Transcriptomic profiling of haloarchaeal denitrification through RNA-Seq analysis.

Denitrification, a crucial biochemical pathway prevalent among haloarchaea in hypersaline ecosystems, has garnered considerable attention in recent years due to its ecological implications. Nevertheless, the underlying molecular mechanisms and genetic regulation governing this respiration/detoxification process in haloarchaea remain largely unexplored. In this study, RNA-sequencing was used to compare the transcriptomes of the haloarchaeon Haloferax mediterranei under oxic and denitrifying conditions, shedding light on the intricate metabolic alterations occurring within the cell, such as the accurate control of the metal homeostasis. Furthermore, the investigation identifies several genes encoding transcriptional regulators and potential accessory proteins with putative roles in denitrification. Among these are bacterioopsin-like transcriptional activators, proteins harboring a domain of unknown function (DUF2249), and cyanoglobin. In addition, the study delves into the genetic regulation of denitrification, finding a regulatory motif within promoter regions that activates numerous denitrification-related genes. This research serves as a starting point for future molecular biology studies in haloarchaea, offering a promising avenue to unravel the intricate mechanisms governing haloarchaeal denitrification, a pathway of paramount ecological importance.IMPORTANCEDenitrification, a fundamental process within the nitrogen cycle, has been subject to extensive investigation due to its close association with anthropogenic activities, and its contribution to the global warming issue, mainly through the release of N2O emissions. Although our comprehension of denitrification and its implications is generally well established, most studies have been conducted in non-extreme environments with mesophilic microorganisms. Consequently, there is a significant knowledge gap concerning extremophilic denitrifiers, particularly those inhabiting hypersaline environments. The significance of this research was to delve into the process of haloarchaeal denitrification, utilizing the complete denitrifier haloarchaeon Haloferax mediterranei as a model organism. This research led to the analysis of the metabolic state of this microorganism under denitrifying conditions and the identification of regulatory signals and genes encoding proteins potentially involved in this pathway, serving as a valuable resource for future molecular studies.

Production of polyhydroxyalkanoates from hydrolysed rapeseed meal by Haloferax mediterranei.

Rapeseed meal (RSM) hydrolysate is a potential low-cost feedstock for the production of polyhydroxyalkanoates (PHAs) by the archaea, Haloferax mediterranei. Acidic and enzymatic hydrolysis were carried out to compare effectiveness. Enzymatic hydrolysis is more effective than acidic hydrolysis for fermentation substrate leading to increased PHA productivity. H. mediterranei didn't grow or produce PHA when acid hydrolysed RSM medium was present in proportions greater than 25% (vol.), potentially due to the effect of inhibitors such as furfural, hydroxymethylfurfural (HMF), etc. However, H. mediterranei was able to grow and produce PHA when using enzymatically hydrolysed RSM medium. The maximum PHA concentration of 0.512 g/L was found at 75% (vol.) in enzymatic RSM hydrolysate medium. The biopolymer obtained had improved thermal and mechanical properties compared to PHB homopolymer. RSM's potential as a low-cost alternative feedstock for improved PHA production under non-sterile conditions was successfully demonstrated, and its usage should be further explored.

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.

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.

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.

Optimising PHBV biopolymer production in haloarchaea via CRISPRi-mediated redirection of carbon flux.

The haloarchaeon Haloferax mediterranei is a potential strain for poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) production, yet the production yield and cost are the major obstacles hindering the use of this archaeal strain. Leveraging the endogenous type I-B CRISPR-Cas system in H. mediterranei, we develop a CRISPR-based interference (CRISPRi) approach that allows to regulate the metabolic pathways related to PHBV synthesis, thereby enhancing PHBV production. Our CRISPRi approach can downregulate the gene expression in a range of 25% to 98% depending upon the target region. Importantly, plasmid-mediated CRISPRi downregulation on the citrate synthase genes (citZ and gltA) improves the PHBV accumulation by 76.4% (from 1.78 to 3.14 g/L). When crRNA cassette integrated into chromosome, this further shortens the PHBV fermentation period and enhances PHA productivity by 165%. Our transcriptome analysis shows that repression of citrate synthase genes redirects metabolic flux from the central metabolic pathways to PHBV synthesis pathway. These findings demonstrate that the CRISPRi-based gene regulation is a transformative toolkit for fine-tuning the endogenous metabolic pathways in the archaeal system, which can be applied to not only the biopolymer production but also many other applications.

Analysis of Haloferax mediterranei Lrp Transcriptional Regulator.

Haloferax mediterranei is an extremely halophilic archaeon, able to live in hypersaline environments with versatile nutritional requirements, whose study represents an excellent basis in the field of biotechnology. The transcriptional machinery in Archaea combines the eukaryotic basal apparatus and the bacterial regulation mechanisms. However, little is known about molecular mechanisms of gene expression regulation compared with Bacteria, particularly in Haloarchaea. The genome of Hfx. mediterranei contains a gene, lrp (HFX_RS01210), which encodes a transcriptional factor belonging to Lrp/AsnC family. It is located downstream of the glutamine synthetase gene (HFX_RS01205), an enzyme involved in ammonium assimilation and amino acid metabolism. To study this transcriptional factor more deeply, the lrp gene has been homologously overexpressed and purified under native conditions by two chromatographic steps, namely nickel affinity and gel filtration chromatography, showing that Lrp behaves asa tetrameric protein of approximately 67 kDa. Its promoter region has been characterized under different growth conditions using bgaH as a reporter gene. The amount of Lrp protein was also analyzed by Western blotting in different nitrogen sources and under various stress conditions. To sum up, regarding its involvement in the nitrogen cycle, it has been shown that its expression profile does not change in response to the nitrogen sources tested. Differences in its expression pattern have been observed under different stress conditions, such as in the presence of hydrogen peroxide or heavy metals. According to these results, the Lrp seems to be involved in a general response against stress factors, acting as a first-line transcriptional regulator.

Functional analysis of Lsm protein under multiple stress conditions in the extreme haloarchaeon Haloferax mediterranei.

The Sm, like-Sm, and Hfq proteins belonging to the Sm superfamily of proteins are represented in all domains of life. These proteins are involved in several RNA metabolism pathways. The functions of bacterial Hfq and eukaryotic Sm proteins have been described, but knowledge about the in vivo functions of archaeal Sm proteins remains limited. This study aims to improve the understanding of Lsm proteins and their role using the haloarchaeon Haloferax mediterranei as a model microorganism. The Haloferax mediterranei genome contains one lsm gene that overlaps with the rpl37e gene. To determine the expression of lsm and rpl37e genes and the co-transcription of both, reverse transcription-polymerase chain reaction (RT-PCR) analyses were performed under different standard and stress conditions. The results suggest that the expression of lsm and rpl37e is constitutive. Co-transcription occurs at sub-optimal salt concentrations and temperatures, depending on the growth phase. The halophilic Lsm protein contains two Sm motifs, Sm1 and Sm2, and the sequence encoding the Sm2 motif also constitutes the promoter of the rpl37e gene. To investigate their biological functions, the lsm deletion mutant and the Sm1 motif deletion mutant, where the Sm2 motif remained intact, were generated and characterised. Comparison of the lsm deletion mutant, Sm1 deletion mutant, and the parental strain HM26 under standard and stress growth conditions revealed growth differences. Finally, swarming assays in complex and defined media showed greater swarming capacity in the deletion mutants.

Haloferax mediterranei Cells as C50 Carotenoid Factories.

Haloarchaea produce C50 carotenoids such as bacterioruberin, which are of biotechnological in-terest. This study aimed to analyze the effect of different environmental and nutritional conditions on the cellular growth and dynamics of carotenoids accumulation in Haloferax mediterranei. The maximum production of carotenoids (40 µg·mL-1) was obtained during the stationary phase of growth, probably due to nutrient-limiting conditions (one-step culture). By seven days of culture, 1 mL culture produced 22.4 mg of dry weight biomass containing 0.18 % (w/w) of carotenoids. On the other hand, carbon-deficient cultures (low C/N ratio) were observed to be optimum for C50 bacterioruberin production by Hfx. mediterranei, but negatively affected the growth of cells. Thus, a two-steps process was evaluated for optimum carotenoids yield. In the first step, a nutri-ent-repleted culture medium enabled the haloarchaea to produce biomass, while in the second step, the biomass was incubated under osmotic stress and in a carbon-deficient medium. Under the conditions used, the obtained biomass contained 0.27% (w/w) of carotenoids after seven days, which accounts for 58.49 µg·mL-1 of carotenoids for a culture with turbidity 14.0.

Production of Polyhydroxyalkanoates (PHA) by Haloferax mediterranei from Food Waste Derived Nutrients for Biodegradable Plastic Applications.

Polyhydroxyalkanoates (PHA) are a family of microbial polyesters that are used as biodegradable plastics in replacement of conventional plastics for various applications. However, the high production cost is the barrier for PHA market expansion. This study aimed to utilize food waste as low-cost feedstock to produce poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) by Haloferax mediterranei. The effects of acetate (Ac), propionate (Pr), butyrate (Bu), and the short-chain carboxylates derived from food waste were examined on the microbial growth and PHBV production. Results showed that a mixture of carboxylates provided a 55% higher PHBV yield than glucose. The food-waste-derived nutrients achieved the yields of 0.41 to 0.54 g PHBV/g Ac from initial loadings of 450 mg/l to 1,800 mg/l Ac of total carboxylates. And the consumption of individual carboxylate varied between different compositions of the carbon source. The present study demonstrates the potential of using food waste as feedstock to produce PHBV by Haloferax mediterranei, which can provide economic benefits to the current PHA industry. Meanwhile, it will also help promote organic waste reduction in landfills and waste management in general.

The metabolic pathways of polyhydroxyalkanoates and exopolysaccharides synthesized by Haloferax mediterranei in response to elevated salinity.

How polymer synthesis is mobilized or activated as a biological response of Haloferax mediterranei against hypertonic conditions remains largely unexplored. This study investigated the protein expression of H. mediterranei in response to high salinity by using isobaric tags for relative and absolute quantitation (iTRAQ)-based proteomic analysis. The microbes were harvested at end of fermentation at the NaCl salinity of 75 and 250 g L-1. Among the identified 2123 proteins, 170 proteins were differentially expressed. Gene ontology annotation revealed that the highest number of proteins was annotated in biological process category, which was responsible for metabolic process, cellular component and catalytic activity. Differentially expressed proteins were belonged to the class of response to stimulus as well as catalytic activity and binding. Under high salinity conditions, three pathways were established as key responses of PHA and EPS production to hypertonic pressure. Two overexpressed proteins, beta-ketoacyl-ACP reductase and 3-hydroxyacyl-CoA dehydrogenase, enhanced the synthesis of PHAs. The serine-pyruvate transaminase and serine-glyoxylate transaminase were upregulated, thereby increasing the conversion of glucose to PHA. Downregulated levels of sulfate-adenylyl transferase and adenylyl-sulfate kinase could cause diminished EPS synthesis. This study could contribute to better understanding of the proteomic mechanisms of the synthesized polymers in defending against salt stress. SIGNIFICANCE: Haloferax mediterranei, a family member of halophilic archaea, is well known for its fermentative production of poly-ß-hydroxyalkanoates (PHAs). PHAs are natural polymers that exhibit great potential in a wide range of applications such as a good alternative to petroleum-based plastics and the biocompatible material. For decades, the functional role of PHAs synthesized by H. mediterranei is deemed to be carbon and energy reservations. The finding proved that differential production of PHA and EPS in H. mediterranei exposed to elevated salinity was caused by differential protein expression. This is the first report on how PHA and EPS synthesized by H. mediterranei is mobilized as the response of increased salinity, contributing to the understanding of halophilic archaea's response to hypertonic stress and the precise control of fermentation production. Despite its advantages as a PHA cell factory, H. mediterranei synthesized EPS simultaneously, thereby lowering the maximum yield of PHA production. Overall, salinity can be used as a vital microbial fermentation parameter to obtain the highest harvest of PHA, as well as the lowest EPS synthesis in industrial fermentation.

Optimization of nitrogen source supply for enhanced biosynthesis and quality of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) by extremely halophilic archaeon Haloferax mediterranei.

The extreme halophilic archaeon, Haloferax mediterranei can accumulate polyhydroxyalkanoate (PHA) from different renewable resources. To enhance the biosynthesis and quality of PHA, H. mediterranei cultivation media was optimized at different C/N ratios using glucose as the main carbon source. Three sets of media (yeast extract [YE], NH4 Cl and combination of YE and NH4 Cl) were prepared at different nitrogen concentrations to achieve C/N ratios of 9, 20, and 35, respectively. The media containing YE (organic nitrogen source) produced a higher growth rate of H. mediterranei than NH4 Cl (inorganic source) at all tested C/N ratios. The highest PHA accumulation (18.4% PHA/cell dry mass) was achieved in a media that combined YE with NH4 Cl at a C/N ratio of 20. Analysis of the produced polymers revealed the production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBHV) with different 3-hydroxyvalerate (3HV) content. The polymers produced from YE and the combined media have greater 3HV content (10 mol%) than those polymers recovered from NH4 Cl (1.5 mol%). Resultingly, PHBHV from YE and the combined media displayed reduced melting points at 144°C. The nitrogen type/concentration was found to also have an impact on the molecular weights and polydispersity indices of the produced biopolymers. Furthermore, the tensile strengths were found to vary with the best tensile strength (14.4 MPa) being recorded for the polymer recovered from YE at C/N = 9. Interestingly, the tensile strength of PHBHV was significantly higher than petroleum-based polyethylene (13.5 MPa), making it a much more suitable bioplastic for industrial application.

New proposal of nitrogen metabolism regulation by small RNAs in the extreme halophilic archaeon Haloferax mediterranei.

The regulatory networks involved in the uptake and metabolism of different nitrogen sources in response to their availability are crucial in all organisms. Nitrogen metabolism pathways have been studied in detail in archaea such as the extreme halophilic archaeon Haloferax mediterranei. However, knowledge about nitrogen metabolism regulation in haloarchaea is very scarce, and no transcriptional regulators involved in nitrogen metabolism have been identified to date. Advances in the molecular biology field have revealed that many small RNAs (sRNAs) are involved in the regulation of a diverse metabolic pathways. Surprisingly, no studies on regulation mediated by sRNAs have focused on the response to environmental fluctuations in nitrogen in haloarchaea. To identify sRNAs involved in the transcriptional regulation of nitrogen assimilation genes in Haloferax mediterranei and, thus, propose a novel regulatory mechanism, RNA-Seq was performed using cells grown in the presence of two different nitrogen sources. The differential transcriptional expression analysis of the RNA-Seq data revealed differences in the transcription patterns of 102 sRNAs according to the nitrogen source, and the molecular functions, cellular locations and biological processes with which the target genes were associated were predicted. These results enabled the identification of four sRNAs that could be directly related to the regulation of genes involved in nitrogen metabolism. This work provides the first proposed regulatory mechanism of nitrogen assimilation-related gene expression by sRNAs in haloarchaea as an alternative to transcriptional regulation mediated by proteins.

Macroalgal biomass subcritical hydrolysates for the production of polyhydroxyalkanoate (PHA) by Haloferax mediterranei.

Non-conventional carbon sources, such as macroalgae, are sustainable alternatives for large-scale production of biopolymers. The present study examined macroalgae-derived carbohydrates, as carbon sources for the production of polyhydroxyalkanoates (PHAs) by Haloferax mediterranei. Simulants of the hydrolysates of seven different macroalgal biomasses were prepared and the PHA production was studied. A maximum biomass concentration with maximum PHA content was detected in medium prepared from green macroalgae. The highest cell dry weight and PHA concentrations were 3.8 ±â€¯0.2 g·L-1 and 2.2 ±â€¯0.12 g·L-1 respectively when Haloferax mediterranei was grown in 25% (w/w) of Ulva sp. hydrolysate, at 42 °C temperature and initial pH of 7.2. Poly(3-hydroxy-butyrate-co-3-hydroxyvalerate was the major PHA constituent. The present study demonstrated that Ulva sp. is a promising feedstock for PHA production.

Denitrifying haloarchaea within the genus Haloferax display divergent respiratory phenotypes, with implications for their release of nitrogenous gases.

Haloarchaea are extremophiles, generally thriving at high temperatures and salt concentrations, thus, with limited access to oxygen. As a strategy to maintain a respiratory metabolism, many halophilic archaea are capable of denitrification. Among them are members of the genus Haloferax, which are abundant in saline/hypersaline environments. Three reported haloarchaeal denitrifiers, Haloferax mediterranei, Haloferax denitrificans and Haloferax volcanii, were characterized with respect to their denitrification phenotype. A semi-automatic incubation system was used to monitor the depletion of electron acceptors and accumulation of gaseous intermediates in batch cultures under a range of conditions. Out of the species tested, only H. mediterranei was able to consistently reduce all available N-oxyanions to N2 , while the other two released significant amounts of NO and N2 O, which affect tropospheric and stratospheric chemistries respectively. The prevalence and magnitude of hypersaline ecosystems are on the rise due to climate change and anthropogenic activity. Thus, the biology of halophilic denitrifiers is inherently interesting, due to their contribution to the global nitrogen cycle, and potential application in bioremediation. This work is the first detailed physiological study of denitrification in haloarchaea, and as such a seed for our understanding of the drivers of nitrogen turnover in hypersaline systems.

Biosynthesis and Characterization of Polyhydroxyalkanoates with Controlled Composition and Microstructure.

Volatile fatty acids (VFA) C2:0 to C6:0 were used as the sole carbon source for poly(3-hydroxybutyrate- co-3-hydroxyvalerate) (PHBV) production with controllable composition and microstructure in Haloferax mediterranei. Feeding carbon-even VFA gave >90 mol % poly(3-hydroxybutyrate) (3HB) PHBV, while carbon-odd VFA generated >87 mol % poly(3-hydroxyvalerate) (3HV) PHBV. Bespoke random, block, and blend copolymers with 0-100 mol % 3HV were synthesized using C4:0/C5:0 mixtures. The copolymer 3HV fraction is proportional to the %C5:0 in the feed mixture, allowing control over copolymer composition. Microstructure depends on the substrate addition order: cofeeding generated random copolymers, while sequential feeding created block and blend copolymers. On average, the PHBV had an ultrahigh molecular weight of 3 × 106 g/mol. 3HV rich copolymers showed lower melting temperatures, enhanced elasticity, and ductility. H. mediterranei is ideal for large-scale production of PHBV due to its inherent bioprocessing advantages, while control over the composition and microstructure of PHBV will facilitate the production of biopolymers capable of meeting industrial criteria for specific applications.

Production of the copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) with varied composition using different nitrogen sources with Haloferax mediterranei.

The extreme haloarchaea Haloferax mediterranei accumulates poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) without the need for specific precursors. In this study, growth kinetics and PHBV synthesis were characterised under nitrogen-excess and nitrogen-limiting conditions in ammonium and, for the first time, nitrate. With excess nitrogen, ammonium and nitrate cultures generated 10.7 g/L biomass containing 4.6 wt% PHBV and 5.6 g/L biomass with 9.3 wt% PHBV, respectively. Copolymer composition varied with the nitrogen source used: PHBV from ammonium cultures had 16.9 mol% 3-hydroxyvalerate (HV), while PHBV from nitrate cultures contained 12.5 mol% HV. Nitrogen limitation was achieved with carbon-to-nitrogen (C/N) molar ratios of 25 or higher. Nitrogen limitation reduced biomass generation and polymer concentration, but polymer accumulation increased to 6.6 and 9.4% for ammonium and nitrate, respectively, with C/N 42. PHBV composition was also affected and cultures with lower C/N ratios produced richer HV polymers. Copolymer formation was not a uniform process: HV was only detected after a minimum accumulation of 0.45 g/L PHB and lasted for a maximum of 48 h. The understanding of copolymer synthesis and the influence of culture conditions such as the nitrogen source will help in designing novel strategies for the production of PHBV with more regular structure and material properties.

Systematic Analysis of Lysine Acetylation in the Halophilic Archaeon Haloferax mediterranei.

Lysine acetylation is a reversible and highly regulated post-translational modification that plays a critical role in regulating many aspects of cellular processes, both in bacteria and in eukaryotes. However, this modification has not been systematically studied in archaea. Herein, we report the lysine acetylome of a model haloarchaeon, Haloferax mediterranei. Using immunoaffinity enrichment and LC-MS/MS analysis, we identified 1017 acetylation sites in 643 proteins, accounting for 17.3% of the total proteins in this haloarchaeon. Bioinformatics analysis indicated that lysine acetylation mainly distributes in cytoplasm (94%) and participates in protein biosynthesis and carbon metabolism. Specifically, the acetylation of key enzymes in PHBV biosynthesis further suggested that acetylation plays a key role in the energy and carbon storage. In addition, a survey of the acetylome revealed a universal rule in acetylated motifs: a positively charged residue (K, R, or H) located downstream of acetylated lysine at the positions +1, +2, or +3. Interestingly, we identified acetylation in several replication initiation proteins Cdc6; mutation on the acetylated site of Cdc6A destroyed the Autonomous Replication Sequence (ARS) activity of its adjacent origin oriC1. Our study indicates that lysine acetylation is an abundant modification in H. mediterranei, and plays key roles in the processes of replication, protein biosynthesis, central metabolism, and carbon storage. This acetylome of H. mediterranei provides opportunities to explore the physiological role of acetylation in halophilic archaea.

A one-stage cultivation process for the production of poly-3-(hydroxybutyrate-co-hydroxyvalerate) from olive mill wastewater by Haloferax mediterranei.

Olive mill wastewater (OMW), a highly polluting waste from the olive oil industry, was utilized as sole carbon source for the production of polyhydroxyalkanoate (PHA) by extremely halophilic Haloferax Mediterranei (H. mediterranei) in a one stage cultivation step. H. mediterranei showed remarkable cell growth and tolerated the inhibitory effect of polyphenols present in medium containing 25% of OMW. H. mediterranei cultivation conditions were optimized in medium containing 15% OMW by investigating several parameters that affect the production of PHA. The highest polymer yield (0.2g/L) and PHA content (43% PHA/cell dry mass) were achieved at 37°C, 170rpm and 22% salt concentration. Analysis of the produced PHA revealed the production of copolyester poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBHV) containing 6.5mol% 3-hydroxyvalerate (3HV). The production of PHBHV was observed without the need for fermentation step or adding external carbon source. The PHBHV displayed reduced melting points at 140.1°C and 154.4°C when compared to homopolymer polyhydroxybutyrate.

Enoyl-CoA hydratase mediates polyhydroxyalkanoate mobilization in Haloferax mediterranei.

Although polyhydroxyalkanoate (PHA) accumulation and mobilization are one of the most general mechanisms for haloarchaea to adapt to the hypersaline environments with changeable carbon sources, the PHA mobilization pathways are still not clear for any haloarchaea. In this study, the functions of five putative (R)-specific enoyl-CoA hydratases (R-ECHs) in Haloferax mediterranei, named PhaJ1 to PhaJ5, respectively, were thoroughly investigated. Through gene deletion and complementation, we demonstrated that only certain of these ECHs had a slight contribution to poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) biosynthesis. But significantly, PhaJ1, the only R-ECH that is associated with PHA granules, was shown to be involved in PHA mobilization in this haloarchaeon. PhaJ1 catalyzes the dehydration of (R)-3-hydroxyacyl-CoA, the common product of PHA degradation, to enoyl-CoA, the intermediate of the ß-oxidation cycle, thus could link PHA mobilization to ß-oxidation pathway in H. mediterranei. This linkage was further indicated from the up-regulation of the key genes of ß-oxidation under the PHA mobilization condition, as well as the obvious inhibition of PHA degradation upon inhibition of the ß-oxidation pathway. Interestingly, 96% of phaJ-containing haloarchaeal species possess both phaC (encoding PHA synthase) and the full set genes of ß-oxidation, implying that the mobilization of carbon storage in PHA through the ß-oxidation cycle would be general in haloarchaea.