Physiology And Biochemistry Of Extremophiles PdfBy Bruno C. In and pdf 28.03.2021 at 22:10 4 min read
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- Protein Adaptations in Archaeal Extremophiles
- Physiology and Biochemistry of Extremophiles
Briefing by: AccessScience Editors. Last reviewed: Extremophiles are microorganisms that thrive in environmental conditions hostile to most life, including extremes of temperature both high and low , acidity or alkalinity, salt concentration, and pressure.
Protein Adaptations in Archaeal Extremophiles
Ectoine and hydroxyectoine are well-recognized members of the compatible solutes and are widely employed by microorganisms as osmostress protectants. A subgroup of the ectoine producers can convert ectoine into 5-hydroxyectoine through a region-selective and stereospecific hydroxylation reaction. This compatible solute possesses stress-protective and function-preserving properties different from those of ectoine.
Hydroxylation of ectoine is carried out by the EctD protein, a member of the non-heme-containing iron II and 2-oxoglutarate-dependent dioxygenase superfamily. We used the signature enzymes for ectoine EctC and hydroxyectoine EctD synthesis in database searches to assess the taxonomic distribution of potential ectoine and hydroxyectoine producers.
Among microbial genomes inspected, species are predicted to produce ectoine and of these, are predicted to synthesize hydroxyectoine as well. Ectoine and hydroxyectoine genes are found almost exclusively in Bacteria. This comprehensive in silico analysis was coupled with the biochemical characterization of ectoine hydroxylases from microorganisms that can colonize habitats with extremes in salinity Halomonas elongata , pH Alkalilimnicola ehrlichii , Acidiphilium cryptum , or temperature Sphingopyxis alaskensis , Paenibacillus lautus or that produce hydroxyectoine very efficiently over ectoine Pseudomonas stutzeri.
These six ectoine hydroxylases all possess similar kinetic parameters for their substrates but exhibit different temperature stabilities and differ in their tolerance to salts. We also report the crystal structure of the Virgibacillus salexigens EctD protein in its apo-form, thereby revealing that the iron-free structure exists already in a pre-set configuration to incorporate the iron catalyst.
Collectively, our work defines the taxonomic distribution and salient biochemical properties of the ectoine hydroxylase protein family and contributes to the understanding of its structure. This is an open-access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Thauer to N. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist. The ability to sensitively detect and respond in a timely manner to changes in the external osmolarity through concerted genetic and physiological adaptation reactions is critical for the wellbeing and growth of most microorganisms  , . The accumulation of compatible solutes is a widely used strategy by members of both the Bacteria and the Archaea to offset the detrimental effects of high osmolarity on cellular hydration and physiology  — .
Compatible solutes are operationally defined as small organic osmolytes, highly water-soluble compounds whose physicochemical properties make them compliant with cellular biochemistry and physiology  — .
As a consequence, microbial cells can build-up compatible solute pools to exceedingly high intracellular levels, either through synthesis or uptake  ,  , and they do this in a manner that is sensitively tied to the degree of the environmentally imposed osmotic stress  , . Accumulation of compatible solutes counteracts the efflux of water under hyperosmotic growth conditions; they thereby stabilize turgor and optimize the solvent properties of the cytoplasm  ,  , .
These processes cooperate in strongly enhancing the growth of high osmolarity challenged cells. Ectoine and its derivative 5-hydroxyectoine are well-recognized members of the compatible solutes  ,  and are effective osmostress protectants for microorganisms  , . The structural genes for the ectoine biosynthetic enzymes are typically organized in an operon ectABC  whose transcription is up-regulated in response to high osmolarity  ,  — .
Enhanced transcription of the ect genes is also triggered in some microorganisms by extremes in growth temperature  ,  as ectoines can also confer protection against both heat and cold stress  — . A subgroup of the ectoine producers also synthesizes a hydroxylated derivative of ectoine, 5-hydroxyectoine  ,  , in a biosynthetic reaction that is catalyzed by the ectoine hydroxylase EctD  ,  , .
In addition to their role in alleviating osmotic stress, ectoines also serve as stabilizers of macromolecules and even entire cells  , . The function-preserving and anti-inflammatory effects of ectoines fostered substantial interest in exploring them for a variety of practical biotechnological applications and potential medical uses  ,  — . Despite their closely related chemical structures, 5-hydroxyectoine often possesses superior stress protecting and function preserving properties than its precursor molecule ectoine  ,  — .
Here, we focus on the ectoine hydroxylase, the enzyme that forms 4 S ,5 S methylhydroxy-1,4,5,6-tetrahydropyrimidinecarboxylic acid from the precursor ectoine through a region-selective and stereospecific hydroxylation reaction  , . The enzymatic characterization of the EctD protein from Virgibacillus salexigens  and Streptomyces coelicolor  identified the ectoine hydroxylase as a member of the non-heme-containing iron II and 2-oxoglutarate-dependent dioxygenase superfamily EC1.
The EctD-mediated hydroxylation of 4 S -ectoine to 4 S ,5 S hydroxyectoine requires O 2 and 2-oxoglutarate as co-substrates, thereby forming CO 2 , succinate, and 5-hydroxyectoine . As seen in other members of the dioxygenase superfamily e. The high-resolution 1. We coupled this comprehensive in silico analysis with the biochemical characterization of six EctD enzymes from phylogenetically widely separated bacteria covering various different lifestyles to define the properties and kinetic parameters of the ectoine hydroxylase on a broad basis.
In addition, the crystal structure of the EctD protein from the salt tolerant moderate halophile V. To assess the prevalence and taxonomic distribution of the ectoine and hydroxyectoine biosynthetic genes in microorganisms, we searched through finished microbial genome sequences at the database of the U.
Department of Energy DOE Joint Genome Institute  for the presence of an ectC ortholog, coding for the signature enzyme of the ectoine biosynthetic pathway, the ectoine synthase . As a search query for this database analysis, we used the amino acid sequence of the V. At the time of the database search, microbial genomes were represented that were derived from members of the Bacteria and members of the Archaea. Excluding closely related strains of the same species for our analysis and using only a single representative, we constructed a phylogentic tree of the EctC sequences Fig.
It is apparent from our database analysis that ectoine is a compatible solute which is synthesized almost exclusively by members of the Bacteria Fig. Genome sequences of strains of Vibrio cholerae are represented among the searched microbial genomes, each of which is predicted to produce ectoine, but only one of them was included in the dataset depicted in Fig. The few predicted archaeal ectoine producers have probably acquired the ectoine biosynthetic genes via lateral gene transfer events, since the exchange of genetic material between members of the kingdoms of the Bacteria and Archaea is a well-documented phenomenon .
These compiled amino acid sequences were then used to assess the phylogenetic distribution of the EctC protein using the iTOL Web-server. Evolutionary distances are not given. The color code indicates the distribution of EctC among members of the Bacteria and Archaea.
The presence of an ectD gene in a given microbial species possessing ectC is indicated by black ectD is part of the ect gene cluster or red circles ectD is located outside of the ect gene cluster. If different strains of the same species were sequenced, only one representative symbolizes them. For instance, there are genomic data of strain of Vibrio cholerae available in the database, each of which possesses an ectABC gene cluster, but only one of these sequences was used for the phylogenetic analysis.
We then assessed the distribution of the ectoine hydroxylase orthologs ectD in bacterial and archaeal genomes by using the V. We found that of the sequenced genomes possessed an ectD gene. Invariably these microorganisms also possessed an ectC gene, a result that is expected from the fact that hydroxyectoine is synthesized directly from the precursor molecule ectoine .
Hence, about two-thirds of the putative ectoine producers are predicted to synthesize hydroxyectoine as well Fig. As expected from the oxygen-dependent reaction of the EctD enzyme, ectD is never present in genomes of obligate anaerobes, although it is not universally present in aerobic or facultative species. Consistently, from the above mentioned archaeal ectoine-producing representatives, only the three aerobic Nitrosopumilus species possess an ectD gene as part of their ect gene clusters, whereas the genome sequences of the two anaerobic Methanosaeta species lacked ectD altogether.
Biochemical properties of native ectoine hydroxylases from V. To determine whether the reported features of these two studied EctD proteins are representative for ectoine hydroxylases in general, we set out to study the characteristics of this type of enzyme on a broader basis.
For these biochemical studies we chose six EctD proteins from the following taxonomically widely separated and mostly extremophilic microorganisms: Halomonas elongata , Acidiphilium cryptum , Alkalilimnicola ehrlichii , Sphingopyxis alaskensis, Paenibacillus lautus , and Pseudomonas stutzeri. The Gammaproteobacterium H. The Alphaproteobacterium S. The Firmicute P. The last studied microorganism was the nitrogen-fixing Gammabacterium Pseudomonas stutzeri strain A that is not an extremophile, as it was isolated from plant roots .
Like the type strain of P. Given the very different habitats of these microorganisms, we wondered if the biochemical properties of their EctD proteins would reflect the preferences of their producers with respect to the salt, pH, and temperature parameters prevalent in their natural habitats. Using the biochemically and structurally well characterized V. To study these EctD enzymes biochemically, we inserted the various ectD genes into an expression vector that allowed the production of the corresponding proteins as recombinant variants with a Strep -tag-II affinity peptide attached to their carboxy-terminus.
These proteins could all be overproduced in an Escherichia coli host strain and isolated with good yields and purities by affinity chromatography on Step-Tactin Superflow material Fig. The amino acid sequences of the native EctD proteins range in length between and amino acids, except for EctD of H. The gel was run at 25 mA for 2.
We note the presence of a overlapping second band in protein sample of the Paenibacillus lautus EctD preparation. This might stem from overloading the gel somewhat or from partial degradation of the purified EctD protein. Since the presence of a correctly complexed iron ligand is critical for EctD-mediated enzyme catalysis  ,  ,  , we determined the iron-content of each of these recombinant proteins and found between 0.
Hence, these recombinant EctD proteins should all be functional. An initial assessment of their enzymatic activities under the same assay conditions as used previously for the ectoine hydroxylases from V.
The data from this set of experiments are summarized in Table 1 and are documented in detail for the S. The data for all other enzymes are summarized in Fig. S1 to Fig. Overall, the basic biochemical parameters of the six newly studied EctD enzymes and the re-analyzed EctD protein from V. However, differences were noted with respect to their resistance to the inhibiting action of increased salt concentrations Table 1.
The enzyme activity of the ectoine hydroxylase from S. In studying the biochemical properties of the ectoine biosynthetic enzymes from H. We did not find any strong stimulating effect of high NaCl concentrations on any of the ectoine hydroxylases we studied here Table 1 , including that of H.
On the contrary, high concentrations of NaCl typically inhibited the enzyme activities of the EctD variants Fig. However, notable stimulating effects [about two- to three-fold Fig. We assessed the quaternary structure of the six newly studied EctD proteins by gel filtration. An example of this analysis is shown in Fig. S6 for the S.
The protein eluted between 72 to 83 ml maximum: Since the calculated molecular mass of the S. The same conclusion was derived for all other analyzed EctD proteins data not shown , including that from V. The studied EctD enzymes have similar temperature optima but differ in the range of temperatures in which they operate naturally Table 1. To investigate this further, we studied their temperature stability. The ectoine hydroxylase from H. The strong temperature resistance of the P.
The considerable heat tolerance of the S. The temperature profiles of the ectoine hydroxylases from H. Each EctD protein was pre-incubated at the indicated temperatures for 15 min before its specific activity was then determined under its optimal assay condition. After having optimized the parameters of the enzyme activity assays for each of the six purified ectoine hydroxylases Table 1 , we determined their apparent kinetic parameters for the co-substrate 2-oxoglutarate and the substrate ectoine Table 2.
This assessment showed that the studied ectoine hydroxylases all possess similar kinetic parameters.
Physiology and Biochemistry of Extremophiles
Ectoine and hydroxyectoine are well-recognized members of the compatible solutes and are widely employed by microorganisms as osmostress protectants. A subgroup of the ectoine producers can convert ectoine into 5-hydroxyectoine through a region-selective and stereospecific hydroxylation reaction. This compatible solute possesses stress-protective and function-preserving properties different from those of ectoine. Hydroxylation of ectoine is carried out by the EctD protein, a member of the non-heme-containing iron II and 2-oxoglutarate-dependent dioxygenase superfamily. We used the signature enzymes for ectoine EctC and hydroxyectoine EctD synthesis in database searches to assess the taxonomic distribution of potential ectoine and hydroxyectoine producers. Among microbial genomes inspected, species are predicted to produce ectoine and of these, are predicted to synthesize hydroxyectoine as well.
Christopher J. Extremophiles, especially those in Archaea, have a myriad of adaptations that keep their cellular proteins stable and active under the extreme conditions in which they live. Rather than having one basic set of adaptations that works for all environments, Archaea have evolved separate protein features that are customized for each environment. We categorized the Archaea into three general groups to describe what is known about their protein adaptations: thermophilic, psychrophilic, and halophilic. Thermophilic proteins tend to have a prominent hydrophobic core and increased electrostatic interactions to maintain activity at high temperatures.
Prokaryotic life has dominated most of the evolutionary history of our planet, evolving to occupy virtually all available environmental niches. Extremophiles, especially those thriving under multiple extremes, represent a key area of research for multiple disciplines, spanning from the study of adaptations to harsh conditions, to the biogeochemical cycling of elements. Extremophile research also has implications for origin of life studies and the search for life on other planetary and celestial bodies. In this article, we will review the current state of knowledge for the biospace in which life operates on Earth and will discuss it in a planetary context, highlighting knowledge gaps and areas of opportunity. Over the past century, the boundary conditions under which life can thrive have been pushed in every possible direction, encompassing broader swaths of temperature, pH, pressure, radiation, salinity, energy, and nutrient limitation. Microorganisms do not only thrive under such a broad spectrum of parameters on Earth, but can also survive the harsh conditions of space, an environment with extreme radiation, vacuum pressure, extremely variable temperature, and microgravity Horneck et al.
Metrics details. Extremophiles are organisms that can grow and thrive in harsh conditions, e. Thermophilic, halophilic and radiation-resistant organisms are all microbes, some of which are able to withstand multiple extremes. Psychrophiles, or cold-loving organisms, include not only microbes, but fish that live in polar waters and animals that can withstand freezing. Extremophiles are structurally adapted at a molecular level to withstand these conditions.
Extremophiles are organisms that can grow and thrive in harsh conditions, e. Thermophilic, halophilic and radiation-resistant organisms are all microbes, some of which are able to withstand multiple extremes. Psychrophiles, or cold-loving organisms, include not only microbes, but fish that live in polar waters and animals that can withstand freezing.
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