Halocin H4 is activated through cleavage by halolysin HlyR4.

Halocins are antimicrobial peptides secreted by haloarchaea capable of inhibiting the growth of other haloarchaea or bacteria. Halocin H4 (HalH4) is secreted by the model halophilic archaeon Haloferax mediterranei ATCC 33500. Despite attempts to express halH4 heterologously in Escherichia coli and subsequent careful renaturation procedures commonly employed for haloarchaeal proteins, no active halocin was obtained. However, it was discovered that the antihaloarchaeal activity of this halocin could be activated through cleavage by halolysin R4 (HlyR4), a serine protease also secreted by Hfx. mediterranei ATCC 33500. Replacement of the cysteine at the number 115 amino acid with glycine and deletion of the internal trans-membrane region (15 aa) markedly abolished HalH4's antihaloarchaeal activity. Compared to the N-terminus, the C-terminal amino acid sequence was found to be more crucial for HalH4 to exert its antihaloarchaeal activity. Mass spectrometry analysis revealed that the biologically active antihaloarchaeal peptide produced after hydrolytic cleavage by HlyR4 was the C-terminus of HalH4, suggesting a potential mechanism of action involving pore formation within competitor species' cell membranes. Taken together, this study offers novel insights into the interplay between halocins and secreted proteases, as well as their contribution to antagonistic interaction within haloarchaea. IMPORTANCE: The antihaloarchaeal function of halocin H4 (HalH4) can be activated by extracellular proteases from haloarchaea, as demonstrated in this study. Notably, we report the first instance of halocin activation through proteolytic cleavage, highlighting its significance in the field. The C-terminus of HalH4 (CTH4) has been identified as the antihaloarchaeal peptide present in hydrolysates generated by HlyR4. The CTH4 exhibited inhibitory activity against a range of haloarchaeal species (Haloarchaeobius spp., Haloarcula spp., Haloferax spp., Halorubellus spp., and Halorubrum spp.), as well as selected bacterial species (Aliifodinibius spp. and Salicola spp.), indicating its broad-spectrum inhibitory potential across domains. The encoding gene of halocin HalH4, halH4, from the model halophilic archaeon Haloferax mediterranei ATCC 33500 can be expressed in Escherichia coli without codon optimization.

Genome-wide transcriptional response to silver stress in extremely halophilic archaeon Haloferax alexandrinus DSM 27206 T.

BACKGROUND: The extremely halophilic archaeon Haloferax (Hfx.) alexandrinus DSM 27206 T was previously documented for the ability to biosynthesize silver nanoparticles while mechanisms underlying its silver tolerance were overlooked. In the current study, we aimed to assess the transcriptional response of this haloarchaeon to varying concentrations of silver, seeking a comprehensive understanding of the molecular determinants underpinning its heavy metal tolerance. RESULTS: The growth curves confirmed the capacity of Hfx. alexandrinus to surmount silver stress, while the SEM-EDS analysis illustrated the presence of silver nanoparticles in cultures exposed to 0.5 mM silver nitrate. The RNA-Seq based transcriptomic analysis of Hfx. alexandrinus cells exposed to 0.1, 0.25, and 0.5 mM silver nitrate revealed the differential expression of multiple sets of genes potentially employed in heavy-metal stress response, genes mostly related to metal transporters, basic metabolism, oxidative stress response and cellular motility. The RT-qPCR analysis of selected transcripts was conducted to verify and validate the generated RNA-Seq data. CONCLUSIONS: Our results indicated that copA, encoding the copper ATPase, is essential for the survival of Hfx. alexandrinus cells in silver-containing saline media. The silver-exposed cultures underwent several metabolic adjustments that enabled the activation of enzymes involved in the oxidative stress response and impairment of the cellular movement capacity. To our knowledge, this study represents the first comprehensive analysis of gene expression in halophillic archaea facing increased levels of heavy metals.

Simultaneous purification and characterization of detergent-stable, solvent-tolerant haloextremozymes protease and lipase from Haloferax sp. strain GUBF 2.

Industrial important proteases and lipases are in increasing demand for various biotechnological applications. In the present study, the concomitantly produced protease and lipase by Haloferax sp. strain GUBF 2 were simultaneously purified as a heterogeneous lipase (45 and 66 kDa) and homogeneous protease (180 kDa); with 28.3 and 31.36 fold purity, respectively using Sephadex G-200. The aforementioned extremozymes were active at pH 3-13, 20-80 °C, 1-5 M NaCl, with optimal activity at pH 6, 70 °C, and 3 M NaCl, thus exhibiting attributes of true haloextremozymes. The Km and Vmax of purified lipase were 3.47 mM and 16.2 U/mL, while protease were 3.29 mg/mL and 28.5 U/mL, respectively. FTIR bands corresponding to the vibrations of amide II and amide III were detected in haloextremozymes which could perhaps be used to determine the secondary structure of the purified proteins. Furthermore, the activity of both enzymes was stimulated by Ca2+ and inhibited by 10 mM Hg2+ and phenylmethyl sulphonyl fluoride (PMSF). Additionally, these haloextremozymes are stable in the presence of detergent additives and organic solvents. In addition, purified protease displayed 74.3 ± 4.85% in-vitro blood clot dissolution activity. Conclusively this study revealed the key features, unusual properties, and possible biomedical applications of detergent-stable and organic solvent-tolerant haloextremozymes from Haloferax sp. strain GUBF 2 to date unexplored.

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.

Production and characterization of a bioactive extracellular homopolysaccharide produced by Haloferax sp. BKW301.

The production of extracellular polysaccharides (EPS) by haloarchaeal members, with novel and unusual physicochemical properties, is of special importance and has the potential for extensive biotechnological exploitation. An extremely halophilic archaeon, Haloferax sp. BKW301 (GenBank Accession No. KT240044) isolated from a solar saltern of Baksal, West Bengal, India has been optimized for the production of EPS under batch culture. It produced a considerable amount (5.95 g/L) of EPS in the medium for halophiles with 15% NaCl, 3% glucose, 0.5% yeast extract, and 6% inoculum under shake flask culture at 120 rpm. The purified EPS, a homopolymer of galactose as revealed by chromatographic methods and Fourier-transform infrared spectroscopy, is noncrystalline (CIxrd , 0.82), amorphous, and could emulsify hydrocarbons like kerosene, petrol, xylene, and so forth. Moreover, the polymer is highly thermostable (up to 420°C) and displayed pseudoplastic rheology. Biologically, the EPS was able to scavenge DPPH (2,2-diphenyl-1-picrylhydrazyl) radical efficiently and inhibit the proliferation of the Huh-7 cell line at an IC50 value of 6.25 µg/ml with a Hill coefficient of 0.844. Large-scale production of this thermostable, pseudoplastic homopolysaccharide, therefore, could find suitable applications in industry and biotechnology.

Physicochemical characterization and emulsifying properties of a novel exopolysaccharide produced by haloarchaeon Haloferax mucosum.

The physicochemical characterization and emulsifying functional properties of a novel exopolysaccharide (EPS) produced by haloarchaea Haloferax mucosum (DSM 27191) were investigated. This biopolymer has a high molecular weight of 152 kDa and important protein content of 10%. Different culture media compositions were investigated taking the ATCC 2185 medium as a base and supplementing with varying concentrations of yeast extract and glucose or sucrose as carbon sources to produce the EPS in a liquid medium. The highest EPS production (7.15 ±â€¯0.44 g/L) was obtained at 96 h. EPS aqueous dispersions showed a non-Newtonian rheological behavior which was well fitted to the Cross equation. The EPS (at 0.32% w/w) was capable of stabilizing water-in-oil emulsions with different nonpolar solvents, including n-hexane, kerosene, chloroform, castor oil and mineral oil. EPS retained its emulsifying activity after to be incubated for one hour in a wide range of temperatures (25, 40, 70 and 100 °C), pH (4, 6.5, 7 and 12) and NaCl concentrations (0, 2.0 and 4.0 M). The viscoelastic behavior and stability of hexane-in-water emulsion were examined through oscillatory shear measurements.

Specificity of protein-DNA interactions in hypersaline environment: structural studies on complexes of Halobacterium salinarum oxidative stress-dependent protein hsRosR.

Interactions between proteins and DNA are crucial for all biological systems. Many studies have shown the dependence of protein-DNA interactions on the surrounding salt concentration. How these interactions are maintained in the hypersaline environments that halophiles inhabit remains puzzling. Towards solving this enigma, we identified the DNA motif recognized by the Halobactrium salinarum ROS-dependent transcription factor (hsRosR), determined the structure of several hsRosR-DNA complexes and investigated the DNA-binding process under extreme high-salt conditions. The picture that emerges from this work contributes to our understanding of the principles underlying the interplay between electrostatic interactions and salt-mediated protein-DNA interactions in an ionic environment characterized by molar salt concentrations.

Division plane placement in pleomorphic archaea is dynamically coupled to cell shape.

One mechanism for achieving accurate placement of the cell division machinery is via Turing patterns, where nonlinear molecular interactions spontaneously produce spatiotemporal concentration gradients. The resulting patterns are dictated by cell shape. For example, the Min system of Escherichia coli shows spatiotemporal oscillation between cell poles, leaving a mid-cell zone for division. The universality of pattern-forming mechanisms in divisome placement is currently unclear. We examined the location of the division plane in two pleomorphic archaea, Haloferax volcanii and Haloarcula japonica, and showed that it correlates with the predictions of Turing patterning. Time-lapse analysis of H. volcanii shows that divisome locations after successive rounds of division are dynamically determined by daughter cell shape. For H. volcanii, we show that the location of DNA does not influence division plane location, ruling out nucleoid occlusion. Triangular cells provide a stringent test for Turing patterning, where there is a bifurcation in division plane orientation. For the two archaea examined, most triangular cells divide as predicted by a Turing mechanism; however, in some cases multiple division planes are observed resulting in cells dividing into three viable progeny. Our results suggest that the division site placement is consistent with a Turing patterning system in these archaea.

Discovery of bacteriorhodopsins in Haloarchaeal species isolated from Indian solar salterns: deciphering the role of the N-terminal residues in protein folding and functional expression.

Interesting optical and photochemical properties make microbial rhodopsin a promising biological material suitable for various applications, but the cost-prohibitive nature of production has limited its commercialization. The aim of this study was to explore the natural biodiversity of Indian solar salterns to isolate natural bacteriorhodopsin (BR) variants that can be functionally expressed in Escherichia coli. In this study, we report the isolation, functional expression and purification of BRs from three pigmented haloarchaea, wsp3 (water sample Pondicherry), wsp5 and K1T isolated from two Indian solar salterns. The results of the 16S rRNA data analysis suggest that wsp3, wsp5 and K1T are novel strains belonging to the genera Halogeometricum, Haloferax and Haloarcula respectively. Overall, the results of our study suggest that 17 N-terminal residues, that were not included in the gene annotation of the close sequence homologues, are essential for functional expression of BRs. The primary sequence, secondary structural content, thermal stability and absorbance spectral properties of these recombinant BRs are similar to those of the previously reported Haloarcula marismortui HmBRI. This study demonstrates the cost-effective, functional expression of BRs isolated from haloarchaeal species using E. coli as an expression host and paves the way for feasibility studies for future applications.

A Cobalamin Activity-Based Probe Enables Microbial Cell Growth and Finds New Cobalamin-Protein Interactions across Domains.

Understanding the factors that regulate microbe function and microbial community assembly, function, and fitness is a grand challenge. A critical factor and an important enzyme cofactor and regulator of gene expression is cobalamin (vitamin B12). Our knowledge of the roles of vitamin B12 is limited, because technologies that enable in situ characterization of microbial metabolism and gene regulation with minimal impact on cell physiology are needed. To meet this need, we show that a synthetic probe mimic of B12 supports the growth of B12-auxotrophic bacteria and archaea. We demonstrate that a B12 activity-based probe (B12-ABP) is actively transported into Escherichia coli cells and converted to adenosyl-B12-ABP akin to native B12 Identification of the proteins that bind the B12-ABP in vivo in E. coli, a Rhodobacteraceae sp. and Haloferax volcanii, demonstrate the specificity for known and novel B12 protein targets. The B12-ABP also regulates the B12 dependent RNA riboswitch btuB and the transcription factor EutR. Our results demonstrate a new approach to gain knowledge about the role of B12 in microbe functions. Our approach provides a powerful nondisruptive tool to analyze B12 interactions in living cells and can be used to discover the role of B12 in diverse microbial systems.IMPORTANCE We demonstrate that a cobalamin chemical probe can be used to investigate in vivo roles of vitamin B12 in microbial growth and regulation by supporting the growth of B12 auxotrophic bacteria and archaea, enabling biological activity with three different cell macromolecules (RNA, DNA, and proteins), and facilitating functional proteomics to characterize B12-protein interactions. The B12-ABP is both transcriptionally and translationally able to regulate gene expression analogous to natural vitamin B12 The application of the B12-ABP at biologically relevant concentrations facilitates a unique way to measure B12 microbial dynamics and identify new B12 protein targets in bacteria and archaea. We demonstrate that the B12-ABP can be used to identify in vivo protein interactions across diverse microbes, from E. coli to microbes isolated from naturally occurring phototrophic biofilms to the salt-tolerant archaea Haloferax volcanii.

Isolation and characterization from solar salterns of North Algeria of a haloarchaeon producing a new halocin.

Halophilic archaea, thriving in hypersaline environments, synthesize antimicrobial substances with an unknown role, called halocins. It has been suggested that halocin production gives transient competitive advantages to the producer strains and represents one of the environmental factors influencing the microbial community composition. Herein, we report on the antibacterial activity of a new haloarchaeon selected from solar salterns of the northern coast of Algeria. A total of 81 halophilic strains, isolated from the microbial consortia, were screened for the production of antimicrobial compounds by interspecies competition test and against a collection of commercial haloarchaea. On the basis of the partial 16S rRNA sequencing, the most efficient halocin producer was recognized as belonging to Haloferax (Hfx) sp., while the best indicator microorganism, showing high sensitivity toward halocin, was related to Haloarcula genus. The main morphological, physiological and biochemical properties of Hfx were investigated and a partial purification of the produced halocin was allowed to identify it as a surface membrane protein with a molecular mass between 30 and 40 kDa. Therefore, in this study, we isolated a new strain belonging to Haloferax genus and producing a promising antimicrobial compound useful for applications in health and food industries.

Activity-guided separation and characterization of new halocin HA3 from fermented broth of Haloferax larsenii HA3.

Haloferax larsenii HA3 was able to grow optimally in HS medium containing 15% NaCl, at pH 7.2 and 42 °C in aerobic conditions. Strain HA3 was found to be round shape, Gram-negative, catalase-positive, sensitive to bile acid, and resistant to chloramphenicol, and could not utilize arginine. The lipid profile revealed the presence of glycerol diether moiety (GDEM) suggesting Haloarchaea characteristics. Phylogenetic analysis based on 16S rRNA gene sequence similarities showed that it was closely related to H. larsenii ZJ206. Interestingly, strain HA3 was found to produce halocin HA3 which was purified using ultrafiltration and chromatography. It was found to be stable up to 80 °C, pH 2.0-10.0, organic solvents, surfactants, and detergents tested. However, the activity of halocin HA3 was completely reduced in the presence of proteinase K and trypsin. It was found to be halocidal against H. larsenii HA10, rupturing cell boundary and leading to cell death. The molecular weight of halocin HA3 was found to be ~13 kDa and MALDI-TOF MS/MS analysis suggested no homology with known halocins. The N-terminal ten amino-acid residues, NH2MNLGIILETN-COOH, suggested a new/novel halocin. These properties of halocin HA3 may be applicable for control of Haloarchaea in environments and salted foods.

Nitrate reduction in Haloferax alexandrinus: the case of assimilatory nitrate reductase.

Haloferax alexandrinus Strain TM JCM 10717T = IFO 16590T is an extreme halophilic archaeon able to produce significant amounts of canthaxanthin. Its genome sequence has been analysed in this work using bioinformatics tools available at Expasy in order to look for genes encoding nitrate reductase-like proteins: respiratory nitrate reductase (Nar) and/or assimilatory nitrate reductase (Nas). The ability of the cells to reduce nitrate under aerobic conditions was tested. The enzyme in charge of nitrate reduction under aerobic conditions (Nas) has been purified and characterised. It is a monomeric enzyme (72 ± 1.8 kDa) that requires high salt concentration for stability and activity. The optimum pH value for activity was 9.5. Effectiveness of different substrates, electron donors, cofactors and inhibitors was also reported. High nitrite concentrations were detected within the culture media during aerobic/microaerobic cells growth. The main conclusion from the results is that this haloarchaeon reduces nitrate aerobically thanks to Nas and may induce denitrification under anaerobic/microaerobic conditions using nitrate as electron acceptor. The study sheds light on the role played by haloarchaea in the biogeochemical cycle of nitrogen, paying special attention to nitrate reduction processes. Besides, it provides useful information for future attempts on microecological and biotechnological implications of haloarchaeal nitrate reductases.

Anaerobic Metabolism in Haloferax Genus: Denitrification as Case of Study.

A number of species of Haloferax genus (halophilic archaea) are able to grow microaerobically or even anaerobically using different alternative electron acceptors such as fumarate, nitrate, chlorate, dimethyl sulphoxide, sulphide and/or trimethylamine. This metabolic capability is also shown by other species of the Halobacteriaceae and Haloferacaceae families (Archaea domain) and it has been mainly tested by physiological studies where cell growth is observed under anaerobic conditions in the presence of the mentioned compounds. This work summarises the main reported features on anaerobic metabolism in the Haloferax, one of the better described haloarchaeal genus with significant potential uses in biotechnology and bioremediation. Special attention has been paid to denitrification, also called nitrate respiration. This pathway has been studied so far from Haloferax mediterranei and Haloferax denitrificans mainly from biochemical point of view (purification and characterisation of the enzymes catalysing the two first reactions). However, gene expression and gene regulation is far from known at the time of writing this chapter.

Isolation and characterization of extremely halophilic CO-oxidizing Euryarchaeota from hypersaline cinders, sediments and soils and description of a novel CO oxidizer, Haloferax namakaokahaiae Mke2.3T, sp. nov.

The phylogenetic affiliations of organisms responsible for aerobic CO oxidation in hypersaline soils and sediments were assessed using media containing 3.8 M NaCl. CO-oxidizing strains of the euryarchaeotes, Haloarcula, Halorubrum, Haloterrigena and Natronorubrum, were isolated from the Bonneville Salt Flats (UT) and Atacama Desert salterns (Chile). A halophilic euryarchaeote, Haloferax strain Mke2.3(T), was isolated from Hawai'i Island saline cinders. Haloferax strain Mke2.3(T) was most closely related to Haloferax larsenii JCM 13917(T) (97.0% 16S rRNA sequence identity). It grew with a limited range of substrates, and oxidized CO at a headspace concentration of 0.1%. However, it did not grow with CO as a sole carbon and energy source. Its ability to oxidize CO, its polar lipid composition, substrate utilization and numerous other traits distinguished it from H. larsenii JCM 13917(T), and supported designation of the novel isolate as Haloferax namakaokahaiae Mke2.3(T), sp. nov (= DSM 29988, = LMG 29162). CO oxidation was also documented for 'Natronorubrum thiooxidans' HG1 (Sorokin, Tourova and Muyzer 2005), N. bangense (Xu, Zhou and Tian 1999) and N. sulfidifaciens AD2(T) (Cui et al. 2007). Collectively, these results established a previously unsuspected capacity for extremely halophilic aerobic CO oxidation, and indicated that the trait might be widespread among the Halobacteriaceae, and occur in a wide range of hypersaline habitats.

Description of a halocin-producing Haloferax larsenii HA1 isolated from Pachpadra salt lake in Rajasthan.

Haloarchaea grow in the extreme environment, such as high salt concentration, and secrete antimicrobial peptides known as halocins. Identification of Haloferax larsenii strain HA1 was carried out using biochemical and molecular methods. Strain HA1 was found as a strict aerobe, catalase positive and Gram negative. It was able to grow optimally at 15 % NaCl (w/v), 42 °C and pH 7.2. Strain HA1 was sensitive to bile acid, was resistant to chloramphenicol and could not utilize arginine. Halocin, produced by strain HA1, was stable up to 100 °C and in a pH range of 5.0-9.0. Antimicrobial activity was not affected by organic solvents, surfactants and detergents, but it was completely lost in the presence of proteinase K, suggesting proteinaceous nature of the compound. It was halocidal against indicator strain Hfx. larsenii HA10. The molecular weight of halocin HA1 was found to be ~14 kDa. These properties of halocin HA1 may be applicable to the preservation of salted foods.

Carotenoids from Haloarchaea and Their Potential in Biotechnology.

The production of pigments by halophilic archaea has been analysed during the last half a century. The main reasons that sustains this research are: (i) many haloarchaeal species possess high carotenoids production availability; (ii) downstream processes related to carotenoid isolation from haloarchaea is relatively quick, easy and cheap; (iii) carotenoids production by haloarchaea can be improved by genetic modification or even by modifying several cultivation aspects such as nutrition, growth pH, temperature, etc.; (iv) carotenoids are needed to support plant and animal life and human well-being; and (v) carotenoids are compounds highly demanded by pharmaceutical, cosmetic and food markets. Several studies about carotenoid production by haloarchaea have been reported so far, most of them focused on pigments isolation or carotenoids production under different culture conditions. However, the understanding of carotenoid metabolism, regulation, and roles of carotenoid derivatives in this group of extreme microorganisms remains mostly unrevealed. The uses of those haloarchaeal pigments have also been poorly explored. This work summarises what has been described so far about carotenoids production by haloarchaea and their potential uses in biotechnology and biomedicine. In particular, new scientific evidence of improved carotenoid production by one of the better known haloarchaeon (Haloferax mediterranei) is also discussed.

Complete genome sequence of Haloferax gibbonsii strain ARA6, a potential producer of polyhydroxyalkanoates and halocins isolated from Araruama, Rio de Janeiro, Brasil.

Haloferax gibbonsii strain ARA6 is a haloarchaea isolated from saline saltern samples from Vermelha lake, located in Araruama region, Rio de Janeiro, Brazil. Its genome displays 66,2% G+C content and is composed by one circular chromosome of 2,945,391 bp and four circular plasmids comprising 993,063 bp. This genomic information shows H. gibbonsii's potential for biotechnological applications and can also contribute to assign evolutionary traits in the genus Haloferax.

Perchlorate and halophilic prokaryotes: implications for possible halophilic life on Mars.

In view of the finding of perchlorate among the salts detected by the Phoenix Lander on Mars, we investigated the relationships of halophilic heterotrophic microorganisms (archaea of the family Halobacteriaceae and the bacterium Halomonas elongata) toward perchlorate. All strains tested grew well in NaCl-based media containing 0.4 M perchlorate, but at the highest perchlorate concentrations, tested cells were swollen or distorted. Some species (Haloferax mediterranei, Haloferax denitrificans, Haloferax gibbonsii, Haloarcula marismortui, Haloarcula vallismortis) could use perchlorate as an electron acceptor for anaerobic growth. Although perchlorate is highly oxidizing, its presence at a concentration of 0.2 M for up to 2 weeks did not negatively affect the ability of a yeast extract-based medium to support growth of the archaeon Halobacterium salinarum. These findings show that presence of perchlorate among the salts on Mars does not preclude the possibility of halophilic life. If indeed the liquid brines that may exist on Mars are inhabited by salt-requiring or salt-tolerant microorganisms similar to the halophiles on Earth, presence of perchlorate may even be stimulatory when it can serve as an electron acceptor for respiratory activity in the anaerobic Martian environment.

Accumulation of polyhydroxyalkanoates by halophilic archaea isolated from traditional solar salterns of India.

Extremely halophilic archaeal isolates obtained from brine and sediment samples of solar salterns of Goa and Tamil Nadu, India were screened for accumulation of polyhydroxyalkanoates (PHA). Seven polymer accumulating haloarchaeal strains (TN4, TN5, TN6, TN7, TN9, TN10 and BBK2) were selected based on their growth and intensity of fluorescence when grown on 20 % NaCl synthetic medium supplemented with 2 % glucose and incorporated with Nile red dye. The polymer was quantified by conversion of PHA to crotonic acid which gave a characteristic absorption maxima at 235 nm. On the basis of phenotypic and genotypic characterization the cultures TN4, TN5, TN6, TN7, TN10 and BBK2 were grouped under genus Haloferax whereas isolate TN9 was grouped under the genus Halogeometricum. Growth kinetics and polymer accumulation studies revealed that the culture Halogeometricum borinquense strain TN9 accumulates PHA maximally at the mid-log phase, i.e. 5th day of growth (approx. 14 wt% PHA of CDW). Analysis of the polymer by IR, (1)H NMR and (13)C NMR confirmed it to be a homopolymer of 3-hydroxybutyrate.