Cold-adapted Bacterium Research Articles

Exploration of novel trehalases from cold-adapted Variovorax sp. PAMC28711: Functional characterization

The cold-adapted bacterium Variovorax sp. PAMC28711 possesses two distinct glycoside hydrolase (GH) families of trehalase, GH15 and GH37. While numerous studies have explored bacterial trehalase, the presence of two different trehalase genes within a single strain has not been reported until now. Interestingly, despite both GH37 and GH15 trehalases serving the same purpose of degrading trehalose, but do not share the sequence similarity. The substrate specificity assay confirmed that Vtre37 and Vtre15 displayed hydrolytic activity on α, α-trehalose. The key catalytic sites were identified as D280 and E469 in Vtre37 and E389 and E554 in Vtre15 through site-directed mutation and confirmed these two enzymes belong to trehalase. In addition, Vtre37 exhibited a relatively high level of enzyme activity of 1306.33 (±53.091) μmolmg−1, whereas Vtre15 showed enzyme activity of 408.39 (±12.503) μmolmg−1. Moreover, Vtre37 performed admirably showing resistance to ethanol (10 %), with high stable at acidic pH range. Furthermore, both prediction and experimental results indicate that validoxylamine A showed a potent inhibitory activity against Vtre37 trehalase with a Ki value of 16.85 nM. Therefore, we postulate that Vtre37 could be utilized as an ethanol enhancer and designed for screening inhibitors related to the trehalose degradation pathway. Additionally, we believe that characterizing these bacterial trehalase contributes to a better understanding of trehalose metabolism and its biological importance in bacteria.

A new family of bacterial ribosome hibernation factors

To conserve energy during starvation and stress, many organisms use hibernation factor proteins to inhibit protein synthesis and protect their ribosomes from damage1,2. In bacteria, two families of hibernation factors have been described, but the low conservation of these proteins and the huge diversity of species, habitats and environmental stressors have confounded their discovery3–6. Here, by combining cryogenic electron microscopy, genetics and biochemistry, we identify Balon, a new hibernation factor in the cold-adapted bacterium Psychrobacter urativorans. We show that Balon is a distant homologue of the archaeo-eukaryotic translation factor aeRF1 and is found in 20% of representative bacteria. During cold shock or stationary phase, Balon occupies the ribosomal A site in both vacant and actively translating ribosomes in complex with EF-Tu, highlighting an unexpected role for EF-Tu in the cellular stress response. Unlike typical A-site substrates, Balon binds to ribosomes in an mRNA-independent manner, initiating a new mode of ribosome hibernation that can commence while ribosomes are still engaged in protein synthesis. Our work suggests that Balon–EF-Tu-regulated ribosome hibernation is a ubiquitous bacterial stress-response mechanism, and we demonstrate that putative Balon homologues in Mycobacteria bind to ribosomes in a similar fashion. This finding calls for a revision of the current model of ribosome hibernation inferred from common model organisms and holds numerous implications for how we understand and study ribosome hibernation.

Whole genome analysis of Pseudomonas mandelii SW-3 and the insights into low-temperature adaptation.

Pseudomonas mandelii SW-3, isolated from the Napahai plateau wetland, can survive in cold environments. The mechanisms underlying the survival of bacteria in low temperatures and high altitudes are not yet fully understood. In this study, the whole genome of SW-3 was sequenced to identify the genomic features that may contribute to survival in cold environments. The results showed that the genome size of strain SW-3 was 6,538,059bp with a GC content of 59%. A total of 67 tRNAs, a 34,110bp prophage sequence, and a large number of metabolic genes were found. Based on 16S rRNA gene phylogeny and average nucleotide identity analysis among P. mandelii, SW-3 was identified as a strain belonging to P. mandelii. In addition, we clarified the mechanisms by which SW-3 survived in a cold environment, providing a basis for further investigation of host-phage interaction. P. mandelii SW-3 showed stress resistance mechanisms, including glycogen and trehalose metabolic pathways, and antisense transcriptional silencing. Furthermore, cold shock proteins and glucose 6-phosphate dehydrogenase may play pivotal roles in facilitating adaptation to cold environmental conditions. The genome-wide analysis provided us with a deeper understanding of the cold-adapted bacterium.

Whole genome analysis and cold adaptation strategies of Pseudomonas sivasensis W-6 isolated from the Napahai plateau wetland

Microbial communities of wetlands play key roles in the earth’s ecology and stability. To elucidate the cold adaptation mechanisms of bacteria in plateau wetlands, we conducted comparative genomic analyses of Pseudomonas sivasensis and closely related lineages. The genome of P. sivasensis W-6, a cold-adapted bacterium isolated from the Napahai plateau wetland, was sequenced and analyzed. The genome length was 6,109,123 bp with a G+C content of 59.5%. Gene prediction yielded 5360 protein-coding sequences, 70 tRNAs, 24 gene islands, and 2 CRISPR sequences. The isolate contained evidence of horizontal gene transfer events during its evolution. Two prophages were predicted and indicated that W-6 was a lysogen. The cold adaptation of the W-6 strain showed psychrophilic rather than psychrotrophic characteristics. Cold-adapted bacterium W-6 can utilize glycogen and trehalose as resources, associated with carbohydrate-active enzymes, and survive in a low-temperature environment. In addition, the cold-adapted mechanisms of the W-6 included membrane fluidity by changing the unsaturated fatty acid profile, the two-component regulatory systems, anti-sense transcription, the role played by rpsU genes in the translation process, etc. The genome-wide analysis of W-6 provided a deeper understanding of cold-adapted strategies of bacteria in environments. We elucidated the adaptive mechanism of the psychrophilic W-6 strain for survival in a cold environment, which provided a basis for further study on host-phage coevolution.

Identification and genome-wide analysis provide insights into the genetic diversity and biotechnological potentials of novel cold-adapted Acinetobacter strain.

Extreme cold environments, such as polar regions or high-altitude mountains, are known for their challenging conditions including low temperatures, high salinity, and limited nutrient availability. Microbes that thrive in these environments have evolved specialized strategies to survive and function under such harsh conditions. The study aims to identify, sequence the genome, perform genome assembly, and conduct a comparative genome-wide analysis of Acinetobacter sp. strain P1, which was isolated from the Batura glacier regions of Pakistan. A basic local alignment search tool of NCBI using 16s RNA gene sequence confirmed the strain Acinetobacter following phylogenetic analysis revealed that strain P1 clustered with Acinetobacter sp. strain AcBz01. The high-throughput Genome sequencing was done by the NovaSeq 6000 sequencing system following de novo genome assembly reported 23 contigs, a genome size of 3,732,502bp containing approximately 3489 genes and 63 RNAs (60 tRNA, 3 rRNA). The comparative genome analysis revealed thatAcinetobacter sp. strain P1 exhibited the highest homology with theAcinetobacter baumannii ATCC 17978 genome and encompassed 1668 indispensable genes, 1280 conserved genes 1821 specific genes suggesting high genomic plasticity and evolutionary diversity. The genes with functional assignments include exopolysaccharide phosphotransferase enzyme, cold-shock proteins, T6SS, membrane modifications, antibiotic resistance, and set of genes related to a wide range of metabolic characteristics such as exopolysaccharides were also present. Moreover, the structural prediction analysis of EPS proteins reveals that structural flexibility allows for conformational modifications during catalysis, which boosts or increases the catalytic effectiveness at lower temperatures. Overall, the identification of Acinetobacter, a cold-adapted bacterium, offers promising applications in bioremediation, enzyme production, food preservation, pharmaceutical development, and astrobiology. Further research and exploration of these microorganisms can unlock their full biotechnological potential and contribute to various industries and scientific endeavors.

Systematic functional analysis and potential application of a serine protease from cold-adapted Planococcus bacterium

In this study, a gene encoding serine protease (PmSpr288) from cold-adapted bacterium, namely Planococcus maritimus XJ11, was cloned and overexpressed in Escherichia coli BL21 (DE3). Bioinformatics analysis revealed that PmSpr288 belongs to serine protease S8 superfamily with a classical catalytic triad comprised by the Asp49, His86 and Ser251. Moreover, PmSpr288 was found to be active over broad alkaline pH and low-moderate temperature, and exhibited wide range of protein substrate specificity. In addition, PmSpr288 was able to hydrolyze the meat proteins actin and myosin, and molecular docking results suggested that the crucial interaction between PmSpr288 and actin/myosin complexes was mainly occupied by hydrogen bonds. The muscle protein hydrolysates of silver carp prepared by PmSpr288 was shown to have antioxidant activity via DPPH radical scavenging assay, which presented an IC50 valve of 1.309 mg/mL. In conclusion, these characteristics imply that PmSpr288 has potential biotechnological application prospect for the production of bioactive peptides.

Metabolic Robustness to Growth Temperature of a Cold- Adapted Marine Bacterium.

Microbial communities experience continuous environmental changes, with temperature fluctuations being the most impacting. This is particularly important considering the ongoing global warming but also in the "simpler" context of seasonal variability of sea-surface temperature. Understanding how microorganisms react at the cellular level can improve our understanding of their possible adaptations to a changing environment. In this work, we investigated the mechanisms through which metabolic homeostasis is maintained in a cold-adapted marine bacterium during growth at temperatures that differ widely (15 and 0°C). We have quantified its intracellular and extracellular central metabolomes together with changes occurring at the transcriptomic level in the same growth conditions. This information was then used to contextualize a genome-scale metabolic reconstruction, and to provide a systemic understanding of cellular adaptation to growth at 2 different temperatures. Our findings indicate a strong metabolic robustness at the level of the main central metabolites, counteracted by a relatively deep transcriptomic reprogramming that includes changes in gene expression of hundreds of metabolic genes. We interpret this as a transcriptomic buffering of cellular metabolism, able to produce overlapping metabolic phenotypes, despite the wide temperature gap. Moreover, we show that metabolic adaptation seems to be mostly played at the level of few key intermediates (e.g., phosphoenolpyruvate) and in the cross talk between the main central metabolic pathways. Overall, our findings reveal a complex interplay at gene expression level that contributes to the robustness/resilience of core metabolism, also promoting the leveraging of state-of-the-art multi-disciplinary approaches to fully comprehend molecular adaptations to environmental fluctuations. IMPORTANCE This manuscript addresses a central and broad interest topic in environmental microbiology, i.e. the effect of growth temperature on microbial cell physiology. We investigated if and how metabolic homeostasis is maintained in a cold-adapted bacterium during growth at temperatures that differ widely and that match measured changes on the field. Our integrative approach revealed an extraordinary robustness of the central metabolome to growth temperature. However, this was counteracted by deep changes at the transcriptional level, and especially in the metabolic part of the transcriptome. This conflictual scenario was interpreted as a transcriptomic buffering of cellular metabolism, and was investigated using genome-scale metabolic modeling. Overall, our findings reveal a complex interplay at gene expression level that contributes to the robustness/resilience of core metabolism, also promoting the use of state-of-the-art multi-disciplinary approaches to fully comprehend molecular adaptations to environmental fluctuations.

Arctic Psychrotolerant Pseudomonas sp. B14-6 Exhibits Temperature-Dependent Susceptibility to Aminoglycosides.

Bacteria can evade antibiotics by acquiring resistance genes, as well as switching to a non-growing dormant state without accompanying genetic modification. Bacteria in this quiescent state are called persisters, and this non-inheritable ability to withstand multiple antibiotics is referred to as antibiotic tolerance. Although all bacteria are considered to be able to form antibiotic-tolerant persisters, the antibiotic tolerance of extremophilic bacteria is poorly understood. Previously, we identified the psychrotolerant bacterium Pseudomonas sp. B14-6 from the glacier foreland of Midtre Lovénbreen in High Arctic Svalbard. Herein, we investigated the resistance and tolerance of Pseudomonas sp. B14-6 against aminoglycosides at various temperatures. This bacterium was resistant to streptomycin and susceptible to apramycin, gentamicin, kanamycin, and tobramycin. The two putative aminoglycoside phosphotransferase genes aph1 and aph2 were the most likely contributors to streptomycin resistance. Notably, unlike the mesophilic Pseudomonas aeruginosa PA14, this cold-adapted bacterium demonstrated reduced susceptibility to all tested aminoglycosides in a temperature-dependent manner. Pseudomonas sp. B14-6 at a lower temperature formed the persister cells that shows tolerance to the 100-fold minimum inhibitory concentration (MIC) of gentamicin, as well as the partially tolerant cells that withstand 25-fold MIC gentamicin. The temperature-dependent gentamicin tolerance appears to result from reduced metabolic activity. Lastly, the partially tolerant Pseudomonas sp. B14-6 cells could slowly proliferate under the bactericidal concentrations of aminoglycosides. Our results demonstrate that Pseudomonas sp. B14-6 has a characteristic ability to form cells with a range of tolerance, which appears to be inversely proportional to its growth rate.

Subtercola endophyticus sp. nov., a cold-adapted bacterium isolated from Abies koreana

A novel Gram-stain-positive, aerobic bacterial strain, designated AK-R2A1-2 T, was isolated from the surface-sterilized needle leaves of an Abies koreana tree. Strain AK-R2A1-2 T had 97.3% and 96.7% 16S rRNA gene sequence similarities with Subtercola boreus K300T and Subtercola lobariae 9583bT, respectively, but formed a distinct phyletic lineage from these two strains. Growth of strain AK-R2A1-2 T was observed at 4–25 °C at pH 5.0–8.0. Strain AK-R2A1-2 T contained menaquinone 9 (MK-9) and menaquinone 10 (MK-10) as the predominant respiratory quinones. The major cellular fatty acids were anteiso-C15:0 and summed feature 8 (C18:1ω7c or/and C18:1ω6c), and the polar lipids included diphosphatidylglycerol (DPG) and three unknown aminolipids, AKL2, AKL3, and AKL4. The complete genome of strain AK-R2A1-2 T was sequenced to understand the genetic basis of its survival at low temperatures. Multiple copies of cold-associated genes involved in cold-active chaperon, stress response, and DNA repair supported survival of the strain at low temperatures. Strain AK-R2A1-2 T was also able to significantly improve rice seedling growth under low temperatures. Thus, this strain represents a novel species of the genus Subtercola, and the proposed name is Subtercola endophyticus sp. nov. The type strain is AK-R2A1-2 T (= KCTC 49721 T = GDMCC 1.2921 T).

Cryoprotectant role of exopolysaccharide ID1 in the vitrification/in-straw warming of in vitro-produced bovine embryos.

The cold-adapted bacterium Pseudomonas sp. ID1 produces the extracellular exopolysaccharide ID1 (EPS ID1) with cryoprotective activity. This study was designed to optimize the vitrification/in-straw warming protocol of in vitro-produced (IVP) blastocysts by adding EPS ID1 to the vitrification media. Day 7-expanded blastocysts were vitrified/warmed using the VitTrans device after the addition of 0 or 100 μg/mL EPS ID1 to the vitrification media. Blastocysts vitrified by the Cryotop method and fresh non-vitrified blastocysts served as controls. Outcomes were assessed in the warmed embryos in terms of survival rates and mRNA relative abundances of BAX, BCL2, GPX1, and CDX2 genes. No differences in survival rates were observed at 3h post-warming between vitrification treatments. At 24 h post-warming, the addition of EPS prior to vitrification with the VitTrans device produced similar survival rates to Cryotop-vitrified embryos and similar hatching rates to fresh non-vitrified or Cryotop-vitrified embryos. No differences emerged in BCL2 gene expression. Lower BAX (p < .05) and higher GPX1 (p < .05) and CDX2 (p < .1) gene expression were observed in expanded and/or hatched blastocysts derived from VitTrans-EPS-vitrified embryos when compared to those from the non-supplemented group. In conclusion, addition of EPS not only promoted blastocyst survival and hatching after VitTrans vitrification/warming but also modified the expression of genes associated with better embryo quality.

Psychrotrophic plant beneficial bacteria from the glacial ecosystem of Sikkim Himalaya: Genomic evidence for the cold adaptation and plant growth promotion

Commercial biofertilizers tend to be ineffective in cold mountainous regions due to reduced metabolic activity of the microbial inoculants under low temperatures. Cold-adapted glacier bacteria with plant growth-promoting (PGP) properties may prove significant in developing cold-active biofertilizers for improving mountain agriculture. With this perspective, the cultivable bacterial diversity was documented from the East Rathong glacier ecosystem lying above 3900 masl of Sikkim Himalaya. A total of 120 bacterial isolates affiliated to Gammaproteobacteria (53.33%), Bacteroidetes (16.66%), Actinobacteria (15.83%), Betaproteobacteria (6.66%), Alphaproteobacteria (4.16%), and Firmicutes (3.33%) were recovered. Fifty-two isolates showed many in vitro PGP activities of phosphate solubilization (9–100 µg/mL), siderophore production (0.3–100 psu) and phytohormone indole acetic acid production (0.3–139 µg/mL) at 10 °C. Plant-based bioassays revealed an enhancement of shoot length by 21%, 22%, and 13% in ERGS5:01, ERMR1:04, and ERMR1:05, and root length by 14%, 17%, 11%, and 22% in ERGS4:06, ERGS5:01, ERMR1:04, and ERMR1:05 treated seeds respectively. An increased shoot dry weight of 4–29% in ERMR1:05 and ERMR1:04, and root dry weight of 42–98% was found in all the treatments. Genome analysis of four bacteria from diverse genera predicted many genes involved in the bacterial PGP activity. Comparative genome study highlighted the presence of PGP-associated unique genes for glucose dehydrogenase, siderophore receptor, tryptophan synthase, phosphate metabolism (phoH, P, Q, R, U), nitrate and nitrite reductase, TonB-dependent receptor, spermidine/putrescine ABC transporter etc. in the representative bacteria. The expression levels of seven cold stress-responsive genes in the cold-adapted bacterium ERGS4:06 using real-time quantitative PCR (RT-qPCR) showed an upregulation of all these genes by 6–17% at 10 °C, and by 3–33% during cold-shock, which indicates the cold adaptation strategy of the bacterium. Overall, this study signifies the psychrotrophic bacterial diversity from an extreme glacier environment as a potential tool for improving plant growth under cold environmental stress.

Physicochemical Approach to Understanding the Structure, Conformation, and Activity of Mannan Polysaccharides.

Extracellular polysaccharides are widely produced by bacteria, yeasts, and algae. These polymers are involved in several biological functions, such as bacteria adhesion to surface and biofilm formation, ion sequestering, protection from desiccation, and cryoprotection. The chemical characterization of these polymers is the starting point for obtaining relationships between their structures and their various functions. While this fundamental correlation is well reported and studied for the proteins, for the polysaccharides, this relationship is less intuitive. In this paper, we elucidate the chemical structure and conformational studies of a mannan exopolysaccharide from the permafrost isolated bacterium Psychrobacter arcticus strain 273-4. The mannan from the cold-adapted bacterium was compared with its dephosphorylated derivative and the commercial product from Saccharomyces cerevisiae. Starting from the chemical structure, we explored a new approach to deepen the study of the structure/activity relationship. A pool of physicochemical techniques, ranging from small-angle neutron scattering (SANS) and dynamic and static light scattering (DLS and SLS, respectively) to circular dichroism (CD) and cryo-transmission electron microscopy (cryo-TEM), have been used. Finally, the ice recrystallization inhibition activity of the polysaccharides was explored. The experimental evidence suggests that the mannan exopolysaccharide from P. arcticus bacterium has an efficient interaction with the water molecules, and it is structurally characterized by rigid-rod regions assuming a 14-helix-type conformation.

Generation of antioxidant peptides from soy protein isolate through psychrotrophic Chryseobacterium sp. derived alkaline broad temperature active protease

Antioxidant peptides confer numerous health benefits. Unexplored niches provide bioprospection opportunities for potential protease producing microorganisms that are useful in releasing antioxidant peptides from protein sources impacting human health. A cold-adapted bacterium Chryseobacterium polytrichastri ERMR1:04 produced an extracellular protease enzyme optimally at 20 °C, pH 8 with 1.25 g/100 mL skim milk inducer. The purified 22.24 kDa protease was active over a broad temperature 5–65 °C (optimum at 37 °C) and pH 6–10 (optimum at pH 8) range, inhibited by EDTA and 1, 10-phenanthroline, enhanced by Ca2+, Mn2+ and hexane, and retained its activity with surfactants and detergents. ERMR1:04 protease-mediated hydrolysis of soy protein generated antioxidant peptides of <6 kDa, with successful identification, showing radical scavenging activities of 77% DPPH, 96% ABTS, 93% ferrous ion chelation, and Abs700 0.159 for reducing power assay. Such promising properties qualify the ERMR1:04 protease as a potent catalyst for high-value functional food production.

Molecular cloning and biochemical characterization of a NAD-dependent sorbitol dehydrogenase from cold-adapted Pseudomonas mandelii.

Sugar alcohols (polyols) have important roles as nutrients, anti-freezing agents and scavengers of free radicals in cold-adapted bacteria, but the characteristics of polyol dehydrogenases in cold-adapted bacteria remain largely unknown. In this study, based on the observation that a cold-adapted bacterium Pseudomonas mandelii JR-1 predominantly utilized d-sorbitol as its carbon source, among the four polyols examined (d-galactitol, d-mannitol, d-sorbitol and d-xylitol), we cloned and characterized a sorbitol dehydrogenase (SDH, EC 1.1.1.14) belonging to the short-chain dehydrogenase/reductase family from this bacterium (the SDH hereafter referred to as PmSDH). PmSDH contained Asn111, Ser140, Tyr153 and Lys157 as catalytic active site residues and existed as an ∼67-kDa dimer in size-exclusion chromatography. PmSDH converted d-sorbitol to d-fructose using nicotinamide adenine dinucleotide (NAD+) as a cofactor and, vice versa, d-fructose to d-sorbitol using nicotinamide adenine dinucleotide reduced (NADH) as a cofactor. PmSDH maintained its conformational flexibility, secondary and tertiary structures, and thermal stability at 4-25°C. These results indicate that PmSDH, which has a flexible structure and a high catalytic activity at colder temperatures, is well suited to sorbitol utilization in the cold-adapted bacterium P. mandelii JR-1.

The nitrogen removal characterization of a cold-adapted bacterium: Bacillus simplex H-b

The removal efficacy of biological nitrogen removal process is inhibited by low temperatures. Herein, a psychrotrophic bacterium strain, Bacillus simplex H-b, was isolated and identified with the potential to conduct heterotrophic nitrification and aerobic denitrification in the temperature range from 5 to 37 °C. At 10 °C, the removal efficiencies of initial nitrate–N (63 mg/L), nitrite–N (10 mg/L) and ammonium–N (60 mg/L) were 67.29%, 78.69% and 82.16%, with the maximum removal rate of 0.56, 0.18 and 0.74 mg/L/h, respectively. Additionally, both the accumulation level of ATP (adenosine triphosphate) and the formation of extracellular polymeric substances was found to increase with the decrease of temperature from 37 °C to 10 °C, indicating strain H-b might resist low temperature stress through its cellular extreme environment resistant mechanism and further suggesting the newly isolated strain could serve as a promising candidate for nitrogen contaminated wastewater treatment, especially under low-temperature condition.

Genome sequencing of cold-adapted Planococcus bacterium isolated from traditional shrimp paste and protease identification.

Psychrophiles have evolved to adapt to freezing environments, and cold-adapted enzymes from these organisms can maintain high catalytic activity at low temperature. The use of cold-adapted enzymes has great potential for the revolution of food and molecular biology industries. In this study, four different strains producing protease were isolated from traditional fermented shrimp paste, one of which, named Planococcus maritimus XJ11 by 16S rRNA nucleotide sequence analysis, exhibited the largest protein hydrolysis clear zone surrounding the colonies. Meanwhile, the strain P. maritimus XJ11 was selected for further investigation because of its great adaptation to low temperature, low salinity and alkaline environment. The enzyme activity assay of P. maritimus XJ11 indicated that the optimum conditions for catalytic activity were pH 10.0 and 40 °C. Moreover, the enzyme also showed an increasing activity with temperatures from 10 to 40 °C and retained more than 67% activity of the maximum over a broad range of salinity (50-150 g L-1 ). Genome sequencing analysis revealed that strain XJ11 possessed one circular chromosome of 3 282 604 bp and one circular plasmid of 67 339 bp, with a total number of 3293 open reading frames (ORFs). Besides, 21 genes encoding protease, including three serine proteases, were identified through the NR database. Cold-adapted bacterium P. maritimus XJ11 was capable of producing alkaline proteases with high catalytic efficiency at low or moderate temperatures. Furthermore, the favorable psychrophilic and enzymatic characters of strain P. maritimus XJ11 seem to have a promising potential for industrial application. © 2020 Society of Chemical Industry.

Pseudarthrobacter psychrotolerans sp. nov., a cold-adapted bacterium isolated from Antarctic soil.

A novel cold-tolerant bacterium, designated strain YJ56T, was isolated from Antarctic soil collected from the Cape Burk area. Phylogenetic analysis through 16S rRNA gene sequence similarity revealed that strain YJ56T was most closely related to the genus Pseudarthrobacter, including Pseudarthrobacter oxydans DSM 20119T (99.06 % similarity), Pseudarthrobacter polychromogenes DSM 20136T (98.98 %) and Pseudarthrobacter sulfonivorans ALLT (98.76 %). The genome size (5.2 Mbp) of strain YJ56T was the largest among all the published genomes of Pseudarthrobacter type strains (4.2-5.0 Mbp). The genomic G+C content of strain YJ56T (64.7 mol%) was found to be consistent with those of other Pseudarthrobacter strains (62.0-71.0 mol%). The average nucleotide identity and average amino acid identity values between strain YJ56T and P. sulfonivorans ALLT were estimated at 84.1 and 84.2 %, respectively. The digital DNA-DNA hybridization value between the two strains was calculated to be 28.0 %. This rod-shaped and obligate aerobic strain exhibited no swimming or swarming motility. It had catalase activity but no oxidase activity. Cells grew at 4-28 °C (optimum, 13 °C) and pH 5.0-11.0 (optimum, pH 7.0) and with 0-6.0 % (w/v) NaCl (optimum, 0%) in Reasoner's 2A medium. MK-9 (H2) was the sole menaquinone. Two-dimensional TLC results revealed that the primary polar lipids were diphosphatidylglycerol, phosphatidylglycerol, two glycolipids and phosphatidylinositol. Fatty acid methyl ester analysis showed that anteiso-C15 : 0, anteiso-C17 : 0, iso-C15 : 0, C16 : 0 and iso-C16 : 0 were the major cellular fatty acids in strain YJ56T. Based on phenotypic and genotypic characteristics, strain YJ56T represents a novel species of the genus Pseudarthrobacter, and thus the name Pseudarthrobacter psychrotolerans sp. nov is proposed. The type strain is YJ56T (=JCM 33881T=KACC 21510T).

Characterisation and bioactivities of an exopolysaccharide from an Antarctic bacterium Shewanella frigidimarina W32–2

The continent of Antarctica breeds numerous special extremophiles that have adapted to the extreme variation of environment with a wide range of novel biodiversity. A cold-adapted bacterium Shewanella frigidimarina W32–2, isolated from Antarctic sediments, was identified to be a promising candidate of exopolysaccharides (EPS) production. In the present study, the physiochemistry and bioactivities of the EPS were investigated to promote its application in aquaculture. Physiological characterisation of this strain was firstly performed by means of combined API 20NE and API ZYM tests. A biochemical analysis revealed that the EPS contained a majority of carbohydrates (56.34 ± 1.29%), uronic acids (8.23 ± 0.55%) and high content of sulphate groups (35.43 ± 1.84%), which were also confirmed by the FTIR spectrum showing various functional groups with potential antioxidant activities. The bioactive properties of the EPS in vitro and in vivo were both evaluated. The effect of the EPS on the non-specific immunity of red tilapia via serum injection exhibited 100% of survival rate. Improvement of activities in different serum enzymes, including AKP, ACP, SOD, CAT and LZM, was observed generally at the early stage of EPS treatment (except LZM at later stage). This study explores novel EPS from an extremophile exhibiting powerful bioactivity with immense potential in aquaculture sustainability.

Structure and Dynamics of a Thermostable Alcohol Dehydrogenase from the Antarctic Psychrophile Moraxella sp. TAE123.

The structure of a recombinant (His-tagged at C-terminus) alcohol dehydrogenase (MoADH) from the cold-adapted bacterium Moraxella sp. TAE123 has been refined with X-ray diffraction data extending to 1.9 Å resolution. The enzyme assumes a homo-tetrameric structure. Each subunit comprises two distinct structural domains: the catalytic domain (residues 1–150 and 288–340/345) and the nucleotide-binding domain (residues 151–287). There are two Zn2+ ions in each protein subunit. Two additional zinc ions have been found in the crystal structure between symmetry-related subunits. The structure has been compared with those of homologous enzymes from Geobacillus stearothermophilus (GsADH), Escherichia coli (EcADH), and Thermus sp. ATN1 (ThADH) that thrive in environments of diverse temperatures. Unexpectedly, MoADH has been found active from 10 to at least 53 °C and unfolds at 89 °C according to circular dichroism spectropolarimetry data. MoADH with substrate ethanol exhibits a small value of activation enthalpy ΔH‡ of 30 kJ mol–1. Molecular dynamics simulations for single subunits of the closely homologous enzymes MoADH and GsADH performed at 280, 310, and 340 K showed enhanced wide-ranging mobility of MoADH at high temperatures and generally lower but more distinct and localized mobility for GsADH. Principal component analysis of the fluctuations of both ADHs resulted in a prominent open–close transition of the structural domains mainly at 280 K for MoADH and 340 K for GsADH. In conclusion, MoADH is a very thermostable, cold-adapted enzyme and the small value of activation enthalpy allows the enzyme to function adequately at low temperatures.

Effects of pH and temperature on (S)-amine activity of transaminase from the cold-adapted bacterium Psychrobacter cryohalolentis.

(7R,8S)-diaminopelargonic acid transaminase from the cold-adapted Gram-negative bacterium Psychrobacter cryohalolentis (Pcryo361) is able to react with unnatural substrates including (S)-( +)-1-phenylethylamine, aldehydes and α-diketones. Additionally, Pcryo361 is active at 0-50°C and retains up to 10% of the maximum activity at 0°C. Here, we report a detailed study on the stability and low temperature activity of Pcryo361. At the optimal pH for (S)-amine activity (pH 10.0), the enzyme was stable at 0-10°C and no decrease in the enzyme activity was observed within 24h in a slightly alkaline medium, pH 8.0, at 35°C. Pcryo361 was solvent stable and was activated in 10% DMSO and DMFA at 35°C. An analysis of the efficiency of catalysis of Pcryo361 at 35°C and 10°C showed that the specificity towards (S)-( +)-1-phenylethylamine dropped at 10°C; however, the specificity towards 2,3-butanedione remained unchanged. Inhibition analysis showed that Pcryo361 activity was not inhibited by acetophenone but inhibited by amines (products of aldehyde amination). The observed pH stability and low temperature activity of Pcryo361 with activated keto substrates are attractive features in the field of development of stereoselective amination at low temperatures.