Effect of Probiotic-Fortified Infant Formula on Infant Gut Health and Microbiota Modulation.

Compared to infant formula, breast milk is the best source of nutrition for infants; it not only improves the neonatal intestinal function, but also regulates the immune system and gut microbiota composition. However, probiotic-fortified infant formula may further enhance the infant gut environment by overcoming the limitations of traditional infant formula. We investigated the probiotic formula administration for one month by comparing 118 Korean infants into the following three groups: infants in each group fed with breast milk (50), probiotic formula (35), or placebo formula-fed group (33). Probiotic formula improved stool consistency and defecation frequency compared to placebo formula-fed group. The probiotic formula helped maintaining the level of secretory immunoglobulin A (sIgA), which had remarkably decreased over time in placebo formula-fed infants (compared to weeks 0 and 4). Moreover, probiotic formula decreased the acidity of stool and considerably increased the butyrate concentration. Furthermore, the fecal microbiota of each group was evaluated at weeks 0 and 4. The microbial composition was distinct between each groups, and the abundance of health-promoting bacteria increased in the probiotic formula compared to the placebo formula-fed group. In summary, supplementation of probiotic infant formula can help optimize the infant gut environment, microbial composition, and metabolic activity of the microbiota, mimicking those of breast milk.

Constructing Marine Bacterial Metabolic Chassis for Potential Biorefinery of Red Algal Biomass and Agaropectin Wastes.

Marine red algal biomass is a promising feedstock for sustainable production of value-added chemicals. However, the major constituents of red algal biomass, such as agar and carrageenan, are not easily assimilated by most industrial metabolic chassis developed to date. Synthetic biology offers a solution by utilizing nonmodel organisms as metabolic chassis for consolidated biological processes. In this study, the marine heterotrophic bacterium Pseudoalteromonas atlantica T6c was harnessed as a metabolic chassis to produce value-added chemicals from the affordable red algal galactans or agaropectin, a byproduct of industrial agarose production. To construct a heterologous gene expression device in P. atlantica T6c, promoters related to agar metabolism were screened from the differentially expressed genes using RNA-Seq analysis. The expression device was built and tested with selected promoters fused to a reporter gene and tuned by incorporation of a cognate repressor predicted from the agar-specific polysaccharide utilization locus. The feasibility of the marine bacterial metabolic chassis was examined by introducing the biosynthetic gene clusters of ß-carotene and violacein. Our results demonstrate that the metabolic chassis platform enables direct conversion of low-cost red algal galactans or industrial waste agaropectin into valuable bioactive pigments without any pretreatment of biomass. The developed marine bacterial chassis could potentially be used in a biorefinery framework to produce value-added chemicals from marine algal galactans.

Complete genome sequence of Limosilactobacillus fermentum JNU532 as a probiotic candidate for the functional food and feed supplements.

Lactic acid bacteria (LAB) have been reported to possess various beneficial properties and are commonly used as probiotics. LAB play a crucial role in milk fermentation, industrial lactic acid fermentation, and health and medicine. Limosilactobacillus fermentum isolated from fermented dairy and food products is considered as 'Generally Recognized as Safe' by FDA. Limosilactobacillus fermentum plays an important role in modulation of the intestinal microbiota, enhancing the host immune system and improving feed digestibility. We isolated a probiotic candidate that was identified and named Limosilactobacillus fermentum JNU532. In a previous report, cell-free culture of L. fermentum JNU532 exhibited anti-melanogenic and antioxidant activities. In this study, we present the complete genome assembly of the bacterial strain JNU532. The final genome consists of one circular chromosome (2,077,416 base pairs) with a guanine + cytosine (GC) ratio of 51.5%.

Metabarcoding of eDNA for tracking the floral and geographical origins of bee honey.

Authentic honey products have a high commercial value and are often falsified via adulteration. Metabarcoding of environmental DNA (eDNA) from bacterial, floral, and entomological sources has recently been proposed as a useful tool for identifying and authenticating floral and geographical origins of bee honey. In this study, eDNA metabarcoding was applied to reveal the bacterial, plant, and honey bee DNA signatures of 48 commercial honey products from six different geographical origins. Bacterial DNA composition in commercial honey showed different relative abundance of Paenibacillus and Bacillus in geographically different samples, and high abundance of Methylobacterium in chestnut honey implying potential use of bacterial DNA composition for honey authentication. Using the chloroplast trnL (UAA) as a DNA marker, floral origins of commercial honey were investigated. Based on floral DNA signatures, 12 monofloral honey samples were identified among the 45 samples tested. Targeted amplicon sequencing of cytochrome oxidase I (COI) gene from entomological DNA identified three different Apis mellifera sequence variants, specific to geographic origin of honey, suggesting that COI can be implemented as a DNA marker to trace the origin of honey. Therefore, the current study demonstrated the potential of eDNA based metabarcoding as a robust tool for evaluating commercial bee honey by exploring their floral and geographical origins.

Enzymatic Process for the Carrageenolytic Bioconversion of Sulfated Polygalactans into ß-Neocarrabiose and 3,6-Anhydro-d-galactose.

Oligosaccharides and anhydro-sugars derived from carrageenan have great potential as functional foods and drugs showing various bioactivities, including antioxidant, anti-inflammatory, antiviral, antitumor, and cytotoxic activities. Although preparation of sulfated carrageenan oligosaccharides by chemical and enzymatic processes has been widely reported, preparation of nonsulfated ß-neocarrabiose (ß-NC2) and the rare sugar 3,6-anhydro-d-galactose (d-AHG) was not reported in the literature. Based on the carrageenan catabolic pathway in marine heterotrophic bacteria, an enzymatic process was designed and constructed with recombinant κ-carrageenase, GH127/GH129 α-1,3 anhydrogalactosidase, and cell-free extract from marine carrageenolytic bacteria Colwellia echini A3T. The process consisted of three successive steps, namely, (i) depolymerization, (ii) desulfation, and (iii) monomerization, by which carrageenan oligosaccharides, ß-NC2, and d-AHG were obtained from κ-carrageenan. Unlike the chemical process, enzymatic hydrolysis yields oligosaccharides with the desired degree of polymerization facilitates specific removal of sulfated groups, free of toxic byproducts, and avoids chemical modifications. The final optimized enzymatic process produced 0.52 g of ß-NC2 and 0.24 g of d-AHG from 1 g of κ-carrageenan. The carrageenolytic process designed for the enzymatic hydrolysis of κ-carrageenan can be scaled up for the mass production of bioactive carrageeno-oligosaccharides.

Complete genome and two plasmids sequences of Lactiplantibacillus plantarum L55 for probiotic potentials.

In this study, we report the complete genome sequence of Lactiplantibacillus plantarum L55, a probiotic strain of lactic acid bacteria isolated from kimchi. The genome consists of one circular chromosome (2,077,416 base pair [bp]) with a guanine cytosine (GC) content of 44.5%, and two circular plasmid sequences (54,267 and 19,592 bp, respectively). We also conducted a comprehensive analysis of the genome, which identified the presence of functional genes, genomic islands, and antibiotic-resistance genes. The genome sequence data presented in this study provide insights into the genetic basis of L. plantarum L55, which could be beneficial for the future development of probiotic applications.

Aurantiacibacter sediminis sp. nov., a marine bacterium isolated from a tidal flat.

A yellow-coloured bacterium, designated as strain JGD-13T, was isolated from a tidal flat in the Republic of Korea. Cells were Gram-stain-negative, aerobic, non-flagellated and rod-shaped. Growth was observed at 4-42 °C (optimum, 30 °C), at pH 6.0-12.0 (pH 7.0-8.0) and at 1-7 % (w/v) NaCl concentration (3 %). The 16S rRNA gene sequence analysis indicated that strain JGD-13T was closely related to Aurantiacibacter gangjinensis K7-2T with a sequence similarity of 98.2 %, followed by Aurantiacibacter aquimixticola JSSK-14T (98.1 %), Aurantiacibacter atlanticus s21-N3T (97.6 %), Aurantiacibacter zhengii V18T (97.6 %) and Aurantiacibacter luteus KA37T (97.5 %). The average nucleotide identity and digital DNA-DNA hybridization values with related strains were 70.3-76.2 % and 18.5-20.3 %. The genomic DNA G+C content was 60.2 mol%. Phylogenetic analysis using the maximum-likelihood method showed that strain JGD-13T formed a clade with A. aquimixticola JSSK-14T and A. gangjinensis K7-2T. The major fatty acids were summed feature 8 (39.7 %) and C17 : 1 ω6c (14.4 %). The predominant respiratory quinone was ubiquinone-10. The polar lipids were phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine, one sphingoglycolipid and three unidentified lipids. On the basis of phylogenetic, phenotypic and chemotaxonomic characteristics, strain JGD-13T represents a novel species within the genus Aurantiacibacter, for which the name Aurantiacibacter sediminis JGD-13Tsp. nov. is proposed. The type strain is JGD-13T (=KCTC 72892T=KACC 21676T=JCM 33995T).

Soil environment reshapes microbiota of laboratory-maintained Collembola during host development.

Collembola are soil-dwelling arthropods that play a key role in the soil ecosystem. Allonychiurus kimi (Lee) (Collembola: Onychiuridae) was isolated from the natural environment and has been maintained for 20 years under laboratory conditions. Though the morphological and physiological features of A. kimi are being widely used to evaluate the impact of pesticides and heavy metals on the soil ecosystem, variations observed in these features might be on account of its microbiota. However, the microbiota composition of the laboratory-maintained A. kimi is undetermined and how the community structure is changing in response to soil environments or interacting with the soil microbiota are still unknown. In this study, we determined the microbiota of laboratory-maintained A. kimi at both adult and juvenile stages and examined how the microbiota of A. kimi is affected by the microbial community in the soil environments. Chryseobacterium, Pandoraea, Sphingomonas, Escherichia-Shigella, and Acinetobacter were the core microbiota of A. kimi. Exposure of the laboratory-maintained A. kimi to different soil microbial communities drove dynamic shifts in the composition of A. kimi microbiota. Microbial association network analysis suggested that gut microbiota of lab-grown A. kimi was affected by exposing to soil microbial community. This study implies that shifts in the bacterial community of adult A. kimi can be utilized as an indicator to evaluate the soil ecosystem.

Altererythrobacter lutimaris sp. nov., a marine bacterium isolated from a tidal flat and reclassification of Altererythrobacter deserti, Altererythrobacter estronivorus and Altererythrobacter muriae as Tsuneonella deserti comb. nov., Croceicoccus estronivorus comb. nov. and Alteripontixanthobacter muriae comb. nov.

A yellow-coloured bacterium, designated strain JGD-16T, was isolated from a tidal flat in Janggu-do, Garorim Bay, Taean-gun, Chungcheongbuk-do, Republic of Korea. Cells were Gram-stain-negative, aerobic, non-flagellated and short ovoid to coccoid-shaped. Growth was observed at 10-37 °C (optimum, 30 °C), pH 6.0-9.0 (pH 8.0) and with 1-5% (w/v) NaCl (2%). Results of 16S rRNA gene sequence analysis indicated that strain JGD-16T was closely related to Altererythrobacter xiamenensis LY02T (97.1 %), Altererythrobacter aurantiacus O30T (96.3 %), Altererythrobacter ishigakiensis JPCCMB0017T (95.8 %), Altererythrobacter epoxidivorans JCS350T (95.7 %) and Altererythrobacter insulae BPTF-M16T (95.3%). Phylogenomic analysis using the maximum-likelihood algorithm showed that strain JGD-16T formed a clade with the genus Altererythrobacter. The genomic DNA G+C content was 57.8 mol%. The predominant respiratory quinone was ubiquinone-10. The major polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine, a sphingoglycolipid, an unidentified glycolipid and an unidentified lipid. The major fatty acids were C18:1 ω7c (31.5 %) and C18:3 ω6c (19.6 %). On the basis of its phylogenomic, physiological and chemotaxonomical characteristics, strain JGD-16T represents a novel species within the genus Altererythrobacter, for which the name Altererythrobacter lutimaris JGD-16Tsp. nov. is proposed. The type strain is JGD-16T (=KCTC 72632T=KACC 21405T=JCM 33750T). We also propose the reclassification of Altererythrobacter deserti as Tsuneonella deserti comb. nov., Altererythrobacter estronivorus as Croceicoccus estronivorus comb. nov. and Altererythrobacter muriae as Alteripontixanthobacter muriae comb. nov.

Metabolism perturbation Causedby the overexpression of carbon monoxide dehydrogenase/Acetyl-CoA synthase gene complex accelerated gas to acetate conversion rate ofEubacterium limosumKIST612.

Microbial conversion of carbon monoxide (CO) to acetate is a promising upcycling strategy for carbon sequestration. Herein, we demonstrate that CO conversion and acetate production rates of Eubacterium limosum KIST612 strain can be improved by in silico prediction and in vivo assessment. The mimicked CO metabolic model of KIST612 predicted that overexpressing the CO dehydrogenase (CODH) increases CO conversion and acetate production rates. To validate the prediction, we constructed mutant strains overexpressing CODH gene cluster and measured their CO conversion and acetate production rates. A mutant strain (ELM031) co-overexpressing CODH, coenzyme CooC2 and ACS showed a 3.1 × increased specific CO oxidation rate as well as 1.4 × increased specific acetate production rate, compared to the wild type strain. The transcriptional and translational data with redox balance analysis showed that ELM031 has enhanced reducing potential from up-regulation of ferredoxin and related metabolism directly linked to energy conservation.

Dietary sophorolipid accelerates growth by modulation of gut microbiota population and intestinal environments in broiler chickens.

BACKGROUND: Gut is a crucial organ for the host's defense system due to its filtering action of the intestinal membrane from hazardous foreign substances. One strategy to strengthen the gut epithelial barrier function is to upregulate beneficial microflora populations and their metabolites. Sophorolipid (SPL), which is a glycolipid bio-surfactant, could increase beneficial microflora and decrease pathogenic bacteria in the gastrointestinal tract. Therefore, herein, we conducted an experiment with broiler chickens to investigate the fortifying effects of SPL on the host's gut defense system by modulating the microbiota population. METHODS: A total of 540 1-day-old chicks (Ross 308) were used, and they were immediately allotted into three treatment groups (6 replications with 30 chicks/pen) according to their initial body weight. The dietary treatments consisted of CON (basal diet), BAM (10 mg/kg bambermycin), and SPL (10 mg/kg SPL). During the experiment, birds freely accessed feed and water, and body weight and feed intake were measured at the end of each phase. On d 35, birds (one bird/pen) were sacrificed to collect jejunum and cecum samples. RESULTS: Dietary SPL and BAM supplementation significantly accelerated birds' growth and also significantly improved feed efficiency compared to CON. Intestinal microbial community was significantly separated by dietary SPL supplementation from that of CON, and dietary SPL supplementation significantly increased Lactobacillus spp. and Akkermansia muciniphila. Moreover, birds fed with dietary SPL also showed the highest concentration of cecal butyrate among all treatment groups. Gut morphological analysis showed that dietary SPL significantly increased villus height, ratio of villus height to crypt depth, goblet cell numbers, and the gene expression levels of claudin-1 and mucin 2. Additionally, dietary SPL significantly decreased the mRNA expression level of pro-inflammatory cytokine, interleukin-6, and increased that of anti-inflammatory cytokine, interleukin-10, compared to other treatments. CONCLUSIONS: Dietary SPL increases the beneficial bacterial population and butyrate concentration, which leads to a strengthened gut barrier function. In addition, the intestinal inflammation was also downregulated by dietary SPL supplementation.

Novosphingobium aureum sp. nov., a marine bacterium isolated from salt flat sediment.

A Gram-stain-negative, aerobic, pale yellow-coloured, rod-shaped marine bacterium designated strain YJ-S2-02T was isolated from salt flat sediment sampled in Yongyu-do, Republic of Korea. Strain YJ-S2-02T grew at pH 6.0-9.0 (optimum, pH 7.0), 10-40 °C (optimum, 30 °C) and with optimum 1 % (w/v) NaCl. The 16S rRNA gene sequence analysis indicated that strain YJ-S2-02T was closely related to Novosphingobium naphthalenivorans NBRC 102051T (97.8 %) followed by Novosphingobium mathurense SM117T (97.5 %), Novosphingobium indicum H25T (97.3 %), Novosphingobium pentaromativorans US6-1T (96.8 %), Novosphingobium fontis STM-14T (96.6 %), Novosphingobium endophyticum EGI60015T (96.5 %), Novosphingobium naphthae D39T (96.5 %) and Novosphingobium malaysiense MUSC 273T (95.9 %). The average nucleotide identity and estimated DNA-DNA hybridization values between YJ-S2-02T and related type strains were 77.0-77.9 % and 19.1-24.0 %. Strain YJ-S2-02T was characterized as having Q-10 as the predominant respiratory quinone and the principal fatty acids (>10 %) were summed feature 8 (C18 : 1 ω6c/ω7c, 20.7 %), C18 : 3 ω6c (16.3 %) and C17 : 1 ω6c (11.8 %). The polar lipids consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, sphingolipids and two unidentified lipids. The DNA G+C content of strain YJ-S2-02T was 65.6 mol%. On the basis of the polyphasic taxonomic evidence presented in this study, YJ-S2-02T should be classified as representing a novel species within the genus Novosphingobium, for which name Novosphingobium aureum is proposed, with the type strain YJ-S2-02T (=KACC 21677T =KCTC 72891T=JCM 33996T).

A Novel Auxiliary Agarolytic Pathway Expands Metabolic Versatility in the Agar-Degrading Marine Bacterium Colwellia echini A3T.

Marine microorganisms encode a complex repertoire of carbohydrate-active enzymes (CAZymes) for the catabolism of algal cell wall polysaccharides. While the core enzyme cascade for degrading agar is conserved across agarolytic marine bacteria, gain of novel metabolic functions can lead to the evolutionary expansion of the gene repertoire. Here, we describe how two less-abundant GH96 α-agarases harbored in the agar-specific polysaccharide utilization locus (PUL) of Colwellia echini strain A3T facilitate the versatility of the agarolytic pathway. The cellular and molecular functions of the α-agarases examined by genomic, transcriptomic, and biochemical analyses revealed that α-agarases of C. echini A3T create a novel auxiliary pathway. α-Agarases convert even-numbered neoagarooligosaccharides to odd-numbered agaro- and neoagarooligosaccharides, providing an alternative route for the depolymerization process in the agarolytic pathway. Comparative genomic analysis of agarolytic bacteria implied that the agarolytic gene repertoire in marine bacteria has been diversified during evolution, while the essential core agarolytic gene set has been conserved. The expansion of the agarolytic gene repertoire and novel hydrolytic functions, including the elucidated molecular functionality of α-agarase, promote metabolic versatility by channeling agar metabolism through different routes. IMPORTANCEColwellia echini A3T is an example of how the gain of gene(s) can lead to the evolutionary expansion of agar-specific polysaccharide utilization loci (PUL). C. echini A3T encodes two α-agarases in addition to the core ß-agarolytic enzymes in its agarolytic PUL. Among the agar-degrading CAZymes identified so far, only a few α-agarases have been biochemically characterized. The molecular and biological functions of two α-agarases revealed that their unique hydrolytic pattern leads to the emergence of auxiliary agarolytic pathways. Through the combination of transcriptomic, genomic, and biochemical evidence, we elucidate the complete α-agarolytic pathway in C. echini A3T. The addition of α-agarases to the agarolytic enzyme repertoire might allow marine agarolytic bacteria to increase competitive abilities through metabolic versatility.

Marinobacter halodurans sp. nov., a halophilic bacterium isolated from sediment of a salt flat.

A Gram-staining-negative, aerobic, cream-coloured, marine bacterium, with rod-shaped cells, designated strain YJ-S3-2T, was isolated from salt flat sediment of Yongyu-do, Republic of Korea. YJ-S3-2T grew at pH 5.0-9.0 (optimum pH 7.0), 4-45 °C (optimum 30 °C) and with 1-18 % (w/v) NaCl (optimum 6 %). The results of 16S rRNA gene sequence analysis indicated that YJ-S3-2T was closely related to Marinobacter segnicrescens SS011B1-4T (97.0 %) followed by, 'Marinobacter nanhaiticus' D15-8W (96.7 %), Marinobacter bryozoorum 50-11T (96.7 %), Marinobacter koreensis DSMZ 179240T T (96.5 %) and Marinobacter bohaiensis T17T (96.5 %). The average nucleotide identity (ANI) and the genome to genome distance calculator (GGDC) estimate values between YJ-S3-2T and related type strains were 73.7-79.8 and 19.9-22.5 %, and also 73.5 and 20.7 % with Marinobacter hydrocarbonoclasticus. YJ-S3-2T was characterized as having Q-9 as the predominant respiratory quinone and the principal fatty acids (>10 %) were C16 : 0 (22.3 %), summed feature 9 (C17 : 1iso ω9c/C16 : 0 10-methyl, 13.8 %) and 3 (C16 : 1ω7c/C16 : 1ω6c, 11.9 %). The polar lipids consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, two unidentified aminolipids and two unidentified phospholipids. The DNA G+C content of YJ-S3-2T is 60.9 mol%. On the basis of the polyphasic taxonomic evidence presented in this study, YJ-S3-2T should be classified as representing a novel species within the genus Marinobacter, for which name Marinobacter halodurans sp. nov. is proposed, with the type strain YJ-S3-2T (=KACC 19883T=KCTC 62937T=JCM 33109T).

Novel Monomeric Fungal Subtilisin Inhibitor from a Plant-Pathogenic Fungus, Choanephora cucurbitarum: Isolation and Molecular Characterization.

The bacterial protease inhibitor domains known as Streptomyces subtilisin inhibitors (SSI) are rarely found in fungi. Genome analysis of a fungal pathogen, Choanephora cucurbitarum KUS-F28377, revealed 11 SSI-like domains that are horizontally transferred and sequentially diverged during evolution. We investigated the molecular function of fungal SSI-like domains of C. cucurbitarum, designated "choanepins." Among the proteins tested, only choanepin9 showed inhibitory activity against subtilisin as the target protease, accounting for 47% of the inhibitory activity of bacterial SSI. However, the binding affinity (expressed as the dissociation constant [Kd ]) of choanepin9 measured via microscale thermophoresis was 21 nM, whereas that for bacterial SSI is 34 nM. The trend of binding and inhibitory activity suggests that the two inhibitors exhibit different inhibitory mechanisms for subtilisin protease. Interestingly, choanepin9 was identified as a monomer in studies in vitro, whereas bacterial SSI is a homodimer. Based on these observations, we constructed a monomeric bacterial SSI protein with decreased binding affinity to abrogate its inhibitory activity. By altering the reactive sites of choanepin9 deduced from the P1 and P4 sites of bacterial SSI, we reestablished that these residues in choanepins are also crucial for modulating inhibitory activity. These findings suggest that the fungal SSI evolved to target specific cognate proteases by altering the residues involved in inhibitory reactivity (reactive sites) and binding affinity (structural integrity). The function of fungal SSI proteins identified in this study provides not only a clue to fungal pathogenesis via protease inhibition but also a template for the design of novel serine protease inhibitors.IMPORTANCE Until recently, Streptomyces subtilisin inhibitors (SSI) were reported and characterized only in bacteria. We found SSI-like domains in a plant-pathogenic fungus, Choanephora cucurbitarum KUS-F28377, which contains 11 sequentially diverged SSI-like domains. None of these fungal SSI-like domains were functionally characterized before. The active form of fungal SSI-like protein is a monomer, in contrast to the homodimeric bacterial SSI. We constructed a synthetic monomer of bacterial SSI to demonstrate the modulation of its activity based on structural integrity and not reactive sites. Our results suggest the duplication and divergence of SSI-like domains of C. cucurbitarum within the genome to inhibit various cognate proteases during evolution by modulating both binding and reactivity. The molecular functional characterization of fungal SSI-like domains will be useful in understanding their biological role and future biotechnological applications.

Muricauda ochracea sp. nov., isolated from a tidal flat in the Republic of Korea.

A yellowish-brown-coloured bacterium, designated strain JGD-17T, was isolated from a tidal flat of Janggu-do, Garorim bay, Taean-gun, Chungcheongbuk-do, Republic of Korea. Cells were Gram-stain-negative, aerobic, non-flagellated and long-rod-shaped. Growth was observed at 20-45 °C (optimum, 25-30 °C), at pH 6.0-10.0 (9.0) and with 1-5 % (w/v) NaCl (1-3 %). Results of 16S rRNA gene sequence analysis indicated that strain JGD-17T was closely related to Muricauda nanhaiensis SM1704T (96.1 %), Muricauda olearia CL-SS4T (95.0 %), Muricauda beolgyonensis BB-My12T (94.9 %), Muricauda marina H19-56T (94.7 %) and Muricauda indica 3PC125-7T (94.5 %). The ranges of values for the average nucleotide identity and digital DNA-DNA hybridization analyses with related strains were 71.3-74.1 % and 16.9-18.2 %. The genomic DNA G+C content was 41.1 mol%. Phylogenetic analysis using the neighbour-joining method showed that strain JGD-17T formed a clade with Muricauda nanhaiensis SM1704T, Muricauda lutaonensis CC-HSB-11T, Muricauda lutea CSW06T and Muricauda pacifica SM027T. The major fatty acids were iso-C15 : 0 (26.9 %), iso-C15 : 1 G (19.5 %) and iso-C17 : 0 3-OH (12.7 %). The predominant respiratory quinone was menaquinone-6. The polar lipids were phosphatidylethanolamine, an unidentified aminolipid, an unidentified phospholipid and two unidentified lipids. On the basis of phylogenetic, phenotypic and chemotaxonomic characteristics, strain JGD-17T represents a novel species within the genus Muricauda, for which the name Muricauda ochracea sp. nov. is proposed. The type strain is JGD-17T (=KCTC 72732T=KACC 21486T=JCM 33817T).

A Multifunctional Polysaccharide Utilization Gene Cluster in Colwellia echini Encodes Enzymes for the Complete Degradation of κ-Carrageenan, ι-Carrageenan, and Hybrid ß/κ-Carrageenan.

Algal cell wall polysaccharides constitute a large fraction in the biomass of marine primary producers and are thus important in nutrient transfer between trophic levels in the marine ecosystem. In order for this transfer to take place, polysaccharides must be degraded into smaller mono- and disaccharide units, which are subsequently metabolized, and key components in this degradation are bacterial enzymes. The marine bacterium Colwellia echini A3T is a potent enzyme producer since it completely hydrolyzes agar and κ-carrageenan. Here, we report that the genome of C. echini A3T harbors two large gene clusters for the degradation of carrageenan and agar, respectively. Phylogenetical and functional studies combined with transcriptomics and in silico structural modeling revealed that the carrageenolytic cluster encodes furcellaranases, a new class of glycoside hydrolase family 16 (GH16) enzymes that are key enzymes for hydrolysis of furcellaran, a hybrid carrageenan containing both ß- and κ-carrageenan motifs. We show that furcellaranases degrade furcellaran into neocarratetraose-43-O-monosulfate [DA-(α1,3)-G4S-(ß1,4)-DA-(α1,3)-G], and we propose a molecular model of furcellaranases and compare the active site architectures of furcellaranases, κ-carrageenases, ß-agarases, and ß-porphyranases. Furthermore, C. echini A3T was shown to encode κ-carrageenases, ι-carrageenases, and members of a new class of enzymes, active only on hybrid ß/κ-carrageenan tetrasaccharides. On the basis of our genomic, transcriptomic, and functional analyses of the carrageenolytic enzyme repertoire, we propose a new model for how C. echini A3T degrades complex sulfated marine polysaccharides such as furcellaran, κ-carrageenan, and ι-carrageenan.IMPORTANCE Here, we report that a recently described bacterium, Colwellia echini, harbors a large number of enzymes enabling the bacterium to grow on κ-carrageenan and agar. The genes are organized in two clusters that encode enzymes for the total degradation of κ-carrageenan and agar, respectively. As the first, we report on the structure/function relationship of a new class of enzymes that hydrolyze furcellaran, a partially sulfated ß/κ-carrageenan. Using an in silico model, we hypothesize a molecular structure of furcellaranases and compare structural features and active site architectures of furcellaranases with those of other GH16 polysaccharide hydrolases, such as κ-carrageenases, ß-agarases, and ß-porphyranases. Furthermore, we describe a new class of enzymes distantly related to GH42 and GH160 ß-galactosidases and show that this new class of enzymes is active only on hybrid ß/κ-carrageenan oligosaccharides. Finally, we propose a new model for how the carrageenolytic enzyme repertoire enables C. echini to metabolize ß/κ-, κ-, and ι-carrageenan.

Preparation of a ribosomally synthesized fungal peptide toxin precursor and its in-vitro cyclization.

Amatoxins are ribosomally synthesized and post-translationally modified peptides (RiPPs) found in poisonous mushrooms. These cyclic peptides are potent inhibitors of RNA polymerase II transcriptional activity. Though the macrocyclization of amatoxin is extensively studied, little is known about its subsequent post-translational modifications. However, studies and the potential use of amatoxins has been deterred by the scarcity of the mushroom biomass. To overcome this issue, we sought to produce the α-amanitin in Escherichia coli. Genes encoding the amanitin precursor peptide (AMA1) and prolyl oligopeptidase (POPB) were separately cloned and expressed in E. coli. Fusion tags were attached to candidate proteins to improve expression and solubility. Purified AMA1 was processed in vitro by POPB, and the formation of cyclic α-amanitin was confirmed by HPLC and MALDI/TOF mass spectroscopy. Our strategy can be applied to the mass production of the α-amanitin, allowing α-amanitin to be investigated as a promising lead compound in drug development.