Abstract
Manganese (Mn) is an essential micronutrient for all photoautotrophic organisms. Two distinct pools of Mn have been identified in the cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis), with 80% of the Mn residing in the periplasm and 20% in cytoplasm and thylakoid lumen (Keren et al., 2002). In this protocol, we describe a method to quantify the periplasmic and intracellular pools of Mn in Synechocystis accurately, using inductively coupled plasma mass spectrometry (ICP-MS).
Keywords: Cyanobacteria, Synechocystis, Manganese, Periplasm, ICP-MS
Background
Mn plays a vital role in the active sites of several enzymes such as the oxygen-evolving complex in photosystem II. In contrast to its role as an important micronutrient, Mn can be toxic when present in excess. It is therefore of crucial importance for cyanobacteria to maintain the intracellular levels of Mn and in particular to avoid free Mn in the cytoplasm. The cyanobacterium Synechocystis addresses this challenge by storing about 80% of the Mn in the periplasm. Only 20% of the cellular content can be detected in the cytoplasm and thylakoid system (Keren et al., 2002), with most of the Mn being incorporated into proteins, leaving virtually no free Mn in the cytoplasm. We recently identified the manganese transport protein Mnx, which resides in the thylakoid membrane and facilitates export of Mn from the cytoplasm into the thylakoid lumen in Synechocystis. According to our study, Mnx is a major player in maintaining the cellular Mn homeostasis (Brandenburg et al., 2017). To analyze the biological significance of Mnx, we developed a protocol to measure the periplasmic and intracellular Mn pools separately. We chose ICP-MS for quantification, since it is a sensitive and reliable method to detect metals in biological samples. Detection limits can be in the range of [ng L-1] and below. Prior to the analysis, all complex molecules of the sample are broken down to atomic compounds by digestion with nitric acid. Subsequently, the sample is ionized by an inductively coupled plasma and analyzed by mass spectrometry. In this protocol, we describe the detailed workflow for subcellular Mn quantification, from sampling to the calculation of Mn concentrations.
Materials and Reagents
Equipment
Procedure
Data analysis
The periplasmic Mn concentration is the sum of the Mn in samples E1 and E2. Sample C2 is the intracellular concentration of Mn, namely cytosol and thylakoid system. Apply equation 1 to calculate the periplasmic Mn concentration using samples E1, E2 as well as volume and cell count from C1. Similarly, use equation 2 to calculate the intracellular Mn concentration in [atoms cell-1] using samples C1 and C2. ICP-MS results usually come in parts per million (ppm), which is equivalent to [µg ml-1] or parts per billion (ppb), which is equivalent to [µg L-1]. Make sure to convert the input data into the correct units. Please note that the molecular weight (MW) in equations 1 and 2 is in [µg mol-1]. Instead of cell counting, you can use OD750 or chlorophyll content for normalization as well. However, we recommend cell counting. As a control, the sum of the Mn in samples C2, E1 and E2 should equal the Mn concentration in sample T2. You can calculate sample T2 in the same way as sample C2. Figure 2 shows an example of the calculations. The numbers used for this calculation were obtained from a WT sample grown at our standard conditions (Note 1). Figure 2. Example calculation of Mn concentration. Make sure the input data is in the correct units. In our case, for example, the ICP-MS results were in [µg L-1]. Please note that the molecular weight (MW) of Mn is in [µg mol-1]. In our experiments, the difference between E1 + E2 + C2 and T2 was in the range of 0.1-10 x 103 [atoms cell-1].
Notes
Recipes
Acknowledgments
This research was supported by the German Science Foundation (EI 945/3-1 to M.E.) and the Israeli Science Foundation (2733/16 to N.K.). The authors declare that there are no conflicts of interest.
References
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