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Citrobacter freundii Characterisation:
Sugar use
An ideal chassis should be able to use various sugars as carbon sources. In order to show that C. freundii is capable of using a variety of carbon sources, we have tested its growth on M9 minimal media plates containing different types of sugars. In addition to plates, we have also assessed its growth in liquid media containing these and other sugars.
While it has not yet been tested by our team, others in the C. French lab has shown that C. freundii grows well on media that contain cellobiose as the sole carbon source, whereas E. coli cannot use cellobiose. Cellobiose is a major component of biomass, so C. freundii can be used well for biomass degradation experiments.
Method (expand)
Small wells were cut in the middle of the plates and 150 μl of 20% sugar solution (glucose, sucrose, lactose, or glycerol) was added to each well. To some plates, powdered sucrose or glucose was added instead. The plates were then streaked with four strains ( E. coli, E. coli + sucrose hydrolase gene, C. freundii NCIMB and C. freundii SBS197) and incubated overnight at 37°C.
A final test involved adding the various sugars to the M9 medium in the bottle as opposed to either adding it to the plate before pouring the agar on top or adding them into the well on the plate. 5x100ml M9 medium bottles were prepared as before and autoclaved. The sugars (1ml) and thiamine hydrochloride (3.4 ml) were added to the bottles prior to the agar getting poured, with two bottles having no sugar added to them. After the agar had set, the plates were inoculated as before. One of the no sugar plates had solid citrate added to its middle, as before with the solid glucose and sucrose, to test the growth of C. freundii on this medium that gives it its name.
For the liquid cultures, M9 minimal medium was used, supplemented with various sugars at a 1% final concentration and chloramphenicol20. The bottles were inoculated with cells containing pSB1C3 grown overnight, and incubated overnight at 37°C
Close the method.
Results - Plates
The results of these experiments can be seen in Figures 1 and 2 below.
Figure 1 - M9 plates with sugars added to wells in the middle of the plates
Figure 2 - Sugars were added to the agar before the plates were poured
From these results, it can be seen that C. freundii SBS197 grows less well on lactose, sucrose and citrate but both strains grow equally well on glycerol and glucose. The E. coli + sucrose hydrolase cells grew well on sucrose even without there being any arsenic (the inducer of the sucrose hydrolase gene) on the plate. For some reason, all bacteria grew weakly on the lactose plates, this might mean that our lactose stock quality needs to be checked.
Liquid Media
To better quantify Citrobacter freundii’s ability to grow using a variety of carbon sources, we have grown them in M9 minimal medium supplemented with these various sources. The growth results can be seen in Figure 3 below.
Figure 3 – The growth of Citrobacter freundii using various carbon sources. Bars in blue show OD measured after overnight incubation at 37°C while bars in red show OD measured after incubation for two days at 37°C (these media did not yield significant results after just one day of incubation).
These results show that Citrobacter freundii can use all but one of the tested sugars as sole carbon sources – the one outlier, xylitol, got us thinking about developing a selectable marker system similar to the sucrose hydrolase system we used for our E. coli cells – one that depends on sugar use. Similarly to how E. coli K12 is unable to use sucrose, Citrobacter freundii cannot grow on xylose (as shown by our experiments and quoted in Bergey’s Manual of Systematic Bacteriology). We have chosen xylitol as the carbon source for this marker and have developed a theoretical protocol for how the development of such a marker could be done. You can read about it on this page.
