopsis, the host plant’s cell wall was mechanically, chemically and enzymatically cleaned. Merely a chemical and enzymatic approach proved to be highly effective. Bryopsis thalli treated with cetyltrimethylammonium bromide (CTAB) lysis buffer, proteinase K and bactericidal cleanser Umonium Master showed no bacterial growth on agar plates or bacterial fluorescence when stained with a DNA fluorochrome. Moreover, the algal cells were intact after sterilization, suggesting endophytic DNA is still present within these algae. This new surface sterilization procedure opens the way to explore endosymbiotic microbial communities of other, even difficult to handle, samples.
KeywordsBryopsisEndosymbiontsEndosymbiotic bacteriaMarine green algaeSurface sterilization
Numerous eukaryotes maintain close associations with bacteria, either on their surface or within their cells. To examine the latter alliance it is essential to remove the bacteria which inhabit the host’s surface and form a main source of contamination. However, the ubiquity of bacterial biofilms prevents the straightforward study of these endosymbionts (Burke et al. 2009). In well established symbiosis models the surface sterilization used is in general quite aggressive. Insect eggs, larvae and adults are treated with hydrogen peroxide, formaldehyde, radiation, antibiotics or highly toxic mercury derivatives like mercuric chloride and thiomersal (Connell 1981; Meyer and Hoy 2008). Land plants or their symbiotic structures (e.g. root nodules) are mostly surface disinfected by means of beads, ethanol or sodium hypochlorite (Lodewyckx et al. 2002). Despite the use of these vigorous techniques, an effective surface sterilization remains a balancing exercise. Few surface disinfection protocols result in complete removal of ectosymbionts without penetrating interior tissues and thereby neutralizing internal bacteria; while an ineffective sterilization may result in outer surface bacteria being mistaken for endosymbionts (Lodewyckx et al. 2002). When the host is delicate, as is the case for the siphonous green alga Bryopsis, finding the right balance becomes even more challenging. Siphonous seaweeds are essentially single giant multinucleate cells surrounded by a xylan-cellulose cell wall, a thin parietal layer of cytoplasm and a huge central vacuole (van den Hoek et al. 1995). Like various other macro-algae (Ashen and Goff 2000; Dawes and Lohr 1978; Turner and Friedmann 1974), Bryopsis has long been suspected to harbor endogenous bacteria in the cytoplasm (Burr and West 1970). The identity of these endosymbionts, however, remains unknown. Further exploration of this algal-bacterial partnership requires an efficient surface sterilization of the Bryopsis host. After all, many seaweeds live in close association with numerous epiphytic bacteria, which control morphological development (Fries and Iwasaki 1976; Marshall et al. 2006; Provasoli and Pintner 1980; Tatewaki et al. 1983) or are linked with various metabolic functions (Chisholm et al. 1996; Croft et al. 2005; Head and Carpenter 1975; Staufenberger et al. 2008; Weinberger et al. 2007), and Bryopsis seems no exception (Kan et al. 1999). Whereas the usage of axenic cultures is quite common for micro-algae, for the study of marine macro-algae this is limited. In general, axenic seaweed cultures are obtained by the addition of antibiotics to the growth medium or a combination of antibiotic use and isolation of reproductive cells (Andersen 2005; Droop 1967; Kooistra et al. 1991). Reported attempts to efficiently remove epiphytes mechanically, chemically or enzymatically from macro-algae are even scarcer. Only a few protocols has been published for the selective extraction and subsequent application of epiphytic DNA from bacteria associated with seaweeds (Burke et al. 2009; Fisher et al. 1998). In addition, siphonous macro-algae, such as Bryopsis, offer some extra options for the elimination of epiphytes due to their giant-cell morphology and regeneration mechanisms: the cytoplasm of these algae can be isolated by centrifugation (Berger and Kaever 1992) and the formation of protoplasts can be easily attained through wounding (Kim et al. 2001). However, the objective of all techniques listed above was never to study the endophytic bacteria within these seaweeds, leaving the effect of these methods on the endophytes unaddressed.
In this study different mechanical, enzymatic and chemical procedures for the complete elimination of epiphytes from Bryopsis plants, in order to study the internal bacterial communities, were compared and evaluated. The aim was to develop a new, highly effective surface sterilization technique which neither lyses the algal cells nor eliminates endophytic DNA, allowing further molecular processing of the endosymbionts.
2 Materials and methods
2.1 Sampling and culturing
A Bryopsis hypnoides strain (BR) was collected from the lower intertidal zone in Roscoff, Brittany, France in July 2008. The plant was grown in sterile 1× modified Provasoli enriched seawater (West and McBride 1999) at 23°C under a 12:12 h Light:Dark cycle with a photon flux rate of 25–30 µE m−2 s−1. Unialgal cultures were achieved by isolating apical fragments of the vegetative thalli under a binocular dissecting microscope. The selected apical fragments were maintained under the same growth conditions as described above. To obtain more material for further applications, unialgal cultures were transferred to sterile 250 ml Erlenmeyer flasks with constant aeration.
List of protocols applied for the surface sterilization of Bryopsis plants
Repeatedly vortex the plants in 0.2 µm filtered ASW with five changes of washing water
Ultrasonic probe sonication
Ultrasonic probe sonication of the samples in sterile ASW for 15 s at 30 kHz
Ultrasonic bath sonication
Ultrasonic bath sonication of the samples in sterile ASW for 15 min at 47 kHz
Use of beads
Add glass beads (0.5 mm, BioSpec Products) to the algal tissue and bead beat the mixture at 30 kHz for 3 × 85 s
Add 10 µl lysozyme (1 mg.mL−1 in 10 mM Tris HCl) and 190 µl sterile ASW to the specimens and incubate for 5 min at room temperature
Incubate the algal thalli in a mixture of 1 µl 20 mg.mL−1 proteinase K and 99 µl ASW for 30 min at 60°C
Rinse plants in 80% ethanol for 5 min
Sterilize algae in 3% sodium hypochlorite for 30 s
Alkaline lysis buffer
Place thalli in 80 µl sterile ASW with 20 µl alkaline lysis buffer (1 M NaOH and 10% sodium dodecyl sulfate) for 15 min at 95°C
Put plants directly into 100 µl CTAB buffer (2 g CTAB, 1 g PEG 8000, 1.5 M NaCl, 0.02 M EDTA and 0.1 M Tris-HCl) for 30 min at 60°C
Place samples in 100 µl UNSET Lysis Buffer (8 M urea, 2% sodium dodecyl sulfate, 0.15 M NaCl, 0.001 M EDTA, 0.1 M Tris pH 7.5) for 15 min at 55°C (Fisher et al. 1998)
Sterilize plants overnight in a 1:1 mixture of 0.2 µm filtered Umonium Master (Huckert’s International) and sterile ASW
1. Place unialgal Bryopsis plants directly into CTAB buffer with 20 mg.mL−1 proteinase K for 30 min at 60°C
2. Wash the Bryopsis thalli with sterile ASW
3. Repeat step 2 two times
4. Incubate overnight the washed thalli in a 1:1 mixture of 0.2 μm filtered Umonium Master and sterile ASW
5. Wash thalli in sterile ASW
6. Repeat step 5 ten times with vigorous vortexing in between the washing steps
2.3 Denaturing gradient gel electrophoresis
To compare the effectiveness of the different sterilization procedures, the remaining bacterial diversity was examined by Denaturing Gradient Gel Electrophoresis (DGGE). Therefore the cleaned Bryopsis plants were placed in liquid nitrogen and ground with a sterile pestle prior to a total DNA extraction following a CTAB protocol modified from Doyle and Doyle (1987). The V3 region of the 16S rRNA gene was amplified by a PCR with the universal bacterial primers F357 (5′-CCTACGGGAGGCAGCAG-3′) and R518 (5′-ATTACCGCGGCTGCTGG-3′) (Temmerman et al. 2003; Yu and Morrison 2004). A GC-clamp was coupled to the forward primer to improve DGGE separation. Amplifications were performed in volumes of 50 μl containing 1 µl of target DNA, 1× PCR buffer (GeneAmp, Applied Biosystems), 100 µM dNTPs, 0.05× BSA, 0.2 µM of both primers, and 1.25 units AmpliTaq DNA polymerase (Applied Biosystems). After an initial denaturing step at 95°C for 1 min, 30 cycles of denaturation (95°C, 30 s), annealing (55°C, 45 s) and extension (72°C, 1 min) were completed, followed by a final amplification step at 72°C for 7 min. Successful amplification of the V3 region was verified through agarose gel electrophoresis. DGGE analysis of PCR amplicons was performed using the DCode Universal Mutation Detection System device (Bio-Rad) as described previously (Temmerman et al. 2003). Optimal electrophoretic separation was obtained using 35–70% denaturing gradient polyacrylamide gels, running for 990 min at 70 V in 1× TAE buffer at a constant temperature of 60°C. The gels were stained with SYBR gold (Molecular Probes, Invitrogen) for 30 min followed by visualization and digital capturing of the profiles via the Molecular Imager Gel Doc XR System (Bio-Rad). Digital images were normalized and processed by means of the BioNumerics software (version 5.1, Applied Maths).
3.2 Denaturing gradient gel electrophoresis
Obtaining axenic macro-algal cultures while maintaining endophytic bacteria is challenging. Established culture techniques relying on the usage of antibiotics are inadequate as antibiotics may penetrate through the algal cell wall and eliminate the endophytes. Observations that antibiotics clearly affect the growth of algae or even kill them indicate such diffusions (Andersen 2005). Also the special features of siphonous seaweeds, e.g. the formation of protoplasts and the ability to isolate their cytoplasm, are insufficient to generate epiphytic-free algal material. Since these extraction techniques both depend on cutting or squeezing the algae, the outer xenic membranes become damaged and give rise to contaminations (personal observations). It has to be mentioned that the development of protoplasts in some larger and therefore easier to manipulate siphonocladous algae, like for example Boergesenia and Ventricaria, shows potential for the formation of epiphytic-free algal material (personal observations, Mine et al. 2008). Hence, traditional algal cultivation and manipulation methods appear inadequate for the removal of bacterial epiphytes from Bryopsis plants without affecting the endophytes. Consequently, different mechanical, enzymatic and chemical surface sterilization protocols were tested and compared. None of these techniques seems on its own able to effectively eliminate the outer surface bacteria. The mechanical and enzymatic methods are highly insufficient, in accordance with observations by Burke et al. (2009). Also the use of various lysis buffers and disinfectants appears to be ineffective, in contrast with previous published studies (Burke et al. 2009; Fisher et al. 1998). In these studies Fisher et al. (1998) successfully sterilized filamentous green algae by placing them directly in UNSET buffer (Table 1), and Burke et al. (2009) fruitfully treated the green and red seaweeds, Ulva australis and Delisea pulchra, with calcium- and magnesium-free artificial seawater (CMFSW) supplemented with EDTA and a rapid multi-enzyme cleaner. These protocols, which were designed for the selective extraction of epiphytic DNA from algae associated bacteria, seem inefficient to completely sterilize Bryopsis externally with the aim of studying the bacterial endophytes. This objective is achieved by a combined chemical and enzymatic approach as presented here. Bryopsis thalli treated with CTAB lysis buffer, proteinase K and the bactericidal cleanser Umonium Master are highly effectively sterilized. They show no bacterial growth on agar plates (Fig. 1f) and no bacterial fluorescence on their cell wall when observed with confocal and epifluorescence microscopy after DAPI staining (Fig. 2p–r). Although often neglected by conventional surface sterilization protocols, the latter verification is essential since only 1% of all known bacteria suspected to be culturable (Amann et al. 1995). For example, the untreated sample and the ethanol and enzymatically cleaned plants all show growth when plated (Fig. 1a–c). On the other hand, fluorescence images allow a more detailed assessment of the outer surface community: the untreated, ethanol-sterilized and enzymatically cleaned samples show, respectively, bacterial biofilms (Fig. 2a–c), reduction of surface biofilms (Fig. 2d–f) and destruction of the biofilms into unattached bacteria (Fig. 2g–i). Much less expected were the results of the DAPI staining of Bryopsis plants sterilized with lysis buffer or Umonium Master. While both samples indicate no bacterial existence on agar (Fig. 1d, e), fluorescence images prove the presence of DNA on the surface of the algae (Fig. 2j–o). Even after vigorously vortexing during the several washing steps, bacterial DNA remains trapped in the degraded algal cell wall which is still clearly outlined by the blue DAPI stain. Despite the fact that these cell walls are gradually more damaged as surface sterilization becomes more effective, they were never fully lysed after the different disinfectant treatments. As shown in Fig. 2 (internal fluorescent foci from nuclei and chloroplasts, a to r) the degraded but intact algal cell walls become permeable for the DAPI fluorochrome after chemical and enzymatic surface sterilization. Not only does this indicate internal algal and bacterial DNA is still present, it also seems suitable for additional molecular processing like PCR amplification and DGGE (Fig. 3). This molecular approach is of great value in further research on the identity and functionality of the—possibly unculturable—endosymbiotic bacteria in Bryopsis algae. Future investigation will probably reveal these bacteria have significant functions within their host. Moreover, some of the compounds produced by these bacterial symbionts may have important applications like for example the production of the anticancer drug kahalalide as suggested by Kan et al. (1999).
Although surface sterilization is a critical step in endosymbiosis research, it remains challenging, especially in delicate organisms such as algae. Certainly in new symbiosis systems, it is worthwhile to test and evaluate conventional sterilization techniques. This study demonstrates that small alterations or combinations of established disinfection protocols permit an efficient sterilization. The protocol presented here will likely be useful in studies of new and difficult to handle hosts, allowing exploration of novel symbiosis systems.
This research was funded by ‘Fonds Wetenschappelijk Onderzoek’ FWO-Flanders project G.0045.08. We sincerely thank Valya Vassileva and Tom Beeckman for confocal microscopy assistance. We acknowledge John West for useful comments.