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This work, combined with that of several others (Carini et al., 2015; Sebastián et al., 2016), has demonstrated that lipid remodelling is an important mechanism by which marine heterotrophic bacteria respond to P scarcity. While such lipid remodelling has been known about for more than 40 years (Minnikin and Abdolrahimzadeh, 1974; Minnikin et al., 1974) and has been described in a wide range of organisms, from bacteria to plants and algae (Tjellström et al., 2008; Van Mooy et al., 2009), little was known about how widely distributed it was amongst bacteria in natural environments. In fact, a previous study had claimed that marine heterotrophic bacteria were likely unable to carry out such remodelling (Van Mooy et al., 2009). This confusion may partly be explained by the patchy distribution of the genes involved – closely related SAR11 strains differ in their ability to remodel their lipids, for example (Carini et al., 2015). The distribution of the lipid remodelling capability, as denoted by the presence of PlcP, indicates that horizontal gene transfer between bacteria adapted to P-depleted marine environments has been a major factor in its proliferation, as seems to be typical for genes related to dealing with P scarcity (Coleman and Chisholm, 2010). It will be interesting to determine whether a similar pattern emerges in other environments, such as fresh water and soil, which are also frequently P-depleted. There is reason to think that this may be the case, since PlcP was first characterised in a soil bacterium, E. meliloti (Zavaleta-Pastor et al., 2010). In this work, I did not attempt to study the enzymology of PlcP, though this will be an important topic for future research. In their original paper describing PlcP in E. meliloti, Zavaleta-Pastor et al. (Zavaleta-Pastor et al., 2010) reported that PlcP was active against the zwitterionic phospholipids PE and PC but not PG. This is in line with my observations that PE in Phaeobacter sp. MED193 was much more greatly reduced in P-starved cells than PG (Chapter 3). Both E. meliloti and Phaeobacter sp.

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MED193 produce DGTS as the major P-free lipid when P is scarce (Geiger et al., 1999; Sebastián et al., 2016), which presumably enables the cells to maintain the overall charge balance of their membranes since both it and PE are zwitterionic. Interestingly, the equivalent gene adjacent to PlcP in SAR11 and many other marine bacteria is a homolog of the bifunctional glycosyltransferase Agt from Agrobacterium

tumefaciens, which can synthesise both the neutral MGDG and the negatively charged

GADG (Semeniuk et al., 2014). This raises the question of whether PlcP from these organisms is also able to degrade PG, or whether they possess some alternative means to degrade PG.

6.1.1 Implications for ocean biogeochemistry

This finding of an extensive capacity for lipid remodelling in certain regions of the ocean potentially has important implications for the biogeochemistry of these regions. The plasticity of P in phytoplankton relative to C and N has been shown to have an important effect on atmospheric CO2 as predicted by oceanic box models

(Galbraith and Martiny, 2015). Models of heterotrophic bacteria have often been assumed them to have relatively inflexible stoichiometry relative to phytoplankton (Wang et al., 2012), studies of lake heterotrophic bacterial communities have shown variations in cellular C:P over three orders of magnitude (Godwin and Cotner, 2015). In light of the accumulating evidence that the stoichiometric flexibility of heterotrophic bacteria has been underestimated, the potential biogeochemical implications of this should be investigated. The inclusion of heterotrophic bacteria in such large-scale ocean-atmosphere model is, however, still in its infancy (Follows and Dutkiewicz, 2011).

It remains unclear how significant a saving of cellular P lipid remodelling represents. Around 20% of cellular P in bacteria is typically bound up within the membrane (Karl and Bjorkman, 2014). Since cells often scale back rRNA production when P is scarce (Elser et al., 2003; Makino et al., 2003), without a means to replace phospholipids it

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seems likely that the membrane would consume an even larger proportion of cellular resources when P is scarce. The potential for a substantial reduction in the cellular P quota should thus provide bacteria capable of lipid remodelling a competitive advantage when P is limiting (Thingstad and Rassoulzadegan, 1999). So far, however, there has been no quantitative assessment of the amount of P that can be spared by lipid remodelling. Indeed, there are remarkably few estimates of lipid P as a proportion of total cellular P. Estimates of the amount of P that can be spared in this way will be important if the kind of stoichiometric flexibility implied by lipid remodelling is to be incorporated into ecological models.

Bacteria that are capable of lipid remodelling often produce substantial quantities of phospholipids when P-replete (Geiger et al., 1999; Carini et al., 2015; Sebastián et al., 2016). This raises the question of why these bacteria do not simply retain membranes comprised primarily of P-free lipids. One suggestion, based on work done on lipid remodelling in plants, is that phospholipids might act as a storage compound for phosphorus, to be mobilised when P becomes scarce (Tjellström et al., 2008). This was based on the observation that lipid remodelling in Arabidopsis thaliana was reversible, with phospholipids rapidly replacing the substitute glycolipids once the P supply was restored. Similar results have also been observed in experiments with diatoms (Martin et al., 2011) although such remodelling has yet to be reported in bacteria. An alternative, or complementary, explanation for these observations could be that a higher proportion of membrane phospholipids is advantageous per se. Strains closely related to those which can remodel in response to P-scarcity but which lack PlcP themselves seem to constitutively produce large amounts of phospholipids (Chapter 4; Carini et al., 2015). Moreover, phospholipids, particularly PE and PG, appear to be more widely distributed through Bacteria than any other major class of membrane lipid (Sohlenkamp and Geiger, 2015). Membrane proteins have evolved alongside their lipid partners (Lee, 2011) and through much of evolutionary history, it appears that the lipid portion of the bacterial membrane has included a substantial fraction of phospholipids. Therefore, it is possible that there is a fitness trade off

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involved in replacing phospholipids with P-free lipids. From studies in E. coli we know that mutants unable to produce a class of phospholipids are viable but exhibit severe phenotypes. Lack of PE, for example, confers a requirement for divalent cations (DeChavigny et al., 1991) and results in the mis-assembly of some proteins such as LacY, which is inverted relative to its conformation in wild type cells (Dowhan and Bogdanov, 2009). There are, however, a number of key differences between cells that have remodelled their lipids in response to P stress and mutants unable to synthesise a phospholipid. Firstly, most bacteria possessing PlcP appear to produce one or more substitute P-free lipids, such as DGTS or MGDG, which can presumably serve as a partial substitute for the phospholipids it replaces. Furthermore, in the strains studied so far, phospholipids are not completely removed after lipid remodelling (See for example Chapter 3; Carini et al., 2015). Thus, there may still be sufficient amounts of these phospholipids remaining to engage in highly specific interactions with membrane proteins (Contreras et al., 2011). These differences explain why the phenotype of cells which have undergone lipid remodelling is much less severe than for mutants entirely lacking a lipid class. However, quantitative differences in the rates of cellular processes, such as nutrient uptake or of respiration, could nonetheless have important implications for the biogeochemistry of regions in the ocean where P is scarce and warrants further investigation.