Physiological Response of Corynebacterium glutamicum to Indole

Abstract

The aromatic heterocyclic compound indole is widely spread in nature. Due to its floral odor indole finds application in dairy, flavor, and fragrance products. Indole is an inter- and intracellular signaling molecule influencing cell division, sporulation, or virulence in some bacteria that synthesize it from tryptophan by tryptophanase. Corynebacterium glutamicum that is used for the industrial production of amino acids including tryptophan lacks tryptophanase. To test if indole is metabolized by C. glutamicum or has a regulatory role, the physiological response to indole by this bacterium was studied. As shown by RNAseq analysis, indole, which inhibited growth at low concentrations, increased expression of genes involved in the metabolism of iron, copper, and aromatic compounds. In part, this may be due to iron reduction as indole was shown to reduce Fe³⁺ to Fe²⁺ in the culture medium. Mutants with improved tolerance to indole were selected by adaptive laboratory evolution. Among the mutations identified by genome sequencing, mutations in three transcriptional regulator genes were demonstrated to be causal for increased indole tolerance. These code for the regulator of iron homeostasis DtxR, the regulator of oxidative stress response RosR, and the hitherto uncharacterized Cg3388. Gel mobility shift analysis revealed that Cg3388 binds to the intergenic region between its own gene and the iolT2-rhcM2D2 operon encoding inositol uptake system IolT2, maleylacetate reductase, and catechol 1,2-dioxygenase. Increased RNA levels of rhcM2 in a cg3388 deletion strain indicated that Cg3388 acts as repressor. Indole, hydroquinone, and 1,2,4-trihydroxybenzene may function as inducers of the iolT2-rhcM2D2 operon in vivo as they interfered with DNA binding of Cg3388 at physiological concentrations in vitro. Cg3388 was named IhtR.

Keywords: Corynebacterium glutamicum; adaptive laboratory evolution; amino acids; aromatic compound catabolism; indole; iron homeostasis; oxidative stress.

Figure 1

Growth of C. glutamicum WT (A) and C1* (B) in the presence of extracellularly added indole. Cultivation with the indicated indole concentrations was performed in the biolector cultivation system. Means and standard deviations of triplicate cultivations are shown.

Figure 2

Growth of C. glutamicum WT (yellow) and C1* (gray) in the presence of varying CuSO₄ (A) and FeSO₄ (B) concentrations, with protocatechuate (PCA) or indole as iron chelators (C) and determination of iron reduction by indole and PCA as assayed with BPS (D). Maximal growth rates (dotted lines) and biomass concentrations (Δbackscatter, filled lines) are depicted as means and standard deviations of duplicate cultivations (A,B). Maximal growth rates of cultivations of WT and C1* without iron chelator (-) or with 195 µM PCA or indole as iron chelator are given as means and standard deviations of triplicates cultivations. The kinetics of Fe³⁺ reduction (D) were monitored using BPS as described in Material and Methods. Means and standard deviations of triplicates are shown.

Figure 3

Adaptive laboratory evolution for fast growth in the presence of indole. The biomass formed after 24 h during the adaptive laboratory evolution at the indicated indole concentration is shown for selected transfers (A). Maximal growth rates (B) and biomass formation (C) of C. glutamicum WT (yellow), IVO20 (bright blue), and IVO38 (dark blue) with various indole concentrations shown as means and standard deviations of triplicate cultivations.

Figure 4

Results of qRT-PCR analysis for expression of dtxR, cg0405, cg3388, and rhcM2 in the ALE strains IVO20 and IVO38 in the presence or absence of 4 mM indole. Comparisons of IVO20 and WT in the absence of indole (A), IVO20 (+) indole vs. without indole (B), IVO38 and WT in the absence of indole (C), and IVO38 (+) indole vs. without indole (D). The log2 fold change of the ΔΔCq value, using the reference gene parA is shown. Means and standard deviations of triplicate cultivations and independent performed qRT-PCRs are shown.

Figure 5

Growth comparison of dtxR (A) and rosR deletion strains (B) and reverse engineered strains (C) in presence of indole. Maximal growth rates of rosR deletions strains (A) and dtxR deletion strains (B) in absence (−) or presence of 4 mM indole (+) and of reverse engineered strains in the presence of 4 mM indole are shown as means and standard deviations of minimum of triplicates. Significance was determined based on a two-sided unpaired Student’s t-test (**: p < 0.05).

Figure 6

Indole-dependent effect on Cg3388. (A) Growth rates of strains WT, Δcg3388, C1*, and C1* cg3388^M1T in CGXII minimal medium with 40 g L⁻¹ glucose as carbon source in absence (−) and presence of 4 mM indole (+) are given as means and experimental imprecision of duplicates. Significance was determined based on a two-sided unpaired Student’s t-test (**: p < 0.05); *** p < 0.01). (B) qRT-PCR analysis of WT and WT Δcg3388 cells grown in presence of 2.5 mM indole, inositol, or resorcinol is shown for cg3388 (dark red), rhcM2 (light red), phe (gray), and creF (green) as means and standard deviations of triplicate cultivations. (C) Electrophoretic mobility shift assays (EMSA) with His-tagged Cg3388 (153 fold molar excess) and the intergenic region (45 ng) between cg3388 and iolT2 with addition of 1,2,4-trihydroxybenzene (0.01–0.025 mM), hydroquinone (0.05–0.05 mM), or indole (1–4 mM) are shown.