Integrating multi-platform assembly to recover MAGs from hot spring biofilms: insights into microbial diversity, biofilm formation, and carbohydrate degradation.

BACKGROUND: Hot spring biofilms provide a window into the survival strategies of microbial communities in extreme environments and offer potential for biotechnological applications. This study focused on green and brown biofilms thriving on submerged plant litter within the Sungai Klah hot spring in Malaysia, characterised by temperatures of 58-74 °C. Using Illumina shotgun metagenomics and Nanopore ligation sequencing, we investigated the microbial diversity and functional potential of metagenome-assembled genomes (MAGs) with specific focus on biofilm formation, heat stress response, and carbohydrate catabolism. RESULTS: Leveraging the power of both Illumina short-reads and Nanopore long-reads, we employed an Illumina-Nanopore hybrid assembly approach to construct MAGs with enhanced quality. The dereplication process, facilitated by the dRep tool, validated the efficiency of the hybrid assembly, yielding MAGs that reflected the intricate microbial diversity of these extreme ecosystems. The comprehensive analysis of these MAGs uncovered intriguing insights into the survival strategies of thermophilic taxa in the hot spring biofilms. Moreover, we examined the plant litter degradation potential within the biofilms, shedding light on the participation of diverse microbial taxa in the breakdown of starch, cellulose, and hemicellulose. We highlight that Chloroflexota and Armatimonadota MAGs exhibited a wide array of glycosyl hydrolases targeting various carbohydrate substrates, underscoring their metabolic versatility in utilisation of carbohydrates at elevated temperatures. CONCLUSIONS: This study advances understanding of microbial ecology on plant litter under elevated temperature by revealing the functional adaptation of MAGs from hot spring biofilms. In addition, our findings highlight potential for biotechnology application through identification of thermophilic lignocellulose-degrading enzymes. By demonstrating the efficiency of hybrid assembly utilising Illumina-Nanopore reads, we highlight the value of combining multiple sequencing methods for a more thorough exploration of complex microbial communities.

Draft genome sequence of Hahella sp. CR1 and its ability in producing cellulases for saccharifying agricultural biomass.

Hahella is a genus that has not been well-studied, with only two identified species. The potential of this genus to produce cellulases is yet to be fully explored. The present study isolated Hahella sp. CR1 from mangrove soil in Tanjung Piai National Park, Malaysia, and performed whole genome sequencing (WGS) using NovaSeq 6000. The final assembled genome consists of 62 contigs, 7,106,771 bp, a GC ratio of 53.5%, and encoded for 6,397 genes. The CR1 strain exhibited the highest similarity with Hahella sp. HN01 compared to other available genomes, where the ANI, dDDH, AAI, and POCP were 97.04%, 75.2%, 97.95%, and 91.0%, respectively. In addition, the CAZymes analysis identified 88 GTs, 54 GHs, 11 CEs, 7 AAs, 2 PLs, and 48 CBMs in the genome of strain CR1. Among these proteins, 11 are related to cellulose degradation. The cellulases produced from strain CR1 were characterized and demonstrated optimal activity at 60 ℃, pH 7.0, and 15% (w/v) sodium chloride. The enzyme was activated by K+, Fe2+, Mg2+, Co2+, and Tween 40. Furthermore, cellulases from strain CR1 improved the saccharification efficiency of a commercial cellulase blend on the tested agricultural wastes, including empty fruit bunch, coconut husk, and sugarcane bagasse. This study provides new insights into the cellulases produced by strain CR1 and their potential to be used in lignocellulosic biomass pre-treatment.

Draft Genome Sequence of Zhouia amylolytica CL16, Isolated from Mangrove Soil.

Zhouia amylolytica CL16 was isolated from the mangrove soil of Tanjung Piai, Malaysia. The present work reports the draft genome sequence of this bacterium. The genome consists of 113 glycoside hydrolases, 40 glycosyltransferases, 4 polysaccharide lyases, 23 carbohydrate esterases, 5 auxiliary activities, and 27 carbohydrate-binding modules, which warrant further investigation.

Draft genome sequence of Joostella atrarenae M1-2T with cellulolytic and hemicellulolytic ability.

The halophilic genus Joostella is one of the least-studied genera in the family of Flavobacteriaceae. So far, only two species were taxonomically identified with limited genomic analysis in the aspect of application has been reported. Joostella atrarenae M1-2T was previously isolated from a seashore sample and it is the second discovered species of the genus Joostella. In this project, the genome of J. atrarenae M1-2T was sequenced using NovaSeq 6000. The final assembled genome is comprised of 71 contigs, a total of 3,983,942 bp, a GC ratio of 33.2%, and encoded for 3,416 genes. The 16S rRNA gene sequence of J. atrarenae M1-2T shows 97.3% similarity against J. marina DSM 19592T. Genome-genome comparison between the two strains by ANI, dDDH, AAI, and POCP shows values of 80.8%, 23.3%, 83.4%, and 74.1% respectively. Pan-genome analysis shows that strain M1-2T and J. marina DSM 19592T shared a total of 248 core genes. Taken together, strain M-2T and J. marina DSM 19592T belong to the same genus but are two different species. CAZymes analysis revealed that strain M1-2T harbors 109 GHs, 40 GTs, 5 PLs, 9 CEs, and 6 AAs. Among these CAZymes, while 5 genes are related to cellulose degradation, 12 and 24 genes are found to encode for xylanolytic enzymes and other hemicellulases that involve majorly in the side chain removal of the lignocellulose structure, respectively. Furthermore, both the intracellular and extracellular crude extracts of strain M1-2T exhibited enzymatic activities against CMC, xylan, pNPG, and pNPX substrates, which corresponding to endoglucanase, xylanase, ß-glucosidase, and ß-xylosidase, respectively. Collectively, description of genome coupled with the enzyme assay results demonstrated that J. atrarenae M1-2T has a role in lignocellulosic biomass degradation, and the strain could be useful for lignocellulosic biorefining.

Draft genome sequence of Thermovorax subterraneus 70BT, a thermophile isolated from a geothermally active underground mine that produces hydrogen.

Thermovorax subterraneus 70BT is a thermophile found in a geothermically active underground mine. The strain 70BT belongs to the class of Clostridia, order of Thermosediminibacterales, and family of Thermosediminibacteraceae. Strain 70BT was the only type strain since the genus was discovered >10 years ago. Strain 70BT was compared to strains from other genera in terms of its phenotypics, chemotaxonomics, and phylogenetics (16S rRNA gene) in previous studies. However, the genome sequence of this strain has not been described. We herein described the genome sequence of strain 70BT. In total, the assembled genome of strain 70BT has a size of 2,451,552 bp, contributed by 44 contigs, with a coverage of 445X, a N50 of 86,294 bp, and a GC% of 43.8. A total of 2,540 genes were encoded in the genome, including 2,431 protein-coding sequences, 46 pseudogenes, and 63 RNA genes. Through the Cluster of Orthologous Groups (COGs) analysis, a total of 2,404 protein-coding genes were functionally assigned to COGs in the genome of strain 70BT. Among the members of Thermosediminibacteraceae family, strain 70BT has the closest relationship to Caldanaerovirga acetigignens JW/SA-NV4T based on the genome-to-genome comparison indexes (i.e., ANI, dDDH, AAI, and POCP). An earlier study reported that strain 70BT could produce hydrogen. We discovered genes encoding [FeFe] hydrogenase through gene mining analysis. For future research, this genome data will be used as a reference for all matters pertaining to the genus Thermovorax and family Thermosediminibacteraceae.

Draft Genome Sequence of Microaerobacter geothermalis Nad S1T, a Microaerophilic Bacterium Isolated from Tenusia Hot Spring.

Microaerobacter geothermalis Nad S1T is a rare Bacillaceae thermophile that grows optimally at 55°C and circumneutral pH. Although strain Nad S1T was discovered >10 years ago, its genome is yet to be described. The release of the Nad S1T genome sequence serves as reference genetic information for subsequent use.

Genome Analysis of Celeribacter sp. PS-C1 Isolated from Sekinchan Beach in Selangor, Malaysia, Reveals Its ß-Glucosidase and Licheninase Activities.

A halophilic marine bacterial strain, PS-C1, was isolated from Sekinchan beach in Selangor, Malaysia. The 16S rRNA gene sequence analysis indicated that strain PS-C1 was associated with the genus Celeribacter. To date, there have been no reports on enzymes from the genus Celeribacter. The present study reports on the cellular features of Celeribacter sp. PS-C1, its annotated genome sequence, and comparative genome analyses of Celeribacter glycoside hydrolase (GH) enzymes. The genome of strain PS-C1 has a size of 3.87 Mbp and a G+C content of 59.10%, and contains 3739 protein-coding genes. Detailed analysis using the Carbohydrate-Active enZYmes (CAZy) database revealed that Celeribacter genomes harboured at least 12 putative genes encoding industrially important GHs that are grouped as cellulases, ß-glucanases, hemicellulases, and starch-degrading enzymes. Herein, the potential applications of these enzymes are discussed. Furthermore, the activities of two types of GHs (ß-glucosidase and licheninase) in strain PS-C1 were demonstrated. These findings suggest that strain PS-C1 could be a reservoir of novel GH enzymes for lignocellulosic biomass degradation.

Thermophiles and carbohydrate-active enzymes (CAZymes) in biofilm microbial consortia that decompose lignocellulosic plant litters at high temperatures.

The SKY hot spring is a unique site filled with a thick layer of plant litter. With the advancement of next-generation sequencing, it is now possible to mine many new biocatalyst sequences. In this study, we aimed to (i) identify the metataxonomic of prokaryotes and eukaryotes in microbial mats using 16S and 18S rRNA markers, (ii) and explore carbohydrate degrading enzymes (CAZymes) that have a high potential for future applications. Green microbial mat, predominantly photosynthetic bacteria, was attached to submerged or floating leaves litter. At the spring head, the sediment mixture consisted of plant debris, predominantly brownish-reddish gelatinous microbial mat, pale tan biofilm, and grey-white filament biofilm. The population in the spring head had a higher percentage of archaea and hyperthermophiles than the green mat. Concurrently, we cataloged nearly 10,000 sequences of CAZymes in both green and brown biofilms using the shotgun metagenomic sequencing approach. These sequences include ß-glucosidase, cellulase, xylanase, α-N-arabinofuranosidase, α-L-arabinofuranosidase, and other CAZymes. In conclusion, this work elucidated that SKY is a unique hot spring due to its rich lignocellulosic material, often absent in other hot springs. The data collected from this study serves as a repository of new thermostable macromolecules, in particular families of glycoside hydrolases.

Draft Genome Sequence of Cellulomonas sp. PS-H5, Isolated from Sekinchan Beach in Selangor, Malaysia.

Cellulomonas sp. PS-H5 was isolated from Sekinchan Beach in Selangor, Malaysia, using an ex situ cultivation method. The present work reports a high-quality draft annotated genome sequence of this strain and suggests its potential glycoside hydrolase enzymes for cellulose, hemicellulose, and starch degradations.

Draft Genome Sequence of Roseovarius sp. PS-C2, Isolated from Sekinchan Beach in Selangor, Malaysia.

Roseovarius sp. PS-C2 is a bacterium that was isolated from Sekinchan Beach in Selangor, Malaysia, using an ex situ cultivation technique. Here, we present a high-quality annotated draft genome of strain PS-C2 and suggest potential applications of this bacterium.

Global Transcriptomic Responses of Roseithermus sacchariphilus Strain RA in Media Supplemented with Beechwood Xylan.

The majority of the members in order Rhodothermales are underexplored prokaryotic extremophiles. Roseithermus, a new genus within Rhodothermales, was first described in 2019. Roseithermus sacchariphilus is the only species in this genus. The current report aims to evaluate the transcriptomic responses of R. sacchariphilus strain RA when cultivated on beechwood xylan. Strain RA doubled its growth in Marine Broth (MB) containing xylan compared to Marine Broth (MB) alone. Strain RA harbors 54 potential glycosyl hydrolases (GHs) that are affiliated with 30 families, including cellulases (families GH 3, 5, 9, and 44) and hemicellulases (GH 2, 10, 16, 29, 31,43, 51, 53, 67, 78, 92, 106, 113, 130, and 154). The majority of these GHs were upregulated when the cells were grown in MB containing xylan medium and enzymatic activities for xylanase, endoglucanase, ß-xylosidase, and ß-glucosidase were elevated. Interestingly, with the introduction of xylan, five out of six cellulolytic genes were upregulated. Furthermore, approximately 1122 genes equivalent to one-third of the total genes for strain RA were upregulated. These upregulated genes were mostly involved in transportation, chemotaxis, and membrane components synthesis.

Draft Genome Sequences of Longimonas halophila KCTC 42399 and Longibacter salinarum KCTC 52045.

Longimonas halophila and Longibacter salinarum are type strains of underexplored genera affiliated with Salisaetaceae Herein, we report the draft genome sequences of two strains of these bacteria, L. halophila KCTC 42399 and L. salinarum KCTC 52045, with the intent of broadening knowledge of this family. Genome annotation and gene mining revealed that both bacteria exhibit amylolytic abilities.

Heterologous expression, purification and biochemical characterization of a new endo-1,4-ß-xylanase from Rhodothermaceae bacterium RA.

Xylanases (EC 3.2.1.8) are essential enzymes due to their applications in various industries such as textile, animal feed, paper and pulp, and biofuel industries. Halo-thermophilic Rhodothermaceae bacterium RA was previously isolated from a hot spring in Malaysia. Genomic analysis revealed that this bacterium is likely to be a new genus of the family Rhodothermaceae. In this study, a xylanase gene (1140 bp) that encoded 379 amino acids from the bacterium was cloned and expressed in Escherichia coli BL21(DE3). Based on InterProScan, this enzyme XynRA1 contained a GH10 domain and a signal peptide sequence. XynRA1 shared low similarity with the currently known xylanases (the closest is 57.2-65.4% to Gemmatimonadetes spp.). The purified XynRA1 achieved maximum activity at pH 8 and 60 °C. The protein molecular weight was 43.1 kDa XynRA1 exhibited an activity half-life (t1/2) of 1 h at 60 °C and remained stable at 50 °C throughout the experiment. However, it was NaCl intolerant, and various types of salt reduced the activity. This enzyme effectively hydrolyzed xylan (beechwood, oat spelt, and Palmaria palmata) and xylodextrin (xylotriose, xylotetraose, xylopentaose, and xylohexaose) to produce predominantly xylobiose. This xylanase is the first functionally characterized enzyme from the bacterium, and this work broadens the knowledge of GH10 xylanases.

Characterizing a Halo-Tolerant GH10 Xylanase from Roseithermus sacchariphilus Strain RA and Its CBM-Truncated Variant.

A halo-thermophilic bacterium, Roseithermus sacchariphilus strain RA (previously known as Rhodothermaceae bacterium RA), was isolated from a hot spring in Langkawi, Malaysia. A complete genome analysis showed that the bacterium harbors 57 glycoside hydrolases (GHs), including a multi-domain xylanase (XynRA2). The full-length XynRA2 of 813 amino acids comprises a family 4_9 carbohydrate-binding module (CBM4_9), a family 10 glycoside hydrolase catalytic domain (GH10), and a C-terminal domain (CTD) for type IX secretion system (T9SS). This study aims to describe the biochemical properties of XynRA2 and the effects of CBM truncation on this xylanase. XynRA2 and its CBM-truncated variant (XynRA2ΔCBM) was expressed, purified, and characterized. The purified XynRA2 and XynRA2ΔCBM had an identical optimum temperature at 70 °C, but different optimum pHs of 8.5 and 6.0 respectively. Furthermore, XynRA2 retained 94% and 71% of activity at 4.0 M and 5.0 M NaCl respectively, whereas XynRA2ΔCBM showed a lower activity (79% and 54%). XynRA2 exhibited a turnover rate (kcat) of 24.8 s-1, but this was reduced by 40% for XynRA2ΔCBM. Both the xylanases hydrolyzed beechwood xylan predominantly into xylobiose, and oat-spelt xylan into a mixture of xylo-oligosaccharides (XOs). Collectively, this work suggested CBM4_9 of XynRA2 has a role in enzyme performance.

Complete genome sequence of Rhodothermaceae bacterium RA with cellulolytic and xylanolytic activities.

Rhodothermaceae bacterium RA is a halo-thermophile isolated from a saline hot spring. Previously, the genome of this bacterium was sequenced using a HiSeq 2500 platform culminating in 91 contigs. In this report, we report on the resequencing of its complete genome using a PacBio RSII platform. The genome has a GC content of 68.3%, is 4,653,222 bp in size, and encodes 3711 genes. We are interested in understanding the carbohydrate metabolic pathway, in particular the lignocellulosic biomass degradation pathway. Strain RA harbors 57 glycosyl hydrolase (GH) genes that are affiliated with 30 families. The bacterium consists of cellulose-acting (GH 3, 5, 9, and 44) and hemicellulose-acting enzymes (GH 3, 10, and 43). A crude cell-free extract of the bacterium exhibited endoglucanase, xylanase, ß-glucosidase, and ß-xylosidase activities. The complete genome information coupled with biochemical assays confirms that strain RA is able to degrade cellulose and xylan. Therefore, strain RA is another excellent member of family Rhodothermaceae as a repository of novel and thermostable cellulolytic and hemicellulolytic enzymes.

Purification and characterization of a novel GH1 beta-glucosidase from Jeotgalibacillus malaysiensis.

Beta-glucosidase (BGL) is an important industrial enzyme for food, waste and biofuel processing. Jeotgalibacillus is an understudied halophilic genus, and no beta-glucosidase from this genus has been reported. A novel beta-glucosidase gene (1344 bp) from J. malaysiensis DSM 28777T was cloned and expressed in E. coli. The recombinant protein, referred to as BglD5, consists of a total 447 amino acids. BglD5 purified using a Ni-NTA column has an apparent molecular mass of 52 kDa. It achieved the highest activity at pH 7 and 65 °C. The activity and stability were increased when CaCl2 was supplemented to the enzyme. The enzyme efficiently hydrolyzed salicin and (1 → 4)-beta-glycosidic linkages such as in cellobiose, cellotriose, cellotetraose, cellopentose, and cellohexanose. Similar to many BGLs, BglD5 was not active towards polysaccharides such as Avicel, carboxymethyl cellulose, Sigmacell cellulose 101, alpha-cellulose and xylan. When BglD5 blended with Cellic® Ctec2, the total sugars saccharified from oil palm empty fruit bunches (OPEFB) was enhanced by 4.5%. Based on sequence signatures and tree analyses, BglD5 belongs to the Glycoside Hydrolase family 1. This enzyme is a novel beta-glucosidase attributable to its relatively low sequence similarity with currently known beta-glucosidases, where the closest characterized enzyme is the DT-Bgl from Anoxybacillus sp. DT3-1.

Use of Megaprimer and Overlapping Extension PCR (OE-PCR) to Mutagenize and Enhance Cyclodextrin Glucosyltransferase (CGTase) Function.

Protein engineering is a very useful tool for probing structure-function relationships in proteins. Specifically, site-directed mutagenized proteins can provide useful insights into structural, binding and catalytic mechanisms of a protein, particularly when coupled with crystallization. In this chapter, we describe two protocols for performing site-directed mutagenesis of any protein-coding sequence, namely, megaprimer PCR and overlapping extension PCR (OE-PCR). We use as an example how these two SDM methods enhanced the function of a cyclodextrin glucosyltransferase (CGTase) from Bacillus lehensis strain G1.

Genome Analysis of a New Rhodothermaceae Strain Isolated from a Hot Spring.

A bacterial strain, designated RA, was isolated from water sample of a hot spring on Langkawi Island of Malaysia using marine agar. Strain RA is an aerophilic and thermophilic microorganism that grows optimally at 50-60°C and is capable of growing in marine broth containing 1-10% (w/v) NaCl. 16S rRNA gene sequence analysis demonstrated that this strain is most closely related (<90% sequence identity) to Rhodothermaceae, which currently comprises of six genera: Rhodothermus (two species), Salinibacter (three species), Salisaeta (one species), Rubricoccus (one species), Rubrivirga (one species), and Longimonas (one species). Notably, analysis of average nucleotide identity (ANI) values indicated that strain RA may represent the first member of a novel genus of Rhodothermaceae. The draft genome of strain RA is 4,616,094 bp with 3630 protein-coding gene sequences. Its GC content is 68.3%, which is higher than that of most other genomes of Rhodothermaceae. Strain RA has genes for sulfate permease and arylsulfatase to withstand the high sulfur and sulfate contents of the hot spring. Putative genes encoding proteins involved in adaptation to osmotic stress were identified which encode proteins namely Na(+)/H(+) antiporters, a sodium/solute symporter, a sodium/glutamate symporter, trehalose synthase, malto-oligosyltrehalose synthase, choline-sulfatase, potassium uptake proteins (TrkA and TrkH), osmotically inducible protein C, and the K(+) channel histidine kinase KdpD. Furthermore, genome description of strain RA and comparative genome studies in relation to other related genera provide an overview of the uniqueness of this bacterium.