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

Wang, Zi-Qian; Yang, Yiling; Zhang, Ju-Yuan; Zeng, Xiaoli; Zhang, Cheng-Cai

doi:10.1038/s42003-023-05012-9

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Published: 15 June 2023

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

Communications Biology volume 6, Article number: 643 (2023) Cite this article

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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.

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Introduction

Transcriptional and translational regulations are critical mechanisms for an organism to adapt to environmental changes. Under certain stress conditions, for example, cells tend to reduce global protein synthesis while upregulating the expression of genes involved in stress resistance¹. Compared to transcriptional control, regulation at the translational level has more direct and rapid impact on protein amounts and activities. Translational control is a complex process, which may occur at multiple levels, such as the regulation of aminoacyl-tRNA synthetase (aaRS) activity, changes of tRNA pool, ribosome heterogeneity, tRNA modification, translational fidelity control, and selective mRNA translation, etc.^1,2,3,4.

Cyanobacteria are the earliest known microorganisms on Earth to produce oxygen through photosynthesis⁵. Like other organisms, cyanobacteria coordinate both transcriptional and translational processes for better adaptation to environmental changes and fitness maintenance. However, translational regulation has been poorly studied in cyanobacteria. Recently, a few studies explored the contribution of post translational modification on environmental adaptability^6,7. Ignacio Luque et al. found that in the cyanobacterium Anabaena sp. strain PCC 7120 (Anabaena hereafter), a tRNA operon (trn) was activated when cells encountering translational stress⁸. Two types of tRNA genes were found in Anabaena. One of these types contains 48 tRNA genes that are scattered on the chromosome and transcribed under standard laboratory conditions. These tRNA genes encode housekeeping tRNAs^8,9. The other type includes 26 tRNA genes that constitute the trn operon, which is on the δ plasmid and is silent under normal laboratory culture conditions, but activated when cells encounter translational stress⁸. The transcription of the trn array was induced by addition of antibiotics targeting translation, and increased trn expression favored survival of Anabaena under translational stress induced by antibiotics⁸. This study proved that a large tRNA array (trn operon), normally silenced, could be activated and participate in the process of translational regulation. However, the underlying mechanism for the regulation of this large trn operon remained unknown⁸.

Amino acids analogs, by disturbing the translational process, have been extensively used for probing translational responses in various organisms^10,11,12. One of such analogs, β-N-methylamino-L-alanine (BMAA), is a non-protein amino acid. BMAA inhibits both the proofreading activity and the alanine aminoacylation activity of the human alanyl-tRNA synthetase, and is also a substrate of the human alanyl-tRNA synthetase by forming BMAA-tRNA^Ala ¹³. In addition, some studies have demonstrated that BMAA can be incorporated into polypeptides during protein synthesis^14,15,16,17. Therefore, BMAA is able to cause translational stress in cells through multiple mechanisms. Previously, we provided evidence that BMAA, as an amino acid analog toxic to cyanobacteria, could be used as a valuable tool for the studies of amino acid transport as well as translational control in cyanobacteria such as Anabaena^18,19. Anabaena is a filamentous and multicellular cyanobacterium. In addition to using combined nitrogen such as nitrate and ammonium as nitrogen sources, it can also fix atmospheric nitrogen through heterocysts, whose differentiation is induced under the condition of combined-nitrogen deficiency²⁰. Previously, using Anabaena as a model organism and BMAA as a molecular tool, through screening of BMAA-resistance mutants and genetic analysis, we explored the toxic mechanisms of BMAA to cyanobacteria and the translational stress response when cells are challenged by BMAA^18,19. By this method, we found that BMAA is imported into Anabaena mainly through N-I and N-II amino acid transport systems, and that the N(6)-threonylcarbamoyl adenosine (t⁶A) modification of tRNA plays an important role in translational regulation^18,19. In addition to BMAA, antibiotics targeting different steps of the translational process are also used for applying the translational stress by inhibiting translation. For example, kasugamycin (Ksg) blocks translation initiation by targeting the 30 S ribosomal subunit²¹, while chloramphenicol (Cm) inhibits translation elongation by binding to the 50 S ribosomal subunit²².

In this study, we report the analysis of several mutations conferring BMAA-resistance, and occurring in all0854, which we annotated as trcR (Translational Control Regulator). TrcR is a transcriptional repressor with a global impact on the expression of genes involved in translational processes. Furthermore, we show that TrcR is a repressor for the transcription of the large trn operon, thus providing a regulation mechanism on the expression of this trn array. The role of TrcR in translational control constitutes a new mechanism for the coupling of transcriptional and translational regulations reported in cyanobacteria.

Results

trcR (all0854), a gene conferring BMAA sensitivity in Anabaena

Previously, we showed that BMAA-resistance mutants allowed us to study the mechanism of BMAA toxicity and translational control in cyanobacteria^18,19. Among the 20 BMAA-resistance mutants obtained, 17 (M1-M17) of them have been described in the previous studies^18,19. Here, we focused on M18, M19, and M20, the remaining three BMAA-resistance mutants that could still grow, despite poorly, in the presence of 50 μM BMAA, while the WT growth was completely inhibited under the same conditions (Supplementary Fig. 1, Supplementary Table 1).

Whole-genome resequencing of M20 revealed that a single transition mutation of T131C occurred in trcR (all0854), resulting in a replacement of Leu at position 44 by a Pro residue in the corresponding protein (Supplementary Table 1 and Supplementary Data 1). Our previous studies on the other BMAA-resistance mutants have identified three genes, alr4167, all1284 and alr2300, which play roles in amino acid uptake (alr4167 and all1284) or t⁶A modification of tRNA (alr2300) (Supplementary Table 1)^18,19. Therefore, for the other two mutants M18 and M19, we checked whether any mutation occurred in trcR, alr4167, all1284 or alr2300 by PCR coupled with sequencing. The results showed that both also had mutations in trcR, with M19 having the same mutation as that in M20, and M18 having a mutation of C215A that resulted in a replacement of Ala72 by an Asp residue in TrcR (Supplementary Table 1).

To further verify that the mutation in trcR is responsible for BMAA resistance, one deletion mutant ΔtrcR was created. We found that ΔtrcR was resistant to BMAA in contrast to the WT (Fig. 1). Furthermore, complementation of M20 and ΔtrcR with trcR (M20-CtrcR or C-trcR) fully restored their BMAA sensitivity to the WT level (Fig. 1). These results confirmed that the mutation of trcR is responsible for BMAA resistance of Anabaena.

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

TrcR is an autoregulated transcriptional repressor

TrcR was annotated as an unknown protein in the data banks. We made a sequence alignment and found that TrcR contains a region with a ribbon-helix-helix (RHH) ___domain (Supplementary Fig. 2). Proteins of the RHH family may bind DNA in a sequence-specific manner, and thus function as transcription factors^23,24,25. To test if TrcR is a transcription factor, we tested whether it binds to its own promoter, since many transcription factors are autoregulated. Sequence alignment of the promoter regions of trcR and its homologous genes from other cyanobacteria revealed the presence of a highly conserved DNA motif (5'-ATACTACACTTGTATTAC-3') (Fig. 2a). Five DNA fragments containing or nearby the promoter region of trcR were then selected as the candidate substrates of TrcR (Fig. 2b). The EMSA results showed that DNA3 and DNA4, two DNA fragments containing an overlapping region, showed retarded migration on the gel due to TrcR binding (Fig. 2c). Further experiment showed that competition for TrcR binding could be observed between unlabeled DNA3 and DNA4 and the respective FAM-labeled fragments. The competition of unlabeled DNA4 with FAM-labeled DNA3, or of unlabeled DNA3 with FAM-labeled DNA4, also occurred；in contrast, no such competition happened with DNA1 (Fig. 2, panels d and e; Supplementary Fig. 3), demonstrating that TrcR specifically binds to DNA3 and DNA4.

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

To determine the binding site of TrcR, truncated forms of DNA3 and DNA4 were tested for their TrcR binding activity by EMSA (Supplementary Fig. 3c, d). The results confirmed that a region of 25 bp from −50 to −25 (relative to the translational start site of trcR) is essential for TrcR binding (Supplementary Fig. 3). Footprinting assay revealed that a DNA fragment from -53 to -28 was protected by TrcR from DNase I digestion (Fig. 2f). As expected, both regions obtained via EMSA and Footprinting assays contain the motif (5'-ATACTACACTTGTATTAC-3') conserved in front of trcR and its homologs in other cyanobacteria (Fig. 2 and Supplementary Fig. 3).

Based on the RNA-Seq results²⁶, the −10 box region ‘TACACT’ and transcription start site (TSS) of trcR overlaps with TrcR binding site, (Fig. 2b), suggesting that TrcR could have an autorepression function. To confirm this hypothesis, a plasmid expressing CFP under the control of the trcR promoter (p_trcRCFP) was transformed into WT, M20 or ΔtrcR. Strong CFP fluorescence was observed in the two mutants (M20::p_trcRCFP and ΔtrcR::p_trcRCFP), while little fluorescence was detected in the WT (WT::p_trcRCFP) (Fig. 3a). This result suggested that TrcR acted as an autorepressor. To further test this hypothesis, we expressed TrcR in the WT or ΔtrcR using a plasmid p_CTTrcRp_trcRCFP in which trcR was controlled by an inducible CT promoter, allowing protein expression only in the presence of inducers (Cu²⁺ and theophylline), and the p_trcRCFP fusion was also present as a reporter on the same plasmid²⁷. As expected, in the absence of inducers, the trcR mutant (ΔtrcR::p_CTTrcR-p_trcRCFP) exhibited stronger CFP fluorescence than that of WT (WT::p_CTTrcR-p_trcRCFP). However, when inducers were supplied in the medium, the fluorescence disappeared in the trcR mutant (ΔtrcR::p_CTTrcR-p_trcRCFP), while no fluorescent change was observed in the same mutant expressing the transcriptional fusion alone (ΔtrcR::p_trcRCFP) (Fig. 3b, c), demonstrating that the production of TrcR in cells led to repression of the trcR promoter. Our results, all together, proved that TrcR binds to its own promoter and acts as a transcriptional repressor.

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

By aligning TrcR with other RHH family proteins, we found that the mutation in M20 resulted in a change from a Leu residue at position 44 to a Pro residue, and this Leu44 is conserved at the helix B in TrcR (Supplementary Fig. 2). This observation suggests that Leu44 is essential for the DNA-binding activity of TrcR. Therefore, we tested the DNA-binding activity of a corresponding mutant form of TrcR (TrcR-L44P, TrcR with Leu²⁸ to Pro²⁸ mutation), and the results indicated that TrcR-L44P lost the binding activity to the promoter of trcR (Fig. 4a–c). These results, together with those obtained with other target-binding sites as described below, demonstrated the binding specificity of TrcR.

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

TrcR regulates transcription of genes involved in translation

Next, to identify genes of the TrcR regulon, we compared the transcriptome of WT and ΔtrcR. RNA-seq data showed that 266 genes were differentially expressed in ΔtrcR, with at least 2-fold changes when compared to WT. Among the 266 genes, 203 genes were upregulated and 63 genes downregulated in ΔtrcR (Supplementary Fig. 4, Supplementary Data 2 and Data 3). We narrowed down the candidate genes to 18 by choosing those with more than 8-fold changes, and all of them were upregulated in ΔtrcR (Table 1), consistent with the idea that TrcR functions mainly as a repressor. EMSA were performed to test the interaction between TrcR and the promoters of the candidate genes, except for the promoter between asr0855-all0854 (Fig. 2b), which was already confirmed (Fig. 2). Note that only the promoter sequence of the first upstream gene was selected for those consecutive genes that may constitute an operon, i.e. alr3301 promoter for alr3301-3303, all0263 promoter for all0261-0263, alr0739 promoter for alr0739-0740 and alr1537 promoter for alr1537-1540. The EMSA results showed that except for the promoter of all5040 that lacks a TrcR binding site, all the tested DNA fragments exhibited band shift in the presence of TrcR (Fig. 4a). Among those target genes, all3526, alr3303 and alr8077 are related to translation. all3526 (rtcB) encodes a widely distributed RNA ligase RtcB, which is involved in tRNA intron splicing in eukaryotes and archaea²⁹, and the repair of cleaved 16 S rRNA and tRNAs in bacteria^30,31. alr8077 (rsgA) encodes the ribosome assembly factor RsgA that is involved in the late stages of 30 S subunit maturation^32,33. alr3303, which is cotranscribed with alr3301 and alr3302 (Supplementary Fig. 5), encodes a ribosome modification protein RimK, an ATP-dependent glutamate ligase that adds glutamate residues to the C-terminus of the ribosomal protein S6³⁴. The post-translational modification of S6 has been shown to be important for translational control and environmental adaptation in cells^35,36,37.

Table 1 List of genes with more than 8-fold increase in the transcriptional levels in ΔtrcR.

Full size table

To further confirm the transcriptome data, qRT-PCR was performed to check the transcript levels of all3526, alr8077, and alr3303 in the WT, ΔtrcR and C-trcR strains (Fig. 4d). The results revealed that the transcription levels of these genes were significantly upregulated when TrcR was absent in the cells. The observed regulation was further validated by using CFP as a reporter (Supplementary Fig. 6). A transcriptional fusion of promoter region of all3526, alr3301 or alr8077 was made and transferred, respectively, into the WT or the ΔtrcR mutant. As expected, all the CFP fusions showed stronger CFP fluorescence in ΔtrcR than that in the WT (Supplementary Fig. 6). Together, these results demonstrate that TrcR is a transcriptional repressor that prevents transcription of all3526, alr3301 and alr8077, genes related to translational process.

Further footprinting assays using the promoter sequences of alr3301, all3526, alr8077, all8564 and alr1537 (Supplementary Fig. 7) revealed regions protected by TrcR. By aligning all the determined binding sites of TrcR, we determined a consensus binding sequence of 8 bp in length for TrcR (Fig. 4e). The position of this motif overlapps with or is located nearby the -10 boxes of the corresponding promoters, consistent with TrcR being a repressor (Fig. 4f).

TrcR is the repressor for the silenced trn gene array

The analysis of transcriptome data did not include long non-coding RNAs. Recently, a trn operon consisting of 26 tRNA genes was found to be activated under translational stress triggered by treatment with the antibiotic Cm⁸. In addition, the same study found also 1496 genes downregulated and 1750 genes upregulated under the same stress conditions⁸. Interestingly, among those upregulated genes, trcR, all0768, all3526, all8564, alr0739, alr0740, alr1537, alr1538, alr1539, alr1554, alr8077, asr0855 and alr0857 were also upregulated by more than 8 folds in the ΔtrcR strain in this study (Table 1). Therefore, TrcR could be the repressor in Anabaena responsible for silencing the expression of the trn operon under standard culture conditions. We first checked by qRT-PCR the expression of trn operon in the WT, the ΔtrcR strain and the complemented strain (Fig. 5). While the expression of this operon remained low in the WT and the complemented strain, a highly activated expression was detected in the deletion mutant (Fig. 5a). As controls, the expression levels of the three housekeeping tRNA genes, allrt06, allrt16 and allrt02, remained relatively constant (Fig. 5b). Consistent with these results, a sequence 5'-TGTAGTAT-3', similar to the consensus-binding site of TrcR overlapps with the -10 box (5'-TAGTAT-3') of the promoter of the trn operon (Fig. 5c). By footpringing experiment, this putative binding site of TrcR was confirmed since TrcR could efficiently protect it against DNase I digestion (Fig. 4f and Supplementary Fig. 7).

**Fig. 5: TrcR represses the *trn* operon expression.**

To further confirm that the trn array is repressed by TrcR, several strategies were used. In vitro, EMSA experiments indicated that TrcR, but not TrcR-L44P, could bind to the promoter region of the trn operon (Fig. 4a and c). In vivo, using CFP as a reporter, the fluorescence intensity of CFP driven by the promoter of the trn operon, was strongly upregulated in the ΔtrcR strain, while it was hardly visible in the WT under the same experimental conditions (Supplementary Fig. 6).

Translational stress relieves the repression of TrcR on its regulatory genes

Previously, it was reported that the trn operon was induced by antibiotics targeting ribosome, and deletion of trn reduced the resistance of Anabaena to this type of antibiotics⁸. To test whether antibiotic treatment could also cause the expression of genes under the control of TrcR, the transcription levels of trcR, all3526, alr3303, alr0877 and trn were visualized via transcriptionally fused CFP gene under each specific promoter in the WT strain upon antibiotic treatment (Fig. 6a and Supplementary Fig. 8). Antibiotics disrupting translational processes were chosen, including Cm, Ksg, and streptomycin (Sp) that inhibits translational elongation by targeting the ribosomal 30 S subunit³⁸, and streptomycin (Sm) that interferes both the selection of aminoacyl-tRNA and the translational proof-reading activity by also targeting 30 S subunit³⁸. Penicillin G (PenG) targeting cell wall biosynthesis was used as a control. All antibiotics were applied at sub-lethal concentrations (Supplementary Fig. 9). Our results showed that Cm, Sp and Sm induced CFP fluorescence in all strains, while PenG and Ksg did not (Fig. 6a and Supplementary Fig. 8). qRT-PCR result confirmed that the transcripts of trcR, all3526, alr3303, alr0877 and trn were significantly increased with time under Cm-induced stress (Supplementary Fig. 10).

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

Unexpectedly, Ksg, as an antibiotic acting on translational initiation, did not cause a derepression of TrcR. To understand this point, we tested the sensitivity of the WT, ΔtrcR and C-trcR to Cm, Ksg, Sp, Sm and PenG. All of them had a similar sensitivity to Cm, Sp, Sm and PenG, but ΔtrcR gained resistance to Ksg (Supplementary Fig. 9). To explore whether the higher resistance of ΔtrcR to Ksg and BMAA is the consequence of gene overexpression, we deleted all3526, alr8077, trn operon and alr3301-alr3303 operon from ΔtrcR, respectively, to create the double mutants ΔtrcRΔall3526, ΔtrcRΔalr8077, ΔtrcRΔtrn and ΔtrcRΔalr3301-03. We found that deletion of these genes from ΔtrcR had no influence on its higher resistance to Ksg and BMAA (Supplementary Figs. 11 and 12). So, further investigation is required to understand the resistance of ΔtrcR to Ksg and BMAA.

Another paradox is that Cm, and to a lesser extent BMAA (Supplementary Figs. 10 and 13), induced the expression of trcR, which normally would lead to stronger repressive effects for the genes of the TrcR regulon; yet, under the same conditions, genes repressed by TrcR were induced. To understand the mechanism of antibiotic-induced derepression from TrcR, we checked the TrcR level in Anabaena exposed to Cm, Ksg and PenG by Western Blot. The results showed that the amount of TrcR significantly decreased with time when 5 μg/mL Cm was added into the medium. Though 5 μg/mL of Ksg and 1.5 μg/mL of PenG also caused a decrease of TrcR, it only lasted for the first 3 h and then was kept at a stable level (Fig. 6b). This observation led to the conclusion that when Anabaena is under translational stress of Cm, the protein level of TrcR will decrease despite the enhanced transcription of trcR under similar conditions. The decreased amount of TrcR resulted in the expression of genes that were normally repressed (Fig. 6c). These results also suggest the existence of a translational or a posttranslational regulation of TrcR for the control of its protein level.

Deletion of trcR lowers translational fidelity in Anabaena

Our previous study showed that BMAA affected translational fidelity in Anabaena¹⁹. Since the inactivation of trcR had a strong impact on the expression of a number of genes involved in translation, we tested the effect of such a misregulation by measuring translational fidelity using a series of plasmids carrying the lacZ gene reporter or its derivatives as previously described¹⁹. Three categories of the lacZ derivatives were used: with the initiation codon AUG of lacZ replaced by AUA, AUC or CUG; with lacZ that has +1 or -1 frameshift mutation at the eighth codon, or with nonsense mutation introduced into lacZ by changing its eighth codon to UAA, UAG or UGA. We found that compared with the WT, the relative β-galactosidase activity was significantly higher in ΔtrcR bearing all three types of LacZ mutant variants (Fig. 7). This result indicated that in the absence of TrcR, the translational machinery was more likely to misread the codons or misincorporate an amino acid residue. Therefore, deletion of trcR leads to decreased translational fidelity, including leaky scanning of initiation codons, frameshift mutation or reading through of stop codons.

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

TrcR regulates the expression of alr1537-alr1540 responsible for BMAA export and resistance

Till now, we know that TrcR is a transcriptional repressor involved in translational control, but it is still unclear why TrcR deletion caused increased resistance to BMAA in Anabaena. From the transcriptome data, we found that alr1538 encoding a DMT family (Drug/Metabolite Transporter Superfamily) transporter was upregulated more than 8 folds in ΔtrcR (Table 1). Proteins of this family are involved in the export of metabolites such as amino acids and their precursors, nucleosides and purine bases³⁹. First, we used RT-PCR experiments to determine that alr1537, alr1538, alr1539 and alr1540 were cotranscribed, thus constituted an operon (Supplementary Fig. 14). Next, we investigated the regulation of TrcR on this operon via EMSA and qRT-PCR. Our EMSA result demonstrated that TrcR bound to the promoter region of alr1537, in contrast to the mutant variant TrcR-L44P that did not (Fig. 8a). The transcription level of alr1537-alr1540 is significantly higher in ΔtrcR compared to the WT and C-trcR, as revealed by qRT-PCR (Fig. 8b).

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

To confirm that the increased BMAA resistance of ΔtrcR is caused by the upregulated expression of alr1537-alr1540, these four genes were deleted, respectively, in ΔtrcR, resulting in the following double mutants: ΔtrcRΔalr1537, ΔtrcRΔalr1538, ΔtrcRΔalr1539 and ΔtrcRΔalr1540. We also constructed a quintuple mutant ΔtrcRΔalr1537-40 by deleting the whole alr1537-alr1540 operon in ΔtrcR. Our results showed that while ΔtrcR kept growing in the presence of 100 μM BMAA, deletion of alr1538 or the entire operon in ΔtrcR restored BMAA sensitivity, with growth inhibited at 25 μM of BMAA, similarly as the WT and C-trcR (Fig. 8c). However, deletion of alr1537, alr1539 or alr1540 in ΔtrcR only partially restored BMAA sensitivity, with no cell growth observed at 100 μM of BMAA and weaker cell growth observed at 50 μM of BMAA as compared to ΔtrcR (Fig. 8c). To see if the increased Ksg resistance of ΔtrcR is also related to alr1537-alr1540 operon, the growth of ΔtrcRΔalr1537-40 was compared to that of ΔtrcR under different Ksg concentrations. The result showed that ΔtrcRΔalr1537-40 still exhibited increased Ksg resistance comparing to the WT, similar as that of ΔtrcR itself (Supplementary Fig. 15).

To confirm that the efflux of BMAA was responsible for BMAA resistance in ΔtrcR, we verified both BMAA import and export abilities of the mutants. Exogenous BMAA was added into the cultures at a final concentration of 50 μM, then cells were harvested at indicated time points for BMAA detection. Our results showed that 15 min incubation resulted in an accumulation of similar amounts of BMAA in ΔtrcR and WT, while BMAA was undetectable in the negative control strain ΔnatAΔbgtA, a double mutant of amino acids transporters required for BMAA uptake (Fig. 8d)¹⁸. This result indicates that deletion of trcR has no influence on BMAA uptake in Anabaena. To further test the export of BMAA, cell samples with 15-min BMAA pre-incubation were prepared at 10, 30 and 60 min after removal of BMAA from the culture medium. Compared to WT and C-trcR, ΔtrcR cells exhibited significantly decreased level of intracellular BMAA over time, concomitantly, the corresponding supernatant had increased amount of BMAA detected (Fig. 8e, f), indicating enhanced ability of BMAA export in the absence of TrcR. However, deletion of alr1538 or alr1537-1540 operon in ΔtrcR fully restored both the amounts of intracellular and secreted BMAA to the WT levels (Fig. 8e, f), strongly suggesting that the protein product of alr1538 participates in BMAA export in the absence of TrcR. Overall, our results demonstrate that alr1537-1540 operon contribute to increased BMAA export, with alr1538 playing a dominant role whose product could function as an efflux pump for BMAA. These results provide a rational for the higher BMAA resistance in the trcR deletion mutant.

Discussion

In the previous study, through BMAA-resistance mutants screening and genetic analysis, we found that tRNA t⁶A modification play an important role in translational control in Anabaena¹⁹. In the present study, by investigating other BMAA-resistance mutants, we identified a gene of unknown function and named here as trcR, with its product acting as a repressor to exert a global control on translational processes. TrcR belongs to the RHH family of transcription factors. Our DNA-binding assays, DNA footprinting, transcriptome, and transcriptional analyses using CFP as a reporter or qRT-PCR, all confirmed that TrcR is a transcription factor. The binding data obtained allowed us to deduce a consensus binding site for TrcR, which could be important for identification of TrcR regulated genes. Most of the TrcR binding sites identified in this study are located near, or overlap with, the promoter regions of the corresponding genes, consistent with its repressive role in gene regulation.

The deletion of trcR led to two major genetic consequences, the resistance to BMAA and the antibiotic Ksg, and derepression of the trn operon and several other genes involved in translation (Fig. 9). The reason for BMAA resistance of ΔtrcR could be determined, which was caused by the derepression of alr1538 encoding an efflux system responsible for the decrease of BMAA levels detected in the cells. However, the reason for Ksg resistance remains unknown. The inactivation of alr1538 or any of those highly expressed genes identified in ΔtrcR mutant could not alleviate the Ksg-resistance effect. Previously, we found that Ksg could cause a translational stress in Anabaena¹⁹. Thus, the Ksg resistance acquired by ΔtrcR could be attributed to either an activated efflux pump or a modification of translational machineries such as ribosomes, making Ksg unable to bind efficiently to its targets. The lack of translational stress caused by Ksg in ΔtrcR, in contrast to other antibiotics targeting translation, is consistent with such possibilities.

**Fig. 9: Cartoon illustrating the function of TrcR.**

The identification of TrcR as the repressor of the trn operon and several other genes involved in translation represents an important advance in our understanding on the translational control in cyanobacteria (Fig. 9). TrcR is the missing link between translational stress induced by antibiotic treatment and the expression of the otherwise silenced trn operon in Anabaena as reported recently⁸. Ignacio Luque et al.⁸ reported that the transcription of trn could be activated by antibiotics targeting the translational process, which increased cell competitiveness and survival in Anabaena. In this study, we found that the regulation of the trn operon was accomplished by TrcR. In addition to the trn operon present on a megaplasmid, several other chromosomal genes such as all3526 (rtcB), alr8077 (rsgA), alr3303 (rimK) involved in translation are also repressed by TrcR. The expression of rtcB can be induced by the accumulation of damaged tRNAs upon translational stress in S. typhimurium⁴⁰, and RtcB plays a role in re-ligation of truncated 16 S rRNA upon stress relief in E. coli³⁰. In E. coli and S. typhimurium, the expression of rtcB was regulated by the σ⁴¹-dependent transcriptional activator RtcR^40,42. No homologous protein of RtcR in Anabaena could be identified, consistent with the absence of alternative sigma factors in cyanobacteria. Instead, our studies indicate that TrcR, belonging to a different family of transcription factors, has a function equivalent to that of RtcR.

Our results demonstrate that in Anabaena, the repression of TrcR on gene expression, including that of trcR itself, was relieved in the presence of antibiotics that inhibit the process of translational elongation (Cm, Sp and Sm). Although trcR transcriptional expression was derepressed under such conditions, the amount of TrcR protein decreased, which provided an explanation on the derepression of genes belonging to the TrcR regulon (Fig. 6b and c). A posttranslational regulation, such as proteolysis or translational inhibition of trcR mRNA, may occur to account for the decreased level of TrcR under translational stress. The signaling processes that regulates both the amount of TrcR and its DNA-binding activity remain to be understood.

In addition to its function in translational control, TrcR also regulates the expression of other genes, such as the alr1537-alr1540 operon, among which alr1538 encodes a DMT family protein at the core of BMAA export machinery. Therefore, TrcR has multiple targets involved in different functions (Fig. 9), consistent with our transcriptome data. TrcR, as well as its binding sites, are highly conserved in many unicellular and filamentous cyanobacterial species, but are missing in the group that forms branching filaments (Supplementary Fig. 16) and some marine picocyanobacteria such as Prochlorococcus. Two homologs of TrcR are found in Synechococcus sp. PCC 7003 (WP_065713646.1) and Synechococcus sp. PCC 11901 (QCS49454.1), which are marine strains^43,44. TrcR appears to represent a lineage of RHH regulators restricted to cyanobacteria, and two homologs found out of the cyanobacterial phylum correspond to those present in two cyanophages, annotated as Nodularia phages vB_NpeS-2AV2 and vB_NspS-kac65v151 in metagenomic data⁴⁵. The transcriptional regulation of translation by TrcR represents an important mechanism for the coupling of the transcriptional and translational regulations in cyanobacteria, and its functional studies will open a new horizon for our understanding of the adaptation mechanisms in cyanobacteria. The resistance to certain antibiotics acquired by some of the mutants may also help us to understand the environmental effects of antibiotics increasingly present in water bodies as a consequence of human activities.

Methods

Strains and growth conditions

BG11 medium⁴⁶ was used to culture Anabaena and its derivatives using conditions as described²⁸. All the cyanobacterial strains used in this study are described in Supplementary Table 2. For growth curve measurement, strains were cultivated in liquid BG11 medium with or without 25 μM BMAA, and the cell density was determined at OD₇₅₀. To test the sensitivity of Anabaena and its derivatives to BMAA, the tested strains were cultured (with a starting OD₇₅₀ of 0.075) in BG11 containing different concentrations of BMAA in the 24-well plates, followed by imaging from the bottom of plates after 7 days. The sensitivity of Anabaena and its derivatives to antibiotics was tested as described in the previous study¹⁹. Briefly, the OD₇₅₀ of the cultures at exponential phase was adjusted to 1.0. After serial dilutions (1/2, 1/4, 1/8, 1/16 and 1/32), 3 μL of cell cultures were spotted onto BG11 agar plates containing specific antibiotics (chloramphenicol (Cm), kasugamycin (Ksg), streptomycin (Sm), spectinomycin (Sp) and penicillin G (PenG)) at different concentrations. The plates were imaged after 10 days of incubation. BMAA was purchased from Wuhan Chuanliu Biotechnology Co., Ltd. (Wuhan, Chian). Ksg was from Shanghai Yuanye Biotechnology Co., Ltd. (shanghai, China), and all the other antibiotics were purchased from Sigma-Aldrich Co., LLC. (St. Louis, MO, USA).

Spontaneous BMAA-resistance mutants screening and genomic sequencing

The methods for spontaneous BMAA-resistance mutants screening in the presence of 100 μM BMAA were described in our previous study¹⁸. Genomic DNA of WT or the mutants was extracted and sequenced by BGI (BGI company, Shenzhen, China) using the next-generation sequencing technique based on Illumina Hiseq 4000 system (Illumine, San Diego, CA, USA). The average sequencing depth was 9.8 million reads per sample with 97-99% coverage of the Anabaena genome. The details of the sequencing and data analysis methods were described previously¹⁹.

Electrophoretic mobility shift assays (EMSA)

The strep-tagged TrcR and TrcR-L44P were heterologously expressed in Escherichia coli BL21 and purified for EMSA. DNA fragments of about 200 bp (predicted promoter regions) were labeled by fluorescent 6-carboxyfluorescein (FAM) tag at both ends. EMSA was carried out in 20 μL of EMSA buffer (1 mM DTT, 20% (v/v) glycerol, 0.1% (v/v) triton-100, 12 mM HEPES at pH 8.0, 4 mM Tris-Cl at pH 8.0, 60 mM KCl, 5 mM MgCl₂, 0.5 mM EDTA, 0.05 μg poly(dI:dC), 300 ng DNA and 0.2 μg TrcR or TrcR-L44P). After incubation at 30 °C for 30 min, samples were loaded on 5% (w/v) native polyacrylamide gels and ran in PAGE buffer (50 mM Tris-Cl at pH 8.0, 380 mM Glycine and 2 mM EDTA at pH 8.0) at 20 mA for 200 min. The gel was then imaged under excitation light of 490 nm that excited FAM. In this work, the promoter regions of studied genes were selected according to the RNA-Seq data published by Mitschke et al.²⁶.

DNase I footprinting

The method used for DNase I footprinting was adapted from that described by Wang et al.⁴⁷. Here, purified TrcR and TrcR-L44P proteins were the same as that for EMSA, but the DNA fragments were labeled with FAM at only one end. The binding of TrcR to DNA fragments was performed in 40 μL reaction buffer (1 mM DTT, 20% (v/v) glycerol, 0.1% (v/v) triton-100, 12 mM HEPES at pH 8.0, 4 mM Tris-Cl at pH8.0, 60 mM KCl, 5 mM MgCl₂, 0.5 mM EDTA, 500 ng DNA and 0.8 μg TrcR) at 30 °C for 30 min. The same reaction system without TrcR was used as a negative control. DNase I (Takara Biomedical Technology Co., Ltd, Beijing, China) was added into the reaction system to the final concentration of 0.005 U/μL. After 1 min of incubation at room temperature, 140 μL stop buffer (200 mM sodium acetate, 30 mM EDTA at pH 8.0 and 0.15% (w/v) SDS) was added into the reaction mixture. Then, the digested DNA was precipitated with ethanol and the dried pellet was dissolved in 10 μL TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0). The samples were subsequently sequenced and analyzed by Tsingke Biotechnology Co., Ltd. (Beijing, China). The sequences protected by TrcR from DNase I digestion were further analyzed on the website MEME (https://meme-suite.org/meme/tools/meme).

RNA preparation and transcriptome analysis

WT and ΔtrcR strains in triplicate were cultured to logarithmic phase in BG11. The cell samples were collected quickly by filtration and soaked in RNAlater (Wuhan Chuanliu Biotechnology Co., Ltd. Wuhan, China) for half an hour. Then, the RNA was isolated from these samples according to the hot phenol procedure⁴⁸. After quality check by agarose gel electrophoresis, the RNA samples were sent to BGI for RNA-seq and data analysis. The details of the procedures for RNA-seq and data analysis were described in our previous publication¹⁹.

Quantitative real-time PCR (qRT-PCR)

qRT-PCR was performed to compare the transcript levels of all3526, alr3303, alr8077 and trn operon among WT, ΔtrcR and C-trcR strains. All strains were cultured to exponential phase before being collected by rapid filtration. To detect changes in gene transcription levels upon Cm treatment, WT were cultured to exponential phase and a sub-lethal concentration (5 μg/mL) of Cm was added into the medium, then cell samples were collected at 0, 3, 6, 9, 24 and 48 h. For comparing the transcription levels of alr1537-alr1540 with or without BMAA treatment, WT, ΔtrcR and C-trcR were cultured to exponential phase. 25 μM of BMAA was added into the medium 24 h before sampling. Samples without BMAA served as control. All strains mentioned above were cultured in triplicate and total RNA of collected samples was extracted for further qRT-PCR analysis.

The kit HiScript II QRT SuperMix for qRCR ( + gDNA wiper) (Vazyme Biotech Co., Ltd, Nanjing, China) was used for reverse transcription following the manufacturer’s instruction. qRT-PCR was performed by C1000 Touch Thermal Cycler (Bio-Rad Laboratories, Inc., Hercules, CA, USA) using ChamQ SYBR qPCR Master Mix (Vazyme Biotech Co., Ltd) with three technical replicates for each sample. The transcript level of allrs04 encoding RNase P served as the internal control. All primers used for qRT-PCR were listed in Supplementary Table 3. The relative transcription levels of genes were obtained according to the 2^-ΔΔCT calculation method⁴⁹. Data were normalized by the transcription level of the corresponding genes in the WT without BMAA treatment.

Western blot

To evaluate the expression of TrcR in Anabaena under the stress caused by the presence of Cm, Ksg or PenG, WT cells in triplicate were cultured to exponential phase, and then antibiotics of sub-lethal concentration (Cm 5 μg/mL, Ksg 5 μg/mL or PenG 1 μg/mL) were added into the medium, respectively. After incubation for 0, 3, 6, 9, 24 and 48 h, cells from 30 mL cultures were collected by filtration for western blot analysis following the procedure as described⁵⁰. The antibody was prepared by injecting recombinant TrcR protein into rabbit (Mabnus Biotech Co., Ltd, Wuhan. China).

Translational fidelity test and β-galactosidase activity measurement

Translational fidelity test was performed as previously described¹⁹. Briefly, 9 plasmids (pTac-lacZ, pTac-lacZ1ATA, pTac-lacZ1ATC, pTac-lacZ1CTG, pTac-lacZ8TAA, pTac-lacZ8TAG, pTac-lacZ8TGA, pTac-lacZ+1shift and pTac-lacZ-1shift) that carried a series of lacZ derivatives as reporters were constructed. pTac-lacZ carried the wild-type lacZ gene. In pTac-lacZ1ATA, pTac-lacZ1ATC and pTac-lacZ1CTG, the initiation codon AUG of lacZ was changed into AUA, AUC and CUG, respectively. In pTac-lacZ+1shift and pTac-lacZ-1shift, frameshifting mutation ( + 1 or -1) was created after the seventh codon. For pTac-lacZ8TAA, pTac-lacZ8TAG and pTac-lacZ8TGA, the eighth codon of lacZ was replaced by stop codon UAA, UAG and UGA, respectively. The transcription of lacZ and its derivatives was under the control of the tac promoter⁵¹. All plasmids were transformed respectively into Anabaena or its derivatives. The translational fidelity of these strains was characterized by β-galactosidase (LacZ) activity¹⁹.

Measurement of BMAA uptake and export

To test the ability of BMAA uptake, WT, ΔtrcR and ΔnatAΔbgtA were cultured to the exponential phase and exogenous BMAA was added into the medium at a final concentration of 50 μM. 20 mL of cell cultures were collected at time 0 (right before BMAA addition), 1, 7 and 15 min (after BMAA addition), respectively. Cell samples were harvested by filtration and washed by BMAA-free medium to remove residual BMAA as soon as possible. The samples were stored at -80 °C for further processing.

To prepare the samples for detecting BMAA export, Anabaena or its derivatives was first incubated with 50 μM BMAA for 15 min, followed by washing with BMAA-free medium for three times. Cell samples from 20 mL cell cultures were harvested by filtration after 0, 10, 30 and 60 min and washed by BMAA-free medium. After 60 min, 500 μL medium of which the cells were removed by 0.2 μm filter was collected to determine the concentration of BMAA exported from the cells. All samples were stored at −80 °C for further processing.

The technique of ultra-high performance liquid chromatography (UPLC) with tandem mass spectrometry detection (UPLC-MS/MS), combined with derivatization using 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC), was used for BMAA detection⁵². Sample preparation, derivatization and the UPLC-MS/MS condition were described in detail in our previous publication¹⁸.

Construction of plasmids and mutants

All the markerless deletion mutants used in this study were constructed using the Cpf1 genome editing system^53,54. The plasmids pCpf1b-Mall0854R246, pCpf1b-Malr1537R372, pCpf1b-Malr1538F404, pCpf1b-Malr1539R169, pCpf1b-Malr1540R179 and pCpf1b-Malr1537-40R674 were used to construct ΔtrcR, Δalr1537, Δalr1538, Δalr1539, Δalr1540 and Δalr1537-40 respectively. The method for the construction of these plasmids followed the previously described procedures⁵⁴. Briefly, the plasmid pCpf1b-sp⁵⁴ was linearized by restriction enzyme BamH I and Bgl II. The upstream and downstream fragments used for homologous recombination were amplified by PCR from genomic DNA of Anabaena PCC 7120. The linearized pCpf1b-sp, the upstream and downstream homologous fragments, were all ligated by the ClonExpress MultiS One Step Cloning Kit (from Vazyme Biotech Co.,Ltd; Nanjing, China) to form a precursor plasmid. Finally, the precursor plasmid was digested by Aar I, and the corresponding guide sequence was ligated into the digested precursor plasmid to complete the construction. The primers used for plasmid construction were listed in Supplementary Table 4.

To construct the mutants used in this study, the plasmids mentioned above were transformed into Anabaena PCC 7120 or ΔtrcR by conjugation^41,55. Positive colonies were screened by using 5 μg/mL spectinomycin and 2.5 μg/mL streptomycin. To get the markerless mutants, positive colonies were continuously screened by using 5% sucrose in BG11 agar plates to remove the Cpf1-based plasmids follow the published procedure⁵⁴. The genotypes of all the constructed mutants were verified by PCR.

The vector for constructing the transcriptional fusion plasmids p_trcRCFP, p_alr1537CFP, p_alr3301CFP, p_alr8077CFP, p_all3526CFP and p_trnCFP was amplified from the plasmid pRLRBS-mTur by primers PpCT-R2979 and PV_16. pRLRBS-mTur, modified from the shuttle plasmid pRL25T⁵⁶, carries the ORF of cfp. The promoter regions of these genes of interests were defined according to the published RNA-Seq data²⁶. The promoter regions amplified from Anabaena PCC 7120 genomic DNA were inserted, respectively, into the pRLRBS-mTur vector through ClonExpress II One Step Cloning Kit (Vazyme Biotech Co.,Ltd; Nanjing, China) to complete the construction.

To construct the complementation plasmid pRL-Call0854, the vector was amplified by PCR from the replicative plasmid pCT²⁷ with primers PpCT-R2979 and PpCT-F3530. The gene all0854 with its native promoter region was amplified by PCR from the genomic DNA of Anabaena PCC 7120. Finally, the vector and the gene fragment were ligated via ClonExpress II One Step Cloning Kit to complete the construction.

The transcriptional fusion plasmids or the complementation plasmid were transformed into Anabaena PCC 7120 or its derivatives by conjugation to get the corresponding transcriptional fusion strain or the complemented strain^41,55.

Statistics and Reproducibility

In qRT-PCR assays, the relative transcriptional levels of the genes of interests were calculated through 2^-ΔΔCT calculation method based on triplicate data⁴⁹. The growth curves data were collected from two parallel cultures and presented as mean values. The relative fluorescence intensity of CFP reporter from stains with transcriptional fusions and the relative integrated density of the Western blot bands were quantified by ImageJ v1.51j8. For BMAA uptake and export detection assays, all the data were collected from 3-6 biological replicates. In this study, all the statistics analysis was carried out by SPSS v20.0 or Origin v2022, and the data are presented as mean±S.D. (standard deviation).

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

RNA-seq data have been deposited at the Gene Expression Omnibus (GEO) database under the accession number GSE218875. The source data for Figs. 1b, 4d, 5a, b, 6a, b, 7, 8b, d–f were shown in Supplementary Data 4–13 respectively. The blot/gel images in Fig. 2c–e, Fig. 4a–c, Fig. 6b, Fig. 8a and Supplementary Figs. 3a, b, d, 5b, 14b were edited from the original pictures shown in Supplementary Figs. 17-23 respectively. Plasmid pCpf1b-sp was deposited at Addgene with ID number #122188. Plasmid pCT was submitted to GenBank with ID number MK948095. All other data are available from the corresponding author upon request.

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Acknowledgements

This work was supported by Youth Program of National Natural Science Foundation of China (grant No. 31900028), The Featured Institute Service Projects from the Institute of Hydrobiology, the Chinese Academy of Sciences [Y85Z061601] and the State Key Laboratory of Freshwater Ecology and Biotechnology [2022FBZ04]. Funding for open access charge: The Featured Institute Service Projects from the Institute of Hydrobiology, the Chinese Academy of Sciences. We thank the staff from the Analysis and Testing Center of the Institute of Hydrobiology, Chinese Academy of Sciences for help with qRT-PCR analysis and BMAA measurement using UPLC-MS/MS.

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Zi-Qian Wang
Present address: State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China

Authors and Affiliations

State Key Laboratory of Freshwater Ecology and Biotechnology and Key Laboratory of Algal Biology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei, 430072, People’s Republic of China
Zi-Qian Wang, Yiling Yang, Ju-Yuan Zhang, Xiaoli Zeng & Cheng-Cai Zhang
University of Chinese Academy of Sciences, Beijing, 100049, People’s Republic of China
Zi-Qian Wang
Institute AMU-WUT, Aix-Marseille Université and Wuhan University of Technology, Wuhan, Hubei, People’s Republic of China
Cheng-Cai Zhang

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Zi-Qian Wang
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Yiling Yang
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Ju-Yuan Zhang
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Xiaoli Zeng
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Cheng-Cai Zhang
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Contributions

C.-C.Z., J.-Y.Z. and Z.-Q.W. planned and designed the research; Z.-Q.W. performed the experiments; Z.-Q.W., Y.Y., J.-Y.Z., X.Z. and C.-C.Z. analyzed the data; Z.-Q.W., Y.Y. and C.-C.Z. wrote the manuscript. Y.Y. and C.-C.Z. edited the manuscript.

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Correspondence to Zi-Qian Wang or Cheng-Cai Zhang.

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Communications Biology thanks Hema Rajaram and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: Gene Chong. A peer review file is available.

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Wang, ZQ., Yang, Y., Zhang, JY. et al. Global translational control by the transcriptional repressor TrcR in the filamentous cyanobacterium Anabaena sp. PCC 7120. Commun Biol 6, 643 (2023). https://doi.org/10.1038/s42003-023-05012-9

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Received: 03 February 2023
Accepted: 02 June 2023
Published: 15 June 2023
DOI: https://doi.org/10.1038/s42003-023-05012-9