Study of excess manganese stress response highlights the central role of manganese exporter Mnx for holding manganese homeostasis in the cyanobacterium Synechocystis sp. PCC 6803

Abstract

Cellular levels of the essential micronutrient manganese (Mn) need to be carefully balanced within narrow borders. In cyanobacteria, a sufficient Mn supply is critical for ensuring the function of the oxygen-evolving complex as the central part of the photosynthetic machinery. However, Mn accumulation is fatal for the cells. The reason for the observed cytotoxicity is unclear. To understand the causality behind Mn toxicity in cyanobacteria, we investigated the impact of excess Mn on physiology and global gene expression in the model organism Synechocystis sp. PCC 6803. We compared the response of the WT and the knock-out mutant in the Mn exporter (Mnx), ∆mnx, which is disabled in the export of surplus Mn and thus functions as a model for toxic Mn overaccumulation. While growth and pigment accumulation in ∆mnx were severely impaired 24 h after the addition of tenfold Mn, the WT was not affected and thus mounted an adequate transcriptional response. RNA-seq data analysis revealed that the Mn stress transcriptomes partly resembled an iron limitation transcriptome. However, the expression of iron limitation signature genes isiABDC was not affected by the Mn treatment, indicating that Mn excess is not accompanied by iron limitation in Synechocystis. We suggest that the ferric uptake regulator, Fur, gets partially mismetallated under Mn excess conditions and thus interferes with an iron-dependent transcriptional response. To encounter mismetallation and other Mn-dependent problems on a protein level, the cells invest in transcripts of ribosomes, proteases and chaperones. In the case of the ∆mnx mutant, the consequences of the disability to export excess Mn from the cytosol manifest in additionally impaired energy metabolism and oxidative stress transcriptomes with a fatal outcome. This study emphasizes the central importance of Mn homeostasis and the transporter Mnx's role in restoring and holding it.

Keywords: RNA-seq; cyanobacteria; manganese; regulation; toxicity; transporter.

Fig. 1.. Effects of Mn treatment on growth and pigment content. (a) Phenotypic appearance of WT and ∆mnx mutant under MnCl₂ standard (9 µM MnCl₂) and excess (90 µM MnCl₂) conditions at different time points in the MC-1000-OD Multi-Cultivator. (b) Representative growth curves of WT and ∆mnx mutant under different MnCl₂ regimes. (c) Chlorophyll a content, (d) phycocyanin content and (e) carotenoid content in WT and ∆mnx cells grown under different MnCl₂ regimes. Pigment levels were normalized to OD₇₅₀. Significance values in (c)–(e) were evaluated with the Student’s t-test. *P≤0.05. **P≤0.01.

Fig. 2.. Effect of MnCl₂ treatment on transcriptomes of WT and ∆mnx. (a) PCA of transcript abundances in WT and ∆mnx cells grown under control and Mn excess (+Mn) conditions. Volcano plots of the global transcriptome responses of WT (b) and ∆mnx (c) towards different MnCl₂ treatments. Shown are log₂-fold changes (log₂FC) of Mn excess (90 µM MnCl₂) versus Mn control (9 µM MnCl₂) conditions. Significant changes (q<0.01; edgeR, [23]) are plotted in green (up) or violet (down). The number of significantly up- and downregulated genes is given for each genotype.

Fig. 3.. Comparison of the transcriptional response of WT and ∆mnx towards Mn excess. The overlap in transcriptional responses upon Mn excess (DEGs with log₂-fold change≤|1| and q≤0.01) is shown with Venn diagrams. The number (#) and percentage (%) of genes, which are shared or specific for ∆mnx and the WT, respectively, are given. (a) The overlap of genes with significantly reduced transcript abundances in the WT and ∆mnx. (b) The overlap of genes with significantly enhanced transcript abundances in the WT and ∆mnx. (c) The overlap of significantly (P≤0.05) enriched KEGG modules and maps in significantly reduced transcripts. (d) The overlap of significantly (P≤0.05) enriched KEGG modules and maps in significantly enhanced transcripts.

Fig. 4.. Transcriptional response of genes involved in Mn homeostasis. Transcriptional response upon Mn excess treatment is presented as a heat map of corresponding log₂-fold change (log₂FC) for the WT and ∆mnx mutant. Statistical differences were evaluated according to Benjamini–Hochberg with q≤0.01 (**). Gene loci and names were obtained from Mills et al. [59].

Fig. 5.. Comparison of Mn excess transcriptional response in WT and ∆mnx with Fe limitation response according to Singh et al. [29] in WT. Colour coding of the boxes indicates the reduced (violet) or increased (green) transcript abundance of the corresponding gene(s) represented by the mean of the log₂-fold changes (log₂FC). Asterisks indicate significant changes with q<0.05 (*) and q<0.01 (**). Values are given in Table S8. Some data points of the Fe limitation set exceed the colour-coded log₂-fold change of |3| and are presented in deep violet and green for better comparison. C., carbohydrate; Ch, chlorophyll a biosynthesis; Cyto. c, cytochrome c biosynthesis.

Fig. 6.. Growth of WT and ∆mnx mutant under different Mn/Fe treatments. Growth of different dilutions (1, 1:10, 1:100, 1 :1000) was investigated on the BG11 medium supplemented with standard Mn/Fe concentrations (9 µM MnCl₂; 23 µM Fe–NH₄–citrate), standard Mn (9 µM MnCl₂) and surplus Fe (230 µM Fe–NH₄–citrate) or on excess Mn (90 µM MnCl₂) and increasing Fe concentrations (23–460 µM Fe–NH₄–citrate). Plates were photographed after 5-d incubation under continuous illumination with 100 µmol photons m⁻² s⁻¹ at 30 °C.

Fig. 7.. Model for an Mn excess response in Synechocystis. Presented are the effects of an Mn excess treatment on the global transcriptome and physiology of Synechocystis WT and ∆mnx mutant, which are defective in Mn efflux. The tile colour indicates whether genes of the functional category were enhanced (green), reduced (violet) or unchanged (white) in transcript abundance 24 h after the application of Mn excess stress in both, the WT and ∆mnx mutant. Responses that are exclusive to the Δmnx mutant are displayed by a colour-framed tile. The response of specific transcripts is given in Fig. 5 and Table S1. Transcription factors related to Fe (Fur), Mn import (ManR) and Fe–S cluster assembly (SufR) are transcriptionally upregulated. Depending on the cytoplasmic Mn status, Fur likely gets activated by mismetallation with Mn instead of Fe and conveys a transcriptional response that is partially overlapping with an Fe acclimation response in Synechocystis. Cellular entrance of both Mn and Fe gets reduced due to the downregulation of Fe and Mn import systems as well as OMPs. Importantly, transcripts of the Fe-deficiency responsive operon isiABCD are not altered, indicating that cells are not suffering from Fe limitation. Fe-dependent haem and chlorophyll biosynthesis is downregulated on a transcript level, overall leading to a lower abundance of photosynthesis, that is light harvesting via PBSs, light reactions and carbon reactions (CBB cycle) and carbohydrate metabolism-related gene transcripts. As an additional negative effect, typically Fe-containing proteins involved in these and further processes are likely mismetallated with Mn and thus inactivated. Together with downregulation of ATPase corresponding gene transcripts, cellular energy levels become depleted, with arrested cell growth as an outcome. To cope with the detrimental effects of Mn excess, several protection mechanisms (D1 turnover, flavodiiron proteins, ROS scavenging, proteases and chaperons) are enhanced on a transcript level to prevent cell death. In the case of the ∆mnx mutant, the Mnx is not operative. The mutant is not able to adjust cytoplasmic Mn homeostasis. Consistently exclusive to ∆mnx mutant cells is the enhanced expression of the sufBCDS operon, involved in Fe–S cluster assembly, which indicates ROS stress in those cells upon Mn stress treatment. The protection mechanisms to deal with ROS and also mismetallation effects are insufficient and lead to cell death. CEF, cyclic electron flow.