Ultrastructural changes in prokaryotic microorganisms caused by long-term exposure to high salt and copper ion concentrations

0568545 - ÚPT 2023 RIV CZ eng A - Abstract
Mrázová, Kateřina - Nováčková, I. - Hrubanová, Kamila - Nebesářová, Jana - Obruča, S. - Krzyžánek, Vladislav
Ultrastructural changes in prokaryotic microorganisms caused by long-term exposure to high salt and copper ion concentrations.
16th Multinational Congress on Microscopy, 16MCM, 04-09 September 2022, Brno, Czech Republic. Book of abstracts. Brno: Czechoslovak Microscopy Society, 2022 - (Krzyžánek, V.; Hrubanová, K.; Hozák, P.; Müllerová, I.; Šlouf, M.). s. 231-232. ISBN 978-80-11-02253-2.
[Multinational Congress on Microscopy /16./. 04.09.2022-09.09.2022, Brno]
R&D Projects: GA TA ČR(CZ) TN01000008
Institutional support: RVO:68081731 ; RVO:60077344
Keywords : Cupriavidus necator * adaptation * heavy metals * osmotic stress * polyhydroxyalkanoates * stress conditions
OECD category: Genetics and heredity (medical genetics to be 3)
https://www.16mcm.cz/wp-content/uploads/2022/09/16MCM-abstract-book.pdf

Cupriavidus necator is a soil bacterium known as a producer of polyhydroxyalkanoates (PHA), polyesters of hydroxyalkanoic acids. PHAs are polymers present in many prokaryotic organisms in the form of intracellular granules and serve as a source of carbon in the cell. Moreover, recent studies indicate, that the presence of PHA in the microbial cells enhances their robustness against various stress factors. Polyhydroxyalkanoates also attract attention as a potential substitute for petrochemical plastics. These biopolymers have similar properties as for example polypropylene, but since they are biodegradable, they will decompose in nature incomparably faster than conventional plastic materials. In our study, we focused on the PHA producer Cupriavidus necator in a long-term experiment, where the bacterial cells were exposed to high concentrations of salt (54 passages) and copper ions (64 passages). After cultivation, bacterial cells were harvested and analysed using the methods of electron microscopy, namely cryo-SEM and TEM. Bacterial cells were centrifuged and fixed using the method of high-pressure freezing. For cryo-SEM analysis, bacterial cells were pipetted on the 0,2 µm side of the 6mm carrier type A and closed with the flat side of carrier type B without any treatment using lecithin or cryoprotectants. Frozen samples were then freeze-fractured and underwent freeze-etching procedure for 7min at -95°C. The cryo-SEM imaging was conducted at -120°C in a scanning electron microscope equipped with a cryo stage. For TEM analysis, 3mm carriers pre-treated with a 1% solution of lecithin were used and the frozen samples underwent freeze-substitution. The chosen freeze-substitution protocol was previously described. Samples cut to ultrathin sections and stained were then imaged in a transmission electron microscope using the electron beam of energy 80kV. In the TEM images of Cu stress, it is possible to observe dense precipitates inside of the cells which can be correlated to cryo-SEM images where cells exposed to Cu+ show small hollows in the cytoplasm of similar shape and size as precipitates in TEM imaging. Also, cells exposed to high osmotic stress appeared to be smaller, than the control cultivation without any stress factors, and of a crescent shape. These findings indicate, that long-term exposure of microbial cells to stress conditions changes not only the production of intracellular polymers but also their morphology and this phenomenon deserves to be studied further.
Permanent Link: https://hdl.handle.net/11104/0339840