The study of microbial communities from extreme environments is a fascinating topic. With every study, biologists and ecologists reveal interesting facts and questions that dispel the old belief that these are inhospitable environments. In this work, we assess the microbial diversity of three hot springs from Neuquén, Argentina, using high-throughput amplicon sequencing. We predicted a distinct metabolic profile in the acidic and the circumneutral samples, with the first ones being dominated by chemolithotrophs and the second ones by chemoheterotrophs. Then, we collected data of the microbial communities of hot springs around the world in an effort to comprehend the roles of pH and temperature as shaping factors. Interestingly, there was a covariation between both parameters and the phylogenetic distance between communities; however, neither of them could explain much of the microbial profile in an ordination model. Moreover, there was no correlation between alpha diversity and these parameters. Therefore, the microbial communities' profile seemed to have complex shaping factors beyond pH and temperature. Lastly, we looked for taxa associated with different environmental conditions. Several such taxa were found. For example, Hydrogenobaculum was frequently present in acidic springs, as was the Sulfolobaceae family; on the other hand, Candidatus Hydrothermae phylum was strongly associated with circumneutral conditions. Interestingly, some singularities related to sites featuring certain taxa were also observed.
The wide use of whole-genome sequencing approach in the modern genomic era has opened a great opportunity to reveal the prospective applications of halophilic bacteria. Robertkochia marina CC-AMO-30DT is one of the halophilic bacteria that was previously taxonomically identified without any inspection on its biotechnological potential from a genomic aspect. In this study, we present the whole-genome sequence of R. marina and demonstrated the ability of this bacterium in solubilizing phosphate by producing phosphatase. The genome of R. marina has 3.57 Mbp and contains 3107 predicted genes, from which 3044 are protein coding, 52 are non-coding RNAs, and 11 are pseudogenes. Several phosphatases such as alkaline phosphatases and pyrophosphatases were mined from the genome. Further genomic study (phylogenetics, sequence analysis, and functional mechanism) and experimental data suggested that the alkaline phosphatase produced by R. marina could potentially be utilized in promoting plant growth, particularly for plants on saline-based agricultural land.
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.
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.
Vitellibacter aquimaris D-24T (=KCTC 42708T=DSM 101732T), a halophilic marine bacterium, was isolated from seawater collected from Desaru beach, Malaysia. Here, we present the draft genome sequence of D-24T with a genome size of approximately 3.1Mbp and G+C content of 39.93%. The genome of D-24T contains genes involved in reducing a potent greenhouse gas (N2O) in the environment and the degradation of proteinaceous compounds. Genome availability will provide insights into potential biotechnological and environmental applications of this bacterium.
Jeotgalibacillus malaysiensis, a moderate halophilic bacterium isolated from a pelagic area, can endure higher concentrations of sodium chloride (NaCl) than other Jeotgalibacillus type strains. In this study, we therefore chose to sequence and assemble the entire J. malaysiensis genome. This is the first report to provide a detailed analysis of the genomic features of J. malaysiensis, and to perform genetic comparisons between this microorganism and other halophiles. J. malaysiensis encodes a native megaplasmid (pJeoMA), which is greater than 600 kilobases in size, that is absent from other sequenced species of Jeotgalibacillus. Subsequently, RNA-Seq-based transcriptome analysis was utilised to examine adaptations of J. malaysiensis to osmotic stress. Specifically, the eggNOG (evolutionary genealogy of genes: Non-supervised Orthologous Groups) and KEGG (Kyoto Encyclopaedia of Genes and Genomes) databases were used to elucidate the overall effects of osmotic stress on the organism. Generally, saline stress significantly affected carbohydrate, energy, and amino acid metabolism, as well as fatty acid biosynthesis. Our findings also indicate that J. malaysiensis adopted a combination of approaches, including the uptake or synthesis of osmoprotectants, for surviving salt stress. Among these, proline synthesis appeared to be the preferred method for withstanding prolonged osmotic stress in J. malaysiensis.
To date, the genus Roseivirga consists of six species with one subspecies and is one of the least-studied genera among the family Flammeovirgaceae. In order to further explore this genus, the genome sequences of five Roseivirga spp. were compared and described in this study. The Roseivirga genomes have similar sizes in the range of 4.08-4.47Mb with an average of 4.22Mb. Several key proteins related to osmotic stress adaptation were identified in Roseivirga spp. including betaine transporter, choline dehydrogenase, and glutamate synthases. Significant amount of proteins associated with amino acid transport and metabolism were also present in Roseivirga genome. All five Roseivirga spp. were able to grow in medium contained casamino acids (mixture of amino acids) as sole carbon or nitrogen sources. Taken together, these findings suggested the potential role of Roseivirga in decomposing organic nitrogen matter in marine environment.
In general, biofuel production involves biomass pretreatment and enzymatic saccharification, followed by the subsequent sugar conversion to biofuel via fermentation. The crucial step in the production of biofuel from biomass is the enzymatic saccharification. Many of the commercial cellulase enzyme cocktails, such as Spezyme(®) CP (Genencor), Acellerase™ 1000 (Genencor), and Celluclast(®) 1.5L (Novozymes), are ineffectively to release free glucose from the pretreated biomass without additional β-glucosidase.
The glycoside hydrolase family 39 (GH39) proteins are renowned for their extremophilic and multifunctional enzymatic properties, yet the molecular mechanisms underpinning these unique characteristics continue to be an active subject of research. In this study, we introduce WsuXyn, a GH39 protein with a molecular weight of 58 kDa, originating from the thermophilic Geobacillus sp. WSUCF1. Previously reported for its exceptional thermostable β-xylosidase activity, WsuXyn has recently demonstrated a significant endoxylanase activity (3752 U·mg-1) against beechwood xylan, indicating towards its bifunctional nature. Physicochemical characterization revealed that WsuXyn exhibits optimal endoxylanase activity at 70 °C and pH 7.0. Thermal stability assessments revealed that the enzyme is resilient to elevated temperatures, with a half-life of 168 h. Key kinetic parameters highlight the exceptional catalytic efficiency and strong affinity of the protein for xylan substrate. Moreover, WsuXyn-mediated hydrolysis of beechwood xylan has achieved 77 % xylan conversion, with xylose as the primary product. Structural analysis, amalgamated with docking simulations, has revealed strong binding forces between xylotetraose and the protein, with key amino acid residues, including Glu278, Tyr230, Glu160, Gly202, Cys201, Glu324, and Tyr283, playing pivotal roles in these interactions. Therefore, WsuXyn holds a strong promise for biodegradation and value-added product generation through lignocellulosic biomass conversion.
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, β-xylosidase, and β-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.
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.
Vibrio sp. PBL-C16 is a bacterium that was isolated from Batu Laut Beach in Selangor, Malaysia. Here, we present a high-quality annotated draft genome of strain PBL-C16 and suggest its potential glycoside hydrolase enzymes for polysaccharide degradation.
Species of Anoxybacillus are widespread in geothermal springs, manure, and milk-processing plants. The genus is composed of 22 species and two subspecies, but the relationship between its lifestyle and genome is little understood. In this study, two high-quality draft genomes were generated from Anoxybacillus spp. SK3-4 and DT3-1, isolated from Malaysian hot springs. De novo assembly and annotation were performed, followed by comparative genome analysis with the complete genome of Anoxybacillus flavithermus WK1 and two additional draft genomes, of A. flavithermus TNO-09.006 and A. kamchatkensis G10. The genomes of Anoxybacillus spp. are among the smaller of the family Bacillaceae. Despite having smaller genomes, their essential genes related to lifestyle adaptations at elevated temperature, extreme pH, and protection against ultraviolet are complete. Due to the presence of various competence proteins, Anoxybacillus spp. SK3-4 and DT3-1 are able to take up foreign DNA fragments, and some of these transferred genes are important for the survival of the cells. The analysis of intact putative prophage genomes shows that they are highly diversified. Based on the genome analysis using SEED, many of the annotated sequences are involved in carbohydrate metabolism. The presence of glycosyl hydrolases among the Anoxybacillus spp. was compared, and the potential applications of these unexplored enzymes are suggested here. This is the first study that compares Anoxybacillus genomes from the aspect of lifestyle adaptations, the capacity for horizontal gene transfer, and carbohydrate metabolism.
A new subfamily of glycosyl hydrolase family GH13 was recently proposed for α-amylases from Anoxybacillus species (ASKA and ADTA), Geobacillus thermoleovorans (GTA, Pizzo, and GtamyII), Bacillus aquimaris (BaqA), and 95 other putative protein homologues. To understand this new GH13 subfamily, we report crystal structures of truncated ASKA (TASKA). ASKA is a thermostable enzyme capable of producing high levels of maltose. Unlike GTA, biochemical analysis showed that Ca(2+) ion supplementation enhances the catalytic activities of ASKA and TASKA. The crystal structures reveal the presence of four Ca(2+) ion binding sites, with three of these binding sites are highly conserved among Anoxybacillus α-amylases. This work provides structural insights into this new GH13 subfamily both in the apo form and in complex with maltose. Furthermore, structural comparison of TASKA and GTA provides an overview of the conformational changes accompanying maltose binding at each subsite.
The Bacillaceae family members are a good source of bacteria for bioprocessing and biotransformation involving whole cells or enzymes. In contrast to Bacillus and Geobacillus, Anoxybacillus is a relatively new genus that was proposed in the year 2000. Because these bacteria are alkali-tolerant thermophiles, they are suitable for many industrial applications. More than a decade after the first report of Anoxybacillus, knowledge accumulated from fundamental and applied studies suggests that this genus can serve as a good alternative in many applications related to starch and lignocellulosic biomasses, environmental waste treatment, enzyme technology, and possibly bioenergy production. This current review provides the first summary of past and recent discoveries regarding the isolation of Anoxybacillus, its medium requirements, its proteins that have been characterized and cloned, bioremediation applications, metabolic studies, and genomic analysis. Comparisons to some other members of Bacillaceae and possible future applications of Anoxybacillus are also discussed.
The genus Meridianimaribacter is one of the least-studied genera within Cytophaga-Flavobacteria. To date, no genomic analysis of Meridianimaribacter has been reported. In this study, Meridianimaribacter sp. strain CL38, a lignocellulose degrading halophile was isolated from mangrove soil. The genome of strain CL38 was sequenced and analyzed. The assembled genome contains 17 contigs with 3.33 Mbp, a GC content of 33.13% and a total of 2982 genes predicted. Lignocellulose degrading enzymes such as cellulases (GH3, 5, 9, 16, 74 and 144), xylanases (GH43 and CE4) and mannanases (GH5, 26, 27 and 130) are encoded in the genome. Furthermore, strain CL38 demonstrated its ability to decompose empty fruit bunch, a lignocellulosic waste residue arising from palm oil industry. The genome information coupled with experimental studies confirmed the ability of strain CL38 to degrade lignocellulosic biomass. Therefore, Meridianimaribacter sp. strain CL38, with its halotolerance, could be useful for seawater based lignocellulosic biorefining.
Anoxybacillus gonensis type strain G2(T) (=NCIMB 13,933(T) =NCCB 100040(T)) has been isolated from the Gönen hot springs in Turkey. This strain produces a number of well-studied, biotechnologically important enzymes, including xylose isomerase, carboxylesterase, and fructose-1,6-bisphosphate aldolase. In addition, this strain is an excellent candidate for the bioremediation of areas with heavy metal pollution. Here, we present a high-quality, annotated, complete genome of A. gonensis G2(T). Furthermore, this report provides insights into several novel enzymes of strain G2(T) and their potential industrial applications.
Desulfotomaculum copahuensis strain CINDEFI1 is a novel spore-forming sulfate-reducing bacterium isolated from the Copahue volcano area, Argentina. Here, we present its draft genome in which we found genes related with the anaerobic respiration of sulfur compounds similar to those present in the Copahue environment.
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 β-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.
Strain Corallo1T was isolated from mucus of red coral (Corallium rubrum) at Punta Pizzaco (Procida island, Naples, Italy). It was characterised as a Gram-stain negative, motile, rod-shaped bacterium. Strain Corallo1T was found to show positive responses for cytochrome-c oxidase, catalase, reduction of nitrate and nitrite, β-galactosidase activity and hydrolysis of starch, xylan, peptone, Tween 40, Tween 80 and casein. Strain Corallo1T was found to be mesophilic, neutrophilic to alkalophilic and slightly halophilic. According to analysis of the almost-complete 16S rRNA gene, strain Corallo1T is closely related to Vibrio celticus (100% sequence similarity), Vibrio gigantis (100%), Vibrio crassostreae (99.7%), Vibrio artabrorum (99.7%) and Vibrio pomeroyi (99.6%). MLSA of five housekeeping genes (atpA, pyrH, recA, rpoA and rpoD) was performed to refine the phylogenetic relationships of strain Corallo1T. A draft genome sequence of strain Corallo1T was obtained. The DNA G+C content of this strain was determined to be 44.5 mol %. The major cellular fatty acids of strain Corallo1T are C16:1, n-C16:0 and C18:1, and the major isoprenoid ubiquinone is Q8. ANI indexes, in silico estimations of DDH values and wet lab DDH values demonstrated that strain Corallo1T represents an independent genomospecies. Based on a polyphasic taxonomic characterisation, strain Corallo1T is concluded to represent a novel species of the genus Vibrio, for which the name Vibrio coralliirubri sp. nov. is proposed. The type strain is Corallo1T (= DSM 27495T = CIP 110630T).