The primary metabolome of UWO241 grown at 4°C responds strongly
to heat stress
To understand the heat-induced changes to psychrophilic metabolism, we
analyzed the primary metabolome of UWO241 exposed to the non-permissive
temperature of 24°C for 6h. PCA analysis of all 771 detected metabolites
demonstrated a separation along both principal components between the
metabolome of the UWO241 cultures grown at 4°C and the metabolome of the
same cultures exposed to 24°C (Figure 6). We also observed a separation
between the metabolomes of UWO241 grown at 10°C and those exposed to
heat, albeit only along PC2. There was minimal separation along either
component between the cultures acclimated to the highest growth
temperature of 15°C before and after 6h heat stress (Figure 6). HCA
(Figure 7A) and volcano plot analysis (Table 3) revealed a strong
response at the level of the metabolome when cultures grown at 4°C were
exposed to 24°C for 6 hrs (222 DAMs, 29%). This heat stress response
was attenuated in cultures acclimated to 10°C, and even more so in the
cultures acclimated to 15°C, with 71 (9%) and 26 (3%) DAMs after heat
exposure, respectively.
Detailed analysis on the 161 positively identified metabolites revealed
that 98 metabolites (54.8%) were significantly different between
control and heat stress samples (ANOVA, p<0.01, Tukey’s post
hoc). We organized the positively identified metabolites by chemical
categories and normalized all control samples to an arbitrary value of
1. We compared the metabolomes from cultures exposed to 24°C to the
appropriate steady-state growth temperature (i.e., 4°C metabolome was
compared to 4°C + 6h heat stress metabolome). We reported the
differences between the metabolite classes (Figure 7B, Supplemental
Dataset S2) and the 20 metabolites that showed the largest abundance
difference between steady-state and heat stress treatments (Table 4).
Most metabolite classes, including carbohydrates, sugar alcohols, amino
acids, lipids and antioxidants, increased in abundance in the cultures
grown at 4°C and exposed to 24°C, with the exception of carboxylic acids
and sugar phosphates, which increased significantly in the 10°C cultures
(but not in those grown at 15°C). Notably, these metabolites are already
present in high amounts in UWO241 acclimated to 4°C (Figure 3B). We also
analyzed the 20 metabolites that showed the most significant change in
accumulation in heat stressed UWO241 (Table 4). Glucose-6-phosphate was
the only compound that showed a small but significant decrease
regardless of the initial culturing temperature (FC 1.7-2.6). Ergosterol
(FC 439.1) and α-tocopherol (FC 308.1) increased at very high amounts in
all cultures exposed to heat, regardless of the initial growth
conditions (Table 5). These increases followed a temperature dependent
pattern, with the highest FC seen in the cultures initially grown at
4°C.