Roseiterribacter gracilis gen. nov., sp. nov., a novel filterable alphaproteobacterium isolated from soil using a gel-filled microwell array device.

Our previous studies indicate the abundant and diverse presence of yet-to-be-cultured microorganisms in the micropore-filtered fractions of various environmental samples. Here, we isolated a novel bacterium (designated as strain TMPK1T) from a 0.45-µm-filtered soil suspension by using a gel-filled microwell array device comprising 900 microwells and characterized its phylogenetic and physiological features. This strain showed low 16S rRNA gene sequence identities (<91%) and low average nucleotide identity values (<70%) to the closest validly described species, and belonged to a novel-family-level lineage within the order Rhodospirillales of Alphaproteobacteria. Strain TMPK1T exhibited small cell sizes (0.08-0.23 µm3) and had a high cyclopropane fatty acid content (>13%), and these characteristics were differentiated from other Rhodospirillales bacteria. A comprehensive habitability search using amplicon datasets suggested that TMPK1T and its close relatives are mainly distributed in soil and plant-associated environments. Based on these results, we propose that strain TMPK1T represents a novel genus and species named Roseiterribacter gracilis gen. nov., sp. nov. (JCM 34627T = KCTC 82790T). We also propose Roseiterribacteraceae fam. nov. to accommodate the genus Roseiterribacter.

A Marine Group A isolate relies on other growing bacteria for cell wall formation.

Most of Earth's prokaryotes live under energy limitation, yet the full breadth of strategies that enable survival under such conditions remain poorly understood. Here we report the isolation of a bacterial strain, IA91, belonging to the candidate phylum Marine Group A (SAR406 or 'Candidatus Marinimicrobia') that is unable to synthesize the central cell wall compound peptidoglycan itself. Using cultivation experiments and microscopy, we show that IA91 growth and cell shape depend on other bacteria, deriving peptidoglycan, energy and carbon from exogenous muropeptide cell wall fragments released from growing bacteria. Reliance on exogenous muropeptides is traceable to the phylum's ancestor, with evidence of vertical inheritance across several classes. This dependency may be widespread across bacteria (16 phyla) based on the absence of key peptidoglycan synthesis genes. These results suggest that uptake of exogenous cell wall components could be a relevant and potentially common survival strategy in energy-limited habitats like the deep biosphere.

Atrimonas thermophila gen. nov., sp. nov., a novel anaerobic thermophilic bacterium of the phylum Atribacterota isolated from deep subsurface gas field and proposal of Atrimonadaceae fam. nov. within the class Atribacteria in the phylum Atribacterota.

A novel anaerobic, thermophilic bacterium of the class Atribacteria, strain M15T, was isolated from a high-temperature gas reservoir, Japan. Cells of strain M15T were gram-negative, short oval-shaped, and lacked flagella. Growth occurred at 45-75 °C (optimum 70-75 °C) and pH 6.5-8.5 (optimum pH 7.5-8.0) and was fast under optimal conditions (doubling time 11.4 h). Yeast extract was required for growth. Fermentative growth with glucose, arabinose, xylose, and cellobiose was observed. The major fermentative end products of glucose were acetate and hydrogen. The major cellular fatty acids were C16:0, iso-C15:0, and C18:0. The genomic G + C content was 46.0 mol%. Fluorescence and electron microscopy observations revealed the intracellular localization of genomic DNA surrounded by a membrane in the cells of strain M15T as reported in a sole validly described species of the class Atribacteria in the phylum Atribacterota, Atribacter laminatus strain RT761T, suggesting that the unique morphological traits are widely shared in this class. Phylogenetic analyses indicated that strain M15T belongs to a distinct family-level lineage in the class Atribacteria and shows low similarities to Atribacter laminatus strain RT761T (16S rRNA gene sequence identity of 90.1 %, average nucleotide identity [ANI] of 66.1 %, average amino acid identity [AAI] of 55.8 %). Phenotypic traits of strain M15T (thermophilic, fast-growing, relatively high G + C content, etc.) were clearly distinct from A. laminatus. Based on these phenotypic and genomic properties, we propose a novel genus and species, Atrimonas thermophila gen. nov., sp. nov. for strain M15T (=JCM39389T, =KCTC25731T) representing a novel family Atrimonadaceae fam., nov. in the class Atribacteria.

Identification and cultivation of anaerobic bacterial scavengers of dead cells.

The cycle of life and death and Earth's carbon cycle(s) are intimately linked, yet how bacterial cells, one of the largest pools of biomass on Earth, are recycled back into the carbon cycle remains enigmatic. In particular, no bacteria capable of scavenging dead cells in oxygen-depleted environments have been reported thus far. In this study, we discover the first anaerobes that scavenge dead cells and the two isolated strains use distinct strategies. Based on live-cell imaging, transmission electron microscopy, and hydrolytic enzyme assays, one strain (designated CYCD) relied on cell-to-cell contact and cell invagination for degrading dead food bacteria where as the other strain (MGCD) degraded dead food bacteria via excretion of lytic extracellular enzymes. Both strains could degrade dead cells of differing taxonomy (bacteria and archaea) and differing extents of cell damage, including those without artificially inflicted physical damage. In addition, both depended on symbiotic metabolic interactions for maximizing cell degradation, representing the first cultured syntrophic Bacteroidota. We collectively revealed multiple symbiotic bacterial decomposition routes of dead prokaryotic cells, providing novel insight into the last step of the carbon cycle.

Metabolic implications for predatory and parasitic bacterial lineages in activated sludge wastewater treatment systems.

Deciphering unclear microbial interactions is key to improving biological wastewater treatment processes. Microbial predation and parasitism in wastewater treatment ecosystems are unexplored survival strategies that have long been known and have recently attracted attention because these interspecies interactions may contribute to the reduction of excess sludge. Here, microbial community profiling of 600 activated sludge samples taken from six industrial and one municipal wastewater treatment processes (WWTPs) was conducted. To identify the shared lineages in the WWTPs, the shared microbial constituents were defined as the family level taxa that had ≥ 0.1% average relative abundance and detected in all processes. The microbial community analysis assigned 106 families as the shared microbial constituents in the WWTPs. Correlation analysis showed that 98 of the 106 shared families were significantly correlated with total carbon (TC) and/or total nitrogen (TN) concentrations, suggesting that they may contribute to wastewater remediation. Most possible predatory or parasitic bacteria belonging to the phyla Bdellovibrionota, Myxococcota, and Candidatus Patescibacteria were found to be the shared families and negatively correlated with TC/TN; thus, they were frequently present in the WWTPs and could be involved in the removal of carbon/nitrogen derived from cell components. Shotgun metagenome-resolved metabolic reconstructions indicated that gene homologs associated with predation or parasitism are conserved in the Bdellovibrionota, Myxococcota, and Ca. Patescibacteria genomes (e.g., host interaction (hit) locus, Tad-like secretion complexes, and type IV pilus assembly proteins). This study provides insights into the complex microbial interactions potentially linked to the reduction of excess sludge biomass in these processes.

Complete Genome Sequence of Dyella sp. Strain GSA-30, a Predominant Endophytic Bacterium of Dendrobium Plants.

We report a complete genome sequence of Dyella sp. strain GSA-30, a predominant endophytic bacterium of Dendrobium plants. The genome consists of a circular 5,501,810-bp chromosome with a G+C content of 61.4%. The genome was predicted to harbor 6 rRNA genes, 51 tRNA genes, and 4,713 coding sequences.

A Fast and Easy Method to Co-extract DNA and RNA from an Environmental Microbial Sample.

We herein propose a fast and easy DNA and RNA co-extraction method for environmental microbial samples. It combines bead beating and phenol-chloroform phase separation followed by the separation and purification of DNA and RNA using the Qiagen AllPrep DNA/RNA mini kit. With a handling time of ~3 h, our method simultaneously extracted high-quality DNA (peak size >10-15| |kb) and RNA (RNA integrity number >6) from lake bacterioplankton filtered samples. The method is also applicable to low-biomass samples (expected DNA or RNA yield <50| |ng) and eukaryotic microbial samples, providing an easy option for more versatile eco-genomic applications.

Complete Genome Sequence of Flavobacterium sp. Strain GSB-24, Isolated from inside Dendrobium Roots.

Here, we report a complete genome sequence of Flavobacterium sp. strain GSB-24, an endophytic bacterium of Dendrobium plants. The genome consists of a circular 5,286,830-bp chromosome with a G+C content of 33.8% and a circular 64,374-bp plasmid with a G+C content of 29.3%.

Novel quantitative method for individual isotopomer of organic acids from 13C tracer experiments determines carbon flow in acetogenesis.

Anaerobic microbial acetogenesis is ubiquitous on Earth, and thus plays an important role in the global carbon cycle. The mechanism of carbon fixation in acetogens has attracted great interest from various studies for combatting climate change, and even for studying ancient metabolic pathways. Here, we developed a new, simple method for investigating carbon flows in the metabolic reaction of acetogen by conveniently and accurately determining the relative abundance of individual acetate- and/or formate-isotopomers formed in 13C labeling experiments. We measured the underivatized analyte by gas chromatography-mass spectrometry (GC-MS) coupled with a direct aqueous sample injection technique. The individual abundance of analyte isotopomers was calculated by the mass spectrum analysis using the least-squares approach. The validity of the method was demonstrated by determining known mixtures of unlabeled and 13C-labeled analytes. The developed method was applied to study the carbon fixation mechanism of the well-known acetogen Acetobacterium woodii grown on methanol and bicarbonate. We provided a quantitative reaction model for methanol metabolism of A. woodii, which indicated that methanol was not the sole carbon precursor of the acetate methyl group and that 20-22% of the methyl group was formed from CO2. In contrast, the carboxyl group of acetate appeared to form exclusively by CO2 fixation. Thus, our simple method, without laborious analytical procedures, has broad utility for the study of biochemical and chemical processes related to acetogenesis on Earth.

Unique H2-utilizing lithotrophy in serpentinite-hosted systems.

Serpentinization of ultramafic rocks provides molecular hydrogen (H2) that can support lithotrophic metabolism of microorganisms, but also poses extremely challenging conditions, including hyperalkalinity and limited electron acceptor availability. Investigation of two serpentinization-active systems reveals that conventional H2-/CO2-dependent homoacetogenesis is thermodynamically unfavorable in situ due to picomolar CO2 levels. Through metagenomics and thermodynamics, we discover unique taxa capable of metabolism adapted to the habitat. This included a novel deep-branching phylum, "Ca. Lithacetigenota", that exclusively inhabits serpentinite-hosted systems and harbors genes encoding alternative modes of H2-utilizing lithotrophy. Rather than CO2, these putative metabolisms utilize reduced carbon compounds detected in situ presumably serpentinization-derived: formate and glycine. The former employs a partial homoacetogenesis pathway and the latter a distinct pathway mediated by a rare selenoprotein-the glycine reductase. A survey of microbiomes shows that glycine reductases are diverse and nearly ubiquitous in serpentinite-hosted environments. "Ca. Lithacetigenota" glycine reductases represent a basal lineage, suggesting that catabolic glycine reduction is an ancient bacterial innovation by Terrabacteria for gaining energy from geogenic H2 even under hyperalkaline, CO2-poor conditions. Unique non-CO2-reducing metabolisms presented here shed light on potential strategies that extremophiles may employ for overcoming a crucial obstacle in serpentinization-associated environments, features potentially relevant to primordial lithotrophy in early Earth.

Improved Cultivation and Isolation of Diverse Endophytic Bacteria Inhabiting Dendrobium Roots by Using Simply Modified Agar Media.

Dendrobium plants are members of the family Orchidaceae, many of which are endangered orchids with ornamental and medicinal values. Dendrobium endophytic microbes have attracted attention for the development of strategies for plant protection and utilization of medicinal principles. However, the role of endophytic bacteria is poorly elucidated due to the lack of their successful cultivation. This study obtained a total of 749 endophytic isolates from Dendrobium roots using solid media prepared by simply modified methods (separate sterilization of phosphate and agar [PS] and use of gellan gum as a gelling reagent [GG]) and by a conventional method of autoclaving the phosphate and agar together (PT method). Notably, based on a comparison of 16S rRNA gene sequences between the isolates and the Dendrobium root endophyte community, we successfully retrieved more than 50% (17 out of 30) of the predominant endophytic bacterial operational taxonomic units (OTUs) using PS and GG media, which is a much higher recovery rate than that of PT medium (16.7%). We further found that a number of recalcitrant bacteria, including phylogenetically novel isolates and members of even the rarely cultivated phyla Acidobacteriota and Verrucomicrobiota, were obtained only when using PS and/or GG medium. Intriguingly, the majority of these recalcitrant bacteria formed colonies faster on PS or GG medium than on PT medium, which may have contributed to their successful isolation. Taken together, this study succeeded in isolating a wide variety of Dendrobium endophytic bacteria, including predominant ones using PS and GG media, and enables performance of future studies to clarify their unknown roles associated with the growth of Dendrobium plants. IMPORTANCE Dendrobium endophytic bacteria are of great interest since their functions may contribute to the protection of endangered orchids with ornamental and medicinal values. To understand and reveal the "true roles" of the endophytes, obtaining those axenic cultures is necessary even in the metagenomic era. However, no effective methods for isolating a variety of endophytic bacteria have been established. This study first demonstrated that the use of simply modified medium is quite effective and indeed allows the isolation of more than half of the predominant endophytic bacteria inhabiting Dendrobium roots. Besides, even phylogenetically novel and/or recalcitrant endophytic bacteria were successfully obtained by the same strategy. The obtained endophytic bacteria could serve as "living material" for elucidating their unprecedented functions related to the conservation of endangered orchid plants. Furthermore, the culture method used in this study may enable the isolation of various endophytic bacteria dominating not only in orchid plants but also in other useful plants.

Granulimonas faecalis gen. nov., sp. nov., and Leptogranulimonas caecicola gen. nov., sp. nov., novel lactate-producing Atopobiaceae bacteria isolated from mouse intestines, and an emended description of the family Atopobiaceae.

Two strictly anaerobic, Gram-stain-positive, non-motile bacteria (strains OPF53T and TOC12T) were isolated from mouse intestines. Strains OPF53T and TOC12T grew at pH 5.5-9.0 and 5.0-9.0, respectively, and at temperatures of 30-45 °C. The cell morphologies of these strains were short rods and rods, respectively, and the cells possessed intracellular granules. The major cellular fatty acids of OPF53T were C18  :  1 cis 9 and C18  :  1 cis 9 dimethyl acetal, whereas those of TOC12T were C18  :  0 and C18  :  1 cis 9. In OPF53T, the main end-products of modified peptone-yeast extract-glucose (PYG) fermentation were lactate, formate and butyrate, whereas, in addition to these acids, TOC12T also produced hydrogen. The genomes of OPF53T and TOC12T were respectively 2.2 and 2.0 Mbp in size with a DNA G+C contents of 69.1 and 58.7 %. The 16S rRNA gene sequences of OPF53T and TOC12T showed the highest similarity to members of the family Atopobiaceae, namely, Olsenella phocaeensis Marseille-P2936T (94.3 %) and Olsenella umbonata KCTC 15140T (93.2 %), respectively. Phylogenetic analyses revealed that both isolates formed distinct lineages from other genera of the family Atopobiaceae. In addition, the two strains were characterized by relatively low 16S rRNA gene sequence similarity (93.4 %) and can be distinguished by their distinctive traits (including cell shape, DNA G+C content, and major fatty acids profiles). On the basis of their polyphasic taxonomic properties, these isolates represent two noel species of two novel genera within the family Atopobiaceae, for which the names Granulimonas faecalis gen. nov., sp. nov. (OPF53T=JCM 35015T=KCTC 25474T) and Leptogranulimonas caecicola gen. nov., sp. nov. (TOC12T=JCM 35017T=KCTC 25472T) are proposed.

Bile Salt Hydrolases with Extended Substrate Specificity Confer a High Level of Resistance to Bile Toxicity on Atopobiaceae Bacteria.

The bile resistance of intestinal bacteria is among the key factors responsible for their successful colonization of and survival in the mammalian gastrointestinal tract. In this study, we demonstrated that lactate-producing Atopobiaceae bacteria (Leptogranulimonas caecicola TOC12T and Granulimonas faecalis OPF53T) isolated from mouse intestine showed high resistance to mammalian bile extracts, due to significant bile salt hydrolase (BSH) activity. We further succeeded in isolating BSH proteins (designated LcBSH and GfBSH) from L. caecicola TOC12T and G. faecalis OPF53T, respectively, and characterized their enzymatic features. Interestingly, recombinant LcBSH and GfBSH proteins exhibited BSH activity against 12 conjugated bile salts, indicating that LcBSH and GfBSH have much broader substrate specificity than the previously identified BSHs from lactic acid bacteria, which are generally known to hydrolyze six bile salt isomers. Phylogenetic analysis showed that LcBSH and GfBSH had no affinities with any known BSH subgroup and constituted a new BSH subgroup in the phylogeny. In summary, we discovered functional BSHs with broad substrate specificity from Atopobiaceae bacteria and demonstrated that these BSH enzymes confer bile resistance to L. caecicola TOC12T and G. faecalis OPF53T.

Isolation of Aquatic Plant Growth-Promoting Bacteria for the Floating Plant Duckweed (Lemna minor).

Plant growth-promoting bacteria (PGPB) can exert beneficial growth effects on their host plants. Little is known about the phylogeny and growth-promoting mechanisms of PGPB associated with aquatic plants, although those of terrestrial PGPB have been well-studied. Here, we report four novel aquatic PGPB strains, MRB1-4 (NITE P-01645-P-01648), for duckweed Lemna minor from our rhizobacterial collection isolated from Lythrum anceps. The number of L. minor fronds during 14 days co-culture with the strains MRB1-4 increased by 2.1-3.8-fold, compared with an uninoculated control; the plant biomass and chlorophyll content in co-cultures also increased. Moreover, all strains possessed an indole-3-acetic acid production trait in common with a plant growth-promoting trait of terrestrial PGPB. Phylogenetic analysis showed that three strains, MRB-1, -3, and -4, were affiliated with known proteobacterial genera (Bradyrhizobium and Pelomonas); this report is the first to describe a plant-growth promoting activity of Pelomonas members. The gammaproteobacterial strain MRB2 was suggested to be phylogenetically novel at the genus level. Under microscopic observation, the Pelomonas strain MRB3 was epiphytic and adhered to both the root surfaces and fronds of duckweed. The duckweed PGPB obtained here could serve as a new model for understanding unforeseen mechanisms behind aquatic plant-microbe interactions.

Long-Read-Resolved, Ecosystem-Wide Exploration of Nucleotide and Structural Microdiversity of Lake Bacterioplankton Genomes.

Reconstruction of metagenome-assembled genomes (MAGs) has become a fundamental approach in microbial ecology. However, a MAG is hardly complete and overlooks genomic microdiversity because metagenomic assembly fails to resolve microvariants among closely related genotypes. Aiming at understanding the universal factors that drive or constrain prokaryotic genome diversification, we performed an ecosystem-wide high-resolution metagenomic exploration of microdiversity by combining spatiotemporal (2 depths × 12 months) sampling from a pelagic freshwater system, high-quality MAG reconstruction using long- and short-read metagenomic sequences, and profiling of single nucleotide variants (SNVs) and structural variants (SVs) through mapping of short and long reads to the MAGs, respectively. We reconstructed 575 MAGs, including 29 circular assemblies, providing high-quality reference genomes of freshwater bacterioplankton. Read mapping against these MAGs identified 100 to 101,781 SNVs/Mb and 0 to 305 insertions, 0 to 467 deletions, 0 to 41 duplications, and 0 to 6 inversions for each MAG. Nonsynonymous SNVs were accumulated in genes potentially involved in cell surface structural modification to evade phage recognition. Most (80.2%) deletions overlapped with a gene coding region, and genes of prokaryotic defense systems were most frequently (>8% of the genes) overlapped with a deletion. Some such deletions exhibited a monthly shift in their allele frequency, suggesting a rapid turnover of genotypes in response to phage predation. MAGs with extremely low microdiversity were either rare or opportunistic bloomers, suggesting that population persistency is key to their genomic diversification. The results concluded that prokaryotic genomic diversification is driven primarily by viral load and constrained by a population bottleneck. IMPORTANCE Identifying intraspecies genomic diversity (microdiversity) is crucial to understanding microbial ecology and evolution. However, microdiversity among environmental assemblages is not well investigated, because most microbes are difficult to culture. In this study, we performed cultivation-independent exploration of bacterial genomic microdiversity in a lake ecosystem using a combination of short- and long-read metagenomic analyses. The results revealed the broad spectrum of genomic microdiversity among the diverse bacterial species in the ecosystem, which has been overlooked by conventional approaches. Our ecosystem-wide exploration further allowed comparative analysis among the genomes and genes and revealed factors behind microbial genomic diversification, namely, that diversification is driven primarily by resistance against viral infection and constrained by the population size.

Features of the gut prokaryotic virome of Japanese patients with Crohn's disease.

BACKGROUND/AIMS: The gut virome is mainly composed of bacteriophages and influences gut homeostasis and pathogenic conditions. In this study, we analyzed the gut prokaryotic virome in Japanese patients with Crohn's disease (CD). MATERIALS/METHODS: We collected 19 fecal samples from CD patients and 16 samples from healthy controls. The gut bacteriome was analyzed by 16S rRNA gene sequencing and the virome was profiled by shotgun metagenomic sequencing. RESULTS: Despite no differences in richness and evenness, there was a significant difference in the overall structure of the gut virome between CD patients and controls (P = 0.013). CrAssphage and Staphylococcus virus, belonging to the order Caudovirales, were dominant in the gut virome of controls and CD patients. The abundance of crAssphage was significantly greater in CD patients than controls (P = 0.021). Lactococcus, Enterococcus and Lactobacillus phages were present only in CD patients, while Xanthomonas and Escherichia phages were unique to the controls. In the gut bacteriome of CD patients, richness and evenness were significantly lower, and a significant difference in the overall structure was observed between groups (P = 0.014). The gut bacteriome of CD patients was characterized by a decrease of the genera Faecalibacterium, Roseburia, and Ruminococcus and an increase of the family Enterobacteriaceae. There were more significant correlations between viruses and bacteria in CD patients than controls. CONCLUSIONS: The gut virome of CD patients was distinct from that of healthy controls in a Japanese population. An altered gut virome may be one of the factors associated with the bacterial dysbiosis of CD.

Polymer-based chemical-nose systems for optical-pattern recognition of gut microbiota.

Gut-microbiota analysis has been recognized as crucial in health management and disease treatment. Metagenomics, a current standard examination method for the gut microbiome, is effective but requires both expertise and significant amounts of general resources. Here, we show highly accessible sensing systems based on the so-called chemical-nose strategy to transduce the characteristics of microbiota into fluorescence patterns. The fluorescence patterns, generated by twelve block copolymers with aggregation-induced emission (AIE) units, were analyzed using pattern-recognition algorithms, which identified 16 intestinal bacterial strains in a way that correlates with their genome-based taxonomic classification. Importantly, the chemical noses classified artificial models of obesity-associated gut microbiota, and further succeeded in detecting sleep disorder in mice through comparative analysis of normal and abnormal mouse gut microbiota. Our techniques thus allow analyzing complex bacterial samples far more quickly, simply, and inexpensively than common metagenome-based methods, which offers a powerful and complementary tool for the practical analysis of the gut microbiome.

Bile Salt Hydrolase Degrades ß-Lactam Antibiotics and Confers Antibiotic Resistance on Lactobacillus paragasseri.

Bile salt hydrolase (BSH) is a well-characterized probiotic enzyme associated with bile detoxification and colonization of lactic acid bacteria in the human gastrointestinal tract. Here, we isolated a putative BSH (LpBSH) from the probiotic bacterium Lactobacillus paragasseri JCM 5343T and demonstrated its bifunctional activity that allows it to degrade not only bile salts but also the antibiotic (penicillin). Although antibiotic resistance and bile detoxification have been separately recognized as different microbial functions, our findings suggest that bifunctional BSHs simultaneously confer ecological advantages to host gut bacteria to improve their survival in the mammalian intestine by attaining a high resistance to bile salts and ß-lactams. Strain JCM 5343T showed resistance to both bile salts and ß-lactam antibiotics, suggesting that LpBSH may be involved in this multi-resistance of the strain. We further verified that such bifunctional enzymes were broadly distributed among the phylogeny, suggesting that the bifunctionality may be conserved in other BSHs of gut bacteria. This study revealed the physiological role and phylogenetic diversity of bifunctional enzymes degrading bile salts and ß-lactams in gut bacteria. Furthermore, our findings suggest that the hitherto-overlooked penicillin-degrading activity of penicillin acylase could be a potential new target for the probiotic function of gut bacteria.

Isolation of a Highly Thermostable Bile Salt Hydrolase With Broad Substrate Specificity From Lactobacillus paragasseri.

Bile salt hydrolase (BSH) enzymes produced by intestinal Lactobacillus species have been recognized as major targets for probiotic studies owing to their weight-loss and cholesterol-lowering effects. In this study, we isolated a highly thermostable BSH with broad substrate specificity, designed as LapBSH (BSH from a probiotic bacterium, Lactobacillus paragasseri JCM 5343 T ). The recombinant LapBSH protein clearly hydrolyzed 12 different substrates, including primary/secondary, major/minor, and taurine/glycine-conjugated bile salts in mammalian digestive tracts. Intriguingly, LapBSH further displayed a highly thermostable ability among all characterized BSH enzymes. Indeed, this enzyme retained above 80% of its optimum BSH activity even after 6 h of incubation at 50-90°C. LapBSH also exerted a functionally stable activity and maintained above 85% of its original activity after pre-heating at 85°C for 2 h. Therefore, LapBSH is a very unique probiotic enzyme with broad substrate specificity and high thermostability. The strain itself, JCM 5343T, was also found to exhibit high heat-resistance ability and could form colonies even after exposure to 85°C for 2 h. As thermostable enzyme/bacterium offers industrial and biotechnological advantages in terms of its productivity and stability improvements, both thermostable LapBSH and thermotolerant L. paragasseri JCM 5343T could be promising candidates for future probiotic research.