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Noda Institute for Scientific Research GRANT2013 Young Investigator Research GrantOutline of Research Results

Analysis of a novel physiological function of the respiratorychain of Zymomonas mobilisTakeshi HayashiDepartment of Food Science and Nutrition, Graduate School of Food Science and Nutrition, Beppu University

Research aims

Results

Zymomonas mobilis is an ethanol-fermenting bacteriumthat is responsible for the fermentation of tequila. In addition to the ethanol fermentation pathway, Z. mobilis alsopossesses a respiratory chain. I previously isolated elevenrespiratory-deficient mutant (RDM) strains of Z. mobilis1)and revealed that five of these strains (RDM-1, -2, -3, -6,and -7) were deficient in cytochrome bd-type ubiquinol oxidase (CydAB), and that five other strains (RDM-4, -5, -8,-9, and -11) were deficient in NADH dehydrogenase(NDH).2) Compared to the wild-type (wt) strain, the RDMstrains exhibited faster growth rates and higher levels ofethanol production at high temperatures, under aerobic conditions. These results indicated that the RDM strains weremore efficient ethanol producers under these conditions. 2), 3)The observed phenotypes of the RDM strains also indicate that the respiratory chain of Z. mobilis is an inhibitoryfactor for growth under aerobic conditions, at high temperatures. As the respiratory chain appears to be unnecessaryin Z. mobilis, it is unclear why the respiratory chain components have been conserved in this bacterium. Recently, itwas proposed that the respiratory chain of Z. mobilis has anovel physiological role.3) To investigate this possibility, Ianalyzed the physiological function of the respiratory chainof Z. mobilis.

I hypothesized that the respiratory chain of Z. mobilisplays a role in abiotic stress tolerance. To address this hypothesis, the growth rates of the RDM strains were compared with that of the wt strain in the presence of variousstressors, including osmolarity, heat, cold, and organic solvents. Among the examined stressors, osmotic stress fromexposure to sodium chloride and potassium chloride, butnot ethylene glycol, impaired the growth of all RDM strainscompared to the wt.
Complementation experiments were performed to examine the apparent association between the respiratory chainand salt tolerance. To complement respiratory defects,pZA22-ndh and pZA22-cydAB expression vectors were introduced into the corresponding RDM strains, and growthof the complemented strains was evaluated in the presenceof salt-containing medium. The complemented RDMstrains exhibited similar growth profiles as the wt strain inmedia containing either 0.25 M sodium chloride or 0.25 Mpotassium chloride (Fig. 1). In addition, the wt strains harboring either the ndh (wt-pZA22-ndh) or cydAB (wtpZA22-cydAB) vector exhibited a greater than 2-fold increase in cell density at 30 h compared to the wt-pZA22control strain (Fig. 1). Interestingly, the cell morphology ofMethodsThe wild type Z. mobilis strain ZM6 (ATCC 29191) wasused to generate the various RDM strains, as previously described.1) The Z. mobilis wt and RDM strains (RDM 1–9,and 11) were cultured in liquid medium containing 0.5%(w/v) yeast extract and 2% (w/v) glucose. Growth curveswere performed in test tubes incubated at 30°C with shaking at 50 rpm, and growth was monitored every 1 h bymeasuring the absorbance at 600 nm (OD-Monitor C&T,Taitec, Koshigaya, Japan). The NDH (pZA22-ndh) andCydAB (pZA22-cydAB) expression vectors used for complementation experiments were constructed previously.2)Intracellular sodium chloride concentrations were determined using Mohr’s method. d-sorbitol levels were measured enzymatically using a F-kit with D-Sorbitol/Xylitol(Roche Diagnostics, Basel, Switzerland).

Fig. 1. Growth rates of RDM strains complemented with NDH, undersalt stress conditions.
The wt (○), RDM-4 (△), RDM-5 (□), and RDM-11 (◇) strains weregrown in liquid medium containing 0.25 M sodium chloride. Open andclosed symbols denote strains possessing the pZA22 and pZA22-ndh vectors, respectively.

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wt-pZA22-ndh strains. After culturing in the high-salt medium, the wt-pZA22-ndh strain displayed a 1.25-fold increase in the amount of accumulated sorbitol compared tothe wt-pZA22 strain.
ConclusionFig. 2. Cell morphology of the RDM-4 strain complemented with NDH,under salt stress conditions. Strains RDM4-pZA22 (a) and RDM4-pZA22ndh (b) were cultivated in liquid medium containing 1.8 M sodium chloride and observed by light microscopy.

the RDM strains was abnormally elongated when grown insalt-containing media (Fig. 2a). This morphology was restored to near wild type appearance, however, in the complemented strains (Fig. 2b). Together, these results suggestthat the respiratory chain of Z. mobilis is involved in salttolerance.4)To elucidate the mechanism by which the respiratorychain of Z. mobilis mediates salt tolerance, the intracellularsodium chloride concentrations of the wt-pZA22 and wtpZA22-ndh strains were compared after culturing in thepresence of 1.8 M sodium chloride. The two strains exhibited similar intracellular sodium chloride concentrations,indicating that the respiratory chain of Z. mobilis is not involved in Na+ transport. It was previously reported that Z.
mobilis accumulates sorbitol as a compatible solute underosmotic stress conditions. Therefore, I compared the intracellular concentrations of D-sorbitol in the wt-pZA22 andThe results of this study indicate the respiratory chain ofZ. mobilis is involved in mediating salt tolerance, and thatthe regulation of sorbitol production may be an integral partof this process.4) My findings also revealed that salt tolerance is enhanced in Z. mobilis by the overexpression of respiratory chain enzymes such as NDH and CydAB.4)References1) Takeshi Hayashi, Yoshifumi Furuta, and Kensuke Furukawa. (2011)Respiration-deficient mutants of Z. mobilis show improved growthand ethanol fermentation under aerobic and high temperature conditions. J. Biosci. Bioeng. 111: 414–419.
2) Takeshi Hayashi, Tsuyoshi Kato, and Kensuke Furukawa. (2012)Respiratory chain analysis of Zymomonas mobilis mutants producinghigh levels of ethanol. Appl. Environ. Microbiol. 78: 5622–5629.
3) Takeshi Hayashi and Kensuke Furukawa. (2012) Respiratory-deficient mutants acquire high ethanol productivity under aerobic condition and thermotolerance. Kagakutoseibutu. 50: 780–781.
4) Takeshi Hayashi, Tsuyoshi Kato, Satoshi Watakabe, Wonjoon Song,Shizuho Aikawa, and Kensuke Furukawa. (2015) The respiratorychain provides salt stress tolerance by maintaining a low NADH/NAD+ ratio in Zymomonas mobilis. Microbiology. 161: 2384–2394.