Recombinant Expression and Functional Assessment of Uricase from a Pertinent Origin of the Enzyme, Streptomyces sp. Strain 17-1.

Background: Uricase or urate oxidase, as a therapeutic enzyme, is extensively applied to oxidize accumulated uric acid in the body to soluble form to treat related illnesses. Objectives: This study was conducted with the aim of searching for potential sources of uricase-producing Streptomyces from Eshtehard salt desert in Alborz province, Iran and heterologous expression, purification and functional assay of the enzyme. Materials and Methods: Main screening was conducted by cultivation of the strains on a medium enriched with 0.3 percent (w/v) uric acid. The uricase gene from the most potent strain was then recombinantly expressed in E. coli BL21 (DL3). Results: Out of the tested strains, only seven showed uricase activity. The highest level of native uricase activity (11.5735 U.mL-1) belonged to strain 17-1, which had the closest similarity to Streptomyces nigra. A recombinant uricase with a molecular mass of approximately 38 kDa was produced. The purified uricase exhibited a specific activity of about 28.29±0.59 U.mg-1, which is among the highest level of uricase activity reported by other studies. Conclusions: This enzyme is a promising candidate for further applicable investigations and large-scale production in terms of its large volume of soluble expression and selective competitive activity.

Toll-like receptor 9 (TLR9) genetic variants rs187084 and rs352140 confer protection from Behcet's disease among Iranians.

BACKGROUND: Behcet's disease (BD) is a multisystem and multifactorial autoimmune disease characterized by relapsing episodes of oral aphthae, genital ulcers, and ocular and skin lesions. Toll-like receptor 9 (TLR9) has pro-inflammatory roles and its genetic variants might be involved in the pathogenesis of inflammatory diseases.  METHODS: Two hundred five BD patients and 207 age and sex-matched healthy controls were evaluated for TLR9 single nucleotide polymorphisms - 1486 T/C (rs187084) and + 2848:G/A (rs352140) using polymerase chain reaction-restriction fragment length polymorphism (RFLP-PCR). RESULTS: Healthy individuals had a significantly higher frequency of rs187084 AG and AG + GG genotypes than BD patients (p = 0.02 and p = 0.018; respectively). Of interest, healthy males had a significantly higher frequency of rs187084 AG + GG genotype and G allele than male BD patients (p = 0.035 and p = 0.045; respectively). However, rs187084 AG genotype and G allele frequencies were significantly higher in male patients with genital aphthous (p = 0.01 and p = 0.046; respectively). Furthermore, a significantly higher frequency of rs352140 CT and TT + CT genotypes was detected in healthy individuals than in BD patients (p = 0.01, and p = 0.032; respectively). Such results were also seen in healthy females than female patients (p = 0.001, and p = 0.004; respectively). Haplotype analysis revealed a significantly higher frequency of A-C and G-C haplotypes among patients and healthy subjects, respectively (p = 0.002 and p = 0.000; respectively). CONCLUSION: Our data suggested that rs187084 AG and AG + GG genotypes and rs352140 CT and TT + CT genotypes protect Iranian individuals from BD but rs187084 AG genotype and G allele predispose male BD individuals to genital aphthous. However, additional studies are required to verify these results.

An optimized method for PCR-based genotyping to detect human APOE polymorphisms.

Background: Apolipoprotein E (APOE) is one of the most polymorphic genes at two single nucleotides (rs429358 and rs7412). The various isoforms of APOE have been associated with a variety of diseases, including neurodegenerative, type 2 diabetes, etc. Hence, predicting the APOE genotyping is critical for disease risk evaluation. The purpose of this study was to optimize the tetra amplification refractory mutation system (Tetra-ARMS) PCR method for the detection of APOE mutations. Material and methods: Here, in our optimized Tetra-ARMS PCR method, different factors like cycle conditions, using HiFidelity enzyme instead of Taq polymerase and setting its best concentration, and the lack of using dimethylsulfoxide (DMSO) for amplifying the GC-regions were set up for all primer pairs. The sensitivity and accuracy were tested. For validation of the assay, the results were compared with known genotypes for the APOE gene that were previously obtained by two independent methods, RFLP and Chip-typing. Results: Successful Tetra-ARMS PCR and genotyping are influenced by multiple factors. Our developed method enabled us to amplify the DNA fragment by 25 cycles without adding any hazardous reagent, like DMSO. Our findings showed 100 % accuracy and sensitivity of the optimized Tetra-ARMS PCR while both criteria were 95 % for RFLP and 100 % for the chip-typing method. In addition, our results showed 91 % and 100 % consistency with RFLP and chip typing methods, respectively. Conclusions: Our current method is a simple and accurate approach for detecting APOE polymorphisms within a large sample size in a short time and can be performed even in low-tech laboratories.

Investigating The Correction of IVS II-1 (G> A) Mutation in HBB Gene in TLS-12 Cell Line Using CRISPR/Cas9 System.

OBJECTIVE: Beta-thalassemia is a group of inherited hematologic. The most HBB gene variant among Iranian beta-thalassemia patients is related to two mutations of IVSII-1 (G>A) and IVSI-5 (G>C). Therefore, our aim of this study is to use the knock in capability of CRISPR Cas9 system to investigate the correction of IVSII-1 (G>A) variant in Iran. MATERIALS AND METHODS: In this experimental study, following bioinformatics studies, the vector containing Puromycin resistant gene (PX459) was cloned individually by designed RNA-guided nucleases (gRNAs), and cloning was confirmed by sequencing. Proliferation of TLS-12 was done. Then, the transfect was set up by the vector with GFP marker (PX458). The PX459 vectors carrying the designed gRNAs together with Single-stranded oligodeoxynucleotides (ssODNs) as healthy DNA pattern were transfected into TLS-12 cells. After taking the single cell clones, molecular evaluations were performed on single clones. Sanger sequencing was then performed to investigate homology directed repair (HDR). RESULTS: The sequencing results confirmed that all three gRNAs were successfully cloned into PX459 vector. In the transfection phase, The TLS-12 containing PX459-gRNA/ssODN was selected. Molecular evaluations showed that the HBB gene was cleaved by the CRISPR/Cas9 system, that indicates that the performance of non-homologous end joining (NHEJ) repair system. Sequencing in some clones cleaved by the T7E1 enzyme showed that HDR was not confirmed in these clones. CONCLUSION: IVS-II-1 (G> A) mutation, which is the most common thalassemia mutation especially in Iran, the CRISPR/ Cas9 system was able to specifically target the HBB gene sequence. This could even lead to a correction in the mutation and efficiency of the HDR repair system in future research.

Cryopreservation of Iranian Markhoz goat fibroblast cells as an endangered national genetic resource.

BACKGROUND: The continuous accessibility of local animals for sustainable use is being eroded annually. Thus, a strategic vision for the conservation of biodiversity is of far-reaching emphasis to deal with unprecedented challenges in the local population extension facing in the future. This study aimed to establish and cryopreserve endangered Markhoz goat (Capra hircus) fibroblast cell lines in vitro. METHODS AND RESULTS: These primary fibroblast cells were isolated from 58 Iranian Markhoz goats and individually cultured by explant technique in DMEM medium supplemented with 10% FBS and 2 mM L-Glutamine, in the presence of Penicillin (200 U/ml)-Streptomycin (200 mg/ml) during the first passage number. The extracted cell lines were confirmed morphologically as fibroblast cells. The population doubling time for DMEM-cultured cells was 23 ± 0.5 h. Chromosomal analysis indicated a total chromosome number of 2n = 60 with > 95% frequency. The cultured cells were checked for bacteria, fungi, yeast, and mycoplasma contaminations and the results were reported negative. The efficiencies of the fluorescent protein encoded by VSV-G (pMDG) and lentiviral pCSGW vectors reported in a range of 65% value. According to the species identification analysis, the goat cell lines were banked and confirmed without any miss- and cross-contamination. CONCLUSIONS: The significant issue in this paper can be concluded about the first report of the establishment of endangered Markhoz goat cell banking inside the country. This study demonstrated the successful establishment of a genetically stable fibroblast bank as a valuable genetic resource for the endangered Iranian Markhoz goat breed.

Supernatant Metabolites from Halophilic Archaea to Reduce Tumorigenesis in Prostate Cancer In-vitro and In-vivo.

Halophilic archaea are known as the novel producers of natural products and their supernatant metabolites could have cytotoxic effects on cancer cells. In the present study, we screened the anticancer potential of supernatant metabolites from eight native haloarchaeal strains obtained from a culture collection in Iran. Five human cancer cell lines including breast, lung, prostate and also human fibroblast cells as the normal control were used in the present study. Moreover, to evaluate the anti-tumor effect of the selected supernatant, inhibition of sphere formation and tumor development was assessed in-vitro and in-vivo, respectively. Among all strains, supernatant metabolites from Halobacterium salinarum IBRC M10715 had the most potent cytotoxic effect on prostate cancer cell lines (IC50 = 0.5 mg/mL) without any effects on normal cells. It significantly increased both early and late apoptosis (about 11% and 9%, respectively) in the androgen-dependent PC3 cell line, reduced sphere formation ability of DU145 and PC3 cells with down-regulation of SOX2 gene expression. Furthermore, our results revealed that tumors developed in nude mice significantly shrank post intratumor injection of metabolites of the haloarchaeal strain. In conclusion, we suggested here for the first time that supernatant metabolites from Halobacterium salinarum IBRC M10715 could be a novel component against prostate cancer in-vitro and in-vivo with remarkable reduction in stem-like properties of tumor.

Aureobasidium mangrovei sp. nov., an ascomycetous species recovered from Hara protected forests in the Persian Gulf, Iran.

A new ascomycetous black yeast-like species was recovered from healthy plant (Avicennia marina) of Hara protected mangrove forests at Qeshm Island, Iran. Morphological, physiological analysis as well as a molecular analysis of the internal transcribed spacer (ITS) and partial large ribosomal subunit (D1/D2 domains) confirmed the placement of this strain in the genus Aureobasidium and based on considerable sequence divergence, distinguishable cardinal growth temperatures and salt tolerance a new species Aureobasidium mangrovei sp. nov. is proposed. However, the type strain micro-morphologically is not clearly distinguishable from other members of the genus. The type strain, Aureobasidium mangrovei was preserved in a metabolically inactive state at the Iranian Biological Resource Centre, Tehran, Iran as IBRC-M 30265T and the ex-type culture is deposited in the CBS yeast collection of the Westerdijk Fungal Biodiversity Institute, Utrecht, The Netherlands as CBS 142205T. The GenBank accession numbers for the nucleotide sequences of the large subunit ribosomal DNA and ITS region are KY089084 and KY089085, respectively. The MycoBank number of the new species is MB 823444.

Simple and fast multiplex PCR method for detection of species origin in meat products.

Identification of animal species is one of the major concerns in food regulatory control and quality assurance system. Different approaches have been used for species identification in animal origin of feedstuff. This study aimed to develop a multiplex PCR approach to detect the origin of meat and meat products. Specific primers were designed based on the conserved region of mitochondrial Cytochrome C Oxidase subunit I (COX1) gene. This method could successfully distinguish the origin of the pig, camel, sheep, donkey, goat, cow, and chicken in one single reaction. Since PCR products derived from each species represent unique molecular weight, the amplified products could be identified by electrophoresis and analyzed based on their size. Due to the synchronized amplification of segments within a single PCR reaction, multiplex PCR is considered to be a simple, fast, and inexpensive technique that can be applied for identification of meat products in food industries. Nowadays, this technique has been considered as a practical method to identify the species origin, which could further applied for animal feedstuffs identification.

Effect of flavonoids rich extract of Capparis spinosa on inflammatory involved genes in amyloid-beta peptide injected rat model of Alzheimer's disease.

OBJECTIVES: Alzheimer's disease (AD) is one of the most common forms of neurodegenerative diseases. Despite vast ongoing researches focusing on the area, little is known about novel treatments. In this study, we aimed to survey the effects of Capparis spinosa (C. spinosa) extract on amyloid-beta peptide (Aß)-injected rat. METHODS: For this purpose, hydroalcoholic extracts of caper leaf and fruit were prepared. Total phenolic content, DPPH, and FRAP assay were accomplished to determine antioxidant activity of C. spinosa. HPLC analysis was conducted to measure rutin and quercetin content of selected parts of the plant. Higher levels of flavonoids were observed in leaves of the plant. Twelve male Wistar Aß-induced rats were randomly divided in four groups of (1) Aß-/DW+: Sham-operated group (2) Aß+/DW+: Aß-injected group (3) Aß+/RU+: Standard rutin treatment (4) Aß+/CS+: C. spinosa extract treatment. After 6 weeks of oral administration, real-time qPCR were conducted to determine APP, BACE-1, PSEN-1, and PSEN-2 genes expression in the hippocampus of rats. RESULTS: HPLC analysis showed high levels of rutin and quercetin in leaves of Capparis. Rutin was 16939.2 ± 0.01 and quercetin was 908.93 ± 0.01 µg/g fresh weight. In fruit, 1019.52 ± 0.01 rutin and 97.86 ± 0.01 µg/g FW quercetin were measured. Expression of BACE-1, APP, PSEN-1, and PSEN-2 genes in comparison with the control group showed significant down regulation. DISCUSSION: Results of the study demonstrated that C. spinosa has the potential to down regulate inflammation-involved genes in AD, due to its high levels of flavonoids and could be beneficial as a dietary complement in AD patients.

Congenital Malformations in Singleton Infants Conceived by Assisted Reproductive Technologies and Singleton Infants by Natural Conception in Tehran, Iran.

BACKGROUND: Multiple pregnancies occur more frequently in assisted reproductive technology (ART) compared to normal conception (NC). It is known that the risk of congenital malformations in a multiple pregnancy are higher than single pregnancy. The aim of this study is to compare congenital malformations in singleton infants conceived by ART to singleton infants conceived naturally. MATERIALS AND METHODS: In this historical cohort study, we performed a historical cohort study of major congenital malformations (MCM) in 820 singleton births from January 2012 to December 2014. The data for this analysis were derived from Tehran's ART linked data file. The risk of congenital malformations was compared in 164 ART infants and 656 NC infants. We performed multiple logistic regression analyses for the independent association of ART on each outcome. RESULTS: We found 40 infants with MCM 29 (4.4%) NC infants and 14 (8.3%) ART infants. In comparison with NC infants, ART infants had a significant 2-fold increased risk of MCM (P=0.046). After adjusting individually for maternal age, infant gender, prior stillbirth, mother's history of spontaneous abortion, and type of delivery, we did not find any difference in risk. In this study the majority (95.1%) of all infants were normal but 4.9% of infants had at least one MCM. We found a difference in risk of MCMs between in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI). We excluded the possible role of genotype and other unknown factors in causing more malformations in ART infants. CONCLUSION: This study reported a higher risk of MCMs in ART singleton infants than in NC singleton infants. Congenital heart disease, developmental dysplasia of the hip (DDH), and urogenital malformations were the most reported major malformations in singleton ART infants according to organ and system classification.

Microbial diversity in the hypersaline Lake Meyghan, Iran.

Lake Meyghan is one of the largest and commercially most important salt lakes in Iran. Despite its inland location and high altitude, Lake Meyghan has a thalassohaline salt composition suggesting a marine origin. Inputs of fresh water by rivers and rainfall formed various basins characterized by different salinities. We analyzed the microbial community composition of three basins by isolation and culturing of microorganisms and by analysis of the metagenome. The basins that were investigated comprised a green ~50 g kg-1 salinity brine, a red ~180 g kg-1 salinity brine and a white ~300 g kg-1 salinity brine. Using different growth media, 57 strains of Bacteria and 48 strains of Archaea were isolated. Two bacterial isolates represent potential novel species with less than 96% 16S rRNA gene sequence identity to known species. Abundant isolates were also well represented in the metagenome. Bacteria dominated the low salinity brine, with Alteromonadales (Gammaproteobacteria) as a particularly important taxon, whereas the high salinity brines were dominated by haloarchaea. Although the brines of Lake Meyghan differ in geochemical composition, their ecosystem function appears largely conserved amongst each other while being driven by different microbial communities.

Jaminaea pallidilutea sp. nov. (Microstromatales), a basidiomycetous yeast isolated from plant material of mangrove forests in Iran.

In the course of an ongoing study aiming to catalogue the natural yeast biodiversity of Iran, a number of yeasts were isolated from plant material collected from mangrove forests on the shoreline of Qeshm Island. Two strains were identified as members of order Microstromatales. Standard phenotypic, biochemical, physiological characterization and a phylogenetic analyses of the combined 26S rRNA gene (D1/D2 domains) and ITS region sequences showed the conspecificity of these isolates and suggest their placement in the genus Jaminaea, close to Jaminaea lanaiensis and Jaminaea angkoriensis. Here, we describe this species as Jaminiaea pallidilutea sp. nov. with IBRC-M 30284T=DSM 104392T=CBS 14684T as the type strain. The Mycobank accession number is MB 819618.

Salinifilum gen. nov., with description of Salinifilum proteinilyticum sp. nov., an extremely halophilic actinomycete isolated from Meighan wetland, Iran, and reclassification of Saccharopolyspora aidingensis as Salinifilum aidingensis comb. nov. and Saccharopolyspora ghardaiensis as Salinifilum ghardaiensis comb. nov.

A Gram-positive, halophilic actinobacterial strain Miq-12T was isolated from Meighan wetland in Iran. Strain Miq-12T was strictly aerobic, catalase positive and oxidase negative. The isolate grew at 12-25 % NaCl, at 30-50 °C and pH 5.5-10.5. The optimum NaCl, temperature and pH for growth were 15-20 %, 40 °C and 7.0-8.0, respectively. The cell wall of strain Miq-12T contained meso-diaminopimelic acid as diagnostic diamino acid and arabinose as whole-cell sugar. The polar lipid pattern consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylcholine and phosphatidylinositol. It synthesized cellular fatty acids of anteiso and iso-branched types, anteiso-C17 : 0, iso-C17:0, iso-C15:0, iso-C16 : 0. The major respiratory quinone was MK-9(H4). The G+C content of its genomic DNA was 72.1 mol%. Phylogenetic analysis based on 16S rRNA gene sequence comparison revealed that strain Miq-12T belongs to the family Pseudonocardiaceae, constituted a separate clade, and showed the closest phylogenetic similarity to Saccharopolyspora aidingensis TRM 46074T (96.99 %) and Saccharopolyspora ghardaiensis CCUG 63370T (96.92 %). On the basis of phylogenetic analysis, phenotypic and chemotaxonomic characteristics, a novel genus and species of the family Pseudonocardiaceae, Salinifilum proteinilyticum gen. nov., sp. nov., are proposed. The type strain is Miq-12T (=IBRCM 11033T=LMG 28390T). We also propose that S. aidingensis and S. ghardaiensis should be transferred to this new genus and be named Salinifilum aidingensis comb. nov. and Salinifilum ghardaiensis comb. nov., respectively. The type strain of Salinifilum aidingensis comb. nov. is TRM 46074T (=CCTCCAA 2012014T=JCM 30185T) and the type strain of Salinifilum ghardaiensis comb. nov. is CCUG 63370T (=DSM 45606T=CECT 8304T).

Natronoarchaeum persicum sp. nov., a haloarchaeon isolated from a hypersaline lake.

A novel halophilic archaeon, designated strain WIIAL99T, was isolated from Lake Meyghan, a hypersaline lake in Iran. Cells of strain WIIAL99T were non-motile, catalase-positive and oxidase-negative. Strain WIIAL99T required at least 2.5 M NaCl and 0.05 M MgCl2 for growth. Optimal growth was achieved at 3.5 M NaCl and 0.1 M MgCl2. The optimum pH and temperature for growth were pH 7.0 and 37-40 °C; it was able to grow at pH 6.0-8.5 and 20-55 °C. Cells lysed in distilled water and the minimal NaCl concentration to prevent cell lysis was 8 % (w/v). The major polar lipids of strain WIIAL99T were phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, disulfated diglycosyl diether and one unidentified glycolipid. The DNA G+C content of strain WIIAL99T was 66.7 mol%. The closest relative was Natronoarchaeum rubrum JCM 17119T with 98.2 % similarity in the orthologous 16S rRNA gene sequence. Analysis of 16S rRNA and rpoB' gene sequences indicated that strain WIIAL99T is a member of the genus Natronoarchaeum in the family Halobacteriaceae and forms a distinct cluster. It was concluded that strain WIIAL99T (=IBRC-M 11062T=LMG 29814T) represents a novel species of the genus Natronoarchaeum, for which the name Natronoarchaeum persicum sp. nov. is proposed.

Natrinema soli sp. nov., a novel halophilic archaeon isolated from a hypersaline wetland.

An extremely halophilic archaeon, designated strain 5-3T, was isolated from a soil sample of Meighan wetland in Iran. Strain 5-3T was strictly aerobic, catalase-positive and oxidase-negative. Cells were Gram-stain-negative, non-motile and ovoid. Colonies of strain 5-3T were cream-coloured. The isolate showed optimum growth at 4.0 M NaCl, 40 °C and pH 7.0. The major polar lipids of the strain were phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, two unknown phospholipids and three glycolipids (including one that was chromatographically identical to S2-DGD). The major respiratory quinone was menaquinone MK-8. The G+C content of the genomic DNA was 61.5 mol%. The closest relative was Natrinema salaciae JCM 17869T with 97.3 % similarity in the orthologous 16S rRNA gene sequence. Analysis of 16S rRNA and rpoB' gene sequences indicated that strain 5-3T is a member of the genus Natrinema in the family Natrialbaceae and forms a distinct cluster. On the basis of phylogenetic analysis, and phenotypic and chemotaxonomic characteristics, a novel species of the family Natrialbaceae, Natrinema soli sp. nov., is proposed. The type strain is 5-3T (=IBRC-M 11063T=LMG 29247T).

Aliidiomarina sedimenti sp. nov., a haloalkaliphilic bacterium in the family Idiomarinaceae.

A novel Gram-staining-negative straight or curved rod-shaped, moderately halophilic and alkaliphilic bacterium, designated strain GBSy1T, was isolated from a sediment sample from the coastal-marine wetland Gomishan in Iran. GBSy1T was motile, and formed non-pigmented, mucoid colonies. Growth occurred with between 1 and 15 % (w/v) NaCl and the isolate grew optimally with 5 % (w/v) NaCl. The optimum pH and temperature for growth were 8.5 and 34 °C, while the strain was able to grow at pH 7.0-10 and 4-40 °C. On the basis of the results of 16S rRNA gene sequence analysis, GBSy1T was shown to represent a member of the genus Aliidiomarina within the class Gammaproteobacteria, family Idiomarinaceae and showed closest phylogenetic similarity to Aliidiomarina marisCF12-14T (97.7 %). The DNA G+C content of GBSy1T was 51.2 mol%. The cells of GBSy1T contained the isoprenoid ubiquinones Q-8, Q-9 and Q-10 (92, 2 and 2 %, respectively). The major cellular fatty acids of the isolate were iso-C11 : 0 3-OH, iso-C15 : 0, iso-C17 : 0 and iso-C17 : 1ω9c and its polar lipid profile comprised phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine and three unknown phospholipids. The level of DNA-DNA relatedness between GBSy1T and Aliidiomarina marisDSM 22154T was 31 %. All these features confirmed the placement of GBSy1T within the genus Aliidiomarina. On the basis of evidence from this study, a novel species of the genus Aliidiomarina, Aliidiomarina sedimenti sp. nov., is proposed, with GBSy1T (=IBRC-M 10764T=CECT 8340T) as the type strain.

Establishment and characterization of Caspian horse fibroblast cell bank in Iran.

Caspian horse, a rare horse breed found in 1965 by Louise Firouz in northern Iran, is a small horse which is reported to be in danger of extinction in its original homeland. There seems to be a great need to prevent extinction of this valuable horse. In this study, 51 fibroblast cell lines from Caspian horse ear marginal tissue were successfully established by sampling 60 horses using primary explant technique. Cells were authenticated and growth curve was plotted. According to results obtained, population doubling time (PDT) was calculated 23 ± 0.5 h for all cell lines. Multiplex polymerase chain reaction (multiplex PCR) revealed that cell lines had no cross-contamination with other species. Bacteria, fungi, and mycoplasma contamination were checked using standard methods such as PCR, direct culture, and Hoechst staining. In addition to providing a valuable source for genomic, postgenomic, and somatic cloning researches, the established cell lines would preserve Caspian horse genetic resources. It will also create an accessible database for researchers.

Soortia roseihalophila gen. nov., sp. nov., a new taxon in the order Balneolales isolated from a travertine spring, and description of Soortiaceae fam. nov.

A novel Gram-stain-negative, slightly halophilic, motile, curved rod with a horseshoe shape, designated strain Bsw-2bT, was isolated from Badab-Soort travertine spring in Iran. Phylogenetic analysis of the 16S rRNA gene sequence revealed that strain Bsw-2bT belongs to the order Balneolales, showing 84.6 % sequence similarity to Gracilimonastropica DSM 19535T and 84.4 % and 83.9 % sequence similarity to Gracilimonas rosea CL-KR2T and Balneola vulgaris DSM 17893T, respectively. In addition, phenotypic and physiological features could clearly differentiate strain Bsw-2bT from species of the most closely related genera, Gracilimonas, Balneola, Aliifodinibius and Fodinibius. The strain was able to grow with 1-3 % (w/v) (optimum at 2 %) NaCl, at temperatures of 28-34 °C (optimum at 30 °C) and between pH 6.0 and 8.0 (optimum at pH 7.0). The major cellular fatty acids of strain Bsw-2bT were iso-C15 : 0, iso-C13 : 0 and iso-C14 : 0. The polar lipid profile of strain Bsw-2bT was composed predominantly of phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylglycerol, an unknown glycolipid and four unknown phospholipids. The DNA G+C content was 40.5 mol%. Based on the evidence from the polyphasic study, strain Bsw-2bT represents a novel species in a novel genus within a new family, for which the name Soortia roseihalophila gen. nov., sp. nov. is proposed, within the new family Soortiaceae fam. nov. The type strain is strain Bsw-2bT (=IBRC-M 10915T=LMG 28547T).

Genome Reconstruction from Metagenomic Data Sets Reveals Novel Microbes in the Brackish Waters of the Caspian Sea.

We present here the findings from a study of the microbiome of the southern basin of the Caspian Sea, the largest water body on Earth disconnected from any ocean and a brackish inland sea. By high-throughput metagenomics, we were able to reconstruct the genomes of representative microbes. The gross community structure (at the phylum level) was different from the structure of typical marine and freshwater communities in temperate open oceans, with the Caspian Sea having freshwater-like amounts of Actinobacteria and Alphaproteobacteria, while Gammaproteobacteria and Betaproteobacteria were present at intermediate levels. We assembled the genomes of several groups and provide detailed descriptions of partial genomes from Actinobacteria, Thaumarchaea, and Alphaproteobacteria. Most belonged to hitherto unknown groups, although they were related to either marine or freshwater groups. The phylogenetic placement of the Caspian genomes indicates that the organisms have multiple and separate phylogenetic origins and that they are related to organisms with both freshwater and marine lineages. Comparative recruitment from global aquatic metagenomes indicated that most Caspian microbes are endemic. However, some Caspian genomes were recruited significantly from either marine water (a member of the Alphaproteobacteria) or freshwater (a member of the Actinobacteria). Reciprocally, some genomes of other origins, such as the marine thaumarchaeon " Candidatus Nitrosopelagicus" or the actinobacterium "Candidatus Actinomarina," were recruited from the Caspian Sea, indicating some degree of overlap with the microbiota of other water bodies. Some of these microbes seem to have a remarkably widespread geographic and environmental distribution.

Wickerhamomyces orientalis f.a., sp. nov.: an ascomycetous yeast species belonging to the Wickerhamomyces clade.

Five closely related yeast strains were isolated from soil in Kharg Island, Persian Gulf, Iran, and from fallen fruits in Galle, Sri Lanka, during separate projects. Morphologically, the strains produced white-coloured yeast colonies, with cells that were ovoid to ellipsoidal, making branched, true hyphae and pseudohyphae. Ascospore formation was not observed. Biochemically, the strains were able to ferment d-glucose and weakly ferment d-galactose. The strains could use a wide variety of carbon sources except methanol and hexadecane. Phylogenetic analyses using combined sequences of the small ribosomal subunit and the D1/D2 domains of the LSU, as well as the internal transcribed spacer regions, suggested that these strains belong to the Wickerhamomyces clade and that together they form one strongly supported phylogenetic clade. Differences in their sequences, biochemistry and morphology suggest they are representatives of distinct species of the genus Wickerhamomyces. Therefore, the name Wickerhamomyces orientalis f.a., sp. nov. is proposed to accommodate these novel strains; the type strain is IBRC-M 30103T (=CBS 13306T). The MycoBank number is MB 807323.