Global translational control by the transcriptional repressor TrcR in the filamentous cyanobacterium Anabaena sp. PCC 7120

Abstract

Transcriptional and translational regulations are important mechanisms for cell adaptation to environmental conditions. In addition to house-keeping tRNAs, the genome of the filamentous cyanobacterium Anabaena sp. strain PCC 7120 (Anabaena) has a long tRNA operon (trn operon) consisting of 26 genes present on a megaplasmid. The trn operon is repressed under standard culture conditions, but is activated under translational stress in the presence of antibiotics targeting translation. Using the toxic amino acid analog β-N-methylamino-L-alanine (BMAA) as a tool, we isolated and characterized several BMAA-resistance mutants from Anabaena, and identified one gene of unknown function, all0854, named as trcR, encoding a transcription factor belonging to the ribbon-helix-helix (RHH) family. We provide evidence that TrcR represses the expression of the trn operon and is thus the missing link between the trn operon and translational stress response. TrcR represses the expression of several other genes involved in translational control, and is required for maintaining translational fidelity. TrcR, as well as its binding sites, are highly conserved in cyanobacteria, and its functions represent an important mechanism for the coupling of the transcriptional and translational regulations in cyanobacteria.

Fig. 1. BMAA sensitivity test in the WT, M20, ΔtrcR, M20-CtrcR and C-trcR.

a Cell growth in 24-well plates containing indicated concentrations of BMAA in BG11 medium. b Growth curves of the same strains with 25 μM BMAA (red) or without BMAA (black). Data represents the mean values of two independent experiments.

Fig. 2. TrcR binds to a conserved DNA motif at its own promoter region.

a Sequence alignment of the promoter regions of trcR and its homologous genes from different cyanobacteria. Nodularia: Nodularia spumigena CCY9414; Chrysosporum: Chrysosporum ovalisporum; Calothrix: Calothrix sp. PCC 7507; Hassallia: Hassallia byssoidea VB512170; Cylindrospermum: Cylindrospermum stagnale PCC 7417; Aphanizomenon: Aphanizomenon flos-aquae 2012/KM1/D3; Tolypothrix: Tolypothrix sp. PCC 7601; Scytonema: Scytonema tolypothrichoides VB-61278; Rivularia: Rivularia sp. PCC 7116; Richelia: Richelia intracellularis; Mastigocladus: Mastigocladus laminosus UU774; Fischerella: Fischerella sp. JSC-11; Hapalosiphon: Hapalosiphon sp. MRB220; Nostoc: Nostoc punctiforme PCC 73102; Anabaena: Anabaena sp. PCC 7120; Chroococcidiopsis: Chroococcidiopsis thermalis PCC 7203; Coleofasciculus: Coleofasciculus chthonoplastes PCC 7420. b Schematic Illustration of the 5 DNA fragments selected for EMSA. −10 box (yellow background) and transcription start site (TSS) of trcR based on the RNA-Seq data from Mitschke et al. are shown in the conserved motif identified in a. c EMSA performed with DNA1 to DNA 5 in the presence or absence of TrcR. d, e EMSA competition with unlabeled DNA. d unlabeled DNA3 competes the binding of TrcR with FAM-labeled DNA3. e unlabeled DNA4 competes the binding of TrcR with FAM-labeled DNA4. f TrcR binding region determined by DNase I footprinting. The TrcR protected region is indicated by a red box with sequences shown below (0.8 μg TrcR, lower panel). As a negative control, the corresponding region without TrcR addition was also shown (upper panel). Letters in red indicates the conserved motif identified in (a).

Fig. 3. TrcR is an autorepressor as shown with microscopic images of CFP reporter fluorescence.

a A plasmid bearing a CFP reporter gene under the control of the trcR promoter (p_trcRCFP) in WT, M20 and ΔtrcR backgrounds. b WT or ΔtrcR bearing a replicative plasmid with the expression of TrcR driven by an inducible system (the CT promoter, P_CT), together with p_trcRCFP as in (a). No inducers were added, thus no induction of TrcR from the plasmid. ΔtrcR::p_trcRCFP as in A was used as a control. c Same experiments performed as in (b) but with addition of inducers (0.6 μM Cu²⁺ + 2 mM Tp) in the growth medium for the expression of trcR from the inducible promoter P_CT, and CFP expressed from the promoter of trcR (p_trcRCFP).

Fig. 4. Regulation of several genes under the control of TrcR.

a EMSA showing the binding of TrcR to the promoter regions of trn and other potential target genes (with more than 8-folds upregulation in ΔtrcR). b Purification and quantification of TrcR and TrcR-L44P (TrcR bearing a mutation with replacement of L44 by P) used for EMSA by SDS-Polyacrylamide Gel Electrophoresis following Coomassie blue staining. c Binding assay of TrcR-L44P to the promoters of trn, alr3301, all3526, alr8077 and all8564 tested by EMSA. No binding to any promoter was detected. d Transcription levels of all3526, alr8077 and alr3303 quantified by qRT-PCR in WT, ΔtrcR and C-trcR. n = 3 biologically independent samples. The experiment was repeated three times for each sample. Data shown are the mean values ± S.D. e The consensus binding motif of TrcR based on the DNA region protected by TrcR in footprint experiments. Analysis was performed by using MEME on website (https://meme-suite.org/meme/tools/meme). f Relative positions of the identified TrcR binding motif (shaded background) and the −10 boxes (red letters) are shown in the promoter regions of trcR, alr1537, alr3301, all3526, alr3077, all8564 and trn. The positions of −10 boxes were deduced based on the RNA-Seq data from Mitschke et al..

Fig. 5. TrcR represses the trn operon expression.

a, b Transcription levels of the selected genes (trn, allrt06, allrt16 and allrt02) quantified by qRT-PCR in WT, ΔtrcR and C-trcR. n = 3 biologically independent samples. The experiment was repeated three times for each sample. Data shown are the mean values ± S.D. c Schematic illustration of the trn operon. The cognate amino acid and the anticodon are used for the naming of tRNA. For example, Asp-tRNA_GUC represents the tRNA whose cognate amino acid is Asp and the anticodon of which is GUC. The name and sites of fragments amplified by qRT-PCR in (a) were indicated below. The positions of −10 box (red letters) and a sequence TGTAGTAT (shaded background), similar to the consensus-binding site of TrcR are also indicated.

Fig. 6. TrcR represses gene expression and its protein level is downregulated by translational stress.

a Gene expression examined through the level of CFP reporter fluorescence by transcriptional fusion. The relative fluorescence intensity of WT::p_trnCFP, WT::p_alr3301CFP, WT::p_all3526CFP, WT::p_alr8077CFP and WT::p_trcRCFP was quantified from microscopic images shown in Fig. S8 using ImageJ. All cells were incubated in BG11 containing one of the antibiotics (PenG 1.5 μg/mL, Cm 5 μg/mL, Ksg 5 μg/mL, Sp 0.3 μg/mL, Sm 0.075 μg/mL) for 48 h before imaging. b Effects of antibiotics (Cm, Ksg and PenG) on TrcR protein levels tested by Western blot. Total proteins of Anabaena collected at indicated time points after antibiotic treatment were separated by SDS-PAGE (upper panel). Western blot was carried out with antibody against TrcR (anti-TrcR, middle panel). Quantification of the relative integrated density (IntDen) of the TrcR band analyzed by ImageJ was shown below. c Illustration for the regulation strategy of TrcR under translational stress.

Fig. 7. Effect of trcR deletion on translational fidelity.

Plasmids carrying lacZ and its derivatives with (a) alternative initiation codons (AUA, AUC and CUG), (b) frameshift mutations (+1 or -1) or (c) nonsense mutations (UGA, UAG and UAA) were transferred into the WT and ΔtrcR mutant, respectively. The level of β-galactosidase activity was used to measure the fidelity of the translational machineries to translate mutant forms of lacZ mRNAs. Data were normalized to the β-galactosidase activity of the WT LacZ in either WT or ΔtrcR. Data shown are the mean values ± S.D. (n = 3).

Fig. 8. TrcR represses the expression of the operon alr1537-alr1540 involved in BMAA export.

a EMSA testing the binding of TrcR and TrcR-L44P to the promoter region of alr1537. The promoter of all0854 (trcR) was used as a control. b Transcription levels of alr1537, alr1538, alr1539 and alr1540 quantified by qRT-PCR in WT, ΔtrcR and C-trcR with or without BMAA treatment. n = 3 biologically independent samples. The experiment was repeated three times for each sample. Data shown are the mean values ± S.D. c BMAA sensitivity of ΔtrcRΔalr1537, ΔtrcRΔalr1538, ΔtrcRΔalr1539, ΔtrcRΔalr1540 and ΔtrcRΔalr1537-40 tested in BG11 liquid medium containing different concentrations of BMAA. d Uptake of BMAA in WT, ΔtrcR and ΔnatAΔbgtA at indicated time points. e, f Quantification of BMAA uptake or secretion in WT, ΔtrcR, C-trcR, ΔtrcRΔalr1537, ΔtrcRΔalr1538, ΔtrcRΔalr1539, ΔtrcRΔalr1540 and ΔtrcRΔalr1537-40. e Amount of intracellular BMAA quantified at indicated time points after transferring into the BMAA-free medium. f Amount of BMAA secreted into the supernatant after transferring into the BMAA-free medium for 60 min. Data shown in (d), (e) and (f) are mean values ± S.D from triplicates.

Fig. 9. Cartoon illustrating the function of TrcR.

As an autoregulated transcriptional repressor, TrcR inhibits the transcription of several genes involved in translational control such as rsgA, rtcB, rimK and trn under normal laboratory culture conditions. However, the gene inhibition was relieved under translational stress induced by antibiotics, probably due to TrcR degradation through an unknown pathway. Besides the translational genes, TrcR also regulates the expression of an operon alr1537-alr1540, whose protein products are responsible for the export of BMAA.