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To investigate the expressional control of branched respiratory chain complexes of the amino-acid producing bacterium Corynebacterium glutamicum according to growth conditions, the expression indexes of the ndh, sdh, qcrCAB, ctaCF, ctaD, ctaE, and cydAB genes were estimated under aerobic and microaerobic, and carbon-rich and -poor conditions. The promoter region of each target gene was cloned upstream of the EGFP gene on expression vector pVK6, and the nine reporter constructs were transformed into C. glutamicum ssp. lactofermentum. The cytochrome content of cellular membranes obtained from each growth phase closely corresponded to the expression indexes based on EGFP fluorescence and cell density, indicating that this rapid and convenient method is suitable for analyzing the expression levels of respiratory chain complexes. Using this method, we demonstrated that a reciprocal change in the expression levels of cytochrome bd-type and aa (3)-type oxidases occurs when C. glutamicum cells are held in stationary phase for extended periods.

Corynebacterium glutamicum contains at least two terminal oxidases in the respiratory chain; cytochrome aa(3)-type cytochrome c oxidase and bd-type menaquinol oxidase. Thus, the chain has two branches of electron flow. The bcc-aa(3) branch translocates three protons per electron transferred, while the bd branch translocates only one. In this study, we constructed two mutant strains, lacking of either subunit I of the cytochrome c oxidase (DeltactaD) or subunits I and II of the quinol oxidase (DeltacydAB), and also plasmids to complement the deficient genes to investigate their effects on energy conservation and cell growth. We measured H(+)/O ratios of C. glutamicum wild-type and mutant cells grown aerobically. The H(+)/O ratio of the wild-type cells grown in the semi-synthetic medium was 3.94 +/- 0.30, while the value was 2.76 +/- 0.25 for the DeltactaD mutant. In contrast, the value was 5.23 +/- 0.36 for the DeltacydAB mutant. The cells grown in the LB medium showed higher value compared to that of cells grown in the semi-synthetic medium. The DeltactaD mutant grew less than the wild-type in LB medium, while they grew about equally in semi-synthetic medium. Correlation between bioenergetics and growth of C. glutamicum was significantly affected by the growth nutrients.