Figure 1.
The methylotrophy specific metabolic network in in M. extorquens AM1.
All genes, except for the mau cluster (gray), are present and >95% identical in M. extorquens PA1. M. extorquens AM1 and M. extorquens PA1 were grown on various C1 and multi-C substrates (blue) for this study. Genes highlighted in red were deleted in M. extorquens PA1 to uncover that the metabolic network involved in methylotrophy in M. extorquens PA1 is identical to M. extorquens AM1. TCA: Tricarboxylic acid Cycle and EMC: Ethyl-malonyl CoA Pathway.
Table 1.
M. extorquens strains and plasmids used in this study.
Figure 2.
Quantitative comparison of growth rates and maximum OD600 values.
A) Growth rates for the Δcel ‘wild-type’ strain of AM1 (filled) versus the Δcel ‘wild-type’ strain of PA1 (open) on C1 substrates (M, 15 mM methanol; MA, 15 mM methylamine; F, 15 mM formate), the joint C1 and multi-C substrate betaine (B, 15 mM), multi-carbon substrates (S, 3.5 mM succinate; P, 5 mM pyruvate; E, 7.5 mM ethanol) and a combination of C1 and multi-carbon substrates (½M+½S, 1.75 mM succinate and 7.5 mM methanol). Error bars represent the 95% C.I. of the average of three biological replicates. The line indicates the approximate detection limit of our automated growth rate measurement device of 0.04 hr−1. Growth rates for PA1 on MA or B, and for AM1 on E were below this detection limit. B) Maximum OD600 for the Δcel ‘wild-type’ strain of AM1 (filled) versus the Δcel ‘wild-type’ strain of PA1 (open) on C1 substrates (M, 15 mM methanol; MA, 15 mM methylamine; F, 15 mM formate), the joint C1 and multi-C substrate betaine (B, 15 mM), multi-carbon substrates (S, 3.5 mM succinate; P, 5 mM pyruvate; E, 7.5 mM ethanol) and a combination of C1 and multi-carbon substrates (½M+½S, 1.75 mM succinate and 7.5 mM methanol). Error bars represent the 95% C.I. (confidence interval) of the average of three biological replicates.
Figure 3.
Heat map depicting the ratio of growth rate of knockout mutants of PA1 relative to the growth rate of the Δcel wild-type strain on C1 or multi-C substrates (same concentrations as in Figure 2).
Undetectable growth is indicated by grey. A significant difference (determined by comparing the mean growth rate of three biological replicates using the t-test) in growth rate with a p-value <0.05 is indicated by a * and a p-value <0.01 is indicated by **.
Figure 4.
Heat map depicting the ratio of maximum OD600 of knockout mutants of PA1 relative to the maximum OD600 of the Δcel wild-type strain on several C1 or multi-C substrates (same concentrations as in Figure 2).
Maximum OD600 values below 0.01 are indicated by grey. A significant difference in maximum OD600 (determined by comparing the mean growth rate of three biological replicates using the t-test) with a p-value <0.05 is indicated by a * and a p-value <0.01 is indicated by **.
Figure 5.
Comparison of growth rates and maximum OD600 values on methanol with succinate versus on succinate alone.
A) Ratio of growth rates, of the Δcel ‘wildtype’ strain of PA1 and various knockout mutants, on a combination of ½M+½S (7.5 mM methanol+1.75 mM succinate) versus S (3.5 mM succinate). The dotted line depicts the expected ratio for growth rate, if no methanol was oxidized in a combination of M+S. B) Ratio of yield (measured as the maximum OD600 value during growth), for the Δcel ‘wildtype’ strain of PA1 and various knockout mutants, on M+S versus S. The dotted line depicts expected ratio for yields, if no methanol was assimilated in a combination of M+S. For all data error bars represent the 95% C.I. of the average ratio of three biological replicates grown in each condition.