Ciprofloxacin triggered glutamate production by Corynebacterium glutamicum

Abstract

Background: Corynebacterium glutamicum is a well-studied bacterium which naturally overproduces glutamate when induced by an elicitor. Glutamate production is accompanied by decreased 2-oxoglutatate dehydrogenase activity. Elicitors of glutamate production by C. glutamicum analyzed to molecular detail target the cell envelope.

Results: Ciprofloxacin, an inhibitor of bacterial DNA gyrase and topoisomerase IV, was shown to inhibit growth of C. glutamicum wild type with concomitant excretion of glutamate. Enzyme assays showed that 2-oxoglutarate dehydrogenase activity was decreased due to ciprofloxacin addition. Transcriptome analysis revealed that this inhibitor of DNA gyrase increased RNA levels of genes involved in DNA synthesis, repair and modification. Glutamate production triggered by ciprofloxacin led to glutamate titers of up to 37 ± 1 mM and a substrate specific glutamate yield of 0.13 g/g. Even in the absence of the putative glutamate exporter gene yggB, ciprofloxacin effectively triggered glutamate production. When C. glutamicum wild type was cultivated under nitrogen-limiting conditions, 2-oxoglutarate rather than glutamate was produced as consequence of exposure to ciprofloxacin. Recombinant C. glutamicum strains overproducing lysine, arginine, ornithine, and putrescine, respectively, secreted glutamate instead of the desired amino acid when exposed to ciprofloxacin.

Conclusions: Ciprofloxacin induced DNA synthesis and repair genes, reduced 2-oxoglutarate dehydrogenase activity and elicited glutamate production by C. glutamicum. Production of 2-oxoglutarate could be triggered by ciprofloxacin under nitrogen-limiting conditions.

Keywords: 2-oxoglutarate; Arginine; Ciprofloxacin; Corynebacterium glutamicum; DNA gyrase; Glutamate; Lysine; Ornithine; Overflow metabolism; Putrescine.

Fig. 1

Colony formation of C. glutamicum wild type in the presence of different ciprofloxacin concentrations. The cells were cultured in CGXII (4 % (w/v) glucose) to an OD₆₀₀ of 15 and ciprofloxacin was added. After five hours of ciprofloxacin exposure, cells were diluted in 0.9 % NaCl to an OD₆₀₀ of 1 and further diluted. Colony forming units (cfu) were determined. Experiments were performed in biological duplicates and colony number determined for two technical replicates

Fig. 2

Biomass formation and glutamate production after ciprofloxacin addition. The C. glutamicum wild type, supplemented with 4 % (w/v) glucose, was grown to an optical density of 15 and ciprofloxacin in different concentrations was applied. After ciprofloxacin addition, cultures were incubated until glucose was consumed and the cell dry weight produced in this phase (∆CDW, open diamonds) and the glutamate concentration (black squares) were determined, after the consumption of the substrate. Values and error bars represent the mean and the experimental imprecision of duplicates

Fig. 3

Difference of ciprofloxacin and biotin limitation after yggB deletion. The strains MB001 (black) and MB001∆yggB (white) were cultured to an optical density of 2 to 5 in CGXII supplemented with 1 % (w/v) glucose and ciprofloxacin was applied. In addition, the MB001 strain (black) was compared to MB001∆yggB (white) during biotin limitation. Therefore the pre-limited cells (in CGXII, 4 % glucose, 0 μg biotin per L) were re-inocculated to CGXII, containing 2 μg biotin per L, supplemented with 1 % (w/v) glucose. The glutamate concentration of both conditions was determined in the culture supernatant after the complete consumption of glucose. Values and error bars represent the mean and the standard error of triplicate cultivations

Fig. 4

Growth of amino acid and diamine producer strains exposed to ciprofloxacin. The strains DM1729 (lysine producer) (a + b), ARG1 (arginine producer) (c + d), ORN1 (ornithine producer) (e + f) and PUT21 (putrescine producer) (g + h) were cultured to an optical density of 2 to 5 in CGXII supplemented with 1 % (w/v) glucose and ciprofloxacin was applied. Graphs on the left side (a, c, e, g) show the growth inhibition due to the addition of ciprofloxacin in concentrations of 0 μg/ml (white tirangles), 4 μg/ml (hatched squares) and 16 μg/ml (black circles). The graphs on the right side (b, d, f, h) show the concentrations of either lysine, arginine, ornithine or putrescine (black bars) and of glutamate (white bars) after the complete consumption of glucose. Values and error bars represent the mean and the standard error of duplicates

Fig. 5

Scheme of the central carbon metabolism and glutamate biosynthesis and relative RNA levels with/without ciprofloxacin treatment. Genes are depicted next to the reaction catalyzed by the encoded enzymes. Relative RNA levels of cells treated with 4 µg/ml ciprofloxacin compared to untreated cells are shown (values in green are below 1, those in red greater than 1), however, unlike the genes listed in Table 2, none of the genes depicted here showed significantly changed expression as determined by Student’s t-test, i.e., p > 0.05. 6PGL: 6-phosphogluconolactone; 6PG: 6-phosphogluconat