Exploring Andean High-Altitude Lake Extremophiles through Advanced Proteotyping.

Quickly identifying and characterizing isolates from extreme environments is currently challenging while very important to explore the Earth's biodiversity. As these isolates may, in principle, be distantly related to known species, techniques are needed to reliably identify the branch of life to which they belong. Proteotyping these environmental isolates by tandem mass spectrometry offers a rapid and cost-effective option for their identification using their peptide profiles. In this study, we document the first high-throughput proteotyping approach for environmental extremophilic and halophilic isolates. Microorganisms were isolated from samples originating from high-altitude Andean lakes (3700-4300 m a.s.l.) in the Chilean Altiplano, which represent environments on Earth that resemble conditions on other planets. A total of 66 microorganisms were cultivated and identified by proteotyping and 16S rRNA gene amplicon sequencing. Both the approaches revealed the same genus identification for all isolates except for three isolates possibly representing not yet taxonomically characterized organisms based on their peptidomes. Proteotyping was able to indicate the presence of two potentially new genera from the families of Paracoccaceae and Chromatiaceae/Alteromonadaceae, which have been overlooked by 16S rRNA amplicon sequencing approach only. The paper highlights that proteotyping has the potential to discover undescribed microorganisms from extreme environments.

Isoptericola haloaureus sp. nov., a dimorphic actinobacterium isolated from mangrove sediments of southeast India, implicating biosaline agricultural significance through nitrogen fixation and salt tolerance genes.

Strain MP-1014T, an obligate halophilic actinobacterium, was isolated from the mangrove soil of Thandavarayancholanganpettai, Tamil Nadu, India. A polyphasic approach was utilized to explore its phylogenetic position completely. The isolate was Gram-positive, filamentous, non-motile, and coccoid in older cultures. Ideal growth conditions were seen at 30 °C and pH 7.0, with 5% NaCl (W/V), and the DNA G + C content was 73.3%. The phylogenic analysis of this strain based upon 16S rRNA gene sequence revealed 97-99.8% similarity to the recognized species of the genus Isoptericola. Strain MP-1014T exhibits the highest similarity to I. sediminis JC619T (99.7%), I. chiayiensis KCTC19740T (98.9%), and subsequently to I. halotolerans KCTC19646T (98.6%), when compared with other members within the Isoptericola genus (< 98%). ANI scores of strain MP-1014T are 86.4%, 84.2%, and 81.5% and dDDH values are 59.7%, 53.6%, and 34.8% with I. sediminis JC619T, I. chiayiensis KCTC19740T and I. halotolerans KCTC19646T respectively. The major polar lipids of the strain MP-1014T were phosphatidylinositol, phosphatidylglycerol, diphosphotidylglycerol, two unknown phospholipids, and glycolipids. The predominant respiratory menaquinones were MK9 (H4) and MK9 (H2). The major fatty acids were anteiso-C15:0, anteiso-C17:0, iso-C14:0, C15:0, and C16:0. Also, initial genome analysis of the organism suggests it as a biostimulant for enhancing agriculture in saline environments. Based on phenotypic and genetic distinctiveness, the strain MP-1014 T represents the novel species of the genus Isoptericola assigned Isoptericola haloaureus sp. nov., is addressed by the strain MP-1014 T, given its phenotypic, phylogenetic, and hereditary uniqueness. The type strain is MP-1014T [(NCBI = OP672482.1 = GCA_036689775.1) ATCC = BAA 2646T; DSMZ = 29325T; MTCC = 13246T].

Halobacterium salinarum: Life with more than a grain of salt.

Halobacterium salinarum is a halophilic (salt-loving) archaeon that grows in salt concentrations near or at saturation. Although isolated from salted fish a century ago, it was the 1971 discovery of bacteriorhodopsin, the light-driven proton pump, that raised interest in Hbt. salinarum across a range of disciplines, including biophysics, chemistry, molecular evolution and biotechnology. Hbt. salinarum have since contributed to numerous discoveries, such as advances in membrane protein structure determination and the first example of a non-eukaryal glycoprotein. Work on Hbt. salinarum, one of the species used to define Archaea, has also elucidated molecular workings in the third domain. Finally, Hbt. salinarum presents creative solutions to the challenges of life in high salt.

Heterologous Expression and Partial Characterization of a Putative Opine Dehydrogenase from a Metagenomic Sequence of Desulfohalobium retbaense.

The aim of this research was to prove the function of the putative opine dehydrogenase from Desulfohalobium retbaense and to characterize the enzyme in terms of functional and kinetic parameters. A putative opine dehydrogenase was identified from a metagenomic library by a sequence-based technique search of the metagenomic library, and afterward was successfully heterologously produced in Escherichia coli. In order to examine its potential for applications in the synthesis of secondary amines, first the substrate specificity of the enzyme towards different amino donors and amino acceptors was determined. The highest affinity was observed towards small amino acids, preferentially L-alanine, and when it comes to α-keto acids, pyruvate proved to be a preferential amino acceptor. The highest activity was observed at pH 6.5 in the absence of salts. The enzyme showed remarkable stability in a wide range of experimental conditions, such as broad pH stability (from 6.0-11.0 after 30 min incubation in buffers at a certain pH), stability in the presence of NaCl up to 3.0 M for 24 h, it retained 80 % of the initial activity after 1 h incubation at 45 °C, and 65 % of the initial activity after 24 h incubation in 30 % dimethyl sulfoxide.

Exploring halophilic environments as a source of new antibiotics.

Microbial natural products from microbes in extreme environments, including haloarchaea, and halophilic bacteria, possess a huge capacity to produce novel antibiotics. Additionally, enhanced isolation techniques and improved tools for genomic mining have expanded the efficiencies in the antibiotic discovery process. This review article provides a detailed overview of known antimicrobial compounds produced by halophiles from all three domains of life. We summarize that while halophilic bacteria, in particular actinomycetes, contribute the vast majority of these compounds the importance of understudied halophiles from other domains of life requires additional consideration. Finally, we conclude by discussing upcoming technologies- enhanced isolation and metagenomic screening, as tools that will be required to overcome the barriers to antimicrobial drug discovery. This review highlights the potential of these microbes from extreme environments, and their importance to the wider scientific community, with the hope of provoking discussion and collaborations within halophile biodiscovery. Importantly, we emphasize the importance of bioprospecting from communities of lesser-studied halophilic and halotolerant microorganisms as sources of novel therapeutically relevant chemical diversity to combat the high rediscovery rates. The complexity of halophiles will necessitate a multitude of scientific disciplines to unravel their potential and therefore this review reflects these research communities.

Shouchella tritolerans sp. nov., a facultative anaerobic bacterium isolated from marine sediments.

An alkali, salt, and thermo-tolerant strain designated FJAT-45399T was isolated from marine sediment in Fujian Province, China. Strain FJAT-45399T was Gram-stain-positive, rod-shaped, and facultatively aerobic. It shared high 16S rRNA gene sequence similarities with the members of the genus Shouchella. Further, the phylogenetic and phylogenomic analysis also suggested strain FJAT-45399T clustered with the members of the genus Shouchella. Growth of strain FJAT-45399T was observed at 15-55 °C (optimum 45-50 °C), pH 7.0-13.0 (optimum 9.0) and 0-15% (w/v) NaCl (optimum 2%). It contained MK-7 as the menaquinone. The polar lipids were diphosphatidylglycerol (DPG), phosphatidylglycerol (PG) and an unidentified glycolipid (UGL) and lipid (UL). The major fatty acids (> 10%) were C16:0 (22.8%), iso-C15:0 (21.3%), and anteiso-C15:0 (14.0%). The genomic DNA G + C content was 44.5%. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between strain FJAT-45399T and the most closely related type strain Shouchella clausii DSM 8716T (ANI 94.1% and dDDH 55.4%) were both below the cut-off level for species delineation. Based on the above results, strain FJAT-45399T represents a novel species of the genus Shouchella, for which the name Shouchella tritolerans sp. nov., is proposed. The type strain is FJAT-45399T (= GDMCC 1.3098T = JCM 35613T).

Marinobacter iranensis sp. nov., a slightly halophilic bacterium from a hypersaline lake.

A novel halophilic bacterium, strain 71-iT, was isolated from Inche-Broun hypersaline lake in Golestan province, in the north of Iran. It was a Gram-stain-negative, non-endospore forming, rod-shaped bacterium. It grew at 4-40 °C (optimum 30 °C), pH 6.0-11.0 (optimum pH 7.5) and with 0.5-15 % (w/v) NaCl [optimum 3 % (w/v) NaCl]. The results of phylogenetic analyses based on the 16S rRNA gene sequence comparison indicated its affiliation to the genus Marinobacter and the low percentage of identity with the most closely related species (97.5 %), indicated its placement as a novel species within this genus. Digital DNA-DNA hybridization (dDDH) values and average nucleotide identity (ANI) analyses of this strain against closely related species confirmed its condition of novel taxon. On the other hand, the percentage of the average amino acid identity (AAI) affiliated strain 71-iT within the genus Marinobacter. The DNA G+C content of this isolate was 57.7 mol%. The major fatty acids were C16 : 0 and C16 : 1ω7c and/or C16 : 1 ω6c. Ubiquinone-9 was the major isoprenoid quinone and diphosphatidylglycerol (DPG), phosphatidylglycerol (PG) and phosphatidylethanolamine (PE) were the main polar lipids of this strain. On the basis of the phylogenomic and phenotypic (including chemotaxonomic) features, we propose strain 71-iT (= IBRC M 11023T = CECT 30160T = LMG 29252T) as the type strain of a novel species within the genus Marinobacter, with the name Marinobacter iranensis sp. nov. Genomic detections of this strain in various metagenomic databases indicate that it is a relatively abundant species in environments with low salinities (approximately 5 % salinity), but not in hypersaline habitats with high salt concentrations.

Identification of novel halophilic/halotolerant bacterial species producing compatible solutes.

Ectoine and hydroxyectoine are compatible solutes with enormous potential for use in the medical and cosmetic industries. Considering the excellent osmoprotective properties of these compatible solutes, we investigate the presence of four compatible solutes (ectoine, hydroxyectoine, proline, and glutamic acid) quantitatively by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in forty-five halophilic/halotolerant bacterial isolates. We determined ectoine production by Marinibacillus sp., Nesterenkonia xinjiangensis, Halobacillus sp., Bacillus patagoniensis, Virgibacillus picturae, Halomonas neptunia, Bacillus patagoniensis, Gracilibacillus sp., Thalassobacillus devorans, Microbacterium sp., Nesterenkonia sp., and Bacillus agaradhaerens, and this production was NaCl dependent. Additionally, the production of hydroxyectoine was observed in six bacterial isolates (Nesterenkonia xinjiangensis, Halobacillus sp., Halomonas neptunia, Thalassobacillus devorans, Nesterenkonia sp., and Bacillus agaradhaerens) which was NaCl and temperature dependent. The study identified new bacterial isolates producing ectoine or hydroxyectoine. While the ectoine production in many different Bacillus members and a few Nesterenkonia have been documented before, ectoine production by Bacillus patagoniensis and Nesterenkonia xinjiangensis has not been shown so far. Further, ectoine production by a member of the genus Thalassobacillus (Thalassobacillus devorans) was demonstrated experimentally for the first time. The findings reported in the study may serve as a basis for the large-scale production of ectoine and hydroxyectoine in the future.

Molecular evolution steered structural adaptations in the DNA polymerase III α subunit of halophilic bacterium Salinibacter ruber.

A significant portion of the earth has a salty environment, and the literature on bacterial survival mechanisms in salty environments is limited. During molecular evolution, halophiles increase acidic amino acid residues on their protein surfaces which leads to a negatively charged surface potential that helps them to maintain the protein integrity and protect them from denaturation by competing with salt ions. Through protein family analysis, we have investigated the molecular-level adaptive features of DNA polymerase III's catalytic subunit (alpha) and its structure-function relationship. This study throws light on the novel understanding of halophilic bacterial replication and the molecular basis of salt adaptation. Comparisons of the amino acid contents and electronegativity of halophilic and mesophilic bacterial proteins revealed adaptations that allow halophilic bacteria to thrive in high salt concentrations. A significantly lower isoelectric point of halophilic bacterial proteins indicates the acidic nature. Also, an abundance of disordered regions in halophiles suggests the requirement of the salt ions that play a crucial role in their stable protein folding. Despite having similar topology, mesophilic and halophilic proteins, a set of very prominent molecular modifications was observed in the alpha subunit of halophiles.

Cytotoxic effects of halophilic archaea metabolites on ovarian cancer cell lines.

BACKGROUND: Ovarian cancer is one of the most frequent and deadly gynaecological cancers, often resistant to platinum-based chemotherapy, the current standard of care. Halophilic microorganisms have been shown to produce a large variety of metabolites, some of which show toxicity to various cancer cell lines. However, none have yet been shown to be active against ovarian cancer cells. Here, we examined the effects of metabolites secreted by the halophilic archaea Halorhabdus rudnickae and Natrinema salaciae on various cancer cell lines, including ovarian cancer cell lines. RESULTS: 1H NMR analyses of Hrd. rudnickae and Nnm. salaciae culture supernatants contain a complex mixture of metabolites that differ between species, and even between two different strains of the same species, such as Hrd. rudnickae strains 64T and 66. By using the MTT and the xCELLigence RTCA assays, we found that the secreted metabolites of all three halophilic strains expressed cytotoxicity to the ovarian cancer cell lines, especially A2780, as well as its cisplatin-resistant derivative A2780cis, in a dose-dependent manner. The other tested cell lines A549, HepG2, SK-OV-3 and HeLa were only minimally, or not at all affected by the archaeal metabolites, and this was only seen with the MTT assay. CONCLUSIONS: The halophilic archaea Hrd. rudnickae and Nnm. salaciae, isolated from a Polish salt mine and Lake Medee in the Mediterranean Sea, respectively, secrete metabolites that are active against ovarian cancer cells, including those that are resistant to cisplatin. This opens potential new possibilities for the treatment of these frequent and deadly gynaecological cancers.

Antimicrobial potential of secondary metabolites and DNA gyrase B blocking molecules produced by a halophilic bacterium Virgibacillus salarius (MML1918).

AIM: The present study aims to determine the antimicrobial potential of Virgibacillus salairus (MML1918) against human pathogens and its in-vitro and in-silico properties. METHODS AND RESULTS: In this present study, totally 63 halophilic bacterial cultures were obtained and cultivated in nutrient broth medium containing 8% NaCl and the metabolites, were extracted using ethyl acetate and screened for their antimicrobial property by cell viability assay against 12 pathogenic bacteria and fungi, among 63 halophilic bacteria the Vir. salaries (MML1918) found to be the best producer for secondary metabolites production against clinical pathogens. The optimization of growth for important physiochemical parameters was characterized and applied for different production media and based on its highest activity as 17.5 ± .07 mm zone of inhibition (ZOI) for Bacillus cereus followed by 17.5 ± 00 mm ZOI for Staphylococcus aureus, the production medium ATCC1097 was chosen for mass production. The mass production of secondary metabolites from Vir. salaries MML1918 was carried out in a fermenter under controlled conditions and crude metabolites was extracted and condensed. The antimicrobial activity of crude metabolites showed B. cereus (19.3 ± 0.5 mm ZOI), Staph. aureus, and Candida albicans (18.3 ± 0.5 mm ZOI) as the highest ZOI in production media for halophilic bacteria ATCC1097. Further, the gas chromatography-mass spectrometry analysis showed 24 compounds present in crude metabolites. Among the 24 compounds, four molecules were found to be important based on molecule percentage in crude and structural similarity. The molecular docking studies show that the selected four molecules effectively bind with the active region DNA gyrase B. CONCLUSION: Virgibacillus salarius (MML1918) effectively showed antimicrobial activity against several pathogenic organisms and can be employed as a suitable candidate for producing novel antimicrobial agents.

Extracellular proteases from halophiles: diversity and application challenges.

Halophilic extracellular proteases offer promising application in various fields. Information on these prominent proteins including the synthesizing organisms, biochemical properties, domain organisation, purification, and application challenges has never been covered in recent reviews. Although extracellular proteases from bacteria pioneered the study of proteases in halophiles, progress is being made in proteases from halophilic archaea. Recent advances in extracellular proteases from archaea revealed that archaeal proteases are more robust and applicable. Extracellular proteases are composed of domains that determine their mechanisms of action. The intriguing domain structure of halophilic extracellular proteases consists of N-terminal domain, catalytic domain, and C-terminal extension. The role of C-terminal domains varies among different organisms. A high diversity of C-terminal domains would endow the proteases with diverse functions. With the development of genomics, culture-independent methods involving heterologous expression, affinity chromatography, and in vitro refolding are deployed with few challenges on purification and presenting novel research opportunities. Halophilic extracellular proteases have demonstrated remarkable potentials in industries such as detergent, leather, peptide synthesis, and biodegradation, with desirable properties and ability to withstand harsh industrial processes. KEY POINTS: • Halophilic extracellular proteases have robust properties suitable for applications. • A high diversity of C-terminal domains may endow proteases with diverse properties. • Novel protease extraction methods present novel application opportunities.

Bioengineering of air-filled protein nanoparticles by genetic and chemical functionalization.

BACKGROUND: Various bacteria and archaea, including halophilic archaeon Halobacterium sp. NRC-1 produce gas vesicle nanoparticles (GVNPs), a unique class of stable, air-filled intracellular proteinaceous nanostructures. GVNPs are an attractive tool for biotechnological applications due to their readily production, purification, and unique physical properties. GVNPs are spindle- or cylinder-shaped, typically with a length of 100 nm to 1.5 µm and a width of 30-250 nm. Multiple monomeric subunits of GvpA and GvpC proteins form the GVNP shell, and several additional proteins are required as minor structural or assembly proteins. The haloarchaeal genetic system has been successfully used to produce and bioengineer GVNPs by fusing several foreign proteins with GvpC and has shown various applications, such as biocatalysis, diagnostics, bioimaging, drug delivery, and vaccine development. RESULTS: We demonstrated that native GvpC can be removed in a low salt buffer during the GVNP purification, leaving the GvpA-based GVNP's shell intact and stable under physiological conditions. Here, we report a genetic engineering and chemical modification approach for functionalizing the major GVNP protein, GvpA. This novel approach is based on combinatorial cysteine mutagenesis within GvpA and genetic expansion of the N-terminal and C-terminal regions. Consequently, we generated GvpA single, double, and triple cysteine variant libraries and investigated the impact of mutations on the structure and physical shape of the GVNPs formed. We used a thiol-maleimide chemistry strategy to introduce the biotechnological relevant activity by maleimide-activated streptavidin-biotin and maleimide-activated SpyTag003-SpyCatcher003 mediated functionalization of GVNPs. CONCLUSION: The merger of these genetic and chemical functionalization approaches significantly extends these novel protein nanomaterials' bioengineering and functionalization potential to assemble catalytically active proteins, biomaterials, and vaccines onto one nanoparticle in a modular fashion.

Novel Antimicrobial Peptides from Saline Environments Active against E. faecalis and S. aureus: Identification, Characterisation and Potential Usage.

Microorganisms inhabiting saline environments have been known for decades as producers of many valuable bioproducts. These substances include antimicrobial peptides (AMPs), the most recognizable of which are halocins produced by halophilic Archaea. As agents with a different modes of action from that of most conventionally used antibiotics, usually associated with an increase in the permeability of the cell membrane as a result of a formation of channels and pores, AMPs are a currently promising object of research focused on the investigation of antibiotics with non-standard modes of action. The aim of this study was to investigate antimicrobial activity against multidrug-resistant human pathogens of three peptides, which were synthetised based on sequences identified in metagenomes from saline environments. The investigations were performed against Enterococcus faecalis, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Escherichia coli and Candida albicans. Subsequently, the cytotoxicity and haemolytic properties of the tested peptides were verified. An in silico analysis of the interaction of the tested peptides with molecular targets for reference antibiotics was also carried out in order to verify whether or not they can act in a similar way. The P1 peptide manifested the growth inhibition of E. faecalis at a MIC50 of 32 µg/mL and the P3 peptide at a MIC50 of 32 µg/mL was shown to inhibit the growth of both E. faecalis and S. aureus. Furthermore, the P1 and P3 peptides were shown to have no cytotoxic or haemolytic activity against human cells.

Microbial adaptation to different environmental conditions: molecular perspective of evolved genetic and cellular systems.

Microorganisms are ubiquitous on Earth and can inhabit almost every environment. In a complex heterogeneous environment or in face of ecological disturbance, the microbes adjust to fluctuating environmental conditions through a cascade of cellular and molecular systems. Their habitats differ from cold microcosms of Antarctica to the geothermal volcanic areas, terrestrial to marine, highly alkaline zones to the extremely acidic areas and freshwater to brackish water sources. The diverse ecological microbial niches are attributed to the versatile, adaptable nature under fluctuating temperature, nutrient availability and pH of the microorganisms. These organisms have developed a series of mechanisms to face the environmental changes and thereby keep their role in mediate important ecosystem functions. The underlying mechanisms of adaptable microbial nature are thoroughly investigated at the cellular, genetic and molecular levels. The adaptation is mediated by a spectrum of processes like natural selection, genetic recombination, horizontal gene transfer, DNA damage repair and pleiotropy-like events. This review paper provides the fundamentals insight into the microbial adaptability besides highlighting the molecular network of microbial adaptation under different environmental conditions.

Microbial communities of halite deposits and other hypersaline environments.

Large regions of Earth's surface are underlain by salt deposits that evaporated from ancient oceans and are populated by extreme halophilic microbes. While the microbiology of ancient evaporites has been well studied, the ecology of halite deposits and more recently formed NaCl "salticle" stalactite structures (speleothems) in a Triassic halite mine are less well characterized. The microbiome of Kilroot Salt Mine was profiled using conventional and enhanced culturing techniques. From this, 89 halophilic archaeal isolates from six known genera, and 55 halophilic or halotolerant bacterial isolates from 18 genera were obtained. Culture-independent metagenomic approaches also revealed that culturing techniques were inadvertently biased toward specific taxa, and the need for optimized isolation procedures are required to enhance cultivation diversity. Speleothems formed from saturated brines are unique structures that have the potential to entomb haloarchaea cells for thousands of years within fluid inclusions. The presence of such fluid inclusions, alongside the high abundance of genes related to glycerol metabolism, biofilm formation, and persister cell formation is highly suggestive of an environmental niche that could promote longevity and survivability. Finally, previous studies reporting the discovery of novel biocatalysts from the Kilroot mine microbiome, suggests that this environment may be an untapped source of chemical diversity with high biodiscovery potential.

Tannery wastewater treatment coupled with bioenergy production in upflow microbial fuel cell under saline condition.

Tannery industry handling huge amount of leather materials release immense amount of saline organic content wastewater. The present research was focused on the treatment of tannery industrial wastewater in UMFC (upflow microbial fuel cell) under saline condition (4%). The UMFC reactor was operated at different organic load (OL) such as 0.6, 1.2, 1.8 and 2.4 gCOD/L respectively. Total chemical oxygen demand (TCOD) removal at 0.6, 1.2, 1.8 gCOD/L was 87 ± 1.2%, 91 ± 1.2% and 93 ± 1.8% respectively. Soluble chemical oxygen demand (SCOD) removal in UMFC at 0.6, 1.2, 1.8 gCOD/L was 85 ± 0.6%, 88 ± 1.2% and 91 ± 1.8% respectively. Total suspended solids (TSS) removal was 49%, 78%, 81% at 0.6, 1.2, 1.8 gCOD/L OL in UMFC. Further, raise in OL to 2.4 gCOD/L showed decrease in TCOD, SCOD (80% and 72%) and TSS (60%) removal. Maximal power production of 854 mV with corresponding PD (power density) of 462 mW/m2 and CD (current density) of 523 mA/m2 was registered at 1.8 gCOD/L OL in UMFC. Increase in OL to 2.4 gCOD/L revealed decline in energy production to 810 mV with PD (385 mW/m2) and CD (438 mA/m2) in UMFC. Maximal bioaccumulation of chromium (95%) was recorded at 1.8 gCOD/L OL. Among different OL used 1.8 gCOD/L OL was optimum for the treatment of tannery wastewater and energy production. Bacterial community analysis in anode of UMFC revealed the dominance of promising electrogenic halophilic strains such as Ochrobactrum, Marinobacter, Rhodococcus and Bacillus in all the OL. Thus, the research clearly revealed the efficacy of halophilic consortium to treat the saline tannery wastewater coupled with bioenergy production in UMFC.

Abundance of integrons in halophilic bacteria.

Integrons are genetic platforms used for expressing open reading frames (ORFs) arranged in gene cassettes. Excision and integration of gene cassettes are controlled by their associated integron integrase (IntI). Using IntegronFinder software, we analyzed all complete halophilic genomes available in the HaloDom database, along with selected partial halophilic genomes. We identified 18 new complete bacterial integrons and 46 clusters of attC sites lacking a neighboring integron integrase (CALINs). Different classes of insertion sequences (ISs) were also identified within and near integrons and CALINs, with an abundance of IS1182 elements and different ISs that can presumably mobilize adjacent genomic structures. Different promoters of intI genes (PintI) showed nearby binding sites for arginine repressor (ArgR), raising the possibility that IntI expression and recombination activity are regulated by these proteins. Our findings revealed the existence of new integrons in halophilic bacteria with possible adaptive roles.

Metabolic Potential of Halophilic Filamentous Fungi-Current Perspective.

Salty environments are widely known to be inhospitable to most microorganisms. For centuries salt has been used as a food preservative, while highly saline environments were considered uninhabited by organisms, and if habited, only by prokaryotic ones. Nowadays, we know that filamentous fungi are widespread in many saline habitats very often characterized also by other extremes, for example, very low or high temperature, lack of light, high pressure, or low water activity. However, fungi are still the least understood organisms among halophiles, even though they have been shown to counteract these unfavorable conditions by producing multiple secondary metabolites with interesting properties or unique biomolecules as one of their survival strategies. In this review, we focused on biomolecules obtained from halophilic filamentous fungi such as enzymes, pigments, biosurfactants, and osmoprotectants.

Identification of Multi-Potent Protein Subtilisin A from halophilic bacterium Bacillus firmus VE2.

Screening of halophiles with antimicrobial activity in saltpan soil samples from Nagapattinam district, Tamil Nadu, revealed isolate VE-2 as the most potent, identified as Bacillus firmus strain VE-2 through 16s rRNA gene sequencing. It had an optimum growth condition (OD 3.1) and antimicrobial protein (AMP) production (450 µg/mL) at 37 °C, pH 8, 25% NaCl, and 36 h incubation. SDS-PAGE analysis of the purified AMP showed the molecular weight of 36 kDa. HPLC analysis of the purified AMP showed different amino acids, such as asparagines, alanine, lysine, proline, threonine, glycine, cysteine, serine, aspartic acid leucine, and valine. Further characterization and identification using FT-IR, 2D-PAGE, MALDI-TOF, and in-silico analysis showed that the isolated AMP had the highest similarity to Subtilisin-A. It showed antibacterial activity against clinical bacterial pathogens like S. aureus, S. pyogenes, C. diphtheria, E. coli, and P. aeruginosa with the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration of 2.5 µg/mL and 20 µg/mL and also against various fungal pathogens such as A. niger, A. flavus, C. albicans, C. tropicalis and C. parapsilosis with the MIC and minimum fungicidal concentrations of 1.25-80 µg/mL. The purified AMP had excellent antioxidant potential, showed a scavenging effect against DPPH and Nitric oxide radicals, and displayed anticancer activity against HeLa cell lines with the IC50 values 53 µg/mL. Hence, the purified bioactive antimicrobial peptides (AMP) could also be used in anticancer therapies.