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.

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.

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.

Complete Genome Sequencing of Polar Arthrobacter sp. PAMC25284, Copper Tolerance Potential Unraveled with Genomic Analysis.

The genus Arthrobacter is a known group of Gram-positive, opportunistic pathogenic bacteria from cold climates, with members that are believed to play a variety of roles at low temperatures. However, their survival mechanisms in frigid environments like the Antarctic are still unknown. We identified a species of Arthrobacter isolated from seawater in the polar region using 16S rRNA sequence analysis. The strain PAMC25284 genome consists of a circular chromosome with a GC content of 65.6% and is projected to contain 3,588 genes, of which 3,150 are protein coding, 366 are pseudogenes, 19 are rRNA coding, and 50 are tRNA coding genes. Using comparative genomics, we showed that PMAC25284 has copper-transporting ATPases, copper chaperone, copper-responsive transcriptional regulator, and multi-copper oxidase domains, which are found in both Gram-positive (like Mycobacterium tuberculosis and Enterococcus hirae) and Gram-negative bacteria (like E. coli and Pseudomonas aeruginosa). The existence of 4 multi-copper oxidase genes, which supplied an additional copper defense mechanism, could be intriguing information regarding Gram-positive bacteria such as Arthrobacter sp. PAMC25284. In addition, our strain PAMC25284 has the same MmcO gene as M. tuberculosis, with a locus tag KY499_RS04055 similarity of 40.61%, which is the highest among the Gram-positive and Gram-negative bacteria studied for this gene. Our cold-adapted Arthrobacter sp. strain PAMC25564 was published previously but did not contain a multi-copper oxidase domain-containing gene, but strain PAMC25284 was studied in this study.

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.

Prediction of trehalose-metabolic pathway and comparative analysis of KEGG, MetaCyc, and RAST databases based on complete genome of Variovorax sp. PAMC28711.

BACKGROUND: Metabolism including anabolism and catabolism is a prerequisite phenomenon for all living organisms. Anabolism refers to the synthesis of the entire compound needed by a species. Catabolism refers to the breakdown of molecules to obtain energy. Many metabolic pathways are undisclosed and many organism-specific enzymes involved in metabolism are misplaced. When predicting a specific metabolic pathway of a microorganism, the first and foremost steps is to explore available online databases. Among many online databases, KEGG and MetaCyc pathway databases were used to deduce trehalose metabolic network for bacteria Variovorax sp. PAMC28711. Trehalose, a disaccharide, is used by the microorganism as an alternative carbon source. RESULTS: While using KEGG and MetaCyc databases, we found that the KEGG pathway database had one missing enzyme (maltooligosyl-trehalose synthase, EC 5.4.99.15). The MetaCyc pathway database also had some enzymes. However, when we used RAST to annotate the entire genome of Variovorax sp. PAMC28711, we found that all enzymes that were missing in KEGG and MetaCyc databases were involved in the trehalose metabolic pathway. CONCLUSIONS: Findings of this study shed light on bioinformatics tools and raise awareness among researchers about the importance of conducting detailed investigation before proceeding with any further work. While such comparison for databases such as KEGG and MetaCyc has been done before, it has never been done with a specific microbial pathway. Such studies are useful for future improvement of bioinformatics tools to reduce limitations.

Complete Genome Sequence and Comparative Genome Analysis of Variovorax sp. Strains PAMC28711, PAMC26660, and PAMC28562 and Trehalose Metabolic Pathways in Antarctica Isolates.

The complete genomes of Variovorax strains were analyzed and compared along with the genomes of Variovorax strains PAMC28711, PAMC28562, and PAMC26660, Antarctic isolates. The genomic information was collected from the NCBI database and the CAZyme database, and Prokka annotation was used to find the genes that encode for the trehalose metabolic pathway. Likewise, CAZyme annotation (dbCAN2 Meta server) was performed to predict the CAZyme family responsible for trehalose biosynthesis and degradation enzymes. Trehalose has been found to respond to osmotic stress and extreme temperatures. As a result, the study of the trehalose metabolic pathway was carried out in harsh environments such as the Antarctic, where bacteria Variovorax sp. strains PAMC28711, PAMC28562, and PAMC26660 can survive in extreme environments, such as cold temperatures. The trehalose metabolic pathway was analyzed via bioinformatics tools, such as the dbCAN2 Meta server, Prokka annotation, Multiple Sequence Alignment, ANI calculator, and PATRIC database, which helped to predict trehalose biosynthesis and degradation genes' involvement in the complete genome of Variovorax strains. Likewise, MEGA X was used for evolutionary and conserved genes. The complete genomes of Variovorax strains PAMC28711, PAMC26660, and PAMC28562 are circular chromosomes of length (4,320,000, 7,390,000, and 4,690,000) bp, respectively, with GC content of (66.00, 66.00, and 63.70)%, respectively. The GC content of these three Variovorax strains is lower than that of the other Variovorax strains with complete genomes. Strains PAMC28711 and PAMC28562 exhibit three complete trehalose biosynthetic pathways (OtsA/OtsB, TS, and TreY/TreZ), but strain PAMC26660 only possesses one (OtsA/OtsB). Despite the fact that all three strains contain trehalose, only strain PAMC28711 has two trehalases according to CAZyme families (GH37 and GH15). Moreover, among the three Antarctica isolates, only strain PAMC28711 exhibits auxiliary activities (AAs), a CAZyme family. To date, although the Variovorax strains are studied for different purposes, the trehalose metabolic pathways in Variovorax strains have not been reported. Further, this study provides additional information regarding trehalose biosynthesis genes and degradation genes (trehalases) as one of the factors facilitating bacterial survival under extreme environments, and this enzyme has shown potential application in biotechnology fields.

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.