Monitoring enzyme expression of a branched respiratory chain of corynebacterium glutamicum using an EGFP reporter gene

Department of Bioscience and Bioinformatics, Kyushu Institute of Technology, 680-4 Kawazu, Iizuka, Fukuoka, 820-8502, Japan.
Journal of Bioenergetics (Impact Factor: 3.21). 06/2011; 43(3):257-66. DOI: 10.1007/s10863-011-9355-6
Source: PubMed


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.

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    • "Although experimental K m values are not available, the oxygen affinity of cytochrome aa 3 oxidase is assumed to be lower than that of cytochrome bd oxidase [18]. Accordingly, cytochrome bd oxidase is presumably required under microaerobic conditions, while the bc 1 –aa 3 supercomplex predominates under oxygen-sufficient conditions [18] [36]. Another difference between the two branches is that cytochrome bd oxidase, in contrast to cytochrome aa 3 oxidase, does not require copper ions for activity. "
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    ABSTRACT: In this study a comparative analysis of three Corynebacterium glutamicum ATCC 13032 respiratory chain mutants lacking either the cytochrome bd branch (ΔcydAB), or the cytochrome bc1-aa3 branch (Δqcr), or both branches was performed. The lack of cytochrome bd oxidase was inhibitory only under conditions of oxygen limitation, whereas the absence of a functional cytochrome bc1-aa3 supercomplex led to decreases in growth rate, biomass yield, respiration and proton-motive force (pmf) and a strongly increased maintenance coefficient under oxygen excess. These results show that the bc1-aa3 supercomplex is of major importance for aerobic respiration. For the first time, a C. glutamicum strain with a completely inactivated aerobic respiratory chain was obtained (ΔcydABΔqcr), named DOOR (devoid of oxygen respiration), which was able to grow aerobically in BHI (brain-heart infusion) glucose complex medium with a 70% reduced biomass yield compared to the wild type. Surprisingly, reasonable aerobic growth was also possible in glucose minimal medium after supplementation with peptone. Under these conditions, the DOOR strain displayed a fermentative type of catabolism with l-lactate as major and acetate and succinate as minor products. The DOOR strain had about 2% of the oxygen consumption rate of the wild type, showing the absence of additional terminal oxidases. The pmf of the DOOR mutant was reduced by about 30% compared to the wild type. Candidates for pmf generation in the DOOR strain are succinate:menaquinone oxidoreductase, which probably can generate pmf in the direction of fumarate reduction, and F1FO-ATP synthase, which can couple ATP hydrolysis to the export of protons.
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    ABSTRACT: Promoters are DNA sequences which function as regulatory signals of transcription initiation catalyzed by RNA polymerase. Since promoters substantially influence levels of gene expression, they have become powerful tools in metabolic engineering. Methods for their localization used in Corynebacterium glutamicum and techniques for the analysis of their function are described in this review. C. glutamicum promoters can be classified according to the respective σ factors which direct RNA polymerase to these structures. C. glutamicum promoters are recognized by holo-RNA polymerase formed by subunits α(2)ββ'ω + σ. C. glutamicum codes for seven different sigma factors: the principal sigma factor σ(A) and alternative sigma factors σ(B), σ(C), σ(D), σ(E), σ(H) and σ(M), which recognize various classes of promoters. The promoters of housekeeping genes recognized by σ(A), which are active during the exponential growth, form the largest described group. These promoters and their mutant derivatives are the most frequently used elements in modulation of gene expression in C. glutamicum. Promoters recognized by alternative sigma factors and their consensus sequences are gradually emerging.
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    ABSTRACT: Corynebacterium glutamicum has a branched respiratory chain: one of the branches is cytochrome bcc complex and cytochrome aa3-type cytochrome c oxidase, and the other is cytochrome bd-type menaquinol oxidase. The factors that influence the expression patterns of these respiratory enzymes remain unclear. To investigate the expressional control mechanism of the enzymes, we have previously constructed a promoter assay system utilizing enhanced green fluorescence protein. Here, we monitored respiratory enzymes' expression by using this system during growth in various culture media, with and without Cu(2+) ion supplementation. The promoter activities of cytochrome aa3 oxidase in the early stationary phase in the media supplemented with Cu(2+) ion at 40 or 400 μM were significantly increased 1.49-fold or 1.99-fold, respectively, as compared to the control. Moreover, the H(+)/O ratio, or the proton-pumping activity of the cells, increased about 1.6 times by the Cu(2+) supplementation. These facts indicate that copper ions can switch the branches.
    Bioscience Biotechnology and Biochemistry 10/2014; 79(2):1-7. DOI:10.1080/09168451.2014.968089 · 1.06 Impact Factor