Cloning, expression in Komagataella phaffii, and biochemical characterization of recombinant sequence variants of Pseudomonas sp. S9 GDSL-esterase

Two recombinant Komagataella phaffii (formerly Pichia pastoris) yeast strains for production of two sequential variants of EstS9 esterase from psychrotolerant bacterium Pseudomonas sp. S9, i.e. αEstS9N (a two-domain enzyme consisting of a catalytic domain and an autotransporter domain) and αEstS9Δ (a single-domain esterase) were constructed. However, only one of recombinant K. phaffii strains, namely Komagataella phaffii X-33/pPICZαestS9Δ, allowed to successfully produce and secrete recombinant αEstS9Δ enzyme outside of the host cell. The purified αEstS9Δ esterase was active towards short-chain p-nitrophenyl esters (C2–C8), with optimal activity for the acetate (C2) ester. The single-domain αEstS9Δ esterase exhibits the highest activity at 60oC and pH 9.5. In addition, the enzyme retains 90% of its activity after 3 hour incubation at 70–90oC. What should be also noted is that αEstS9Δ esterase produced in the K. phaffii expression system has a much higher specific activity (0.069 U/mg of protein) than the recombinant EstS9Δ esterase produced in an E. coli expression system (0.0025 U/mg of protein) (Wicka et al., 2016, Acta Biochim Pol 63: 117–125. https://doi.org/10.18388/abp.2015_1074).

Draft Genome Sequence of the Chitin-Degrading Psychrotolerant Bacterium Pedobacter jejuensis TN23, Isolated from Antarctic Soil

Pedobacter jejuensis TN23 was isolated from soil from Terra Nova Bay, Victoria Land, Antarctica. The assembled draft genome size is 4,795,808 bp, and it contains a total of 4,095 genes with 3,970 coding sequences, including genes putatively involved in the degradation of chitin.

Description and Genome Analysis of Microvirga Antarctica Sp. Nov., A Novel Pink-Pigmented Psychrotolerant Bacterium Isolated From Antarctic Soil

During the investigation of exploring potential psychrotolerant species from Antarctica soil, a novel pink-pigmented bacterium designated strain 3D7T was isolated. Cells of the isolate were observed to be rod-shaped (0.7–0.9×1.0–2.2 µm), Gram-stain negative and non-motile. It was able to grow at 4–32 ℃, pH 7.0–10.0 and in the presence of 0–3 % (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain 3D7T belongs to the genus Microvirga and was most closely related to ‘Microvirga brassicacearum’ CDVBN77T (98.3 %), Microvirga subterranea DSM 14364T (96.8 %), Microvirga guangxiensis 25BT (96.5 %) and Microvirga aerophila DSM 21344T (96.5 %). The predominant quinone was ubiquinone 10 (Q-10), and the major fatty acids were summed feature 8 (C18:1ω7c and/or C18:1ω6c) and C19:0 cyclo ω8c. The predominant polar lipids were phosphatidylcholine and phosphatidylethanolamine. The genomic DNA G + C content of strain 3D7T was 63.5 mol%. Its genome sequence showed genes encoding phosphatases and lipases. Genetic machinery related to carbohydrate-active enzymes and secondary metabolites were also observed. The average nucleotide identity and digital DNA–DNA hybridization values based on whole genome sequences of strain 3D7T and its closely related species were below the threshold range for species determination. Phenotypic, chemotaxonomic, phylogenetic and genomic analyses suggested that strain 3D7T represents a novel species of the genus Microvirga, for which the name Microvirga antarctica sp. nov. is proposed. The type strain is 3D7T (= CGMCC 1.13821T = KCTC 72465T).

Physiological response of the moderately halophilic psychrotolerant strain Chromohalobacter sp. N1 to salinity change and low temperature

The available information on de novo synthesized compatible solutes in response to high medium salinity by bacteria of the Chromohalobacter genus is limited to studies of the mesophilic moderately halophilic strain Chromohalobacter salexigens DSM 3043T. Therefore, there is a need for studies of representatives of other species of the Chromohalobacter genus of the Halomonadaceae family. A moderately halophilic psychrotolerant bacterium, strain N1, closely related to the species Chromohalobacter japonicus was isolated from the salt crust of a rock salt waste pile in Berezniki, Perm Krai, Russia. An intracellular pool of compatible solutes of strain N1 was investigated by NMR spectroscopy. Cells grown in the presence of 5% NaCl at optimal growth temperature (28 °C) accumulated ectoine, glutamate, N(4)-acetyl-l-2,4-diaminobutyrate (NADA), alanine, trehalose, hydroxyectoine, and valine. Such a combination of compatible solutes is unique and distinguishes the strain from C. salexigens DSM 3043T. Hyperosmotic stress induced by 15% NaCl caused the accumulation of ectoine, NADA, and hydroxyectoine but led to a decrease in the amount of alanine, valine, and trehalose. The intracellular pool of glutamate was not significantly changed. A reduction of the growth temperature from 28 to 5 °C led to an increase in the amount of ectoine, NADA, trehalose, and hydroxyectoine. Ectoine was the major compatible solute.

Whole-Genome Shotgun Sequence of Bacillus mycoides Strain U53, a Psychrotolerant Bacterium Isolated from the Sakhalin Region in Russia

ABSTRACT The shotgun genome sequence of Bacillus mycoides strain U53, a psychrotolerant bacterium that was isolated from soil in Russia, is characterized. The bacterial genome is 5,710,017 bp long and is predicted to have 5,847 genes. The GC content is 35.4%.

Hymenobacter polaris sp. nov., a psychrotolerant bacterium isolated from an Arctic station

A pink-pigmented, non-motile, Gram-stain-negative, rod-shaped bacterium, designated RP-2-7T, was obtained from soil sampled at the Arctic station, Spitsbergen, Svalbard, Norway. Cells were strictly aerobic, psychrotolerant, grew optimally at 15–20 °C and hydrolysed CM-cellulose. Phylogenetic analysis based on its 16S rRNA gene sequence revealed that strain RP-2-7T formed a lineage within the family Hymenobacteraceae and clustered with members of the genus Hymenobacter . Its closest relative was Hymenobacter marinus KJ035T (97.6 % sequence similarity). The sequence similarities to other strains were ≤96.9 %. The principal respiratory quinone was MK-7 and the major polar lipids were phosphatidylethanolamine and an unidentified aminophospholipid. The predominant cellular fatty acids were summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c), anteiso-C15 : 0, iso-C15 : 0, C16 : 1 ω5c and summed featured 4 (iso-C17 : 1 I and/or anteiso-C17 : 1 B). The DNA G+C content was 62.8 mol%. In addition, the average nucleotide identity and in silico DNA–DNA hybridization relatedness values between strain RP-2-7T and closely related strains were lower than species demarcation thresholds. Based on the resuls of genomic, chemotaxonomic, phenotypic and phylogenetic analyses, strain RP-2-7T represents novel species in the genus Hymenobacter , for which the name Hymenobacter polaris sp. nov. is proposed. The type strain is RP-2-7T (=KACC 21670T=NBRC 114391T).