A novel Microbacterium strain SRS2 promotes the growth of Arabidopsis and MicroTom (S. lycopersicum) under normal and salt stress conditions.

MAIN CONCLUSION: Microbacterium strain SRS2 promotes growth and induces salt stress resistance in Arabidopsis and MicroTom in various growth substrates via the induction of the ABA pathway. Soil salinity reduces plant growth and development and thereby decreases the value and productivity of soils. Plant growth-promoting rhizobacteria (PGPR) have been shown to support plant growth such as in salt stress conditions. Here, Microbacterium strain SRS2, isolated from the root endosphere of tomato, was tested for its capability to help plants cope with salt stress. In a salt tolerance assay, SRS2 grew well up to medium levels of NaCl, but the growth was inhibited at high salt concentrations. SRS2 inoculation led to increased biomass of Arabidopsis and MicroTom tomato in various growth substrates, in the presence and in the absence of high NaCl concentrations. Whole-genome analysis revealed that the strain contains several genes involved in osmoregulation and reactive oxygen species (ROS) scavenging, which could potentially explain the observed growth promotion. Additionally, we also investigated via qRT-PCR, promoter::GUS and mutant analyses whether the abscisic acid (ABA)-dependent or -independent pathways for tolerance against salt stress were involved in the model plant, Arabidopsis. Especially in salt stress conditions, the plant growth-promotion effect of SRS2 was lost in aba1, abi4-102, abi3, and abi5-1 mutant lines. Furthermore, ABA genes related to salt stress in SRS2-inoculated plants were transiently upregulated compared to mock under salt stress conditions. Additionally, SRS2-inoculated ABI4::GUS and ABI5::GUS plants were slightly more activated compared to the uninoculated control under salt stress conditions. Together, these assays show that SRS2 promotes growth in normal and in salt stress conditions, the latter possibly via the induction of ABA-dependent and -independent pathways.

Microbacterium humicola sp. nov., Microbacterium terrisoli sp. nov., Paenibacillus pedocola sp. nov., Paenibacillus silviterrae sp. nov., Flavobacterium terrisoli sp. nov., and Aquabacterium humicola sp. nov., isolated from soil.

Four Gram-stain-positive and two Gram-stain-negative bacterial strains, designated as W4T, FW7T, TW48T, UW52T, PT-3T, and RJY3T, were isolated from soil samples collected from the Republic of Korea. The 16S rRNA gene sequence analysis showed that strains W4T and FW7T belonged to the genus Microbacterium, strains TW48T and UW52T were affiliated to the genus Paenibacillus, strain PT-3T was related to the genus Flavobacterium, and strain RJY3T was associated with the genus Aquabacterium. The closest phylogenetic taxa to W4T, FW7T, TW48T, UW52T, PT-3T, and RJY3T were Microbacterium bovistercoris NEAU-LLET (97.7 %), Microbacterium protaetiae DFW100M-13T (97.9 %), Paenibacillus auburnensis JJ-7T (99.6 %), Paenibacillus allorhizosphaerae JJ-447T (95.7 %), Flavobacterium buctense T7T (97.1 %), and Aquabacterium terrae S2T (99.5 %), respectively. Average nucleotide identity and digital DNA-DNA hybridization values between the novel strains and related reference type strains were <95.0 % and <70.0 %, respectively. The major cellular fatty acid in strains W4T, FW7T TW48T, and UW52T was antiso-C15 : 0. Similarly, strain PT-3T revealed iso-C15 : 0, iso-C15 : 1 G, iso-C17 : 0 3-OH, and iso-C15 : 0 3-OH as its principal fatty acids. On the other hand, RJY3T exhibited summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c), C16 : 0, summed feature 8 (C18 : 1 ω7c and/or C18 : 1 ω6c), and C12 : 0 as its predominant fatty acids. Overall, the polyphasic taxonomic data indicated that strains W4T, FW7T, TW48T, UW52T, PT-3T, and RJY3T represent novel species within the genera Microbacterium, Paenibacillus, Flavobacterium, and Aquabacterium. Accordingly, we propose the names Microbacterium humicola sp. nov., with the type strain W4T (=KCTC 49888T=NBRC 116001T), Microbacterium terrisoli sp. nov., with the type strain FW7T (=KCTC 49859T=NBRC 116000T), Paenibacillus pedocola sp. nov., with the type strain TW48T (=KCTC 43470T=NBRC 116017T), Paenibacillus silviterrae sp. nov., with the type strain UW52T (=KCTC 43477T=NBRC 116018T), Flavobacterium terrisoli sp. nov., with the type strain PT-3T (=KCTC 92106T=NBRC 116012T), and Aquabacterium humicola sp. nov., with the type strain RJY3T (=KCTC 92105T=NBRC 115831T).

Discovery of a new bacterium, Microbacterium betulae sp. nov., in birch wood associated with hypersensitivity pneumonitis in woodworkers.

A Gram-positive, aerobic, rod-shaped mesophilic bacterium was isolated from birch wood, referred to as the AB strain. Allergological tests suggest that this strain may cause allergic alveolitis in sawmill workers. Employing a polyphasic taxonomic approach, the AB strain's 16S rRNA gene sequence showed high similarity to Microbacterium barkeri and M. oryzae, with 97.25% and 96.91%, respectively, a finding supported by rpoB and gyrB sequence analysis. Further genome sequence comparison with the closely related M. barkeri type strain indicated a digital DNA-DNA hybridization value of 25.5% and an average nucleotide identity of 82.52%. The AB strain's cell wall peptidoglycan contains ornithine, and its polar lipids comprise diphosphatidylglycerol, phosphatidylglycerol, and unidentified glycolipids. Its major fatty acids include anteiso C15:0, anteiso C17:0, and iso C16:0, while MK-10 is its predominant respiratory quinone. Comprehensive analysis through 16S rRNA, whole-genome sequencing, phenotyping, chemotaxonomy, and MALDI-TOF MS profiling indicates that the AB strain represents a new species within the Microbacterium genus. It has been proposed to name this species Microbacterium betulae sp. nov., with ABT (PCM 3040T = CEST 30706T) designated as the type strain.

Microbacterium algihabitans sp. nov., Microbacterium phycohabitans sp. nov., and Microbacterium galbum sp. nov., isolated from dried beach seaweeds.

Three actinobacterial strains, KSW2-21T, KSW2-29T and KSW4-17T, were isolated from dried seaweeds collected around Gwakji Beach in Jeju, Republic of Korea. Their taxonomic positions were determined based on genomic, physiological and morphological characteristics. The isolates were Gram-positive, aerobic, non-motile, rod-shaped bacteria characterized by the following chemotaxonomic features: ornithine as the cell wall diamino acid, the N-glycolyl type of murein, MK-11 as the predominant menaquinone, polar lipids including diphosphatidylglycerol, phosphatidylglycerol, two unidentified glycolipids and four unidentified phospholipids, with anteiso-C15 : 0, iso-C16 : 0 and anteiso-C17 : 0 as the the major fatty acids. The 16S rRNA gene phylogeny showed that the novel strains formed three distinct sublines within the genus Microbacterium. Strain KSW4-17T formed a tight cluster with the type strain of Microbacterium hydrothermale, while strains KSW2-21T and KSW2-29T occupied distinct positions between the type strains of M. hydrothermale and Microbacterium testaceum. Strains KSW4-17T and KSW2-29T showed 99.9 % rRNA gene sequence similarity to M. hydrothermale CGMCC 1.12512T, while strain KSW2-21T revealed 99.4 % 16S rRNA gene sequence similarity to the type strains of M. hydrothermale and M. testaceum. The genome sizes and genomic G+C contents of the three isolates ranged from 3.44 to 3.74 Mbp and from 70.3 to 70.8 mol%, respectively. The phylogenomic tree based on 92 core gene sequences exhibited similar topologies to the 16S rRNA gene phylogeny. The comparison of overall genomic relatedness indices, such as average nucleotide indentity and digital DNA-DNA hybridization, supported that the isolates represent three new species of the genus Microbacterium. Based on the results obtained here, Microbacterium algihabitans sp. nov. (type strain, KSW2-21T=KACC 23322T=DSM 116381T), Microbacterium phycohabitans sp. nov. (type strain KSW2-29T=KACC 22350T=NBRC 115221T) and Microbacterium galbum sp. nov. (type strain, KSW4-17T=KACC 23323T=DSM 116383T) are proposed.

Microbacterium horticulturae sp. nov., a novel actinobacterium isolated from flowerpot soil.

Strain CJN36-1NT, a Gram-stain-positive, non-flagellated, strictly aerobic and short rod-shaped bacterium, was isolated from flowerpot soil sampled in the Jeonju region of the Republic of Korea. Based on 16S rRNA gene sequences and the resulting phylogenetic tree, the strain belonged to the genus Microbacterium. Strain CJN36-1NT contained a chromosome of 3.6 Mbp with a G+C content of 68.5 mol%. The strain grew at 10-37 °C (optimally at 28 °C), at pH 5.0-8.0 (optimally at pH 8.0), and in the presence of 0-7 % NaCl (w/v; optimally with 0 % NaCl). Digital DNA-DNA hybridization, average nucleotide identity and average amino acid identity values between strain CJN36-1NT and its closest related species, Microbacterium protaetiae DFW100M-13T, were 82.0, 81.2, and 23.2 %, respectively. We propose naming this novel species Microbacterium horticulturae sp. nov., with CJN36-1NT (=KACC 23027T=NBRC 116065T) as the type strain.

Metabolism of Inulin via Difructose Anhydride I Pathway in Microbacterium flavum.

Difructose anhydride I (DFA-I) can be produced from inulin, with DFA-I-forming inulin fructotransferase (IFTase-I). However, the metabolism of inulin through DFA-I remains unclear. To clarify this pathway, several genes of enzymes related to this pathway in the genome of Microbacterium flavum DSM 18909 were synthesized, and the corresponding enzymes were encoded, purified, and investigated in vitro. After inulin is decomposed to DFA-I by IFTase-I, DFA-I is hydrolyzed to inulobiose by DFA-I hydrolase. Inulobiose is then hydrolyzed by ß-fructofuranosidase to form fructose. Finally, fructose enters glycolysis through fructokinase. A ß-fructofuranosidase (MfFFase1) clears the byproducts (sucrose and fructo-oligosaccharides), which might be partially hydrolyzed by fructan ß-(2,1)-fructosidase/1-exohydrolase and another fructofuranosidase (MfFFase2). Exploring the DFA-I pathway of inulin and well-studied enzymes in vitro extends our basic scientific knowledge of the energy-providing way of inulin, thereby paving the way for further investigations in vivo and offering a reference for further nutritional investigation of inulin and DFA-I in the future.

Effect of subtherapeutic and therapeutic sulfamethazine concentrations on transcribed genes and translated proteins involved in Microbacterium sp. C448 resistance and degradation.

Microbacterium sp. C448, isolated from a soil regularly exposed to sulfamethazine (SMZ), can use various sulphonamide antibiotics as the sole carbon source for growth. The basis for the regulation of genes encoding the sulphonamide metabolism pathway, the dihydropteroate synthase sulphonamide target (folP), and the sulphonamide resistance (sul1) genes is unknown in this organism. In the present study, the response of the transcriptome and proteome of Microbacterium sp. C448 following exposure to subtherapeutic (33 µM) or therapeutic (832 µM) SMZ concentrations was evaluated. Therapeutic concentration induced the highest sad expression and Sad production, consistent with the activity of SMZ degradation observed in cellulo. Following complete SMZ degradation, Sad production tended to return to the basal level observed prior to SMZ exposure. Transcriptomic and proteomic kinetics were concomitant for the resistance genes and proteins. The abundance of Sul1 protein, 100-fold more abundant than FolP protein, did not change in response to SMZ exposure. Moreover, non-targeted analyses highlighted the increase of a deaminase RidA and a putative sulphate exporter expression and production. These two novel factors involved in the 4-aminophenol metabolite degradation and the export of sulphate residues formed during SMZ degradation, respectively, provided new insights into the Microbacterium sp. C448 SMZ detoxification process.

Microbacterium elymi sp. nov., Isolated from the Rhizospheric Soil of Elymus tsukushiensis, a Plant Native to the Dokdo Islands, Republic of Korea.

Microbacterium elymi KUDC0405T was isolated from the rhizosphere of Elymus tsukushiensis from the Dokdo Islands. The KUDC0405T strain was Gram-stain-positive, non-spore forming, non-motile, and facultatively anaerobic bacteria. Strain KUDC0405T was a rod-shaped bacterium with size dimensions of 0.3-0.4 × 0.7-0.8 µm. Based on 16S rRNA gene sequences, KUDC0405T was most closely related to Microbacterium bovistercoris NEAU-LLET (97.8%) and Microbacterium pseudoresistens CC-5209T (97.6%). The dDDH (digital DNA-DNA hybridization) values between KUDC0405T and M. bovistercoris NEAU-LLET and M. pseudoresistens CC-5209T were below 17.3% and 17.5%, respectively. The ANI (average nucleotide identity) values among strains KUDC0405T, M. bovistercoris NEAU-LLET, and M. pseudoresistens CC-5209T were 86.6% and 80.7%, respectively. The AAI (average amino acid identity) values were 64.66% and 64.97%, respectively, between KUDC0405T and its closest related type strains. The genome contained 3,596 CDCs, three rRNAs, 46 tRNAs, and three non-coding RNAs (ncRNAs). The genomic DNA GC content was 70.4%. The polar lipids included diphosphatydilglycerol, glycolipid, phosphatydilglycerol, and unknown phospholipid, and the major fatty acids were anteiso-C17:0 and iso-C16:0. Strain KUDC0405T contained MK-12 as the major menaquinone. Based on genotypic, phylogenetic, and phenotypic properties, strain KUDC0405T should be considered a novel species within the genus Microbacterium, for which we propose the name M. elymi sp. nov., and the type strain as KUDC0405T (=KCTC 49411T, =CGMCC1.18472T).

Description of Microbacterium neungamense sp. nov. isolated from a hot spring.

The Gram-positive, nonmotile, rod-shaped bacterium EF45044T was isolated from a hot spring in Chungju, South Korea. The strain was able to grow at concentrations of 0‒5% (w/v) NaCl, at pH 6.0‒10.0 and in the temperature range of 18‒50 °C. Strain EF45044T showed the highest 16S rRNA gene sequence similarity (98.2%) with Microbacterium ketosireducens DSM 12510T, and the digital DNA‒DNA hybridization (dDDH), average amino acid identity (AAI), and average nucleotide identity (ANI) values were all lower than the accepted species threshold. Strain EF45044T contained MK‒12 and MK‒13 as the predominant respiratory quinones and anteiso‒C17:0, anteiso‒C15:0, and iso‒C16:0 as the major fatty acids. Diphosphatidylglycerol, phosphatidylglycerol, and glycolipid were detected as the major polar lipids. The cell-wall peptidoglycan contained ornithine. The DNA G + C content was 71.4 mol%. Based on the polyphasic data, strain EF45044T (= KCTC 49703T) presents a novel species of the genus Microbacterium, for which the name Microbacterium neungamense sp. nov. is proposed.

Novel Cluster AZ Arthrobacter phages Powerpuff, Lego, and YesChef exhibit close functional relationships with Microbacterium phages.

Bacteriophages exhibit a vast spectrum of relatedness and there is increasing evidence of close genomic relationships independent of host genus. The variability in phage similarity at the nucleotide, amino acid, and gene content levels confounds attempts at quantifying phage relatedness, especially as more novel phages are isolated. This study describes three highly similar novel Arthrobacter globiformis phages-Powerpuff, Lego, and YesChef-which were assigned to Cluster AZ using a nucleotide-based clustering parameter. Phages in Cluster AZ, Microbacterium Cluster EH, and the former Microbacterium singleton Zeta1847 exhibited low nucleotide similarity. However, their gene content similarity was in excess of the recently adopted Microbacterium clustering parameter, which ultimately resulted in the reassignment of Zeta1847 to Cluster EH. This finding further highlights the importance of using multiple metrics to capture phage relatedness. Additionally, Clusters AZ and EH phages encode a shared integrase indicative of a lysogenic life cycle. In the first experimental verification of a Cluster AZ phage's life cycle, we show that phage Powerpuff is a true temperate phage. It forms stable lysogens that exhibit immunity to superinfection by related phages, despite lacking identifiable repressors typically required for lysogenic maintenance and superinfection immunity. The ability of phage Powerpuff to undergo and maintain lysogeny suggests that other closely related phages may be temperate as well. Our findings provide additional evidence of significant shared phage genomic content spanning multiple actinobacterial host genera and demonstrate the continued need for verification and characterization of life cycles in newly isolated phages.

Characterization and genomic analysis of an efficient dibutyl phthalate degrading bacterium Microbacterium sp. USTB-Y.

A promising bacterial strain for biodegrading dibutyl phthalate (DBP) was successfully isolated from activated sludge and characterized as a potential novel Microbacterium sp. USTB-Y based on 16S rRNA sequence analysis and whole genome average nucleotide identity (ANI). Initial DBP of 50 mg/L could be completely biodegraded by USTB-Y both in mineral salt medium and in DBP artificially contaminated soil within 12 h at the optimal culture conditions of pH 7.5 and 30 â„ƒ, which indicates that USTB-Y has a strong ability in DBP biodegradation. Phthalic acid (PA) was identified as the end-product of DBP biodegraded by USTB-Y using GC/MS. The draft genome of USTB-Y was sequenced by Illumina NovaSeq and 29 and 188 genes encoding for putative esterase/carboxylesterase and hydrolase/alpha/beta hydrolase were annotated based on NR (non redundant protein sequence database) analysis, respectively. Gene3781 and gene3780 from strain USTB-Y showed 100% identity with dpeH and mpeH from Microbacterium sp. PAE-1. But no phthalate catabolic gene (pht) cluster was found in the genome of strain USTB-Y. The results in the present study are valuable for obtaining a more holistic understanding on diverse genetic mechanisms of PAEs biodegrading Microbacterium sp. strains.

Description of Microbacterium luteum sp. nov., Microbacterium cremeum sp. nov., and Microbacterium atlanticum sp. nov., three novel C50 carotenoid producing bacteria.

We have identified three Microbacterium strains, A18JL200T, NY27T, and WY121T, that produce C50 carotenoids. Taxonomy shows they represent three novel species. These strains shared < 98.5% 16S rRNA gene sequence identity with each other and were closely related to Microbacterium aquimaris JCM 15625T, Microbacterium yannicii JCM 18959T, Microbacterium ureisolvens CFH S00084T, and Microbacterium hibisci CCTCC AB 2016180T. Digital DNA-DNA hybridization (dDDH) values and average nucleotide identity (ANI) showed differences among the three strains and from their closest relatives, with values ranging from 20.4% to 34.6% and 75.5% to 87.6%, respectively. These values are below the threshold for species discrimination. Both morphology and physiology also differed from those of phylogenetically related Microbacterium species, supporting that they are indeed novel species. These strains produce C50 carotenoids (mainly decaprenoxanthin). Among the three novel species, A18JL200T had the highest total yield in carotenoids (6.1 mg/L or 1.2 mg/g dry cell weight). Unusual dual isoprenoid biosynthetic pathways (methylerythritol phosphate and mevalonate pathways) were annotated for strain A18JL200T. In summary, we found strains of the genus Microbacterium that are potential producers of C50 carotenoids, but their genome has to be investigated further.

Microbacterium helvum sp. nov., a novel actinobacterium isolated from cow dung.

A Gram-positive, aerobic, non-motile, non-spore-forming, short rod-shaped strain, NEAU-LLCT, was isolated from cow dung in Shangzhi City, Heilongjiang Province, Northeast China and identified by a polyphasic taxonomic study. Colonies was light yellow, round, with entire margin. Strain NEAU-LLCT was grown at 15-45 â„ƒ and pH 6.0-10.0. NaCl concentration ranged from 0 to 5% (W/V). The 16S rRNA gene sequence of NEAU-LLCT showed the high similarities with Microbacterium kyungheense JCM 18735T (98.5%), Microbacterium trichothecenolyticum JCM 1358T (98.3%) and Microbacterium jejuense JCM 18734T (98.2%). The whole-cell sugars were glucose, rhamnose and ribose. The menaquinones contained MK-12 and MK-13. Ornithine, glutamic acid, lysine and a small amount of alanine and glycine were the amino acids in the hydrolyzed products of the cell wall. The major fatty acids were iso-C16:0, iso-C18:0, anteiso-C15:0 and anteiso-C17:0. The main polar lipids were diphosphatidylglycerol, phosphatidylglycerol and an unidentified glycolipid. The genome of NEAU-LLCT was 4,369,375 bp and G + C content is 70.28 mol%. A combination of DNA-DNA hybridization result and some phenotypic characteristics demonstrated that strain NEAU-LLCT could be distinguished from its closely related strains. Therefore, the strain NEAU-LLCT was considered to represent a novel species, which was named Microbacterium helvum sp. (Type strain NEAU-LLCT = CCTCC AA 2018026T = JCM 32661T).

Genomic analysis of Microbacterium sediminis YLB-01T reveals backgrounds related to its deep-sea environment adaptation.

Microbacterium sediminis YLB-01T, a piezotolerant and psychrotolerant actinomycete, was isolated from deep-sea sediment of the South-West Indian Ocean and could be a good model for understanding the adaptation of extremophiles to the benthic piezosphere. Here, we report the analysis of the complete genome sequence of strain YLB-01T. The genome sequence consists of a single circular chromosome comprising 2,792,195 bp and a linear plasmid comprising 127,669 bp with G + C content of 71.76 and 68.49 mol%, respectively. In this regard, strain YLB-01T possesses the smallest genome size but the highest G + C content among the genus Microbacterium sequenced to date. As the first complete genome sequence of the genus Microbacterium isolated from deep-sea environment, the strain YLB-01T genome is unique or enriched in genes involved in xenobiotics biodegradation and metabolism, compatible solutes, and transposases, some of which might be related to bacterial enhancement of ecological fitness in the deep sea.

Functional genomic analysis of bacterial lignin degraders: diversity in mechanisms of lignin oxidation and metabolism.

Although several bacterial lignin-oxidising enzymes have been discovered in recent years, it is not yet clear whether different lignin-degrading bacteria use similar mechanisms for lignin oxidation and degradation of lignin fragments. Genome sequences of 13 bacterial lignin-oxidising bacteria, including new genome sequences for Microbacterium phyllosphaerae and Agrobacterium sp., were analysed for the presence of lignin-oxidising enzymes and aromatic degradation gene clusters that could be used to metabolise the products of lignin degradation. Ten bacterial genomes contain DyP-type peroxidases, and ten bacterial strains contain putative multi-copper oxidases (MCOs), both known to have activity for lignin oxidation. Only one strain lacks both MCOs and DyP-type peroxidase genes. Eleven bacterial genomes contain aromatic degradation gene clusters, of which ten contain the central ß-ketoadipate pathway, with variable numbers and types of degradation clusters for other aromatic substrates. Hence, there appear to be diverse metabolic strategies used for lignin oxidation in bacteria, while the ß-ketoadipate pathway appears to be the most common route for aromatic metabolism in lignin-degrading bacteria.

Symbiotic riboflavin degradation by Microbacterium and Nocardioides bacteria.

Unlike its biosynthetic mechanisms and physiological function, current understanding of riboflavin degradation in soil is limited to a few bacteria that decompose it to lumichrome. Here, we isolated six Microbacterium and three Nocardioides strains. These strains utilized riboflavin and lumichrome, respectively, as carbon sources. Among these strains, we identified Microbacterium paraoxydans R16 (R16) and Nocardioides nitrophenolicus L16 (L16), which were isolated form the same enrichment culture. Co-cultured R16 and L16 reconstituted a riboflavin-degrading interspecies consortium, in which the R16 strain degraded riboflavin to lumichrome and ᴅ-ribose. The L16 strain utilized the lumichrome as a carbon source, indicating that R16 is required for L16 to grow in the consortium. Notably, rates of riboflavin degradation and growth were increased in co-cultured, compared with monocultured R16 cells. These results indicated that a beneficial symbiotic interaction between M. paraoxydans R16 and N. nitrophenolicus L16 results in the ability to degrade riboflavin.

Characterization of cold-tolerant trehalose-6-phosphate synthase from the deep-sea bacterium Microbacterium sediminis YLB-01.

A gene encoding the enzyme trehalose-6-phosphate synthase (TPS), which is part of the TPS trehalose synthesis pathway, was cloned from the deep-sea psychrotolerant bacterium Microbacterium sediminis YLB-01 and expressed in Escherichia coli BL21. The exogenously expressed TPS exhibited highest similarity (80.93% identity) to Microbacterium sp. TPS. The purified recombinant TPS was cold-tolerant, with low thermostability. The optimum temperature for TPS activity was 40°C, and the enzyme retained 72.6% of its maximal activity at 4°C. The optimum pH was 7.5. TPS activity was cation-dependent, with Mg2+, Co2+, or Ba2+ being essential for maximum activity. The kinetic constants of the recombinant TPS reaction rates confirmed that it was cold-tolerant. Molecular dynamics analysis showed that TPS was more flexible (0.8741Å) at 4°C than 1GZ5, its homolog in the mesophilic bacterium E. coli, and superposition of the 3D enzyme structures supported this.