Evidential deep learning for trustworthy prediction of enzyme commission number.

The rapid growth of uncharacterized enzymes and their functional diversity urge accurate and trustworthy computational functional annotation tools. However, current state-of-the-art models lack trustworthiness on the prediction of the multilabel classification problem with thousands of classes. Here, we demonstrate that a novel evidential deep learning model (named ECPICK) makes trustworthy predictions of enzyme commission (EC) numbers with data-driven domain-relevant evidence, which results in significantly enhanced predictive power and the capability to discover potential new motif sites. ECPICK learns complex sequential patterns of amino acids and their hierarchical structures from 20 million enzyme data. ECPICK identifies significant amino acids that contribute to the prediction without multiple sequence alignment. Our intensive assessment showed not only outstanding enhancement of predictive performance on the largest databases of Uniprot, Protein Data Bank (PDB) and Kyoto Encyclopedia of Genes and Genomes (KEGG), but also a capability to discover new motif sites in microorganisms. ECPICK is a reliable EC number prediction tool to identify protein functions of an increasing number of uncharacterized enzymes.

Elucidation of bacterial trehalose-degrading trehalase and trehalose phosphorylase: physiological significance and its potential applications.

Bacteria possess diverse metabolic and genetic processes, resulting in the inability of certain bacteria to degrade trehalose. However, some bacteria do have the capability to degrade trehalose, utilizing it as a carbon source, and for defense against environmental stress. Trehalose, a disaccharide, serves as a carbon source for many bacteria, including some that are vital for pathogens. The degradation of trehalose is carried out by enzymes like trehalase (EC 3.2.1.28) and trehalose phosphorylase (EC 2.4.1.64/2.4.1.231), which are classified under the glycoside hydrolase families GH37, GH15, and GH65. Numerous studies and reports have explored the physiological functions, recombinant expression, enzymatic characteristics, and potential applications of these enzymes. However, further research is still being conducted to understand their roles in bacteria. This review aims to provide a comprehensive summary of the current understanding of trehalose degradation pathways in various bacteria, focusing on three key areas: (i) identifying different trehalose-degrading enzymes in Gram-positive and Gram-negative bacteria, (ii) elucidating the mechanisms employed by trehalose-degrading enzymes belonging to the glycoside hydrolases GH37, GH15, and GH65, and (iii) discussing the potential applications of these enzymes in different sectors. Notably, this review emphasizes the bacterial trehalose-degrading enzymes, specifically trehalases (GH37, GH15, and GH65) and trehalose phosphorylases (GH65), in both Gram-positive and Gram-negative bacteria, an aspect that has not been highlighted before.

Complete genome of Nakamurella sp. PAMC28650: genomic insights into its environmental adaptation and biotechnological potential.

The mechanisms underlying the survival of bacteria in low temperature and high radiation are not yet fully understood. Nakamurella sp. PAMC28650 was isolated from a glacier of Rwenzori Mountain, Uganda, which species belonged to Nakamurella genus based on 16S rRNA phylogeny, ANI (average nucleotide identity), and BLAST Ring Image Generator (BRIG) analysis among Frankineae suborder. We conducted the whole genome sequencing and comparative genomics of Nakamurella sp. PAMC28650, to understand the genomic features pertaining to survival in cold environment, along with high UV (ultraviolet) radiation. This study highlights the role of polysaccharide in cold adaptation, mining of the UV protection-related secondary metabolites and other related to cold adaptation mechanism through different bioinformatics tools, and providing a brief overview of the genes present in DNA repair systems. Nakamurella sp. PAMC28650 contained glycogen and cellulose metabolism pathways, mycosporine-like amino acids and isorenieratene-synthesizing gene cluster, and a number of DNA repair systems. Also, the genome analysis showed osmoregulation-related genes and cold shock proteins. We infer these genomic features are linked to bacterial survival in cold and UV radiation.

A computational approach to identify CRISPR-Cas loci in the complete genomes of the lichen-associated Burkholderia sp. PAMC28687 and PAMC26561.

The genus Burkholderia and its strains PAMC28687 and PAMC26561 are lichen-associated bacteria isolated from the Antarctic region. Our study is the first to provide the genome sequence of the Burkholderia sp. PAMC26561 strain. The genus Burkholderia includes bacteria that are pathogenic to plants, animals, and humans. Computational analysis of complete genomes of strains from the uncategorized Burkholderia group was performed using the NCBI databank and PATRIC (for genome sequence information) and CRISPRCasFinder (online and offline versions) software in order to predict the CRISPR loci and Cas genes. The RNAfold Webserver online software was used to predict RNA secondary structures. Our study showed that strain MSMB0852 (plasmid) possesses CRISPR-Cas system Class 2, and two lichen-associated strains, PAMC28687 (chromosome I) and PAMC26561 (chromosome I), possess CRISPR-Cas system Class 1. Additionally, only the two lichen-associated strains possess a variety of Cas genes.

Complete genome sequence of Arthrobacter sp. PAMC25564 and its comparative genome analysis for elucidating the role of CAZymes in cold adaptation.

BACKGROUND: The Arthrobacter group is a known set of bacteria from cold regions, the species of which are highly likely to play diverse roles at low temperatures. However, their survival mechanisms in cold regions such as Antarctica are not yet fully understood. In this study, we compared the genomes of 16 strains within the Arthrobacter group, including strain PAMC25564, to identify genomic features that help it to survive in the cold environment. RESULTS: Using 16 S rRNA sequence analysis, we found and identified a species of Arthrobacter isolated from cryoconite. We designated it as strain PAMC25564 and elucidated its complete genome sequence. The genome of PAMC25564 is composed of a circular chromosome of 4,170,970 bp with a GC content of 66.74 % and is predicted to include 3,829 genes of which 3,613 are protein coding, 147 are pseudogenes, 15 are rRNA coding, and 51 are tRNA coding. In addition, we provide insight into the redundancy of the genes using comparative genomics and suggest that PAMC25564 has glycogen and trehalose metabolism pathways (biosynthesis and degradation) associated with carbohydrate active enzyme (CAZymes). We also explain how the PAMC26654 produces energy in an extreme environment, wherein it utilizes polysaccharide or carbohydrate degradation as a source of energy. The genetic pattern analysis of CAZymes in cold-adapted bacteria can help to determine how they adapt and survive in such environments. CONCLUSIONS: We have characterized the complete Arthrobacter sp. PAMC25564 genome and used comparative analysis to provide insight into the redundancy of its CAZymes for potential cold adaptation. This provides a foundation to understanding how the Arthrobacter strain produces energy in an extreme environment, which is by way of CAZymes, consistent with reports on the use of these specialized enzymes in cold environments. Knowledge of glycogen metabolism and cold adaptation mechanisms in Arthrobacter species may promote in-depth research and subsequent application in low-temperature biotechnology.

Complete genome sequencing and comparative CAZyme analysis of Rhodococcus sp. PAMC28705 and PAMC28707 provide insight into their biotechnological and phytopathogenic potential.

Study of carbohydrate-active enzymes (CAZymes) can reveal information about the lifestyle and behavior of an organism. Rhodococcus species is well known for xenobiotic metabolism; however, their carbohydrate utilization ability has been less discussed till date. This study aimed to present the CAZyme analysis of two Rhodococcus strains, PAMC28705 and PAMC28707, isolated from lichens in Antarctica, and compare them with other Rhodococcus, Mycobacterium, and Corynebacterium strains. Genome-wide computational analysis was performed using various tools. Results showed similarities in CAZymes across all the studied genera. All three genera showed potential for significant polysaccharide utilization, including starch, cellulose, and pectin referring their biotechnological potential. Keeping in mind the pathogenic strains listed across all three genera, CAZymes associated to pathogenicity were analyzed too. Cutinase enzyme, which has been associated with phytopathogenicity, was abundant in all the studied organisms. CAZyme gene cluster of Rhodococcus sp. PAMC28705 and Rhodococcus sp. PAMC28707 showed the insertion of cutinase in the cluster, further supporting their possible phytopathogenic properties.

Complete Genome Sequence of Pedobacter sp. PAMC26386 and Their Low Temperature Application in Arabinose-containing Polysaccharides Degradation.

Pedobacter are a representative genus of soil-associated bacteria. Here we have provided the complete genome sequence of Pedobacter sp. PAMC26386 isolated from Antarctic soil, and functionally annotated the genome, describing the unique features of carbohydrate active enzymes (CAZymes) and α-L-arabinofuranosidase (α-L-ABF). The genome of Pedobacter sp. PAMC26386 is circular and comprises 4,796,773 bp, with a 38.2% GC content. The genome encodes 4,175 genes, including 7 rRNA and 44 tRNA genes. We identified 172 genes (8 auxiliary activities, 8 carbohydrate binding modules, 23 carbohydrate esterases, 86 glycoside hydrolases, 42 glycosyl transferases, and 5 polysaccharide lyases) related to CAZymes using the dbCAN2 tool. We checked enzyme activity on 11 substrates using the AZCL assay and obtained strong activity for arabinooligosaccharide and hemicellulose. This includes information regarding α-L-ABF, which is active at low temperatures, based on the annotation results. Our findings on Pedobacter sp. PAMC26386 provide the basis for research in the future. The favorable properties of Pedobacter sp. PAMC26386 make it a good candidate for industrial applications involving low temperatures.

Comparative Genomic Study of Polar Lichen-Associated Hymenobacter sp. PAMC 26554 and Hymenobacter sp. PAMC 26628 Reveals the Presence of Polysaccharide-Degrading Ability Based on Habitat.

The genus Hymenobacter is classified in the family Hymenobacteraceae under the phylum Bacteroidetes. They have been isolated from diverse environments, such as air, soil, and lichen, along with extreme polar environments, including the Arctic and Antarctic regions. The polar regions have attracted intense research interest for the discovery of novel microorganisms and their functions. Analysis of the polysaccharide utilization-related carbohydrate-active enzyme among the two lichen-associated polar organisms Hymenobacter sp. PAMC 26554 and Hymenobacter sp. PAMC 26628 was performed, along with its comparison with the complete genome of the same genus available in the NCBI database. The study was conducted relying on the AZCL screening data for the two polar lichen-associated species. While comparing with eight other complete genomes, differences in polysaccharide preferences based on the isolation environment and biosample source were discovered. All the species showed almost similar percentage of cellulose synthesis and degradation genes. However, the polar lichen-associated microorganism was found to have a high percentage of hemicellulose degradation genes, and less starch and laminarin degradation. The Hymenobacter species with higher number of hemicellulose degradation genes was found to have a lower number of starch and laminarin degradation genes and vice versa, highlighting the differences in polysaccharide utilization among the species.

Complete genome sequencing of Shigella sp. PAMC 28760: Identification of CAZyme genes and analysis of their potential role in glycogen metabolism for cold survival adaptation.

Shigella sp. PAMC 28760 (isolated from Himantormia sp. lichen in Antarctica) is a gram-negative, non-sporulating bacterium that has cellulolytic and amylolytic characteristics as well as glycogen metabolic pathways. In this study, we isolated S. sp. PAMC 28760 from Antarctic lichen, and present the complete genome sequence with annotations describing its unique features. The genome sequence has 58.85% GC content, 4,278 coding DNA sequences, 85 tRNAs, and 22 rRNA operons. 16S rRNA gene sequence analyses revealed strain PAMC 28760 as a potentially new species of genus Shigella, showing various differences from pathogenic bacteria reported previously. dbCAN2 analyses revealed 91 genes related to carbohydrate-metabolizing enzymes. S. sp. PAMC 28760 likely degrades polysaccharide starch to obtain glucose for energy conservation. This study provides a foundation for understanding Shigella survival adaptation mechanisms under extremely cold Antarctic conditions.

Genomic insight into O-demethylation of 4-methoxybenzoate by a two-component system from Amycolatopsis magusensis KCCM40447.

Cytochrome P450 monooxygenases perform a multitude of roles, including the generation of hydroxylated aromatic compounds that might be utilized by microorganisms for their survival. WGS data of Amycolatopsis magusensis KCCM40447 revealed a complete circular genome of 9,099,986 base pairs and functionally assigned 8601 protein-encoding genes. Genomic analysis confirmed that the gene for 4-methoxybenzoate monoxygenase (CYP199A35) was conserved in close proximity to the gene for 4-hydroxybenzoate transporter (PcaK). The co-localized genes encoding CYP199A35, and ferredoxin-NAD(P) reductase (Mbr) represent a two-component system for electron transfer. CYP199A35 was specific for O-demethylation of para O-methyl substituted benzoic acid derivatives, 4-methoxybenzoate (4 MB), and 4-methoxycinnamic acid (4MCA) using the native redox partner (Mbr); two-component system and non-physiological redox partners (Pdr/Pdx); three-component system. The catalytic efficiency for O-demethylation of 4 MB using Mbr and Pdr/Pdx was 0.02 ± 0.006 min-1 µM-1 and 0.07 ± 0.02 min-1 µM-1 respectively. Further, sequence annotation and function prediction by RAST and KEEG analysis revealed a complete catabolic pathway for the utilization of 4 MB by strain KCCM40447, which was also proved experimentally.

Comparative Genome Analysis of Polar Mesorhizobium sp. PAMC28654 to Gain Insight into Tolerance to Salinity and Trace Element Stress.

In this study, Mesorhizobium sp. PAMC28654 was isolated from a soil sample collected from the polar region of Uganda. Whole-genome sequencing and comparative genomics were performed to better understand the genomic features necessary for Mesorhizobium sp. PAMC28654 to survive and thrive in extreme conditions and stresses. Additionally, diverse sequence analysis tools were employed for genomic investigation. The results of the analysis were then validated using wet-lab experiments. Genome analysis showed trace elements' resistant proteins (CopC, CopD, CzcD, and Acr3), exopolysaccharide (EPS)-producing proteins (ExoF and ExoQ), and nitrogen metabolic proteins (NarG, NarH, and NarI). The strain was positive for nitrate reduction. It was tolerant to 100 mM NaCl at 15 °C and 25 °C temperatures and resistant to multiple trace elements (up to 1 mM CuSO4·5H2O, 2 mM CoCl2·6H2O, 1 mM ZnSO4·7H2O, 0.05 mM Cd(NO3)2·4H2O, and 100 mM Na2HAsO4·7H2O at 15 °C and 0.25 mM CuSO4·5H2O, 2 mM CoCl2·6H2O, 0.5 mM ZnSO4·7H2O, 0.01 mM Cd(NO3)2·4H2O, and 100 mM Na2HAsO4·7H2O at 25 °C). This research contributes to our understanding of bacteria's ability to survive abiotic stresses. The isolated strain can be a potential candidate for implementation for environmental and agricultural purposes.

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.

In Silico Analysis and Biochemical Characterization of Streptomyces PET Hydrolase with Bis(2-Hydroxyethyl) Terephthalate Biodegradation Activity.

Polyethylene terephthalate (PET), one of the most widely used plastics in the world, causes serious environmental problems. Recently, scientists have been focused on the enzymatic degradation of PET, an environmentally friendly method that offers an attractive approach to the degradation and recycling of PET. In this work, PET hydrolase from Streptomyces sp. W2061 was biochemically characterized, and the biodegradation of PET was performed using the PET model substrate bis (2-hydroxyethyl terephthalate) (BHET). PET hydrolase has an isoelectric point of 5.84, and a molecular mass of about 50.31 kDa. The optimum pH and temperature were 7.0 and 40°C, respectively. LC-MS analysis of the enzymatic products showed that the PET hydrolase successfully degraded a single ester bond of BHET, leading to the formation of MHET. Furthermore, in silico characterization of the PET hydrolase protein sequence and its predicted three-dimensional structure was designed and compared with the well-characterized IsPETase from Ideonella sakaiensis. The structural analysis showed that the (Gly-x1-Ser-x2-Gly) serine hydrolase motif and the catalytic triad (Ser, Asp, and His) were conserved in all sequences. In addition, we integrated molecular dynamics (MD) simulations to analyze the variation in the structural stability of the PET hydrolase in the absence and presence of BHET. These simulations showed the formation of a stable complex between the PET hydrolase and BHET. To the best of our knowledge, this is the first study on Streptomyces sp. W2061 to investigate the BHET degradation activity of PET hydrolase, which has potential application in the biodegradation of plastics in the environment.

Complete Genome Analysis of Subtercola sp. PAMC28395: Genomic Insights into Its Potential Role for Cold Adaptation and Biotechnological Applications.

This study reports the complete genome sequence of Subtercola sp. PAMC28395, a strain isolated from cryoconite in Uganda. This strain possesses several active carbohydrate-active enzyme (CAZyme) genes involved in glycogen and trehalose metabolism. Additionally, two specific genes associated with α-galactosidase (GH36) and bacterial alpha-1,2-mannosidase (GH92) were identified in this strain. The presence of these genes indicates the likelihood that they can be expressed, enabling the strain to break down specific polysaccharides derived from plants or the shells of nearby crabs. The authors performed a comparative analysis of CAZyme patterns and biosynthetic gene clusters (BGCs) in several Subtercola strains and provided annotations describing the unique characteristics of these strains. The comparative analysis of BGCs revealed that four strains, including PAMC28395, have oligosaccharide BGCs, and we confirmed that the pentose phosphate pathway was configured perfectly in the genome of PAMC28395, which may be associated with adaptation to low temperatures. Additionally, all strains contained antibiotic resistance genes, indicating a complex self-resistance system. These results suggest that PAMC28395 can adapt quickly to the cold environment and produce energy autonomously. This study provides valuable information on novel functional enzymes, particularly CAZymes, that operate at low temperatures and can be used for biotechnological applications and fundamental research purposes.

Insights into group-specific pattern of secondary metabolite gene cluster in Burkholderia genus.

Burkholderia is a versatile strain that has expanded into several genera. It has been steadily reported that the genome features of Burkholderia exhibit activities ranging from plant growth promotion to pathogenicity across various isolation areas. The objective of this study was to investigate the secondary metabolite patterns of 366 Burkholderia species through comparative genomics. Samples were selected based on assembly quality assessment and similarity below 80% in average nucleotide identity. Duplicate samples were excluded. Samples were divided into two groups using FastANI analysis. Group A included B. pseudomallei complex. Group B included B. cepacia complex. The limitations of MLST were proposed. The detection of genes was performed, including environmental and virulence-related genes. In the pan-genome analysis, each complex possessed a similar pattern of cluster for orthologous groups. Group A (n = 185) had 14,066 cloud genes, 2,465 shell genes, 682 soft-core genes, and 2,553 strict-core genes. Group B (n = 181) had 39,867 cloud genes, 4,986 shell genes, 324 soft-core genes, 222 core genes, and 2,949 strict-core genes. AntiSMASH was employed to analyze the biosynthetic gene cluster (BGC). The results were then utilized for network analysis using BiG-SCAPE and CORASON. Principal component analysis was conducted and a table was constructed using the results obtained from antiSMASH. The results were divided into Group A and Group B. We expected the various species to show similar patterns of secondary metabolite gene clusters. For in-depth analysis, a network analysis of secondary metabolite gene clusters was conducted, exemplified by BiG-SCAPE analysis. Depending on the species and complex, Burkholderia possessed several kinds of siderophore. Among them, ornibactin was possessed in most Burkholderia and was clustered into 4,062 clans. There was a similar pattern of gene clusters depending on the species. NRPS_04014 belonged to siderophore BGCs including ornibactin and indigoidine. However, it was observed that each family included a similar species. This suggests that, besides siderophores being species-specific, the ornibactin gene cluster itself might also be species-specific. The results suggest that siderophores are associated with environmental adaptation, possessing a similar pattern of siderophore gene clusters among species, which could provide another perspective on species-specific environmental adaptation mechanisms.

Identification and evaluation of CAZyme genes, along with functional characterization of a new GH46 chitosanase from Streptomyces sp. KCCM12257.

The genomic analysis of Streptomyces sp. KCCM12257 presented 233 CAZyme genes with a predominant glycosyl hydrolase family. This contributes degradation of various polysaccharides including chitin and chitosan, and other promising candidates for the production of different oligosaccharides. We screened the strain providing different polysaccharides as a sole source of carbon and strain KCCM12257, showed higher activity towards colloidal chitosan. Further, we identified and characterized a new chitosanase (MDI5907146) of GH46 family. There was no activity towards chitin, carboxymethylcellulose, or even with chitosan powder. This enzyme acts on colloidal chitosan and hydrolyzes it down into monoacetyl chitobiose, which consists of two glucosamine units with an acetyl group attached to them. The maximum enzyme activity was observed at pH 6.5 and 40 °C using colloidal chitosan as a substrate. The Co2+ metal ions almost double the reaction as compared to other metal ions. The dissociation constant (Km) and of colloidal chitosan (≥90 % and ≥75%DD) were 3.03 mg/ml and 5.01 mg/ml respectively, while maximum velocity (Vmax) values were found to be 36 mg/ml, and 30 µM/µg/min, respectively. Similarly, catalytic efficiency (Kcat/Km) of colloidal chitosan with ≥90 %DD was 1.9 fold higher than colloidal chitosan with ≥75%DD.

Comparative Genomic Analysis of Biofilm-Forming Polar Microbacterium sp. Strains PAMC22086 and PAMC21962 Isolated from Extreme Habitats.

The members of Microbacterium isolated from different environments are known to form peptidoglycan. In this study, we compared the biofilm-forming abilities of Microbacterium sp. PAMC22086 (PAMC22086), which was isolated from the soil in the South Shetland Islands and Microbacterium sp. PAMC21962 (PAMC21962), which was isolated from algae in the South Shetland Islands. The analysis of average nucleotide identity and phylogeny of PAMC22086 revealed a 97% similarity to Microbacterium oxydans VIU2A, while PAMC21962 showed a 99.1% similarity to Microbacterium hominis SGAir0570. For the comparative genomic analysis of PAMC22086 and PAMC21962, the genes related to biofilm formation were identified using EggNOG and KEGG pathway databases. The genes possessed by both PAMC22086 and PAMC21962 are cpdA, phnB, rhlC, and glgC, which regulate virulence, biofilm formation, and multicellular structure. Among the genes indirectly involved in biofilm formation, unlike PAMC21962, PAMC22086 possessed csrA, glgC, and glgB, which are responsible for attachment and glycogen biosynthesis. Additionally, in PAMC22086, additional functional genes rsmA, which is involved in mobility and polysaccharide production, and dksA, GTPase, and oxyR, which play roles in cell cycle and stress response, were identified. In addition, the biofilm-forming ability of the two isolates was examined in vivo using the standard crystal violet staining technique, and morphological differences in the biofilm were investigated. It is evident from the different distribution of biofilm-associated genes between the two strains that the bacteria can survive in different niches by employing distinct strategies. Both strains exhibit distinct morphologies. PAMC22086 forms a biofilm that attaches to the side, while PAMC21962 indicates growth starting from the center. The biofilm formation-related genes in Microbacterium are not well understood. However, it has been observed that Microbacterium species form biofilm regardless of the number of genes they possess. Through comparison between different Microbacterium species, it was revealed that specific core genes are involved in cell adhesion, which plays a crucial role in biofilm formation. This study provides a comprehensive profile of the Microbacterium genus's genomic features and a preliminary understanding of biofilm in this genus, laying the foundation for further research.

Elucidation of cold adaptation in Glaciimonas sp. PAMC28666 with special focus on trehalose biosynthesis.

Glaciimonas sp. PAMC28666, an extremophilic bacterium thriving in Antarctic soil and belonging to the Oxalobacteraceae family, represents the only complete genome of its genus available in the NCBI database. Its genome measures 5.2 Mb and comprises 4,476 genes (4,350 protein-coding and 72 non-coding). Phylogenetic analysis shows the strain PAMC28666 in a unique branch within the genus Glaciimonas, closely related to Glaciimonas alpine Cr9-12, supported by robust bootstrap values. In addition, strain PAMC28666 showed 77.08 and 23.3% ANI and DDH, respectively, with Glaciimonas sp. PCH181.This study focuses on how polar strain PAMC28666 responds to freeze-thaw conditions, Experimental results revealed a notable survival rate of 47.28% when subjected to a temperature of 15°C for a period of 10 days. Notably, two genes known to be responsive to cold stress, Trehalose 6-phosphate synthase (otsA) and Trehalose 6-phosphate phosphatase (otsB), exhibited increased expression levels as the temperature shifted from 25°C to 15°C. The upregulation of otsAB and the consequent synthesis of trehalose play pivotal roles in enhancing the cold resistance of strain PAMC28666, offering valuable insights into the correlation between trehalose production and adaptation to cold stress. Furthermore, research into this neglected cold-adapted variation, like Glaciimonas sp. PAMC28666, has the potential to shed light on how trehalose is produced in cold-adapted environments Additionally, there is potential to extract trehalose compounds from this strain for diverse biotechnological applications, including food and cosmetics, with ongoing research exploring its unique properties.

Comparative analysis of genome-based CAZyme cassette in Antarctic Microbacterium sp. PAMC28756 with 31 other Microbacterium species.

BACKGROUND: The genus Microbacterium belongs to the family Microbacteriaceae and phylum Actinobacteria. A detailed study on the complete genome and systematic comparative analysis of carbohydrate-active enzyme (CAZyme) among the Microbacterium species would add knowledge on metabolic and environmental adaptation. Here we present the comparative genomic analysis of CAZyme using the complete genome of Antarctic Microbacterium sp. PAMC28756 with other complete genomes of 31 Microbacterium species available. OBJECTIVE: The genomic and CAZyme comparison of Microbacterium species and to rule out the specific features of CAZyme for the environmental and metabolic adaptation. METHODS: Bacterial source were collected from NCBI database, CAZyme annotation of Microbacterium species was analyzed using dbCAN2 Meta server. Cluster of orthologous groups (COGs) analysis was performed using the eggNOG4.5 database. Whereas, KEGG database was used to compare and obtained the functional genome annotation information in carbohydrate metabolism and glyoxylate cycle. RESULTS: Out of 32 complete genomes of Microbacterium species, strain No. 7 isolated from Activated Sludge showed the largest genomic size at 4.83 Mb. The genomic size of PAMC28756 isolated from Antarctic lichen species Stereocaulons was 3.54 Mb, the G + C content was 70.4% with 3,407 predicted genes, of which 3.36% were predicted CAZyme. In addition, while comparing the Glyoxylate cycle among 32 bacteria, except 10 strains, all other, including our strain have Glyoxylate pathway. PAMC28756 contained the genes that degrade cellulose, hemicellulose, amylase, pectinase, chitins and other exo-and endo glycosidases. Utilizing these polysaccharides can provides source of energy in an extreme environment. In addition, PAMC28756 assigned the (10.15%) genes in the carbohydrate transport and metabolism functional group closely related to the CAZyme for polysaccharides degradation. CONCLUSIONS: The genomic content and CAZymes distribution was varied in Microbacterium species. There was the presence of more than 10% genes in the carbohydrate transport and metabolism functional group closely related to the CAZyme for polysaccharides degradation. In addition, occurrence of glyoxylate cycle for alternative utilization of carbon sources suggest the adaptation of PAMC28756 in the harsh microenvironment.

In silico analysis and a comparative genomics approach to predict pathogenic trehalase genes in the complete genome of Antarctica Shigella sp. PAMC28760.

Although four Shigella species (S. flexneri, S. sonnei, S. dysenteriae, and S. boydii) have been reported, S. sp. PAMC 28760, an Antarctica isolate, is the only one with a complete genome deposited in NCBI database as an uncharacterized isolate. Because it is the world's driest, windiest, and coldest continent, Antarctica provides an unfavourable environment for microorganisms. Computational analysis of genomic sequences of four Shigella species and our uncategorized Antarctica isolates Shigella sp. PAMC28760 was performed using MP3 (offline version) program to predict trehalase encoding genes as a pathogenic or non-pathogenic form. Additionally, we employed RAST and Prokka (offline version) annotation programs to determine locations of periplasmic (treA) and cytoplasmic (treF) trehalase genes in studied genomes. Our results showed that only 56 out of 134 Shigella strains had two different trehalase genes (treF and treA). It was revealed that the treF gene tends to be prevalent in Shigella species. In addition, both treA and treF genes were present in our strain S. sp. PAMC28760. The main objective of this study was to predict the prevalence of two different trehalase genes (treF and treA) in the complete genome of Shigella sp. PAMC28760 and other complete genomes of Shigella species. Till date, it is the first study to show that two types of trehalase genes are involved in Shigella species, which could offer insight on how the bacteria use accessible carbohydrate like glucose produced from the trehalose degradation pathway, and importance of periplasmic trehalase involvement in bacterial virulence.