Depletion of m-type thioredoxin impairs photosynthesis, carbon fixation, and oxidative stress in cyanobacteria.

Thioredoxins (Trxs) are disulfide oxidoreductases that regulate many biological processes. The m-type thioredoxin (TrxA) is the only Trx present in all oxygenic photosynthetic organisms. Extensive biochemical and proteomic analyses have identified many TrxA target proteins in different photosynthetic organisms. However, the precise function of this essential protein in vivo is still poorly known. In this study, we generated a conditional Synechocystis sp. PCC 6803 mutant strain (STXA2) using an on-off promoter that is able to survive with only 2% of the TrxA level of the wild-type (WT) strain. STXA2 characterization revealed that TrxA depletion results in growth arrest and pronounced impairment of photosynthesis and the Calvin-Benson-Bassham (CBB) cycle. Analysis of the in vivo redox state of the bifunctional enzyme fructose-1,6-bisphosphatase/sedoheptulose-1,7-bisphosphatase showed higher levels of oxidation that affected enzyme activity in STXA2. This result implies that TrxA-mediated redox regulation of the CBB cycle is conserved in both cyanobacteria and chloroplasts, although the targets have different evolutionary origins. The STXA2 strain also accumulated more reactive oxygen species and was more sensitive to oxidative stress than the WT. Analysis of the in vivo redox state of 2-Cys peroxiredoxin revealed full oxidation, corresponding with TrxA depletion. Overall, these results indicate that depletion of TrxA in STXA2 greatly alters the cellular redox state, interfering with essential processes such as photosynthetic machinery operativity, carbon assimilation, and oxidative stress response. The TrxA regulatory role appears to be conserved along the evolution of oxygenic photosynthetic organisms.

The glutathione/glutaredoxin system is essential for arsenate reduction in Synechocystis sp. strain PCC 6803.

Arsenic resistance in Synechocystis sp. strain PCC 6803 is mediated by an operon of three genes in which arsC codes for an arsenate reductase with unique characteristics. Here we describe the identification of two additional and nearly identical genes coding for arsenate reductases in Synechocystis sp. strain PCC 6803, which we have designed arsI1 and arsI2, and the biochemical characterization of both ArsC (arsenate reductase) and ArsI. Functional analysis of single, double, and triple mutants shows that both ArsI enzymes are active arsenate reductases but that their roles in arsenate resistance are essential only in the absence of ArsC. Based on its biochemical properties, ArsC belongs to a family that, though related to thioredoxin-dependent arsenate reductases, uses the glutathione/glutaredoxin system for reduction, whereas ArsI belongs to the previously known glutaredoxin-dependent family. We have also analyzed the role in arsenate resistance of the three glutaredoxins present in Synechocystis sp. strain PCC 6803 both in vitro and in vivo. Only the dithiolic glutaredoxins, GrxA (glutaredoxin A) and GrxB (glutaredoxin B), are able to donate electrons to both types of reductases in vitro, while GrxC (glutaredoxin C), a monothiolic glutaredoxin, is unable to donate electrons to either type. Analysis of glutaredoxin mutant strains revealed that only those lacking the grxA gene have impaired arsenic resistance.

A comprehensive analysis of the peroxiredoxin reduction system in the Cyanobacterium Synechocystis sp. strain PCC 6803 reveals that all five peroxiredoxins are thioredoxin dependent.

Cyanobacteria perform oxygenic photosynthesis, which gives rise to the continuous production of reactive oxygen species, such as superoxide anion radicals and hydrogen peroxide, particularly under unfavorable growth conditions. Peroxiredoxins, which are present in both chloroplasts and cyanobacteria, constitute a class of thiol-dependent peroxidases capable of reducing hydrogen peroxide as well as alkyl hydroperoxides. Chloroplast peroxiredoxins have been studied extensively and have been found to use a variety of endogenous electron donors, such as thioredoxins, glutaredoxins, or cyclophilin, to sustain their activities. To date, however, the endogenous reduction systems for cyanobacterial peroxiredoxins have not been systematically studied. We have expressed and purified all five Synechocystis sp. strain PCC 6803 peroxiredoxins, which belong to the classes 1-Cys Prx, 2-Cys Prx, type II Prx (PrxII), and Prx Q, and we have examined their capacities to interact with and receive electrons from the m-, x-, and y-type thioredoxins from the same organism, which are called TrxA, TrxB, and TrxQ, respectively. Assays for peroxidase activity demonstrated that all five enzymes could use thioredoxins as electron donors, whereas glutathione and Synechocystis sp. strain PCC 6803 glutaredoxins were inefficient. The highest catalytic efficiency was obtained for the couple consisting of PrxII and TrxQ thioredoxin. Studies of transcript levels for the peroxiredoxins and thioredoxins under different stress conditions highlighted the similarity between the PrxII and TrxQ thioredoxin expression patterns.

Genomic responses to arsenic in the cyanobacterium Synechocystis sp. PCC 6803.

Arsenic is a ubiquitous contaminant and a toxic metalloid which presents two main redox states in nature: arsenite [As(III)] and arsenate [As(V)]. Arsenic resistance in Synechocystis sp. strain PCC 6803 is mediated by the arsBHC operon and two additional arsenate reductases encoded by the arsI1 and arsI2 genes. Here we describe the genome-wide responses to the presence of arsenate and arsenite in wild type and mutants in the arsenic resistance system. Both forms of arsenic produced similar responses in the wild type strain, including induction of several stress related genes and repression of energy generation processes. These responses were transient in the wild type strain but maintained in time in an arsB mutant strain, which lacks the arsenite transporter. In contrast, the responses observed in a strain lacking all arsenate reductases were somewhat different and included lower induction of genes involved in metal homeostasis and Fe-S cluster biogenesis, suggesting that these two processes are targeted by arsenite in the wild type strain. Finally, analysis of the arsR mutant strain revealed that ArsR seems to only control 5 genes in the genome. Furthermore, the arsR mutant strain exhibited hypersentivity to nickel, copper and cadmium and this phenotype was suppressed by mutation in arsB but not in arsC gene suggesting that overexpression of arsB is detrimental in the presence of these metals in the media.

Redox regulation of glycogen biosynthesis in the cyanobacterium Synechocystis sp. PCC 6803: analysis of the AGP and glycogen synthases.

Glycogen constitutes the major carbon storage source in cyanobacteria, as starch in algae and higher plants. Glycogen and starch synthesis is linked to active photosynthesis and both of them are degraded to glucose in the dark to maintain cell metabolism. Control of glycogen biosynthesis in cyanobacteria could be mediated by the regulation of the enzymes involved in this process, ADP-glucose pyrophosphorylase (AGP) and glycogen synthase, which were identified as putative thioredoxin targets. We have analyzed whether both enzymes were subjected to redox modification using purified recombinant enzymes or cell extracts in the model cyanobacterium Synechocystis sp. PCC 6803. Our results indicate that both AGP and glycogen synthases are sensitive to copper oxidation. However, only AGP exhibits a decrease in its enzymatic activity, which is recovered after reduction by DTT or reduced thioredoxin (TrxA), suggesting a redox control of AGP. In order to elucidate the role in redox control of the cysteine residues present on the AGP sequence (C45, C185, C320, and C337), they were replaced with serine. All AGP mutant proteins remained active when expressed in Synechocystis, although they showed different electrophoretic mobility profiles after copper oxidation, reflecting a complex pattern of cysteines interaction.

Metals in cyanobacteria: analysis of the copper, nickel, cobalt and arsenic homeostasis mechanisms.

Traces of metal are required for fundamental biochemical processes, such as photosynthesis and respiration. Cyanobacteria metal homeostasis acquires an important role because the photosynthetic machinery imposes a high demand for metals, making them a limiting factor for cyanobacteria, especially in the open oceans. On the other hand, in the last two centuries, the metal concentrations in marine environments and lake sediments have increased as a result of several industrial activities. In all cases, cells have to tightly regulate uptake to maintain their intracellular concentrations below toxic levels. Mechanisms to obtain metal under limiting conditions and to protect cells from an excess of metals are present in cyanobacteria. Understanding metal homeostasis in cyanobacteria and the proteins involved will help to evaluate the use of these microorganisms in metal bioremediation. Furthermore, it will also help to understand how metal availability impacts primary production in the oceans. In this review, we will focus on copper, nickel, cobalt and arsenic (a toxic metalloid) metabolism, which has been mainly analyzed in model cyanobacterium Synechocystis sp. PCC 6803.