Role of the Cell Surface Oxidation System in the Cell Growth ofAcetic Acid Bacteria
Toshiharu YAKUSHI
Yamaguchi University
Research objective
Acetic acid bacteria have a unique metabolic system. In acetic acid fermentation, the oxidation
reaction from ethanol to acetic acid, the essence of acetic acid fermentation, occurs in theperiplasm, which is called the cell surface. The resulting reducing power is used to reduce
oxygen to water via ubiquinone on the cell membrane (Fig. 1). Therefore, the oxidation
reaction on the cell surface is connected to the respiratory chain electron transport system,obtains energy in the form of a
proton driving force, and isused for ATP synthesis (1). As
described above, cell surface
oxidation
onlyoxidizessubstances;
therefore,
it
contributes only to energy
production. In contrast, theanabolic metabolism necessary
for cell division is performed
separately inside the cell. In
summary, during the growth ofacetic acid bacteria, this cellsurface oxidation metabolism
provides energy for the
intracellular metabolism that
assimilates carbon sources (1).
The physiological significanceof cell surface metabolism has
notyetbeen
validatedexperimentally; therefore, it
was investigated in this study.
It is difficult toseparate and analyze the two
metabolic systems of acetic

Figure 1. Overview of glycerol and glucose metabolism
in the wild-type and mutant strains of Gluconobacter
thailandicus, and the strategy used in this study.
It is not easy to separately examine the cell surface andintracellular metabolism in wild-type strain (top).
Using a mutant strain (Gly- DHA- ∆GDH, bottom) thatblocks the intracellular metabolism of glycerol anddihydroxyacetone (DHA) as well as the cell surface
metabolism of glucose (Gly- DHA- ∆GDH), the twometabolic systems can be separately examined.

acid bacteria, namely the cell surface and intracellular metabolic systems. Glycerol
dehydrogenase (GLDH) oxidizes glycerol to produce dihydroxyacetone (DHA), which is acell surface metabolism, but it requires a small amount of glycerol or DHA and uses it for cell
growth (Fig. 1). Therefore, we blocked the intracellular metabolism of glycerol and DHA andconstructed a mutant strain (Gly- DHA-) in which glycerol underwent only cell surface
oxidation (2).
Based on this Gly- DHA- mutant strain, we introduced mutations that allowed us toexamine the growth of substances that undergo only intracellular metabolism, without cellsurface metabolism. We focused on glucose, which is presumably the most well-understood
intracellular metabolite of Gluconobacter sp. Glucose is oxidized on the cell surface byglucose dehydrogenase (GDH) in the wild-type strain. Even GDH-disrupted strains of
Gluconobacter sp. take up glucose, metabolize it, and grow. Therefore, in this study, wedisrupted the GDH gene (gdhM) in Gly- DHA- mutant to create a Gly- DHA- ∆GDH mutant.
Methods
We used the Gluconobacter thailandicus strain (NBRC3255) and Gly- DHA- mutant strain(KAO-7), in which the glycerol kinase gene (glpK) and two DHA kinase genes (dhaK andderK) were eliminated (2). This mutant strain produced DHA from glycerol, but could not
grow on glycerol medium. We disrupted the gdhM gene of the Gly- DHA- mutant using
homologous recombination to generate the Gly- DHA- ∆GDH mutant. In addition, GLDH gene(sldBA) of the Gly- DHA- mutant was disrupted via homologous recombination to generate
the Gly- DHA- ΔGLDH mutant.
For growth experiments, preculture was performed in a medium containing bothglucose and glycerol. The preculture was transferred to a medium containing only glucose,
only glycerol, or both glucose and glycerol, and cultured with shaking at 30 °C.
Results
Growth behavior of the mutant strains was investigated (Fig. 2). Gly- DHA- mutant did not
grow on the glycerol medium. Since glucose metabolism is healthy, it grew on the glucosemedium. The addition of glycerol improved its growth (Fig. 2A). This improvement is thought
to be due to the energy obtained from the cell surface metabolism of glycerol by GLDH. To
test this hypothesis, we compared the growth on glucose–glycerol medium with the Gly- DHAΔGLDH mutant and found that the growth was worse than that of the Gly- DHA- mutant (Fig.
2B). This decrease is thought to be derived from the energy obtained by cell surface
metabolism of glycerol by GLDH, supporting the hypothesis that cell surface metabolism
helps growth.
Finally, we obtained results that indicated the importance of cell surface metabolism

in the Gly- DHA- ∆GDH mutant strain. This strain could not grow on the glucose or glycerol
medium, but grew on the glucose–glycerol medium (Fig. 2C). These results indicate that thisstrain cannot grow by cytoplasmic metabolism alone, and requires energy from cell surface
metabolism. These results strongly support the hypothesis of this study. Cell surface
metabolism supports intracellular metabolism and promotes cell proliferation.

Figure 2. Growth of Gluconobacter thailandicus mutants on the glucose-only medium,glycerol-only medium, and medium containing both glucose and glycerol. Preculture
was performed in a glucose–glycerol medium. A, Growth of the Gly- DHA- mutant on
glucose medium (red), glycerol medium (white), and glucose–glycerol medium (black).
B, Growth comparison of Gly- DHA- mutant (black) and Gly- DHA- ∆GLDH mutant(green) on glucose–glycerol medium. C, Growth of the Gly- DHA- ∆GDH mutant strain(The symbols are the same as those used in A).
Conclusion
In acetic acid bacteria, cell surface oxidation supplies the energy required for anabolic
metabolism and cell proliferation.
References
1） Matsushita, K., Toyama, H., and Adachi, O. (1994) Respiratory chains and bioenergetics ofacetic acid bacteria. Adv. Microb. Physiol. 36: 247-301.
2）Kataoka, N., Hirata, K., Matsutani, M., Ano, Y., Nguyen, T. M., Adachi, O., Matsushita, K.,and Yakushi, T. (2021) Three ATP-dependent phosphorylating enzymes in the first committed
step of dihydroxyacetone metabolism in Gluconobacter thailandicus NBRC3255. Appl.
Microbiol. Biotechnol. 105: 1227-1236.