Isolation and characterization of a main porin from the outer membrane of Salinibacter ruber.

Salinibacter ruber is an extremophilic bacterium able to grow in high-salts environments, such as saltern crystallizer ponds. This halophilic bacterium is red-pigmented due to the production of several carotenoids and their derivatives. Two of these pigment molecules, salinixanthin and retinal, are reported to be essential cofactors of the xanthorhodopsin, a light-driven proton pump unique to this bacterium. Here, we isolate and characterize an outer membrane porin-like protein that retains salinixanthin. The characterization by mass spectrometry identified an unknown protein whose structure, predicted by AlphaFold, consists of a 8 strands beta-barrel transmembrane organization typical of porins. The protein is found to be part of a functional network clearly involved in the outer membrane trafficking. Cryo-EM micrographs showed the shape and dimensions of a particle comparable with the ones of the predicted structure. Functional implications, with respect to the high representativity of this protein in the outer membrane fraction, are discussed considering its possible role in primary functions such as the nutrients uptake and the homeostatic balance. Finally, also a possible involvement in balancing the charge perturbation associated with the xanthorhodopsin and ATP synthase activities is considered.

Structural and Functional Analysis of Bacterial Sulfonosphingolipids and Rosette-Inducing Factor 2 (RIF-2) by Mass Spectrometry-Guided Isolation and Total Synthesis.

We have analyzed the abundance of bacterial sulfonosphingolipids, including rosette-inducing factors (RIFs), in seven bacterial prey strains by using high-resolution tandem mass spectrometry (HRMS2 ) and molecular networking (MN) within the Global Natural Product Social Molecular Networking (GNPS) web platform. Six sulfonosphingolipids resembling RIFs were isolated and their structures were elucidated based on comparative MS and NMR studies. Here, we also report the first total synthesis of two RIF-2 diastereomers and one congener in 15 and eight synthetic steps, respectively. For the total synthesis of RIF-2 congeners, we employed a decarboxylative cross-coupling reaction to synthesize the necessary branched α-hydroxy fatty acids, and the Garner-aldehyde approach to generate the capnine base carrying three stereogenic centers. Bioactivity studies in the choanoflagellate Salpingoeca rosetta revealed that the rosette inducing activity of RIFs is inhibited dose dependently by the co-occurring sulfonosphingolipid sulfobacins D and F and that activity of RIFs is specific for isolates obtained from Algoriphagus.

Not All Photoactive Yellow Proteins Are Built Alike: Surprises and Insights into Chromophore Photoisomerization, Protonation, and Thermal Reisomerization of the Photoactive Yellow Protein Isolated from Salinibacter ruber.

We demonstrate that the Halorhodospira halophila (Hhal) photoactive yellow protein (PYP) is not representative of the greater PYP family. The photodynamics of the PYP isolated from Salinibacter ruber (Srub) is characterized with a comprehensive range of spectroscopic techniques including ultrafast transient absorption, photostationary light titrations, Fourier transform infrared, and cryokinetics spectroscopies. We demonstrate that the dark-adapted pG state consists of two subpopulations differing in the protonation state of the chromophore and that both are photoactive, with the protonated species undergoing excited-state proton transfer. However, the primary I0 photoproduct observed in the Hhal PYP photocycle is absent in the Srub PYP photodynamics, which indicates that this intermediate, while important in Hhal photodynamics, is not a critical intermediate in initiating all PYP photocycles. The excited-state lifetime of Srub PYP is the longest of any PYP resolved to date (∼30 ps), which we ascribe to the more constrained chromophore binding pocket of Srub PYP and the absence of the critical Arg52 residue found in Hhal PYP. The final stage of the Srub PYP photocycle involves the slowest known thermal dark reversion of a PYP (∼40 min vs 350 ms in Hhal PYP). This property allowed the characterization of a pH-dependent equilibrium between the light-adapted pB state with a protonated cis chromophore and a newly resolved pG' intermediate with a deprotonated cis chromophore and pG-like protein conformation. This result demonstates that protein conformational changes and chromophore deprotonation precede chromophore reisomerization during the thermal recovery of the PYP photocycle.

Bacteroidetocins Target the Essential Outer Membrane Protein BamA of Bacteroidales Symbionts and Pathogens.

Bacteroidetocins are a family of antibacterial peptide toxins that are produced by and target members of the phylum Bacteroidetes. To date, 19 bacteroidetocins have been identified, and four have been tested and shown to kill diverse Bacteroidales species (M. J. Coyne, N. Béchon, L. M. Matano, V. L. McEneany, et al., Nat Commun 10:3460, 2019, https://doi.org/10.1038/s41467-019-11494-1). Here, we identify the target and likely mechanism of action of the bacteroidetocins. We selected seven spontaneous mutants of four different genera, all resistant to bacteroidetocin A (Bd-A) and found that all contained mutations in a single gene, bamA. Construction of three of these bamA mutants in the wild-type (WT) strains confirmed they confer resistance to Bd-A as well as to other bacteroidetocins. We identified an aspartate residue of BamA at the beginning of exterior loop 3 (eL3) that, when altered, renders strains resistant to Bd-A. Analysis of a panel of diverse Bacteroidales strains showed a correlation between the presence of this aspartate residue and Bd-A sensitivity. Fluorescence microscopy and transmission electron microscopy (TEM) analysis of Bd-A-treated cells showed cellular morphological changes consistent with a BamA defect. Transcriptomic analysis of Bd-A-treated cells revealed gene expression changes indicative of cell envelope stress. Studies in mice revealed that bacteroidetocin-resistant mutants are outcompeted by their WT strain in vivo. Analyses of longitudinal human gut isolates showed that bamA mutations leading to bacteroidetocin resistance do not become fixed in the human gut, even in bacteroidetocin-producing strains and nonproducing coresident strains. Together, these data lend further support to the applicability of the bacteroidetocins as therapeutic peptides in the treatment of maladies involving Bacteroidales species. IMPORTANCE The bacteroidetocins are a newly discovered class of bacteriocins specific to Bacteroidetes with a spectrum of targets extending from symbiotic gut Bacteroides, Parabacteroides, and Prevotella species to pathogenic oral and vaginal Prevotella species. We previously showed that one such bacteroidetocin, Bd-A, is active at nanomolar concentrations, is water soluble, and is bactericidal, all desirable features in a therapeutic antibacterial peptide. Here, we identify the target of several of the bacteroidetocins as the essential outer membrane protein BamA. Although mutations in bamA can be selected in bacteria grown in vitro, we show both in a mouse model and in human gut ecosystems that bamA mutants leading to Bd-A resistance are fitness attenuated and are not selected. These features further support the potential usefulness of the bacteroidetocins as therapeutics for maladies associated with pathogenic Prevotella species, such as recurrent bacterial vaginosis, for which there are few effective treatments.

Real-time identification of two substrate-binding intermediates for the light-driven sodium pump rhodopsin.

Membrane transport proteins undergo critical conformational changes during substrate uptake and release, as the substrate-binding site is believed to switch its accessibility from one side of the membrane to the other. Thus, at least two substrate-binding intermediates should appear during the process, that is, after uptake and before the release of the substrate. However, this view has not been verified for most transporters because of the difficulty in detecting short-lived intermediates. Here, we report real-time identification of these intermediates for the light-driven outward current-generating Na+-pump rhodopsin. We triggered the transport cycle of Na+-pump rhodopsin using a short laser pulse, and subsequent formation and decay of various intermediates was detected by time-resolved measurements of absorption changes. We used this method to analyze transport reactions and elucidated the sequential formation of the Na+-binding intermediates O1 and O2. Both intermediates exhibited red-shifted absorption spectra and generated transient equilibria with short-wavelength intermediates. The equilibria commonly shifted toward O1 and O2 with increasing Na+ concentration, indicating that Na+ is bound to these intermediates. However, these equilibria were formed independently; O1 reached equilibrium with preceding intermediates, indicating Na+ uptake on the cytoplasmic side. In contrast, O2 reached equilibrium with subsequent intermediates, indicating Na+ release on the extracellular side. Thus, there is an irreversible switch in "accessibility" during the O1 to O2 transition, which could represent one of the key processes governing unidirectional Na+ transport.

Detection of 2-hydroxy-fatty acids and 2-hydroxy-fatty acid-containing ceramides in a gliding marine bacterium Aureispira marina.

The cellular fatty acid composition of Aureispira marina IAM 15389T (JCM 23197T), a gliding bacterium isolated from the coastline of Thailand, was re-examined by using a standard MIDI method based on alkaline hydrolysis, and two other methods. The direct transesterification using 5% HCl/methanol or 4 M HCl hydrolysis followed by methyl esterification revealed that 2-hydroxy-15-methyl-hexadecanoic acid (2-OH-iso-C17:0) and 2-hydroxy-15-methyl-hexadecenoic acid (2-OH-iso-C17:1), which were not reported in a previous paper, were found to be major cellular fatty acids of this bacterium, and the amount of 2-OH-iso-C17:1 was even higher than that of arachidonic acid (C20:4), a characteristic polyunsaturated fatty acid present in this bacterium. These 2-hydroxy-fatty acids were contained in two cellular lipids that were relatively stable against alkaline hydrolysis. One of them was analyzed by mass spectrometry, 1H-nuclear magnetic resonance, and other chemical methods, and identified as a ceramide composed of 2-hydroxy-fatty acid and sphingosine of 19 carbons with three double bonds. A minor ceramide containing 18 carbon sphingosine with three double bonds was also detected.

Low-temperature Raman spectroscopy of sodium-pump rhodopsin from Indibacter alkaliphilus: insight of Na+ binding for active Na+ transport.

We carried out the low-temperature Raman measurement of a sodium pump rhodopsin from Indibacter alkaliphilus (IaNaR) and examined the primary structural change for the light-driven Na+ pump. We observed that photoexcitation of IaNaR produced the distorted 13-cis retinal chromophore in the presence of Na+, while the structural distortion was significantly relaxed in the absence of Na+. This structural difference of the chromophore with/without Na+ was attributed to the Na+ binding to the protein, which alters the active site. Using the spectral sensitivity to the ion binding, we found that IaNaR had a second Na+ binding site in addition to the one already specified on the extracellular surface. To date, the Na+ binding has not been considered as a prerequisite for Na+ transport. However, this study provides insight that the protein structural change induced by the ion binding involved the formation of an R108-D250 salt bridge, which has critical importance in the active transport of Na+.

Adhaeribacter soli sp. nov., a bacterium isolated from soil in Korea.

Strain MA2T was isolated from a soil sample from Gijang-gun, Busan in Korea. The strain, a Gram-stain-negative aerobic bacterium, is non-motile, ovoid- or rod-shaped, catalase- and oxidase-positive, and grows at NaCl concentrations 1% (w/v), at 15-30 °C (optimum 25 °C) and at pH 6-8.5 (optimum pH 7.5). The 16S rRNA gene sequence indicates that it belongs to the genus Adhaeribacter in the family Hymenobacteraceae. Phylogenetically, its closest relatives are Adhaeribacter terrae HY02T and Adhaeribacter terreus DNG6T, to which the strain shows 16S rRNA gene sequence similarity values of 96.6 and 96.0%, respectively. The major fatty acids (> 5% of the total fatty acids) of strain MA2T are C15:0 iso, C15:0 iso-G and summed feature 4 (anteiso-C17:1 B and/or iso-C17:1 I). The only detected isoprenoid quinone of strain MA2T is MK-7. The major polar lipid was phosphatidylethanolamine. The draft genome sequence of strain MA2T has a size of 4.9 Mkb. The genomic DNA G + C content was 46.9 mol%. Based on the phylogenetic, genotypic, phenotypic and chemotaxonomic data, the strain represents a novel species of the genus Adhaeribacter, for which the name Adhaeribacter soli sp. nov. is proposed. Strain MA2T (= KCTC 72630T = NBRC 114192T) is the type strain.

"Watching" a Molecular Twist in a Protein by Raman Optical Activity.

Light-absorbing chromophores in photoreceptors contain a π-electron system and are intrinsically planar molecules. However, within a protein environment these cofactors often become nonplanar and chiral in a manner that is widely believed to be functionally important. When the same chromophore is out-of-plane distorted in opposite directions in different members of a protein family, such conformers become a set of enantiomers. In techniques using chiral optical spectroscopy such as Raman optical activity (ROA), such proteins are expected to show opposite signs in their spectra. Here we use two microbial rhodopsins, Gloeobacter rhodopsin and sodium ion pump rhodopsin (NaR), to provide the first experimental and theoretical evidence that the twist direction of the retinal chromophore indeed determines the sign of the ROA spectrum. We disrupt the hydrogen bond responsible for the distortion of the retinal in NaR and show that the sign of the ROA signals of this nonfunctional mutant is flipped. The reported ROA spectra are monosignate, a property that has been seen for a variety of photoreceptors, which we attribute to an energetically favorable gradual curvature of the chromophore.

Description of Maribellus sediminis sp. nov., a marine nitrogen-fixing bacterium isolated from sediment of cordgrass and mangrove.

Two marine bacterial strains designated Y2-1-60T and GM1-28 were isolated from sediments of cordgrass and mangrove along the Luoyang estuary in Quanzhou Bay, China, respectively. Both strains were Gram-staining-negative, straight rod-shaped, non-flagellum, facultatively anaerobic, nitrogen-fixing, and did not contain carotenoid pigment. Catalase activities were found to be weak positive and oxidase activities negative. The 16S rRNA gene sequences of the two strains were identical and had maximum similarity of 98.0% with Maribellus luteus XSD2T, and of <94.5% with other species. ANI value (96.9%) and DDH estimate (71.5%) between the two strains supported that they belonged to the same species. ANI value and DDH estimate between the two strains and M. luteus XSD2T was 74.3% and 19.4%, respectively, indicating that they represent a novel species. Phylogenetic analysis based on 16S rRNA gene and phylogenomic analysis indicated that strains Y2-1-60T and GM1-28 formed a monophyletic branch within the genus Maribellus. The respiratory quinone was menaquinone MK-7. The major fatty acid (>10%) consisted of iso-C15:0, and iso-C17:0 3-OH. The polar lipids consisted of phosphatidylethanolamine and several unidentified lipids. The genomic G+C contents were 41.9-42.0mol%. Gene annotation revealed that strains Y2-1-60T and GM1-28 contained a set of nif gene cluster (nifHDKENB) responsible for nitrogen fixation. Based on the above characteristics, strains Y2-1-60T and GM1-28 represent a novel species within the genus Maribellus. Thus, Maribellus sediminis sp. nov. is proposed with type strain Y2-1-60T (=MCCC 1K04285T=KCTC 72884T), isolated from cordgrass sediment and strain GM1-28 (=MCCC 1K04384=KCTC 72880), isolated from mangrove sediment.

Bacteroidetes can be a rich source of novel lanthipeptides: The case study of Pedobacter lusitanus.

Lanthipeptides are intriguing peptides known since 1928, the year of penicillin's discovery. At that time, they were known as lantibiotics due to their (methyl)lanthionine amino acids and antibacterial activity. Their body of knowledge expanded tremendously over the last few years. Our analysis reveals that Bacteroidetes has a high state of clusters encoding the biosynthesis of class I lanthipeptides. We show that some strains of Pedobacter have a number of LanBs/genome comparable to that of some Actinobacteria. The case study selected was Pedobacter lusitanus NL19. Its clusters identified encode LanBs associated with LanCs as well as orphan LanBs. The first are concomitant with LanT transporters typical of class II lanthipeptides (and not class I), making their clusters into a hybrid class I and class II type. So far, this kind of operon was described only once and is involved in the production of pinensins, the first lanthipeptide with antifungal activity. A particular feature of pinensins is their splitted LanBs and we found that these enzymes are also widely encoded in Bacteroides. The function of a high percentage of proteins predicted to play a role in the production of Pedobacter lanthipeptides is unknown. Other major fraction of these proteins is expected to be enrolled in signal-transduction pathways. We demonstrate that the occurrence of lanthipeptides clusters in the genomes of Gram-negative bacteria is higher than previously reported. More importantly, we show that their genetic background is highly diverse, which is an undeniable foreshadowing of novel peptide structures, biochemistry and biological function.

Arthrospiribacter ruber gen. nov., sp. nov., a novel bacterium isolated from Arthrospira cultures.

Polyphasic analysis of ten isolates of the red-pigmented bacteria isolated from ten Arthrospira cultures originating from different parts of the world is described. The 16S rRNA analysis showed <95 % identity with the known bacteria on public databases, therefore, additional analyses of fatty acids profiles, MALDI-TOF/MS, genome sequencing of the chosen isolate and following phylogenomic analyses were performed. Gram-stain-negative, strictly aerobic rods were positive for catalase, negative for oxidase, proteolytic and urease activity. Major fatty acids were 15 : 0 iso, 17:0 iso 3 OH and 17:1 iso w9c/16:0 10-methyl. The whole phylogenomic analyses revealed that the genomic sequence of newly isolated strain DPMB0001 was most closely related to members of Cyclobacteriaceae family and clearly indicated distinctiveness of newly isolated bacteria. The average nucleotide identity and in silico DNA-DNA hybridisation values were calculated between representative of the novel strains DPMB0001 and its phylogenetically closest species, Indibacter alkaliphilus CCUG57479 (LW1)T (ANI 69.2 % is DDH 17.2 %) and Mariniradius saccharolyticus AK6T (ANI 80.02 % isDDH 26.1 %), and were significantly below the established cut-off <94 % (ANI) and <70 % (isDDH) for species and genus delineation. The obtained results showed that the analysed isolates represent novel genus and species, for which names Arthrospiribacter gen nov. and Arthrospiribacter ruber sp. nov. (type strain DPMB0001=LMG 31078=PCM 3008) is proposed.

Evidence in the Japan Sea of microdolomite mineralization within gas hydrate microbiomes.

Over the past 15 years, massive gas hydrate deposits have been studied extensively in Joetsu Basin, Japan Sea, where they are associated primarily with active gas chimney structures. Our research documents the discovery of spheroidal microdolomite aggregates found in association with other impurities inside of these massive gas hydrates. The microdolomites are often conjoined and show dark internal cores occasionally hosting saline fluid inclusions. Bacteroidetes sp. are concentrated on the inner rims of microdolomite grains, where they degrade complex petroleum-macromolecules present as an impurity within yellow methane hydrate. These oils show increasing biodegradation with depth which is consistent with the microbial activity of Bacteroidetes. Further investigation of these microdolomites and their contents can potentially yield insight into the dynamics and microbial ecology of other hydrate localities. If microdolomites are indeed found to be ubiquitous in both present and fossil hydrate settings, the materials preserved within may provide valuable insights into an unusual microhabitat which could have once fostered ancient life.

Genome Mining and Biosynthesis of Primary Amine-Acylated Desferrioxamines in a Marine Gliding Bacterium.

Genome mining of Fulvivirga sp. W222 revealed a desferrioxamine-like biosynthetic gene cluster containing an unknown gene fulF that is conserved in many Bacteroidetes species. A series of primary amine-acylated desferrioxamine G1 analogues, fulvivirgamides, were identified, and fulvivirgamides A2, B2, B3, and B4 (1-4) were purified and characterized. The function of FulF, which is a novel acyltransferase for the acylation of the primary amine of Desferrioxamine G1, was verified by heterologous expression and feeding experiments.

Mucilaginibacter hurinus sp. nov., isolated from briquette warehouse soil.

A novel bacterial strain, designated ZR32T, was isolated from briquette warehouse soil in Ulsan (Korea). The strain was aerobic, showing pink-colored colonies on R2A agar. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain ZR32T was closely related to Mucilaginibacter soli R9-65T (97.0%), Mucilaginibacter gynuensis YC7003T (96.9%), and Mucilaginibacter lutimaris BR-3T (96.8%). The values of DNA-DNA relatedness related two highest strains M. soli R9-65T and M. gynuensis YC7003T were 31.2 ± 6.9% and 19.7 ± 0.3%, respectively. Its genome size was 3.9 Mb, comprising 3402 predicted genes. The DNA G+C content of strain ZR32T was 43.0 mol%. The major cellular fatty acids (> 5% of total) were summed feature 3 (C16:1ω6c and/or C16:1ω7c), C16:0, C16:1ω5c, iso-C15:0, iso-C17:0 3-OH, and C17:1ω9c. The major respiratory quinine was menaquinone-7 (MK-7). The major polar lipids were phosphatidylethanolamine, two unidentified phospholipids, one unidentified sphingolipid, and one unidentified polar lipid. Strain ZR32T showed distinctive characteristics such as the temperature and pH for growth ranges, being positive for ß-glucosidase, salicin production, negative for N-acetyl-glucosamine assimilation, being resistant to carbenicillin and piperacillin to related species. On the basis of phenotypic, chemotaxonomic, and phylogenetic data, strain ZR32T represents a novel species of the genus Mucilaginibacter, for which the name Mucilaginibacter hurinus sp. nov. is proposed. The type strain is ZR32T (= KCTC 62193 = CCTCC AB 2017285).

Pontibacter beigongshangensis sp. nov., Isolated from the Mash of Wine.

A Gram-negative, aerobic, oval-shaped, and light red pigmented bacterium, designated T6-1T, was isolated from the mash of wine collected from a wine-making laboratory simulated fermenter located in Beijing, China. The optimal growth of T6-1T occurred at 30 °C, pH 7.0 with 1% NaCl. The sole respiratory quinone was menaquinone-7 (MK-7). The principal cellular fatty acids (>5%) were iso-C15:0, iso-C17:0 3OH, C16:1 ω5c, and iso-C17:0. The major polar lipids were PE (phosphatidylethanolamine), PL (unidentified phospholipid), and L1-2 (unidentified lipids). 16S rRNA phylogenetic analysis indicated that strain T6-1T belonged to the genus Pontibacter. The 16S rRNA gene sequence of strain T6-1T was most similar to Pontibacter amylolyticus 9-2T (95.92%). The genomic DNA G+C content of strain T6-1 was 50.34 mol%. The digital DNA-DNA relatedness and average nucleotide identity value between T6-1T and 9-2T was 20.20% and 74.18%, respectively. Polyphasic taxonomy analysis indicated that strain T6-1T represents a novel species of the genus Pontibacter, for which the name Pontibacter beigongshangensis sp. nov. is proposed, with the type strain T6-1T (= CGMCC 1.17104T = KCTC 72413T).

X-ray Crystallographic Structure and Oligomerization of Gloeobacter Rhodopsin.

Gloeobacter rhodopsin (GR) is a cyanobacterial proton pump which can be potentially applied to optogenetics. We solved the crystal structure of GR and found that it has overall similarity to the homologous proton pump from Salinibacter ruber, xanthorhodopsin (XR). We identified distinct structural characteristics of GR's hydrogen bonding network in the transmembrane domain as well as the displacement of extracellular sides of the transmembrane helices relative to those of XR. Employing Raman spectroscopy and flash-photolysis, we found that GR in the crystals exists in a state which displays retinal conformation and photochemical cycle similar to the functional form observed in lipids. Based on the crystal structure of GR, we selected a site for spin labeling to determine GR's oligomerization state using double electron-electron resonance (DEER) spectroscopy and demonstrated the pH-dependent pentamer formation of GR. Determination of the structure of GR as well as its pentamerizing propensity enabled us to reveal the role of structural motifs (extended helices, 3-omega motif and flipped B-C loop) commonly found among light-driven bacterial pumps in oligomer formation. Here we propose a new concept to classify these pumps based on the relationship between their oligomerization propensities and these structural determinants.

Comparison of microbial source tracking efficacy for detection of cattle fecal contamination by quantitative PCR.

Identification of fecal contamination sources in surface water has become heavily dependent on quantitative PCR (qPCR) because this technique allows for the rapid enumeration of fecal indicator bacteria as well as the detection and quantification of fecal source-associated genetic markers in the environment. Identification of contamination sources in impaired waters is a prerequisite for developing best management practices to reduce future pollution. Proper management decisions rely on the quality and interpretation of qPCR data. In this study, we developed a method to determine analytical and process lower limits of detection (LLOD) and quantification (LLOQ) using two cattle-associated genetic markers targeting Bacteroidales. Analytical LLOD (ALLOD) for both CowM2 and CowM3 genetic markers in the qPCR assay were five gene copies per reaction. Using composite fecal DNA, the analytical LLOQ (ALLOQ) determined for CowM2 and CowM3 were 78 and 195 gene copies/reaction, respectively. When plasmid DNA was used, the ALLOQ for CowM2 and CowM3 were 46 and 20 gene copies/reaction, respectively. The process LLOD (PLLOD) for CowM2 and CowM3 were 0.4 and 0.02 mg feces/filter (wet weight), respectively. Using the standard deviation value of 0.25 as a cut-off point for LLOQ in regression analysis, the process LLOQ (PLLOQ) for CowM2 and CowM3 were 3.2 and 0.3 mg feces/filter, respectively. These results indicate that CowM3 exhibited superior performance characteristics compared with CowM2 for fecal samples collected from our geographical region. Moreover, the method for calculating LLOD and LLOQ developed here can be applied to other microbial source tracking studies.

Mucilaginibacter terrigena sp. nov. sp., A Novel Member of the Family Sphingobacteriaceae.

A bacterial strain, 17JY9-4T, was isolated from a soil sample collected on Jeju Island, South Korea. Colonies grown on R2A agar are pale pink in color, and cells are Gram-stain negative, short, and rod-shaped. Analysis of 16S rRNA gene sequences identified this strain as a member of the genus Mucilaginibacter in the family Sphingobacteriaceae, with high levels of 16S rRNA sequence similarity shared with Mucilaginibacter lutimaris BR-3T (98.0%), Mucilaginibacter rigui WPCB133T (98.0%), Mucilaginibacter phyllosphaerae PP-F2F-G21T (97.0%), Mucilaginibacter amnicola TAPP7T (96.8%), and Mucilaginibacter soli R9-65T (96.7%). Growth of strain 17JY9-4T occurs at 10-30 °C, pH 6-8, and in the presence of 0-1.0% NaCl. The genomic G+C content is 44.38 mol%. The predominant respiratory quinone of the isolate is MK-7; the major fatty acids are summed feature 3 (C16:1ω7c/C16:1ω6c) (39.7%), iso-C15:0 (22.8%), iso-C17:0 3-OH (7.8%), and C16:0 (7.7%); and the major polar lipid is phosphatidylethanolamine. The phenotypic and chemotaxonomic data support the placement of strain 17JY9-4T within the genus Mucilaginibacter. However, the DNA-DNA relatedness between the isolate and M. rigui, M. lutimaris, M. phyllosphaerae, M. amnicola, and M. soli were 44.3 ± 3.0%, 38.6 ± 3.7%, 23.2 ± 2.9%, 21.9 ± 3.1%, and 18.6 ± 3.7%, respectively. The results of 16S rRNA gene sequence similarity analysis, DNA-DNA hybridization analysis, and the observed differentiating phenotypic properties from other closely related taxa clearly indicate that strain 17JY9-4T represents a novel species in the genus Mucilaginibacter, for which the name Mucilaginibacter terrigena sp. nov. is proposed. The type strain is 17JY9-4T (= KCTC 62294T = JCM 33049T).

Purification, characterization and functional properties of exopolysaccharide from a novel halophilic Natronotalea sambharensis sp. nov.

An exopolysaccharide producing strain AK103T was isolated from Sambharlake, Rajasthan, India. Based on polyphasic taxonomy, the strain AK103T was found to be a novel species for which the name Natronotalea sambharensis sp. nov. is proposed. The strain was able to secrete (1.2 gL-1) EPS under optimum conditions. The FT-IR spectroscopy revealed that EPS had carboxyl, hydroxyl, carbonyl and sulfate ester groups. The high performance liquid chromatography (HPLC), gel permeation chromatography (GPC) and NMR revealed that the exopolysaccharide was composed of mannose, glucose and glucuronic acid with a molecular weight of 4.6 × 106 KDa. This novel EPS was designated as Nat-103. Interestingly, the EPS was found to have antioxidant activity and in vitro antioxidant studies showed that DPPH (2,2-diphenyl-1-picrylhydrazyl) activity increased in a dose dependent manner. Furthermore, EPS Nat-103 was able to produce gold nanoparticles which were further characterized by FT-IR, DLS and Zeta potential (ZP).