Figure S1. Photosystem II apparent electron transfer rate (ETR) as a function of the
−2 −1
s ). Results are means and standard
deviations of at least three independent experiments. Measurements were determined
in cells grown in liquid cultures under mixotrophic conditions.
Figure S2. Biomass composition. (a) chlorophyll content (pg cell−1), (b) starch
content (pg cell−1), (c) protein content (pg cell−1), (d) total fatty acid content (pg
cell−1), (e) neutral lipid content (relative), (f) cell dry weight (pg cell−1). Asterisks
indicate statistically significantly differences between icl and WT or iclC using
Student’s t test, with a significance threshold of 0.05. For each strain, at least three
biological replicates were included. Error bars indicate standard deviations. The
measurements of all the parameters were determined in cells grown in liquid cultures
under phototrophic conditions.
Figure S3. One-dimensional gel of proteins separation of mixed WT and icl strains.
Whole cells samples were mixed based on equal chlorophyll content (labeled:
unlabeled = 1:1 with 5 μg of chlorophyll each). Mixed samples were fractionated by
SDS-PAGE. The proteins size of the molecular weight marker and the sixty-seven
bands excised from the mix of icl14N/WT15N on the gel are annotated.
Figure S4. Key for visualization of protein expression levels via heat maps.
In heat maps protein expression levels of all conditions (icl14N/WT15N and
iclC14N/WT15N and swapped labels) are compared. The shown mean ratios are log2
transformed and the standard deviation of a ratio is reflected in its box size (see
legend). In those heat maps, proteins of individual bioenergetic pathways are grouped
with
the
mitochondrial
electron
transport
chain
shown
in
a,
the
gluconeogenesis/glyoxylate cycle and glycolysis in b, lipid synthesis and degradation
in c, the TCA cycle in d, the oxidative pentose phosphate pathway (OPP) in e, the
amino acid synthesis pathways in f, the redox enzymes in g. For precise values of
protein ratios see Tables S2-5.
Figure S5. Key for explaining correlation of the number of identified and quantified
proteins with their respective ratios.
The ratios for all quantified proteins for icl14N/WT15N and iclC14N/WT15N (a and b)
as well as for switched labels (c and d) are shown in a log scale, respectively.
The quantitation results were normalized to the PRP8 protein. Error bars indicate
standard deviation between different combinations of peptide, band and charge states
(PBC combination). The value of 1 (e0) indicates unchanged protein ratios. Ratios
below 1 (e0) indicate down-regulated protein expression in icl or iclC, respectively.
Protein ratios above 1(e0) indicate up-regulated protein expression in icl or iclC,
respectively.
The protein number, given in the lower part of each figure, represents the sum of
proteins, which were quantified with only 1 PBC (blue graph) and proteins with more
than 1 PBC (grey graph). For detailed information of protein ratios see Tables S2-5.
Figure S6. Characteristics of strains grown under phototrophic conditions. (a) free
amino acid content (relative value), (b) Growth test analysis on solid medium in
Legends of Supplemental Tables
Table S1. Percentage of the main fatty acids (determined by GC/MS analysis of
FAMEs) in WT, icl and iclC strains. For each strain, three biological replicates
including three technical replicates were performed. Intervals correspond to standard
errors. Asterisks indicate statistically significant differences between icl and WT or
iclC using Student’s t test, with a significance threshold of 0.05. Measurements were
determined in cells grown in liquid cultures under phototrophic conditions.
Table S2. List of all quantified proteins in a mixture of icl14N/WT15N (whole cells)
normalized to PRP8 protein.
PBC count and scan count indicate the number of peptide, band and charge
combinations and number of scans in which a peptide has been identified and
quantified, respectively. The Ratio mean is determined as described in Materials and
Methods by calculating the mean and standard deviation (Ratio sd) of all PBC ratios
leading to one protein. The Ratio RSD indicates the relative standard deviation.
Table S3. List of all quantified proteins in a mixture of iclC14N/WT15N (whole cells)
normalized to PRP8 protein. For details see Table S1.
Table S4. List of all quantified proteins in a mixture of icl15N/WT14N (whole cells)
normalized to PRP8 protein. For details see Table S1.
Table S5. List of all quantified proteins in a mixture of iclC15N/WT14N (whole cells)
normalized to PRP8 protein. For details see Table S1.
Supplemental Methods
Methods S1
Cells were harvested during exponential growth phase (3-5 × 106 cells ml−1) and
suspended at a concentration of 10 µg chlorophyll ml−1 in TAP medium. In vivo
fluorescence kinetics measurements were performed at room temperature using a JTS
spectrophotometer (Biologic, France). The quantum yield of PSII (FPSII) was
calculated as (Fm-Fs)/Fm, where Fm is the maximum fluorescence emission level
induced by a 150 ms pulse of saturating light, and Fs is the steady-state level of
fluorescence emission after 30 s of continuous illumination. Actinic light was
provided by a red source (640 nm) which was switched off transiently (< 1 ms) while
measuring fluorescence in the near Infra Red (IR) region (> 680 nm) in response to a
~10 µs analytical blue pulse. The apparent photosystem II electron transport rate
(ETRPSII) was be calculated as FPSII x I (Genty et al., 1989), where I is the intensity
of the continuous actinic light.
Genty, B., Briantais, J.M. and Baker, N.R. (1989) The relationship between the
quantum yield of photosynthetic electron and photochemical quenching of
chlorophyll fluorescence. Biochim. Biophys. Acta, 990, 87-92.