Molecular Advances in Microbial Metabolism 2.0.

The advances in molecular biology techniques and omics approaches have made it possible to take giant steps in applied research in life sciences [...].

Denitrification in hypersaline and coastal environments.

As the association of denitrification with global warming and nitrogen removal from ecosystems has gained attention in recent decades, numerous studies have examined denitrification rates and the distribution of denitrifiers across different environments. In this minireview, reported studies focused on coastal saline environments, including estuaries, mangroves, and hypersaline ecosystems, have been analysed to identify the relationship between denitrification and saline gradients. The analyses of the literature and databases stated the direct effect of salinity on the distribution patterns of denitrifiers. However, few works do not support this hypothesis thus making this topic controversial. The specific mechanisms by which salinity influences denitrifier distribution are not fully understood. Nevertheless, several physical and chemical environmental parameters, in addition to salinity, have been shown to play a role in structuring the denitrifying microbial communities. The prevalence of nirS or nirK denitrifiers in ecosystems is a subject of debate in this work. In general terms, in mesohaline environments, the predominant nitrite reductase is NirS type and, NirK is found predominantly in hypersaline environments. Moreover, the approaches used by different researchers are quite different, resulting in a huge amount of unrelated information, making it difficult to establish comparative analysis. The main techniques used to analyse the distribution of denitrifying populations along salt gradients have been also discussed.

Haloarchaeal carotenoids exert an in vitro antiproliferative effect on human breast cancer cell lines.

Oxidative stress has been linked to the onset and progression of different neoplasia. Antioxidants might help prevent it by modulating biochemical processes involved in cell proliferation. Here, the aim was to evaluate the in vitro cytotoxic effect of Haloferax mediterranei bacterioruberin-rich carotenoid extracts (BRCE) (0-100 µg/ml) in six BC cell lines, representative of the intrinsic phenotypes and a healthy mammary epithelium cell line. Cell index values were obtained using xCELLigence RTCA System. Furthermore, cell diameter, viability, and concentration were measured at 12 h, 24 h, and 30 h. We found that BC cells were selectively affected by BRCE (SI > 1, p < 0.005). After 30 h, the population of BC cells exposed to 100 µg/ml was 11.7-64.6% of the control (p = 0.0001-0.0009). Triple-negative cells were significantly affected [MDA-MB-231 (IC50 51.8 µg/ml, p < 0.0001) and MDA-MB-468 (IC50 63.9 µg/ml, p < 0.0001)]. Cell size was also reduced after 30 h treatment in 3.8 (± 0.1) µm and 3.3 (± 0.02) µm for SK-BR-3 (p < 0.0001) and MDA-MB-468 (p < 0.0001), respectively. In conclusion, Hfx. mediterranei BRCE exerts a cytotoxic effect on BC cell lines representative of all studied intrinsic subtypes. Furthermore, results obtained for MDA-MB-231 and MDA-MB-468 are very promising, considering the aggressive behaviour of the triple-negative BC subtype.

Molecular Advances in Microbial Metabolism.

Climate change, global pollution due to plastics, greenhouse gasses, or heavy metals among other pollutants, as well as limited natural sources due to unsustainable lifestyles and consumption patterns, are revealing the need for more research to understand ecosystems, biodiversity, and global concerns from the microscale to the macroscale [...].

Analysis of the external signals driving the transcriptional regulation of the main genes involved in denitrification in Haloferax mediterranei.

Haloferax mediterranei is the model microorganism for the study of the nitrogen cycle in haloarchaea. This archaeon not only assimilate N-species such as nitrate, nitrite, or ammonia, but also it can perform denitrification under low oxygen conditions, using nitrate or nitrite as alternative electron acceptors. However, the information currently available on the regulation of this alternative respiration in this kind of microorganism is scarce. Therefore, in this research, the study of haloarchaeal denitrification using H. mediterranei has been addressed by analyzing the promoter regions of the four main genes of denitrification (narGH, nirK, nor, and nosZ) through bioinformatics, reporter gene assays under oxic and anoxic conditions and by site-directed mutagenesis of the promoter regions. The results have shown that these four promoter regions share a common semi-palindromic motif that plays a role in the control of the expression levels of nor and nosZ (and probably nirK) genes. Regarding the regulation of the genes under study, it has been concluded that nirK, nor, and nosZ genes share some expression patterns, and therefore their transcription could be under the control of the same regulator whereas nar operon expression displays differences, such as the activation by dimethyl sulfoxide with respect to the expression in the absence of an electron acceptor, which is almost null under anoxic conditions. Finally, the study with different electron acceptors demonstrated that this haloarchaea does not need complete anoxia to perform denitrification. Oxygen concentrations around 100 µM trigger the activation of the four promoters. However, a low oxygen concentration per se is not a strong signal to activate the promoters of the main genes involved in this pathway; high activation also requires the presence of nitrate or nitrite as final electron acceptors.

Bioactive molecules from haloarchaea: Scope and prospects for industrial and therapeutic applications.

Marine environments and salty inland ecosystems encompass various environmental conditions, such as extremes of temperature, salinity, pH, pressure, altitude, dry conditions, and nutrient scarcity. The extremely halophilic archaea (also called haloarchaea) are a group of microorganisms requiring high salt concentrations (2-6 M NaCl) for optimal growth. Haloarchaea have different metabolic adaptations to withstand these extreme conditions. Among the adaptations, several vesicles, granules, primary and secondary metabolites are produced that are highly significant in biotechnology, such as carotenoids, halocins, enzymes, and granules of polyhydroxyalkanoates (PHAs). Among halophilic enzymes, reductases play a significant role in the textile industry and the degradation of hydrocarbon compounds. Enzymes like dehydrogenases, glycosyl hydrolases, lipases, esterases, and proteases can also be used in several industrial procedures. More recently, several studies stated that carotenoids, gas vacuoles, and liposomes produced by haloarchaea have specific applications in medicine and pharmacy. Additionally, the production of biodegradable and biocompatible polymers by haloarchaea to store carbon makes them potent candidates to be used as cell factories in the industrial production of bioplastics. Furthermore, some haloarchaeal species can synthesize nanoparticles during heavy metal detoxification, thus shedding light on a new approach to producing nanoparticles on a large scale. Recent studies also highlight that exopolysaccharides from haloarchaea can bind the SARS-CoV-2 spike protein. This review explores the potential of haloarchaea in the industry and biotechnology as cellular factories to upscale the production of diverse bioactive compounds.

Evaluation of Haloferax mediterranei Strain R4 Capabilities for Cadmium Removal from Brines.

Haloferax mediterranei has revealed a high bioremediation potential for several inorganic anions (e.g., nitrates and nitrites) and metals from hypersaline waters and brines. However, it is unclear, to date, whether this microorganism allows Cd (II) bioremediation. Consequently, the main objective of this work was to assess the Cd (II) bioremediation potential of Hfx. mediterranei R4. To this end, Hfx. mediterranei cell growth rate and metal bioaccumulation were investigated using different culture media (complex, CM, and defined medium, DM) containing Cd (II) up to 1 mM. In addition, the elemental profile of the biomass (i.e., Al, Ba, Ca, Co, Cu, Fe, K, Mg, Mn, Na, Ni, Sr and Zn) has also been monitored to gain insight into the metabolic processes that may be taking place at the intracellular level for Cd (II) removal. Because of the formation of CdS precipitate, CM is not a suitable culture media for evaluating Cd bioremediation since metal concentration could not be appropriately controlled. When operating in DM, it was observed that the cell doubling time increases three times in the presence of Cd (II). Hfx. mediterranei can bioaccumulate Cd, showing the highest significant accumulation at concentrations of 0.4 mM (108 ± 12 mg Cd/g dry tissue). Finally, the presence of Cd (II) affects the content of K, Mg, Mn and Zn in the biomass, by increasing K levels up to 27 ± 18% and Mn up to 310 ± 140% and reducing Mg levels up to 55 ± 36% and Zn up to 30 ± 4%. These results suggest that different mechanisms are involved in Cd (II) tolerance by Hfx. mediterranei, resulting in increasing the cell concentration of stress-tolerant elements in the biomass (K and Mn), while lowering the concentration of elements which Cd (II) competes with (Mg and Zn), and that all affects the physiological response of the organism by decreasing its growth rate.

Characterization of Polyhydroxybutyrate, PHB, Synthesized by Newly Isolated Haloarchaea Halolamina spp.

This work aims to characterize the haloarchaeal diversity of unexplored environmental salty samples from a hypersaline environment on the southern coast of Jeddah, Saudi Arabia, looking for new isolates able to produce polyhydroxyalkanoates (PHAs). Thus, the list of PHA producers has been extended by describing two species of Halolamina; Halolamina sediminis sp. strain NRS_35 and unclassified Halolamina sp. strain NRS_38. The growth and PHA-production were investigated in the presence of different carbon sources, (glucose, sucrose, starch, carboxymethyl cellulose (CMC), and glycerol), pH values, (5-9), temperature ranges (4-65 °C), and NaCl concentrations (100-350 g L-1). Fourier-transform infra-red analysis (FT-IR) and Liquid chromatography-mass spectrometry (LC-MS) were used for qualitative identification of the biopolymer. The highest yield of PHB was 33.4% and 27.29% by NRS_35 and NRS_38, respectively, using starch as a carbon source at 37 °C, pH 7, and 25% NaCl (w/v). The FT-IR pattern indicated sharp peaks formed around 1628.98 and 1629.28 cm-1, which confirmed the presence of the carbonyl group (C=O) on amides and related to proteins, which is typical of PHB. LC-MS/MS analysis displayed peaks at retention times of 5.2, 7.3, and 8.1. This peak range indicates the occurrence of PHB and its synthetic products: Acetoacetyl-CoA and PHB synthase (PhaC). In summary, the two newly isolated Halolamina species showed a high capacity to produce PHB using different sources of carbon. Further research using other low-cost feedstocks is needed to improve both the quality and quantity of PHB production. With these results, the use of haloarchaea as cell factories to produce PHAs is reinforced, and light is shed on the global concern about replacing plastics with biodegradable polymers.

Carbon Source Influences Antioxidant, Antiglycemic, and Antilipidemic Activities of Haloferax mediterranei Carotenoid Extracts.

Haloarchaeal carotenoids have attracted attention lately due to their potential antioxidant activity. This work studies the effect of different concentrations of carbon sources on cell growth and carotenoid production. Carotenoid extract composition was characterized by HPLC-MS. Antioxidant activity of carotenoid extracts obtained from cell cultures grown under different nutritional conditions was determined by 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and 1,1-diphenyl-2-picrylhydrazyl (DPPH), Ferric Reducing Ability Power (FRAP) and ß-carotene bleaching assays. The ability of these carotenoid extracts to inhibit α-glucosidase, α-amylase, and lipase enzymes was also assessed to determine if they could be used to reduce blood glucose and lipid absorption. The maximum production of carotenoids (92.2 µg/mL) was observed combining 12.5% inorganic salts and 2.5% of glucose/starch. Antioxidant, hypoglycemic, and antilipidemic studies showed that higher carbon availability in the culture media leads to changes in the extract composition, resulting in more active haloarchaeal carotenoid extracts. Carotenoid extracts obtained from high-carbon-availability cell cultures presented higher proportions of all-trans-bacterioruberin, 5-cis-bacterioruberin, and a double isomeric bacterioruberin, whereas the presence 9-cis-bacterioruberin and 13-cis-bacterioruberin decreased. The production of haloarchaeal carotenoids can be successfully optimized by changing nutritional conditions. Furthermore, carotenoid composition can be altered by modifying carbon source concentration. These natural compounds are very promising in food and nutraceutical industries.

Hypersaline environments as natural sources of microbes with potential applications in biotechnology: The case of solar evaporation systems to produce salt in Alicante County (Spain).

Extremophilic microbes show a unique metabolism due to the adaptations they display to deal with extreme environmental parameters characterizing the extreme ecosystems that they inhabit (high salt concentration, high temperatures, and extreme pH values, high exposure to solar radiation etc.). Halophilic microorganisms characterised and isolated from saltmarshes, brines, salted ponds, salty lagoons etc. have recently attracted attention due to their potential biotechnological applications (as whole cells used for different purposes like wastewater treatments, or their biomolecules: enzymes, antibiotics, carotenoids, bioplastics). Alicante county (southeast of Spain) accounts for a significant number of salty environments like coastal or inland salty ponds from where sodium chloride (NaCl)is obtained, marshes, salty lagoons, etc. The best system characterised so far from a microbiological point of view is "Salinas de Santa Pola", also termed "Salinas Bras del Port". However, there are many other salty environments to be explored, like the natural park of Torrevieja and la Mata lagoons, salty lagoon located in Calpe city or inland salted ponds like those located in the northwest of the county. This review summarises the most relevant biotechnological applications of halophilic microbes described up to now. In addition, special attention is focused on ecosystems such as the lagoons of Torrevieja or inland salt marshes as natural environments whose microbial biodiversity is worthy of being studied in search of new strains and species with the aim to analyze their potential biotechnological applications (pharmaceutical, food industry, biomedicine, etc.).

Biosynthesis of Silver Chloride Nanoparticles (AgCl-NPs) from Extreme Halophiles and Evaluation of Their Biological Applications.

The biosynthesis of nanoparticles (NPs) has gained an overwhelming interest due to their biological applications. However, NPs synthesis by pigmented extreme halophiles remains underexplored. The NPs synthesis using pigmented halophiles is inexpensive and less toxic than other processes. In this study, pigmented halophilic microorganisms (n = 77) were screened to synthesize silver chloride nanoparticles (AgCl-NPs) with silver nitrate as metal precursors, and their biological applications were assessed. The synthesis of AgCl-NPs was possible using the crude extract from cellular lysis (CECL) of six extreme halophiles. Two of the AgCl-NPs viz. AK2-NPs and MY6-NPs synthesized by the CECL of Haloferax alexandrinus RK_AK2 and Haloferax lucentense RK_MY6, respectively, exhibited antimicrobial, antioxidative, and anti-inflammatory activities. The surface plasmon resonance of the AgCl-NPs was determined with UV spectroscopy. XRD analysis of AK2-NPs and MY6-NPs confirmed the presence of silver in the form of chlorargyrite (silver chloride) having a cubic structure. The crystallite size of AK2-NPs and MY6-NPs, estimated with the Scherrer formula, was 115.81 nm and 137.50 nm. FTIR analysis verified the presence of diverse functional groups. Dynamic light-scattering analysis confirmed that the average size distribution of NPs was 71.02 nm and 117.36 nm for AK2-NPs and MY6-NPs, respectively, with monodisperse nature. The functional group in 1623-1641 cm-1 indicated the presence of protein ß-sheet structure and shifting of amino and hydroxyl groups from the pigmented CECL, which helps in capping and stabilizing nanoparticles. The study provides evidence that CECL of Haloferax species can rapidly synthesize NPs with unique characteristics and biological applications.

Assessment of Haloferax mediterranei Genome in Search of Copper-Molecular Machinery With Potential Applications for Bioremediation.

Heavy metals are essential micronutrients at low concentrations, serving as cofactors for relevant microbial enzymes (i.e., respiratory nitrate and nitrite reductases NADH dehydrogenase-2, amine oxidase, etc.), but they become harmful cellular intoxicants at significant low concentrations compared to other chemical compounds. The increasing need to incorporate bioremediation in the removal of heavy metals and other contaminants from wastewaters has led extremophiles to the spotlight of research. The haloarchaeon Haloferax mediterranei has promising physiological characteristics regarding bioremediation. However, little is known about how haloarchaea manage to resist high concentrations of heavy metals in the environment. The aim of this work is to develop bioinformatics research as the first step for further omics-based studies to shed light on copper metabolism in haloarchaea by analyzing H. mediterranei genome (strain ATCC 33500). To reach this aim, genome and protein databases have been consulted, and copper-related genes have been identified. BLAST analysis has been carried out to find similarities between copper resistance genes described from other microorganisms and H. mediterranei genes. Plausible copper importer genes, genes coding for siderophores, and copper exporters belonging to P1B-type ATPase group have been found apart from genes encoding copper chaperones, metal-responsive transcriptional regulators, and several proteins belonging to the cupredoxin superfamily: nitrite reductase, nitrous oxide reductases, cytochrome c oxidases, multicopper oxidases, and small blue copper proteins from the amicyanin/pseudoazurin families as halocyanins. As the presence of heavy metals causes oxidative stress, genes coding for proteins involved in antioxidant mechanisms have been also explored: thioredoxin, glutaredoxin, peroxiredoxin, catalase, and γ-glutamylcysteine as an analog of glutathione. Bioinformatic-based analysis of H. mediterranei genome has revealed a set of genes involved in copper metabolism that could be of interest for bioremediation purposes. The analysis of genes involved in antioxidative mechanisms against heavy metals makes it possible to infer the capability of H. mediterranei to synthesize inorganic polyphosphate granules against oxidative stress.

Controversy on the toxic nature of deep eutectic solvents and their potential contribution to environmental pollution.

Deep eutectic solvents (DES) are promising reaction media where interesting catalytic processes can be carried out. In theory, most of these mixtures are environmentally friendly, being an alternative to traditionally pollutant organic solvents used in several processes related to organic chemistry and biotechnology. However, recent studies show contradictory results regarding their toxicity. The method selected to perform toxicity studies could be significantly conditioned by some of the physical properties displayed by the DESs. Also, the metabolic capabilities of the organisms/cells used to monitor their toxicity are influenced by their physical properties. In this review, relevant physical-chemical properties for toxicity studies are summarized. The advantages/disadvantages of the used tests to monitor their toxicity and biodegradability in connection with the chosen organisms/cells are discussed, shedding light on their limitations. These findings could be taken as a starting point for designing more accurate DESs toxicity studies covering a wider spectrum of organisms and cells to be used as biomodels to monitor environmental pollution caused by DESs.

Halophilic Carotenoids and Breast Cancer: From Salt Marshes to Biomedicine.

Breast cancer is the leading cause of death among women worldwide. Over the years, oxidative stress has been linked to the onset and progression of cancer. In addition to the classical histological classification, breast carcinomas are classified into phenotypes according to hormone receptors (estrogen receptor-RE-/progesterone receptor-PR) and growth factor receptor (human epidermal growth factor receptor-HER2) expression. Luminal tumors (ER/PR-positive/HER2-negative) are present in older patients with a better outcome. However, patients with HER2-positive or triple-negative breast cancer (TNBC) (ER/PR/HER2-negative) subtypes still represent highly aggressive behavior, metastasis, poor prognosis, and drug resistance. Therefore, new alternative therapies have become an urgent clinical need. In recent years, anticancer agents based on natural products have been receiving huge interest. In particular, carotenoids are natural compounds present in fruits and vegetables, but algae, bacteria, and archaea also produce them. The antioxidant properties of carotenoids have been studied during the last years due to their potential in preventing and treating multiple diseases, including cancer. Although the effect of carotenoids on breast cancer during in vitro and in vivo studies is promising, clinical trials are still inconclusive. The haloarchaeal carotenoid bacterioruberin holds great promise to the future of biomedicine due to its particular structure, and antioxidant activity. However, much work remains to be performed to draw firm conclusions. This review summarizes the current knowledge on pre-clinical and clinical analysis on the use of carotenoids as chemopreventive and chemotherapeutic agents in breast cancer, highlighting the most recent results regarding the use of bacterioruberin from haloarchaea.

Personalized Diet in Obesity: A Quasi-Experimental Study on Fat Mass and Fat-Free Mass Changes.

Considering that the prevalence of overweight and obesity in Southeast of Spain is high, the aim of this work was to analyze the relation between the adherence to a personalized diet and the effectiveness of changes in the body composition in overweight and obese adults in this region. This quasi-experimental study presents the following selection criteria: attendance at the consultation between 2006 and 2012, subjects ≥ 19 years of age with overweight or obesity. In total, 591 overweight or obese individuals were involved in this study, attending 4091 clinic consultations in total. Most of the sample consisted of subjects who attended >3 consultations (>1.5 months), and were females aged 19-64 years who obtained clinically significant changes in fat mass (FM, ≥5%) and recommended changes in fat-free mass (FFM, ≥0%). Based on the results obtained and the experience gained from this research, the following recommendations are established: (i) record fat mass and fat-free mass index as a complement to body mass index; (ii) use FM and FFM to evaluate effectiveness of interventions with the aim of obtaining body composition changes; (iii) use personalized diet to achieve significant changes in FM and avoid non-recommended changes in FFM.

Ubiquitousness of Haloferax and Carotenoid Producing Genes in Arabian Sea Coastal Biosystems of India.

This study presents a comparative analysis of halophiles from the global open sea and coastal biosystems through shotgun metagenomes (n = 209) retrieved from public repositories. The open sea was significantly enriched with Prochlorococcus and Candidatus pelagibacter. Meanwhile, coastal biosystems were dominated by Marinobacter and Alcanivorax. Halophilic archaea Haloarcula and Haloquandratum, predominant in the coastal biosystem, were significantly (p < 0.05) enriched in coastal biosystems compared to the open sea. Analysis of whole genomes (n = 23,540), retrieved from EzBioCloud, detected crtI in 64.66% of genomes, while cruF was observed in 1.69% Bacteria and 40.75% Archaea. We further confirmed the viability and carotenoid pigment production by pure culture isolation (n = 1351) of extreme halophiles from sediments (n = 410 × 3) sampling at the Arabian coastline of India. All red-pigmented isolates were represented exclusively by Haloferax, resistant to saturated NaCl (6 M), and had >60% G + C content. Multidrug resistance to tetracycline, gentamicin, ampicillin, and chloramphenicol were also observed. Our study showed that coastal biosystems could be more suited for bioprospection of halophiles rather than the open sea.

Distribution of Denitrification among Haloarchaea: A Comprehensive Study.

Microorganisms from the Halobacteria class, also known as haloarchaea, inhabit a wide range of ecosystems of which the main characteristic is the presence of high salt concentration. These environments together with their microbial communities are not well characterized, but some of the common features that they share are high sun radiation and low availability of oxygen. To overcome these stressful conditions, and more particularly to deal with oxygen limitation, some microorganisms drive alternative respiratory pathways such as denitrification. In this paper, denitrification in haloarchaea has been studied from a phylogenetic point of view. It has been demonstrated that the presence of denitrification enzymes is a quite common characteristic in Halobacteria class, being nitrite reductase and nitric oxide reductase the enzymes with higher co-occurrence, maybe due to their possible role not only in denitrification, but also in detoxification. Moreover, copper-nitrite reductase (NirK) is the only class of respiratory nitrite reductase detected in these microorganisms up to date. The distribution of this alternative respiratory pathway and their enzymes among the families of haloarchaea has also been discussed and related with the environment in which they constitute the major populations. Complete denitrification phenotype is more common in some families like Haloarculaceae and Haloferacaceae, whilst less common in families such as Natrialbaceae and Halorubraceae.

In Silico Analysis of the Enzymes Involved in Haloarchaeal Denitrification.

During the last century, anthropogenic activities such as fertilization have led to an increase in pollution in many ecosystems by nitrogen compounds. Consequently, researchers aim to reduce nitrogen pollutants following different strategies. Some haloarchaea, owing to their denitrifier metabolism, have been proposed as good model organisms for the removal of not only nitrate, nitrite, and ammonium, but also (per)chlorates and bromate in brines and saline wastewater. Bacterial denitrification has been extensively described at the physiological, biochemical, and genetic levels. However, their haloarchaea counterparts remain poorly described. In previous work the model structure of nitric oxide reductase was analysed. In this study, a bioinformatic analysis of the sequences and the structural models of the nitrate, nitrite and nitrous oxide reductases has been described for the first time in the haloarchaeon model Haloferax mediterranei. The main residues involved in the catalytic mechanism and in the coordination of the metal centres have been explored to shed light on their structural characterization and classification. These results set the basis for understanding the molecular mechanism for haloarchaeal denitrification, necessary for the use and optimization of these microorganisms in bioremediation of saline environments among other potential applications including bioremediation of industrial waters.