The Role of Stress Proteins in Haloarchaea and Their Adaptive Response to Environmental Shifts

Over the years, in order to survive in their natural environment, microbial communities have acquired adaptations to nonoptimal growth conditions. These shifts are usually related to stress conditions such as low/high solar radiation, extreme temperatures, oxidative stress, pH variations, changes in salinity, or a high concentration of heavy metals. In addition, climate change is resulting in these stress conditions becoming more significant due to the frequency and intensity of extreme weather events. The most relevant damaging effect of these stressors is protein denaturation. To cope with this effect, organisms have developed different mechanisms, wherein the stress genes play an important role in deciding which of them survive. Each organism has different responses that involve the activation of many genes and molecules as well as downregulation of other genes and pathways. Focused on salinity stress, the archaeal domain encompasses the most significant extremophiles living in high-salinity environments. To have the capacity to withstand this high salinity without losing protein structure and function, the microorganisms have distinct adaptations. The haloarchaeal stress response protects cells against abiotic stressors through the synthesis of stress proteins. This includes other heat shock stress proteins (Hsp), thermoprotectants, survival proteins, universal stress proteins, and multicellular structures. Gene and family stress proteins are highly conserved among members of the halophilic archaea and their study should continue in order to develop means to improve for biotechnological purposes. In this review, all the mechanisms to cope with stress response by haloarchaea are discussed from a global perspective, specifically focusing on the role played by universal stress proteins

Lrp as a potential transcriptional regulator involved in stress response in Haloferax mediterranei

The Archaea domain consists of a heterogeneous group of microorganisms with unique physiological properties that occupy a wide variety of niches in nature. Haloferax mediterranei is an extremely halophilic archaeon classified in the Phylum Euryarchaeota, which requires a high concentration of inorganic salts for optimal growth. In haloarchaea, transcription factors play a fundamental role in an adequate adaptation to environmental and nutritional changes, preserving the survival and integrity of the organism. To deepen knowledge of the Lrp/AsnC transcriptional regulator family, a lrp gene (HFX_RS01210) from this family has been studied. Site-directed mutagenesis has allowed us to identify the TATA-box and two potential sites of the transcriptional factor (TF) to its own promoter and autoregulate itself. Several approaches were carried out to elucidate whether this transcriptional regulator is involved in stresses due to heavy metals and limited nitrogen conditions. Characterization of the lrp deletion mutant and the Lrp overexpressed strain, suggests that the level of lrp expression depends on the nitrogen source and the presence of cobalt. The most striking results were obtained in the presence of nitrate as a nitrogen source due to the inability of the deletion mutant to grow. All these results confirm that Lrp is a powerful candidate for a regulatory role in the stress response, particularly under N-limiting conditions and the presence of cobalt.This work was supported by Programa Propio para el Fomento de la I + D + I del Vicerrectorado de Investigación y Transferencia de Conocimiento” of the University of Alicante (VIGRO-016)

Archaea: current and potential biotechnological applications

Archaea are microorganisms with great ability to colonize some of the most inhospitable environments in nature, managing to survive in places with extreme characteristics for most microorganisms. Its proteins and enzymes are stable and can act under extreme conditions in which other proteins and enzymes would degrade. These attributes make them ideal candidates for use in a wide range of biotechnological applications. This review describes the most important applications, both current and potential, that archaea present in Biotechnology, classifying them according to the sector to which the application is directed. It also analyzes the advantages and disadvantages of its use

Towards the Elucidation of Assimilative nasABC Operon Transcriptional Regulation in Haloferax mediterranei

The assimilatory pathway of the nitrogen cycle in the haloarchaeon Haloferax mediterranei has been well described and characterized in previous studies. However, the regulatory mechanisms involved in the gene expression of this pathway remain unknown in haloarchaea. This work focuses on elucidating the regulation at the transcriptional level of the assimilative nasABC operon (HFX_2002 to HFX_2004) through different approaches. Characterization of its promoter region using β-galactosidase as a reporter gene and site-directed mutagenesis has allowed us to identify possible candidate binding regions for a transcriptional factor. The identification of a potential transcriptional regulator related to nitrogen metabolism has become a real challenge due to the lack of information on haloarchaea. The investigation of protein–DNA binding by streptavidin bead pull-down analysis combined with mass spectrometry resulted in the in vitro identification of a transcriptional regulator belonging to the Lrp/AsnC family, which binds to the nasABC operon promoter (p.nasABC). To our knowledge, this study is the first report to suggest the AsnC transcriptional regulator as a powerful candidate to play a regulatory role in nasABC gene expression in Hfx. mediterranei and, in general, in the assimilatory nitrogen pathway.This research was funded by Universidad de Alicante, VIGROB-016

Novel Glutamate–Putrescine Ligase Activity in Haloferax mediterranei: A New Function for glnA-2 Gene

The genome of the halophilic archaea Haloferax mediterranei contains three ORFs that show homology with glutamine synthetase (GS) (glnA-1, glnA-2, and glnA-3). Previous studies have focused on the role of GlnA-1, suggesting that proteins GlnA-2 and GlnA-3 could play a different role to that of GS. Glutamine synthetase (EC 6.3.1.2) belongs to the class of ligases, including 20 subclasses of other different enzymes, such as aspartate–ammonia ligase (EC 6.3.1.1), glutamate–ethylamine ligase (EC 6.3.1.6), and glutamate–putrescine ligase (EC 6.3.1.11). The reaction catalyzed by glutamate–putrescine ligase is comparable to the reaction catalyzed by glutamine synthetase (GS). Both enzymes can bind a glutamate molecule to an amino group: ammonium (GS) or putrescine (glutamate–putrescine ligase). In addition, they present the characteristic catalytic domain of GS, showing significant similarities in their structure. Although these proteins are annotated as GS, the bioinformatics and experimental results obtained in this work indicate that the GlnA-2 protein (HFX_1688) is a glutamate–putrescine ligase, involved in polyamine catabolism. The most significant results are those related to glutamate–putrescine ligase’s activity and the analysis of the transcriptional and translational expression of the glnA-2 gene in the presence of different nitrogen sources. This work confirms a new metabolic pathway in the Archaea domain which extends the knowledge regarding the utilization of alternative nitrogen sources in this domain.This research was funded by Universidad de Alicante, VIGROB-016

Microbial Small RNAs – The Missing Link in the Nitrogen Cycle?

Non-coding small RNAs (sRNAs) regulate a wide range of physiological processes in microorganisms that allow them to rapidly respond to changes in environmental conditions. sRNAs have predominantly been studied in a few model organisms, however it is becoming increasingly clear that sRNAs play a crucial role in environmentally relevant pathways. Several sRNAs have been shown to control important enzymatic processes within the nitrogen cycle and many more have been identified in model nitrogen cycling organisms that remain to be characterized. Alongside these studies meta-transcriptomic data indicates both known and putative sRNA are expressed in microbial communities and are potentially linked to changes in environmental processes in these habitats. This review describes the current picture of the function of regulatory sRNAs in the nitrogen cycle. Anthropogenic influences have led to a shift in the nitrogen cycle resulting in an increase in microbial emissions of the potent greenhouse gas nitrous oxide (N2O) into the atmosphere. As the genetic, physiological, and environmental factors regulating the microbial processes responsible for the production and consumption of N2O are not fully understood, this represents a critical knowledge gap in the development of future mitigation strategies.This work was funded by the Biotechnology and Biological Sciences Research Council (United Kingdom) (BB/L022796/1, BB/M00256X/1, BB/S008942/1) and a University of East Anglia studentship as well as a Generalitat Valenciana (Spain) studentship (grant ACIF/2018/200), “Programa Propio para el Formento de la I+D+I del Vicerrectorado de Investigación y Transferencia de Conociemiento (GRE20-02-C)” University of Alicante

Essentiality of the glnA gene in Haloferax mediterranei: gene conversion and transcriptional analysis

Glutamine synthetase is an essential enzyme in ammonium assimilation and glutamine biosynthesis. The Haloferax mediterranei genome has two other glnA-type genes (glnA2 and glnA3) in addition to the glutamine synthetase gene glnA. To determine whether the glnA2 and glnA3 genes can replace glnA in nitrogen metabolism, we generated deletion mutants of glnA. The glnA deletion mutants could not be generated in a medium without glutamine, and thus, glnA is an essential gene in H. mediterranei. The glnA deletion mutant was achieved by adding 40 mM glutamine to the selective medium. This conditional HM26-ΔglnA mutant was characterised with different approaches in the presence of distinct nitrogen sources and nitrogen starvation. Transcriptomic analysis was performed to compare the expression profiles of the strains HM26-ΔglnA and HM26 under different growth conditions. The glnA deletion did not affect the expression of glnA2, glnA3 and nitrogen assimilation genes under nitrogen starvation. Moreover, the results showed that glnA, glnA2 and glnA3 were not expressed under the same conditions. These results indicated that glnA is an essential gene for H. mediterranei and, therefore, glnA2 and glnA3 cannot replace glnA in the conditions analysed.This work was funded by MICINN Grant Number BIO2013-42921P (to MJB), Generalitat Valenciana Grant Number ACIF/2018/200 (to GP) and Universidad de Alicante (VIGROB-016)

New Uses of Haloarchaeal Species in Bioremediation Processes

The extreme conditions under which haloarchaea survive make them good bioremediation agents in water treatment processes and in saline and hypersaline environments contaminated with toxic compounds such as nitrate, nitrite and ammonia, chlorine compounds such as perchlorate and chlorate, heavy metals, and aromatic compounds. New advances in the understanding of haloarchaea metabolism, biochemistry, and molecular biology suggest that general biochemical pathways related to nitrogen (Nitrogen cycle), metals (iron, mercury), hydrocarbons, or phenols can be used for bioremediation proposals

Mejora en la docencia en biociencias mediante la metodología basada en resolución de problemas

La nueva metodología docente propuesta se basa en trabajar los conceptos de la asignatura de Bioquímica, en el Grado de Nutrición Humana y Dietética, mediante vídeos colgados en Youtube. Estos vídeos recogen la parte teórica de la asignatura, explicada mediante PowerPoint. En clase podemos trabajar cuestionarios sobre los diferentes temas. El objetivo es no centrar la clase teórica en la presentación de los diferentes temas que componen el temario de la asignatura. La presentación primera la hacemos mediante los vídeos, para poder centrar el valioso tiempo de las clases en profundizar en los contenidos mediantes cuestiones y otros puntos de interés, que van sugiriendo los propios alumnos. Los vídeos, en cierta forma, sustituyen a la clase magistral clásica. En resumen, la metodología docente se basa en un sistema de presentación de los temas online y una profundización de los contenidos en clase, buscando siempre la participación activa del alumnado, de forma que el profesor no sea sólo alguien que enseña una materia, sino que sea alguien que acompaña en el proceso de aprendizaje del alumno. La parte activa del aprendizaje se basa en el trabajo del alumnado y no en el profesor