Growth experiments

I carried out a number of growth experiments during the course of this work. These used the strains and growth conditions described above (Section 2.1) and in most cases lipid extraction was carried out as described. There were, however, many small modifications between the growth experiments so I describe each below, including a reference to the figure in which the results are reported.

2.4.1 Characterisation of ΔplcP

Phaeobacter sp. MED193 wild type and ΔplcP were initially grown to late exponential phase in PCR-S11 (six cultures for each strain). Cells were then pelleted by centrifugation at 9,000 x g for 5 minutes. Cell pellets were resuspended in the same volume of either PCR-S11 medium with added P (50 μM) or with no added P (3 replicates per condition for both strains). The cultures were tracked for 4 days following resuspension. On each day, cell density was measured using the optical density at 540 nm (OD540) and a 5 mL aliquot of culture was collected for lipid

analysis. Alkaline phosphatase activity was also measured. Lipid extraction was performed as described above, however the method for LC-MS analysis differed. Samples were resuspended in 1:1 methanol to dichloromethane prior to separation

60

on a 150 mm Nucleosphere HILIC column (Macherey-Nagel) at 30 °C, with a flow rate of 150 μL min-1_{. Samples were run on a gradient of 95% acetonitrile to 28% 10}

mM ammonium acetate, with 2 and 5 minute holds at the start and end of each run, respectively. Following ionisation under conditions which were the same as those described previously, selected masses were targeted for MS2 _{fragmentation, using the}

SmartFrag functionality of the Bruker TrapControl software to select an appropriate voltage. Masses selected for fragmentation were those identified as corresponding to DGTS (738.7 and 764.7) and the PC internal standard (762.7). The relative abundance of DGTS was expressed as the ratio of the sum of the peak areas for DGTS to the peak area for the PC internal standard, normalised to the OD540 of the culture. These results

are reported in Figure 3.1.

This method for lipid analysis had a number of shortcomings, including the presence of a highly intense contaminant ion at m/z 590, which appeared to result from column bleed. This resulted in reduced sensitivity for analyte ions and was eliminated by replacing the column with the BEH-Amide column. Also, the biomass collected for lipid extraction was not standardised, which can result in variable amounts of ion suppression affecting the intensities of target ions in unpredictable ways. Therefore all subsequent lipid analyses were carried out as described in Section 2.3.

2.4.2 Growth of Phaeobacter sp. MED193 for lipidomics analysis

Wild type MED193 was grown as described in Section 2.4.1, with 6 biological replicates for each condition. Three technical replicate samples were collected for lipid analysis 2 days following resuspension in PCRS11 medium with or without added phosphate. The selection of this time point was based on the results of the previous resuspension experiment (Section 2.4.1; Figure 3.1) and supported by the results of alkaline phosphatase assays, which indicated that cultures in the medium lacking P were P-stressed. Lipids from these samples were extracted and analysed according to Section 2.3. Results reported in Figure 3.2 show the ratio of the mean

61

relative abundance of each major lipid class in cultures grown without added phosphate to that of cultures grown with added phosphate.

2.4.3 Identification of glutamine lipid and characterisation of ΔolsB2 and ΔolsA in Ruegeria pomeroyi DSS-3

R. pomeroyi wild type, ΔolsB2 and ΔolsA were grown overnight in ½YTSS, at which point cells were harvested for lipid extraction and analysis, as described in Section 2.3. Three replicates were grown for each strain and results reported in Figures 4.2 and 4.4. The abundances of QL, OL and PE were reported relative to the abundance of the PE internal standard so that all values would be on the same scale.

2.4.4 Growth rate and lipid comparison of R. pomeroyi wild type and mutants

Three biological replicates of R. pomeroyi wild type, ΔolsB2 and ΔolsA were grown in MAMS with either 0.5 mM or 5 mM phosphate. The lower phosphate concentration of 0.5 mM was chosen based on previous preliminary growth experiments in which

ΔolsA had failed to grow at lower phosphate concentrations. Growth was tracked by

measuring OD540 at regular intervals and alkaline phosphatase activity was measured

prior to the collection of samples for lipid analysis, once the cultures were estimated to have entered late exponential phase. These samples, taken as three technical replicates from each culture, were extracted and analysed as described in Section 2.3. Growth rates were calculated by fitting a linear equation to the log transformed growth data from exponential phase cultures, using an implementation of the GrowthRates program (Hall et al., 2014) in the R statistical software package (R Core Team, 2017). Pairwise comparisons of the growth rates of each strain grown at high and low P concentrations, as well as comparisons of the growth rates between strains grown at the same P concentration, were made using Student’s t-test. Maximum yield was calculated as the mean of the maximum OD540 obtained for each strain-phosphate

combination. Lipid class abundances were reported as the ratio of the abundance of in 0.5 mM P compared to 5 mM P. Since ΔolsA again failed to grow in 0.5 mM P, this strain was omitted from this comparison. These results are reported in Figure 4.5.

62

2.4.5 Growth of Δspo3687

R. pomeroyi DSS-3 Δspo3687 was grown overnight in MAMS at which points samples

were collected for lipid extraction and analysis as described in Section 2.3. Lipid extracts were analysed for the presence of the putative homotaurine lipid as shown in Figure 5.3 and compared to results for the wild type obtained previously.