Understanding Fungi in Glacial and Hypersaline Environments.

Hypersaline waters and glacial ice are inhospitable environments that have low water activity and high concentrations of osmolytes. They are inhabited by diverse microbial communities, of which extremotolerant and extremophilic fungi are essential components. Some fungi are specialized in only one of these two environments and can thrive in conditions that are lethal to most other life-forms. Others are generalists, highly adaptable species that occur in both environments and tolerate a wide range of extremes. Both groups efficiently balance cellular osmotic pressure and ion concentration, stabilize cell membranes, remodel cell walls, and neutralize intracellular oxidative stress. Some species use unusual reproductive strategies. Further investigation of these adaptations with new methods and carefully designed experiments under ecologically relevant conditions will help predict the role of fungi in hypersaline and glacial environments affected by climate change, decipher their stress resistance mechanisms and exploit their biotechnological potential.

Low-temperature features of the psychrophilic chaperonin from Pseudoalteromonas haloplanktis.

Chaperonins from psychrophilic bacteria have been shown to exist as single-ring complexes. This deviation from the standard double-ring structure has been thought to be a beneficial adaptation to the cold environment. Here we show that Cpn60 from the psychrophile Pseudoalteromonas haloplanktis (Ph) maintains its double-ring structure also in the cold. A strongly reduced ATPase activity keeps the chaperonin in an energy-saving dormant state, until binding of client protein activates it. Ph Cpn60 in complex with co-chaperonin Ph Cpn10 efficiently assists in protein folding up to 55 °C. Moreover, we show that recombinant expression of Ph Cpn60 can provide its host Escherichia coli with improved viability under low temperature growth conditions. These properties of the Ph chaperonin may make it a valuable tool in the folding and stabilization of psychrophilic proteins.

Characterization of selected phages for biocontrol of food-spoilage pseudomonads.

Pseudomonas spp., such as P. fluorescens group, P. fragi, and P. putida, are the major psychrophilic spoilage bacteria in the food industry. Bacteriophages (phages) are a promising tool for controlling food-spoilage and food-poisoning bacteria; however, there are few reports on phages effective on food-spoilage bacteria such as Pseudomonas spp. In this study, 12 Pseudomonas phages were isolated from chicken and soil samples. Based on the host range and lytic activity at 30 °C and 4 °C and various combinations of phages, phages vB_PflP-PCS4 and vB_PflP-PCW2 were selected to prepare phage cocktails to control Pseudomonas spp. The phage cocktail consisting of vB_PflP-PCS4 and vB_PflP-PCW2 showed the strongest lytic activity and retarded regrowth of P. fluorescens and P. putida at 30 °C, 8 °C, and 4 °C at a multiplicity of infection of 100. Nucleotide sequence analysis of the genomic DNA indicated that vB_PflP-PCS4 and vB_PflP-PCW2 phages were lytic phages of the Podoviridae family and lacked tRNA, toxin, or virulence genes. A novel endolysin gene was found in the genomic DNA of phage vB_PflP-PCS4. The results of this study suggest that the phage cocktail consisting of vB_PflP-PCS4 and vB_PflP-PCW2 is a promising tool for the biocontrol of psychrophilic food-spoilage pseudomonads during cold storage and distribution.

Autochthonous psychrophilic hydrocarbonoclastic bacteria and its ecological function in contaminated cold environments.

Petroleum hydrocarbon (PH) pollution has mostly been caused by oil exploration, extraction, and transportation activities in colder regions, particularly in the Arctic and Antarctic regions, where it serves as a primary source of energy. Due to the resilience feature of nature, such polluted environments become the realized ecological niches for a wide community of psychrophilic hydrocarbonoclastic bacteria (PHcB). In contrast, to other psychrophilic species, PHcB is extremely cold-adapted and has unique characteristics that allow them to thrive in greater parts of the cold environment burdened with PHs. The stated group of bacteria in its ecological niche aids in the breakdown of litter, turnover of nutrients, cycling of carbon and nutrients, and bioremediation. Although such bacteria are the pioneers of harsh colder environments, their growth and distribution remain under the influence of various biotic and abiotic factors of the environment. The review discusses the prevalence of PHcB community in colder habitats, the metabolic processes involved in the biodegradation of PH, and the influence of biotic and abiotic stress factors. The existing understanding of the PH metabolism by PHcB offers confirmation of excellent enzymatic proficiency with high cold stability. The discovery of more flexible PH degrading strategies used by PHcB in colder environments could have a significant beneficial outcome on existing bioremediation technologies. Still, PHcB is least explored for other industrial and biotechnological applications as compared to non-PHcB psychrophiles. The present review highlights the pros and cons of the existing bioremediation technologies as well as the potential of different bioaugmentation processes for the effective removal of PH from the contaminated cold environment. Such research will not only serve to investigate the effects of pollution on the basic functional relationships that form the cold ecosystem but also to assess the efficacy of various remediation solutions for diverse settings and climatic conditions.

Biochemical and Structural Characterization of a Novel Psychrophilic Laccase (Multicopper Oxidase) Discovered from Oenococcus oeni 229 (ENOLAB 4002).

Recently, prokaryotic laccases from lactic acid bacteria (LAB), which can degrade biogenic amines, were discovered. A laccase enzyme has been cloned from Oenococcus oeni, a very important LAB in winemaking, and it has been expressed in Escherichia coli. This enzyme has similar characteristics to those previously isolated from LAB as the ability to oxidize canonical substrates such as 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 2,6-dimethoxyphenol (2,6-DMP), and potassium ferrocyanide K4[Fe(CN6)], and non-conventional substrates as biogenic amines. However, it presents some distinctiveness, the most characteristic being its psychrophilic behaviour, not seen before among these enzymes. Psychrophilic enzymes capable of efficient catalysis at low temperatures are of great interest due to their potential applications in various biotechnological processes. In this study, we report the discovery and characterization of a new psychrophilic laccase, a multicopper oxidase (MCO), from the bacterium Oenococcus oeni. The psychrophilic laccase gene, designated as LcOe 229, was identified through the genomic analysis of O. oeni, a Gram-positive bacterium commonly found in wine fermentation. The gene was successfully cloned and heterologously expressed in Escherichia coli, and the recombinant enzyme was purified to homogeneity. Biochemical characterization of the psychrophilic laccase revealed its optimal activity at low temperatures, with a peak at 10 °C. To our knowledge, this is the lowest optimum temperature described so far for laccases. Furthermore, the psychrophilic laccase demonstrated remarkable stability and activity at low pH (optimum pH 2.5 for ABTS), suggesting its potential for diverse biotechnological applications. The kinetic properties of LcOe 229 were determined, revealing a high catalytic efficiency (kcat/Km) for several substrates at low temperatures. This exceptional cold adaptation of LcOe 229 indicates its potential as a biocatalyst in cold environments or applications requiring low-temperature processes. The crystal structure of the psychrophilic laccase was determined using X-ray crystallography demonstrating structural features similar to other LAB laccases, such as an extended N-terminal and an extended C-terminal end, with the latter containing a disulphide bond. Also, the structure shows two Met residues at the entrance of the T1Cu site, common in LAB laccases, which we suggest could be involved in substrate binding, thus expanding the substrate-binding pocket for laccases. A structural comparison of LcOe 229 with Antarctic laccases has not revealed specific features assigned to cold-active laccases versus mesophilic. Thus, further investigation of this psychrophilic laccase and its engineering could lead to enhanced cold-active enzymes with improved properties for future biotechnological applications. Overall, the discovery of this novel psychrophilic laccase from O. oeni expands our understanding of cold-adapted enzymes and presents new opportunities for their industrial applications in cold environments.

The effect of hydrostatic pressure on the activity and community composition of hydrocarbon-degrading bacteria in Arctic seawater.

IMPORTANCE: Increased ship traffic in the Arctic region raises the risk of oil spills. With an average sea depth of 1,000 m, there is a growing concern over the potential release of oil sinking in the form of marine oil snow into deep Arctic waters. At increasing depth, the oil-degrading community is exposed to increasing hydrostatic pressure, which can reduce microbial activity. However, microbes thriving in polar regions may adapt to low temperature by modulation of membrane fluidity, which is also a well-known adaptation to high hydrostatic pressure. At mild hydrostatic pressures up to 8-12 MPa, we did not observe an altered microbial activity or community composition, whereas comparable studies using deep-sea or sub-Arctic microbial communities with in situ temperatures of 4-5°C showed pressure-induced effects at 10-15 MPa. Our results suggest that the psychrophilic nature of the underwater microbial communities in the Arctic may be featured by specific traits that enhance their fitness at increasing hydrostatic pressure.

New temperature-switchable acyl homoserine lactone-regulated expression vector.

Bacterial expression systems play an indispensable role in the biosynthesis of recombinant proteins. Different proteins and the tasks associated with them may require different systems. The purpose of this work is to make an expression vector that allows switching on and off the expression of the target gene during cell incubation. Several expression vectors for use in Escherichia coli cells were developed using elements of the luxR/luxI type quorum sensing system of psychrophilic bacterium Aliivibrio logei. These vectors contain A. logei luxR2 and (optionally) luxI genes and LuxR2-regulated promoter, under the control of which a target gene is intended to be inserted. The synthesis of the target protein depends directly on the temperature: gene expression starts when the temperature drops to 22 °C and stops when it rises to 37 °C, which makes it possible to fix the desired amount of the target protein in the cell. At the same time, the expression of the target gene at a low temperature depends on the concentration of the autoinducer (L-homoserine N-(3-oxohexanoyl)-lactone, AI) in the culture medium in a wide range from 1 nM to 10 µM, which makes it possible to smoothly regulate the rate of target protein synthesis. Presence of luxI in the vector provides the possibility of autoinduction. Constructed expression vectors were tested with gfp, ardA, and ardB genes. At maximum, we obtained the target protein in an amount of up to 33% of the total cellular protein. KEY POINTS: • A. logei quorum sensing system elements were applied in new expression vectors • Expression of target gene is inducible at 22 °C and it is switched off at 37 °C • Target gene expression at 22 °C is tunable by use different AI concentrations.

Comparative genomic analysis provides insights into taxonomy and temperature adaption of Aeromonas salmonicida.

Aeromonas salmonicida has long been known as psychrophiles since it is mainly isolated from cold water fish, and recent reports have revealed the existence of mesophilic strains isolated from warm sources. However, the genetic differences between mesophilic and psychrophilic strains remain unclear due to few complete genomes of mesophilic strain are available. In this study, six A. salmonicida (2 mesophilic and 4 psychrophilic) were genome-sequenced, and comparative analyses of 25 A. salmonicida complete genomes were conducted. The ANI values and phylogenetic analysis revealed that 25 strains formed three independent clades, which were referred as typical psychrophilic, atypical psychrophilic and mesophilic groups. Comparative genomic analysis showed that two chromosomal gene clusters, related to lateral flagella and outer membrane proteins (A-layer and T2SS proteins), and insertion sequences (ISAs4, ISAs7 and ISAs29) were unique to the psychrophilic groups, while the complete MSH type IV pili were unique to the mesophilic group, all of which may be considered as lifestyle-related factors. The results of this study not only provide new insights into the classification, lifestyle adaption and pathogenic mechanism of different strains of A. salmonicida, but also contributes to the prevention and control of disease caused by psychrophilic and mesophilic A. salmonicida.

Insights into methanogenesis of mesophilic-psychrophilic varied anaerobic digestion of municipal sludge with antibiotic stress.

Methane production through anaerobic digestion (AD) of municipal sludge is economic and eco-friendly, which is commonly affected by temperature and pollutants residues. However, little is known about methanogenesis in psychrophilic AD (PAD) with temperature variations that simulating seasonal variations and with antibiotic stress. Here, two groups of AD systems with oxytetracycline (OTC) were operated with temperature maintained at 35 °C and 15 °C or variation to explore the influence to methanogenesis. The acetic acid was noticeably accumulated in OTC groups initially (P < 0.001). Methane production was noticeably inhibited initially in PAD with OTC, but had been stimulated later at 35 °C. The dominant acetoclastic methanogens Methanosaeta gradually decreased to 15.48% and was replaced by methylotrophic Methanomethylovorans (73.43%) in PAD with OTC. Correspondingly, the abundances of functional genes related to methylotrophic methanogenesis were also higher in these groups. Besides, genes involving in methane oxidation had over 50 times higher abundances in PAD with OTC groups in the second phase. Further investigation is essential to understand the main dynamics of methanogenesis in PAD and to clear the related molecular mechanism for future methane production regulation in sludge systems.

Temperature effect on polymerase fidelity.

The discovery of extremophiles helped enable the development of groundbreaking technology such as PCR. Temperature variation is often an essential step of these technology platforms, but the effect of temperature on the error rate of polymerases from different origins is underexplored. Here, we applied high-throughput sequencing to profile the error rates of DNA polymerases from psychrophilic, mesophilic, and thermophilic origins with single-molecule resolution. We found that the reaction temperature substantially increases substitution and deletion error rates of psychrophilic and mesophilic DNA polymerases. Our motif analysis shows that the substitution error profiles cluster according to phylogenetic similarity of polymerases, not the reaction temperature, thus suggesting that the reaction temperature increases the global error rate of polymerases independent of the sequence context. Intriguingly, we also found that the DNA polymerase I of psychrophilic bacteria exhibits higher polymerization activity than its mesophilic ortholog across all temperature ranges, including down to -19 °C, which is well below the freezing temperature of water. Our results provide a useful reference for how the reaction temperature, a crucial parameter of biochemistry, can affect DNA polymerase fidelity in organisms adapted to a wide range of thermal environments.

Antarctic Polyester Hydrolases Degrade Aliphatic and Aromatic Polyesters at Moderate Temperatures.

Polyethylene terephthalate (PET) is one of the most widely used synthetic plastics in the packaging industry, and consequently has become one of the main components of plastic waste found in the environment. However, several microorganisms have been described to encode enzymes that catalyze the depolymerization of PET. While most known PET hydrolases are thermophilic and require reaction temperatures between 60°C and 70°C for an efficient hydrolysis of PET, a partial hydrolysis of amorphous PET at lower temperatures by the polyester hydrolase IsPETase from the mesophilic bacterium Ideonella sakaiensis has also been reported. We show that polyester hydrolases from the Antarctic bacteria Moraxella sp. strain TA144 (Mors1) and Oleispira antarctica RB-8 (OaCut) were able to hydrolyze the aliphatic polyester polycaprolactone as well as the aromatic polyester PET at a reaction temperature of 25°C. Mors1 caused a weight loss of amorphous PET films and thus constitutes a PET-degrading psychrophilic enzyme. Comparative modeling of Mors1 showed that the amino acid composition of its active site resembled both thermophilic and mesophilic PET hydrolases. Lastly, bioinformatic analysis of Antarctic metagenomic samples demonstrated that members of the Moraxellaceae family carry candidate genes coding for further potential psychrophilic PET hydrolases. IMPORTANCE A myriad of consumer products contains polyethylene terephthalate (PET), a plastic that has accumulated as waste in the environment due to its long-term stability and poor waste management. One promising solution is the enzymatic biodegradation of PET, with most known enzymes only catalyzing this process at high temperatures. Here, we bioinformatically identified and biochemically characterized an enzyme from an Antarctic organism that degrades PET at 25°C with similar efficiency to the few PET-degrading enzymes active at moderate temperatures. Reasoning that Antarctica harbors other PET-degrading enzymes, we analyzed available data from Antarctic metagenomic samples and successfully identified other potential enzymes. Our findings contribute to increasing the repertoire of known PET-degrading enzymes that are currently being considered as biocatalysts for the biological recycling of plastic waste.

Cold-adaptive mechanism of psychrophilic bacteria in food and its application.

Psychrophilic bacteria are a type of microorganisms that normally grow in low-temperature environments. They are usually found in extremely cold environments. However, as people's demand for low-temperature storage of food becomes higher, psychrophilic bacteria have also begun to appear in cold storage and refrigerators, which has become a food safety hazard. In this paper, the optimal cooling strategies of psychrophilic bacteria are reviewed from the aspects of the cell membrane, psychrophilic enzymes, antifreeze proteins, cold shock proteins, gene regulation, metabolic levels and antifreeze agents, and the principle of psychrophilic mechanism is briefly described. The application of thermophilic bacteria and its products adapted to cold environments in food fields are analyzed. The purpose of this paper is to provide ideas for future research on psychrophilic bacteria based on the mechanism and application of psychrophilic bacteria.

Active human full-length CDKL5 produced in the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125.

BACKGROUND: A significant fraction of the human proteome is still inaccessible to in vitro studies since the recombinant production of several proteins failed in conventional cell factories. Eukaryotic protein kinases are difficult-to-express in heterologous hosts due to folding issues both related to their catalytic and regulatory domains. Human CDKL5 belongs to this category. It is a serine/threonine protein kinase whose mutations are involved in CDKL5 Deficiency Disorder (CDD), a severe neurodevelopmental pathology still lacking a therapeutic intervention. The lack of successful CDKL5 manufacture hampered the exploitation of the otherwise highly promising enzyme replacement therapy. As almost two-thirds of the enzyme sequence is predicted to be intrinsically disordered, the recombinant product is either subjected to a massive proteolytic attack by host-encoded proteases or tends to form aggregates. Therefore, the use of an unconventional expression system can constitute a valid alternative to solve these issues. RESULTS: Using a multiparametric approach we managed to optimize the transcription of the CDKL5 gene and the synthesis of the recombinant protein in the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125 applying a bicistronic expression strategy, whose generalization for recombinant expression in the cold has been here confirmed with the use of a fluorescent reporter. The recombinant protein largely accumulated as a full-length product in the soluble cell lysate. We also demonstrated for the first time that full-length CDKL5 produced in Antarctic bacteria is catalytically active by using two independent assays, making feasible its recovery in native conditions from bacterial lysates as an active product, a result unmet in other bacteria so far. Finally, the setup of an in cellulo kinase assay allowed us to measure the impact of several CDD missense mutations on the kinase activity, providing new information towards a better understanding of CDD pathophysiology. CONCLUSIONS: Collectively, our data indicate that P. haloplanktis TAC125 can be a valuable platform for both the preparation of soluble active human CDKL5 and the study of structural-functional relationships in wild type and mutant CDKL5 forms. Furthermore, this paper further confirms the more general potentialities of exploitation of Antarctic bacteria to produce "intractable" proteins, especially those containing large intrinsically disordered regions.

Electricity production and key exoelectrogens in a mixed-culture psychrophilic microbial fuel cell at 4 °C.

The electricity production via psychrophilic microbial fuel cell (PMFC) for wastewater treatment in cold regions offers an alternative to avoid the unwanted methane dissolution of traditional anaerobic fermentation. But, it is seldom reported by mixed-culture, especially closed to 0 °C. Thus, a two-chamber mixed-culture PMFC at 4 °C was successfully operated in this study using acetate as an electron donor. The main results demonstrated a good performance of PMFC, including the maximum voltage of 513 mV at 1000 Ω, coulombic efficiency of 53%, and power density of 689 mW/m2. The cyclic voltammetry curves of enriched biofilm showed a direct electron transfer pathway. These good performances of mixed-culture PMFC were due to the high psychrophilic activity of enriched biofilm, including exoelectrogens genera of Geobacter (6.1%), Enterococcus (17.5%), and Clostridium_sensu_stricto_12 (3.8%). Consequently, a mixed-culture PMFC provides a reasonable strategy to enrich exoelectrogens with high activity. For low-temperature regions, the mixed-culture PMFC involved biotechnologies shall benefit energy generation and valuable chemical production in the future. KEY POINTS: • PMFC showed a maximum voltage of around 513 mV under a resistance of 1000 Ω. • The coulombic efficiency was 53% and the max power density was 689 mW/m2. • Geobacter, Enterococcus, and Clostridium_sensu_stricto_12 were key exoelectrogens.

Unusual Tetrahydropyridoindole-Containing Tetrapeptides with Human Nicotinic Acetylcholine Receptors Targeting Activity Discovered from Antarctica-Derived Psychrophilic Pseudogymnoascus sp. HDN17-933.

Chemical investigation of the psychrophilic fungus Pseudogymnoascus sp. HDN17-933 derived from Antarctica led to the discovery of six new tetrapeptides psegymamides A-F (1-6), whose planar structures were elucidated by extensive NMR and MS spectrometric analyses. Structurally, psegymamides D-F (4-6) possess unique backbones bearing a tetrahydropyridoindoles unit, which make them the first examples discovered in naturally occurring peptides. The absolute configurations of structures were unambiguously determined using solid-phase total synthesis assisted by Marfey's method, and all compounds were evaluated for their inhibition of human (h) nicotinic acetylcholine receptor subtypes. Compound 2 showed significant inhibitory activity. A preliminary structure-activity relationship investigation revealed that the tryptophan residue and the C-terminal with methoxy group were important to the inhibitory activity. Further, the high binding affinity of compound 2 to hα4ß2 was explained by molecular docking studies.

Biochemical Characterization and Elucidation of the Hybrid Action Mode of a New Psychrophilic and Cold-Tolerant Alginate Lyase for Efficient Preparation of Alginate Oligosaccharides.

Alginate lyases with unique biochemical properties have irreplaceable value in food and biotechnology industries. Herein, the first new hybrid action mode Thalassotalea algicola-derived alginate lyase gene (TAPL7A) with both psychrophilic and cold-tolerance was cloned and expressed heterologously in E. coli. With the highest sequence identity (43%) to the exolytic alginate lyase AlyA5 obtained from Zobellia galactanivorans, TAPL7A was identified as a new polysaccharide lyases family 7 (PL7) alginate lyase. TAPL7A has broad substrate tolerance with specific activities of 4186.1 U/mg, 2494.8 U/mg, 2314.9 U/mg for polyM, polyG, and sodium alginate, respectively. Biochemical characterization of TAPL7A showed optimal activity at 15 °C, pH 8.0. Interestingly, TAPL7A exhibits both extreme psychrophilic and cold tolerance, which other cold-adapted alginate lyase do not possess. In a wide range of 5-30 °C, the activity can reach 80-100%, and the residual activity of more than 70% can still be maintained after 1 h of incubation. Product analysis showed that TAPL7A adopts a hybrid endo/exo-mode on all three substrates. FPLC and ESI-MS confirmed that the final products of TAPL7A are oligosaccharides with degrees of polymerization (Dps) of 1-2. This study provides excellent alginate lyase candidates for low-temperature environmental applications in food, agriculture, medicine and other industries.

Cold-Active Lipases and Esterases: A Review on Recombinant Overexpression and Other Essential Issues.

Cold environments characterised by diverse temperatures close to or below the water freezing point dominate about 80% of the Earth's biosphere. One of the survival strategies adopted by microorganisms living in cold environments is their expression of cold-active enzymes that enable them to perform an efficient metabolic flux at low temperatures necessary to thrive and reproduce under those constraints. Cold-active enzymes are ideal biocatalysts that can reduce the need for heating procedures and improve industrial processes' quality, sustainability, and cost-effectiveness. Despite their wide applications, their industrial usage is still limited, and the major contributing factor is the lack of complete understanding of their structure and cold adaptation mechanisms. The current review looked at the recombinant overexpression, purification, and recent mechanism of cold adaptation, various approaches for purification, and three-dimensional (3D) crystal structure elucidation of cold-active lipases and esterase.

Purification and refolding protocol for cold-active recombinant esterase AaSGNH1 from Aphanizomenon flos-aquae expressed as insoluble inclusion bodies.

Microbial esterases are a highly desirable tool for numerous biosynthetic and biotechnological applications requiring ester bond cleavage. Once identified, microbial esterases are often produced recombinantly in Escherichia coli to enhance yield and ease of purification. In this study a polyhistidine-tagged SGNH esterase gene (AaSGNH1), originating from the cyanobacterium Aphanizomenon flos-aquae, was cloned into an over-expression plasmid and expressed in BL21(DE3) cells. The recombinant esterase enzyme was produced as inactive inclusion bodies which were insoluble in 8 M urea but readily solubilized by the detergent Empigen BB®. Crucially, the procurement of active enzyme required controlled removal of detergent during column chromatography and dialysis steps. The refolded esterase was characterized with respect to its ability to catalyze the cleavage of p-nitrophenol esters of different chain lengths (C2, C8, C16). In addition, the temperature and pH optima were determined and it was found that the enzyme was most active at low temperatures (5-15 °C) and under alkaline conditions (pH 8-10). It was found that the kinetic properties of AaSGNH1 were remarkably similar to other SGNH esterases described thereby validating that the protein was effectively refolded. Overall, this study provides a simple strategy for isolating cold-active recombinant esterase enzyme when expressed as inclusion bodies.

Perchlorate-reducing bacteria from Antarctic marine sediments.

Perchlorate is a contaminant that can persist in groundwater and soil, and is frequently detected in different ecosystems at concentrations relevant to human health. This study isolated and characterised halotolerant bacteria that can potentially perform perchlorate reduction. Bacterial microorganisms were isolated from marine sediments on Deception, Horseshoe and Half Moon Islands of Antarctica. The results of the 16S ribosomal RNA (rRNA) gene sequence analysis indicated that the isolates were phylogenetically related to Psychrobacter cryohalolentis, Psychrobacter urativorans, Idiomarina loihiensis, Psychrobacter nivimaris, Sporosarcina aquimarina and Pseudomonas lactis. The isolates grew at a sodium chloride concentration of up to 30% and a perchlorate concentration of up to 10,000 mg/L, which showed their ability to survive in saline conditions and high perchlorate concentrations. Between 21.6 and 40% of perchlorate was degraded by the isolated bacteria. P. cryohalolentis and P. urativorans degraded 30.3% and 32.6% of perchlorate, respectively. I. loihiensis degraded 40% of perchlorate, and P. nivimaris, S. aquimarina and P. lactis degraded 22%, 21.8% and 21.6% of perchlorate, respectively. I. loihiensis had the highest reduction in perchlorate, whereas P. lactis had the lowest reduction. This study is significant as it is the first finding of P. cryohalolentis and. P. lactis on the Antarctic continent. In conclusion, these bacteria isolated from marine sediments on Antarctica offer promising resources for the bioremediation of perchlorate contamination due to their ability to degrade perchlorate, showing their potential use as a biological system to reduce perchlorate in high-salinity ecosystems.

Crystal structure of a novel putative sugar isomerase from the psychrophilic bacterium Paenibacillus sp. R4.

Sugar isomerases (SIs) catalyze the reversible conversion of aldoses to ketoses. A novel putative SI gene has been identified from the genome sequence information on the psychrophilic bacterium Paenibacillus sp. R4. Here, we report the crystal structure of the putative SI from Paenibacillus sp. R4 (PbSI) at 2.98 Å resolution. It was found that the overall structure of PbSI adopts the triose-phosphate isomerase (TIM) barrel fold. PbSI was also identified to have two heterogeneous metal ions as its cofactors at the active site in the TIM barrel, one of which was confirmed as a Zn ion through X-ray anomalous scattering and inductively coupled plasma mass spectrometry analysis. Structural comparison with homologous SI proteins from mesophiles, hyperthermophiles, and a psychrophile revealed that key residues in the active site are well conserved and that dimeric PbSI is devoid of the extended C-terminal region, which tetrameric SIs commonly have. Our results provide novel structural information on the cold-adaptable SI, including information on the metal composition in the active site.