Approaches to isolate novel PPEs from AMT
5.5 Conclusions and future directions
Whilst most of the phytoplankton strains isolated during this study were represented in the clone libraries constructed along AMT18 (chapter 4), most are likely fast growing ‘r’ selected species. Thus, no isolates representative of the plethora of
Chrysochromulina or environmental chrysophyte sequences found in the oligotrophic gyres along AMT18 (chapter 4) were obtained. Clearly, then there is still a significant gap between the extensive diversity of PPE sequences found in environmental surveys compared to the number of PPEs isolated into culture. The use of classical cultivation methods have led to a bias towards the fast growing ‘r’ selected species, whereas the ‘k’ selected species, which are environmentally important in areas such as the oligotrophic gyres, have so far been resistant to cultivation attempts (Andersen, 2005).
It is possible that an integration of knowledge, from the cell to the ecosystem, will be required, in order to further efforts into the cultivation of PPEs. An increase in the knowledge of the desired species through both metagenomic techniques and culture studies, will lead to a better understanding of how these species interact with their environment. With greater knowledge of these interactions, the cultivation techniques can be optimised towards the cultivation of novel organisms.
The ability to isolate novel organisms in the future will certainly require a greater knowledge of the biotic and abiotic factors which control the distribution of PPEs in
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situ, as well as the use of new culturing techniques such as those currently being applied to the cultivation of bacterioplankton (see Alain and Querellou, 2009 for a review).
One technique successfully used to isolate marine bacterioplankton into culture utilises a combination of flow cytometry and single cell encapsulation (Zengler et al., 2002). Cell agarose mixtures are emulsified producing gel microdroplets (GMD), and then grown in culture medium for at least five weeks for the formation of colonies to occur in the GMDs. Since all the encapsulated cells are cultivated together and the gel matrix is large, metabolites and other molecules, such as signalling molecules, are allowed to freely pass in the media, more accurately simulating environmental conditions (Zengler et al., 2002). GMD culturing is performed in an open continuously fed system which does not allow the build up of toxic end products, thus more closely replicating the open ocean conditions found in natural environments. GMD-containing colonies could then be distinguished from empty GMDs, or free living cells and could be sorted into 96 well microtiter plates for further characterisation. However, the isolation of GMDs into microtiter plates disrupts the cell to cell interactions which were present before isolation. Alain and Querellou (2009) proposed a modification of this method, where GMDs are sorted into microbioreactors, directly linking them to the GMD culture community, and keeping cell-cell interactions intact. This production of mixed microbial communities offers further potential to ‘cultivate the uncultivated’. Certainly, it can be expected that in the natural environment many organisms will live as part of a inter-linked ‘community’, with cells exchanging dedicated signalling molecules, trading metabolites and competing for limited resources (West et al., 2007). Mixed microbial assemblages also have the capacity to perform multi-step functions that are
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not possible for individual species. In some cases cooperation within the microbial community involves the sharing of metabolic intermediates or micronutrients. When these dependencies are identified they can be reproduced experimentally allowing for the growth of the desired organism in monoculture (Alain and Querellou, 2009). In the current study a higher diversity of strains was identified in the mixed start cultures than could be subsequently isolated (e.g. E. huxleyi). This could be due simply to the limitations of growing PPEs on agar plates, as not all algae are able to grow on plates (Andersen, 2005), but could clearly also be due to their dependencies on shared products which would not exist in isolation.
Further evidence of the importance of signalling molecules to the cultivation of marine bacterioplankton was obtained by Bruns et al., (2002). Although the addition of signalling molecules, to improve cultivation, has not been explored systematically it was found that marine bacterioplankton have high affinity uptake systems for cyclic AMP (cAMP). In laboratory cultures, which have been starved of nutrients, the addition of cAMP has been shown to prevent substrate accelerated death (Bruns
et al., 2002). The addition of cAMP, which is part of the crp activation system in enteric bacteria, enables the cultivation of novel strains of bacterioplankton which were at a low concentration in the community (Bruns et al., 2002). The use of signalling molecules and shared metabolic intermediaries has not been undertaken with PPEs but the approach is a possible way in which novel algae can be isolated into culture.
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Figure 5.3. Flow diagram showing possible integrations of techniques to further understand environmentally important species (Taken from Tyson and Banfield, 2005)
As community genomic databases increase, the possibility of bypassing bottlenecks in cultivation may also be possible. With the characterisation of cultures which are presently available, combined with analysis of community genomics, reconstruction of metabolic pathways, important in the environment can be achieved. From knowledge of the specific metabolic pathways active in the environment, selective isolation strategies can be identified, to enable isolation of previously uncultured organisms (Figure 5.3). Tyson et al., (2004), undertook a random shotgun sequence analysis of a low diversity acid mine drainage system, to reconstruct genomes of the bacteria and Archaea present in the system. In acid mine drainage nitrogen fixation is an essential process and genome analysis found that the only nitrogen fixation operon present in the environment was assigned to Leptospirillum group III. Subsequently, a diluted environmental sample was inoculated into nitrogen-free media, resulting in the growth of only the target organisms (Tyson and Banfield, 2005). This study highlights the possibility of how deductions in functional partitioning, between community members, can aid in the ability to culture novel
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organisms. As the number of PPE genomic studies increases, this approach could be utilised in the future to obtain novel cultures.
Further attempts at culturing novel PPEs will certainly need to be undertaken, in order that physiological studies can be undertaken to reveal the metabolic capabilities of PPEs, and thus a greater understanding of biogeochemical cycles in the ocean. At present, failures in our ability to do so can only potentially be met using metatranscriptomic approaches (see chapters 6 and 7).
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