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Polyhydroxyalkanoate involvement in stress-survival of two psychrophilic bacterial strains from the High Arctic
(Springer, 2024-05-04) Grzesiak, Jakub; Rogala, Małgorzata Marta; Gawor, Jan; Kouřilová, Xenie; Obruča, Stanislav
An ever-growing body of literature evidences the protective role of polyhydroxyalkanoates (PHAs) against a plethora of mostly physical stressors in prokaryotic cells. To date, most of the research done involved bacterial strains isolated from habitats not considered to be life-challenging or extremely impacted by abiotic environmental factors. Polar region microorganisms experience a multitude of damaging factors in combinations rarely seen in other of Earth's environments. Therefore, the main objective of this investigation was to examine the role of PHAs in the adaptation of psychrophilic, Arctic-derived bacteria to stress conditions. Arctic PHA producers: Acidovorax sp. A1169 and Collimonas sp. A2191, were chosen and their genes involved in PHB metabolism were deactivated making them unable to accumulate PHAs (Delta phaC) or to utilize them (Delta i-phaZ) as a carbon source. Varying stressors were applied to the wild-type and the prepared mutant strains and their survival rates were assessed based on CFU count. Wild-type strains with a functional PHA metabolism were best suited to survive the freeze-thaw cycle - a common feature of polar region habitats. However, the majority of stresses were best survived by the Delta phaC mutants, suggesting that the biochemical imbalance caused by the lack of PHAs induced a permanent cell-wide stress response thus causing them to better withstand the stressor application. Delta i-phaZ mutants were superior in surviving UV irradiation, hinting that PHA granule presence in bacterial cells is beneficial despite it being biologically inaccessible. Obtained data suggests that the ability to metabolize PHA although important for survival, probably is not the most crucial mechanism in the stress-resistance strategies arsenal of cold-loving bacteria. Key points PHA metabolism helps psychrophiles survive freezing PHA-lacking psychrophile mutants cope better with oxidative and heat stresses PHA granule presence enhances the UV resistance of psychrophiles
Quantitative Raman Spectroscopy Analysis of Polyhydroxyalkanoates Produced by Cupriavidus necator H16
(MDPI, 2016-10-28) Obruča, Stanislav; Sedláček, Petr; Benešová, Pavla; Kučera, Dan; Márová, Ivana
We report herein on the application of Raman spectroscopy to the rapid quantitative analysis of polyhydroxyalkanoates (PHAs), biodegradable polyesters accumulated by various bacteria. This theme was exemplified for quantitative detection of the most common member of PHAs, poly(3-hydroxybutyrate) (PHB) in Cupriavidus necator H16. We have identified the relevant spectral region (800-1800 cm(-1)) incorporating the Raman emission lines exploited for the calibration of PHB (PHB line at 1736 cm-1) and for the selection of the two internal standards (DNA at 786 cm(-1) and Amide I at 1662 cm(-1)). In order to obtain quantitative data for calibration of intracellular content of PHB in bacterial cells reference samples containing PHB amounts determined by gas chromatography from 12% to 90% (w/w) were used. Consequently, analytical results based on this calibration can be used for fast and reliable determination of intracellular PHB content during biotechnological production of PHB since the whole procedure from bacteria sampling, centrifugation, and sample preparation to Raman analysis can take about 12 min. In contrast, gas chromatography analysis takes approximately 8 h.
Influence of Culture Media on Microbial Fingerprints Using Raman Spectroscopy
(MDPI, 2015-11-24) Mlynáriková, Katarína; Samek, Ota; Bernatová, Silvie; Růžička, František; Ježek, Jan; Němcová, Andrea; Šiler, Martin; Zemánek, Pavel; Holá, Veronika
Our work here monitors the influence of culture media on the Raman spectra of clinically important microorganisms
Accumulation of Poly(3-hydroxybutyrate) Helps Bacterial Cells to Survive Freezing
(PLoS, 2016-06-17) Obruča, Stanislav; Sedláček, Petr; Mravec, Filip; Kučera, Dan; Benešová, Pavla; Márová, Ivana
Accumulation of polyhydroxybutyrate (PHB) seems to be a common metabolic strategy adopted by many bacteria to cope with cold environments. This work aimed at evaluating and understanding the cryoprotective effect of PHB. At first a monomer of PHB, 3-hydroxybutyrate, was identified as a potent cryoprotectant capable of protecting model enzyme (lipase), yeast (Saccharomyces cerevisiae) and bacterial cells (Cupriavidus necator) against the adverse effects of freezing-thawing cycles. Further, the viability of the frozen-thawed PHB accumulating strain of C. necator was compared to that of the PHB non-accumulating mutant. The presence of PHB granules in cells was revealed to be a significant advantage during freezing. This might be attributed to the higher intracellular level of 3-hydroxybutyrate in PHB accumulating cells (due to the action of parallel PHB synthesis and degradation, the so-called PHB cycle), but the cryoprotective effect of PHB granules seems to be more complex. Since intracellular PHB granules retain highly flexible properties even at extremely low temperatures (observed by cryo-SEM), it can be expected that PHB granules protect cells against injury from extracellular ice. Finally, thermal analysis indicates that PHB-containing cells exhibit a higher rate of transmembrane water transport, which protects cells against the formation of intracellular ice which usually has fatal consequences.
The First Insight into Polyhydroxyalkanoates Accumulation in Multi-Extremophilic Rubrobacter xylanophilus and Rubrobacter spartanus
(MDPI, 2021-05-01) Kouřilová, Xenie; Schwarzerová, Jana; Buchtíková, Iva; Sedlář, Karel; Mrázová, Kateřina; Krzyžánek, Vladislav; Nebesářová, Jana; Obruča, Stanislav
Actinobacteria belonging to the genus Rubrobacter are known for their multi-extremophilic growth conditions-they are highly radiation-resistant, halotolerant, thermotolerant or even thermophilic. This work demonstrates that the members of the genus are capable of accumulating polyhydroxyalkanoates (PHA) since PHA-related genes are widely distributed among Rubrobacter spp. whose complete genome sequences are available in public databases. Interestingly, all Rubrobacter strains possess both class I and class III synthases (PhaC). We have experimentally investigated the PHA accumulation in two thermophilic species, R. xylanophilus and R. spartanus. The PHA content in both strains reached up to 50% of the cell dry mass, both bacteria were able to accumulate PHA consisting of 3-hydroxybutyrate and 3-hydroxyvalerate monomeric units, none other monomers were incorporated into the polymer chain. The capability of PHA accumulation likely contributes to the multi-extremophilic characteristics since it is known that PHA substantially enhances the stress robustness of bacteria. Hence, PHA can be considered as extremolytes enabling adaptation to extreme conditions. Furthermore, due to the high PHA content in biomass, a wide range of utilizable substrates, Gram-stain positivity, and thermophilic features, the Rubrobacter species, in particular Rubrobacter xylanophilus, could be also interesting candidates for industrial production of PHA within the concept of Next-Generation Industrial Biotechnology.

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