Halobellus ruber sp. nov., a deep red-pigmented extremely halophilic archaeon isolated from a Korean solar saltern

A novel halophilic archaeon, strain MBLA0160^T, was isolated from a solar saltern in Sorae, Republic of Korea. The cells are deep-red pigmented, Gram-negative, rod shaped, motile, and lysed in distilled water. The strain MBLA0160^T grew at 25–45 °C (optimum 37 °C), in 15–30% (w/v) NaCl (optimum 20%) and 0.1–1.0 M MgCl₂ (optimum 0.3–0.5 M) at pH 5.0–9.0 (optimum 7.0). Phylogenetic analysis based on the 16S rRNA sequence showed that this strain was related to two species within the genus Halobellus (Hbs.), with 98.4% and 95.8% similarity to Hbs. salinus CSW2.24.4 ^T and Hbs. clavatus TNN18^T, respectively. The major polar lipids of the strain MBLA160^T were phosphatidylglycerol, phosphatidylglycerol sulfate, and phosphatidylglycerol phosphate methyl ester. The genome size, G?+?C content, and N50 value of MBLA0160^T were 3.49 Mb, 66.5 mol%, and 620,127 bp, respectively. According to predicted functional proteins of strain MBLA0160^T, the highest category was amino acid transport and metabolism. Genome rapid annotation showed that amino acid and derivatives was the most subsystem feature counts. Pan-genomic analysis showed that strain MBLA0160^T had 97 annotated unique KEGG, which were mainly included metabolism and environmental information processing. Ortholog average nucleotide identities (OrthoANI) and in silico DNA-DNA hybridization (isDDH) values between the strain MBLA0160^T and other strains of the genus Halobellus were under 84,4% and 28.1%, respectively. The genome of strain MBLA0160^T also contain the biosynthetic gene cluster for C₅₀ carotenoid as secondary metabolite. Based on the phylogenetic, phenotypic, chemotaxonomic properties, and comparative genomic analyses, strain MBLA0160^T is considered to represent a novel species of the genus Halobellus, for which the name Halobellus ruber sp. nov. is proposed. The type strain is MBLA0160^T (=?KCTC 4291 ^T?=?JCM 34172 ^T).

     

Halocafeteria seosinensis gen. et sp. nov. (Bicosoecida), a halophilic bacterivorous nanoflagellate isolated from a solar saltern

Recently, heterotrophic nanoflagellates (HNF) have been reported to actively ingest prokaryotes in high salinity waters. We report the isolation and culture of an HNF from a Korean saltern pond of 300‰ salinity. The organism is biflagellated with an acronematic anterior flagellum and never glides on surfaces. The mitochondria have tubular cristae. Neither transitional helix nor spiral fiber were observed in the transition zones of the flagella. The cell has a cytostome supported by an arc of eight microtubules, suggesting that our isolate is a bicosoecid. Our isolate had neither mastigonemes, lorica, body scales, nor cytopharynx and thus could not be placed in any of the presently described bicosoecid genera. Phylogenetic analysis of 18S rRNA gene sequences from stramenopiles confirmed the bicosoecid affinities of our isolate, but did not place it within any established genus or family. Its closest relatives include Caecitellus and Cafeteria. The optimal range of growth temperature was 30–35°C. The isolated HNF grew optimally at 150‰ salinity and tolerated up to 363‰ salinity, but it failed to grow below 75‰ salinity, indicating that it could be a borderline extreme halophile. On the basis of its morphological features and position in 18S rRNA trees we propose a novel genus for our isolate; Halocafeteria, n. gen. The species name Halocafeteria seosinensis sp. nov. is proposed.     

Natronomonas gomsonensis sp. nov., isolated from a solar saltern

A halophilic archaeal strain, SA3^T, was isolated from sediment of a solar saltern in Gomso Bay, Republic of Korea. Cells of strain SA3^T were observed to be coccoid-shaped, to lyse in distilled water, Gram stain-negative and to form red-pigmented colonies. Strain SA3^T was found to require at least 18 % (w/v) NaCl for growth. Optimal growth was observed at 24 % (w/v) NaCl and 6 % (w/v) MgCl₂. The optimum pH and temperature for growth were determined to be pH 7.0 and 40 °C, respectively, while the strain was found to grow within pH and temperature ranges of 5.5–8.0 and 20–45 °C, respectively. The polar lipids were determined to consist of phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, unidentified phosphoglycolipids and unidentified phospholipids. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain SA3^T was most closely related to the members of the genus Natronomonas, Natronomonas moolapensis JCM 14361^T (95.2 %) and Natronomonas pharaonis JCM 8858^T (95.1 %). The genomic DNA G+C content (61.8 mol%) determined for strain SA3^T was slightly lower than those of N. moolapensis JCM 14361^T (63.4 mol%) and N. pharaonis JCM 8858^T (64.3 mol%). DNA–DNA hybridization values between N. moolapensis JCM 14361^T and N. pharaonis JCM 8858^T and strain SA3^T were <20 %. Based on phenotypic, chemotaxonomic and phylogenetic properties, we describe a new species of the genus Natronomonas, represented by strain SA3^T (=JCM 17867^T = KCTC 4088^T), for which we propose the name Natronomonas gomsonensis sp. nov.     

Rhodospirillum salinarum sp. nov., a halophilic photosynthetic bacterium isolated from a Portuguese saltern

A new species of halophilic photosynthetic bacteria, Rhodospirillum salinarum, has been isolated and described. Its natural habitat are the terminal crystallization ponds of solar salt production plants. R. salinarum grows optimally at 42°C in the presence of 6–18% NaCl (w/v). Growth requirements are complex, yeast extract and peptone being required both for aerobic heterotrophic and for anaerobic phototrophic growth. Increasing concentrations of NaCl in the growth media did not give rise to any corresponding increase in intracellular concentrations of K⁺, Na⁺, polyalcohols or amino acids. Malate dehydrogenase from R. salinarum is not halophilic, being inhibited even at low concentrations of Na⁺ or K⁺. The GC mol % of DNA from R. salinarum is markedly higher than that for DNA from R. salexigens, the only previously described halophilic species of the genus Rhodospirillum.     

Genomic analysis of heavy metal-resistant Halobacterium salinarum isolated from Sfax solar saltern sediments

Extremophiles - We studied the fungal DNA present in a lake sediment core obtained from Trinity Peninsula, Hope Bay, north-eastern Antarctic Peninsula, using metabarcoding through high-throughput...     

Diversity of halophilic archaea in the crystallizers of an Adriatic solar saltern

Haloarchaeal diversity in the crystallizers of Adriatic Secovlje salterns was investigated using gene fragments encoding 16S rRNA and bacteriorhodopsin as molecular markers. Screening of 180 clones from five gene libraries constructed for each gene targeted revealed 15 different 16S rRNA and 10 different bacteriorhodopsin phylotypes, indicating higher haloarchaeal diversity than previously reported in such hypersaline environments. Furthermore, results of rarefaction analysis indicated that analysis of an increasing number of clones would have revealed additional diversity. Finally, most sequences from the crystallizers grouped within the Halorubrum branch, whereas square-shaped 'Haloquadratum' relatives, repeatedly reported to dominate crystallizer communities, were rare. Presence of such special and diverse haloarchaeal community could be attributed to the Secovlje salterns rare continuous short-cycling salt production mechanism.     

Pontibacter jeungdoensis sp. nov., isolated from a solar saltern in Korea

A Gram-staining-negative, rod-shaped and red-pigmented bacterial strain, HMD3125^T, was isolated from a solar saltern in Jeungdo, Republic of Korea. A phylogenetic tree based on 16S rRNA gene sequences showed that strain HMD3125^T formed a lineage within the genus Pontibacter and was similar to Pontibacter salisaro (96.1%) and P. korlensis (95.3%). The major fatty acids of strain HMD3125^T were summed feature 4 (comprising iso-C_17:1 I and/or anteiso-C_17:1 B; 30.4%), iso-C_15:0 (20.4%) and iso-C_17:0 3OH (17.2%). The polar lipid profile of HMD3125^T consisted of the phosphatidylethanolamine, four unidentified polar lipids, unidentified phospholipid, unidentified aminolipid and unidentified aminophospholipid. Strain HMD3125^T contained MK-7 as the predominant menaquinone and sym-homospermidine as the major polyamine. The DNA G+C content of strain HMD3125^T was 45.6 mol%. Strain HMD3125^T assigned as a novel species in the genus Pontibacter, for which the name Pontibacter jeungdoensis sp. nov. is proposed. The type strain is HMD3125^T (=KCTC 23156^T =CECT 7710^T).     

Halobacterium rubrum sp. nov., isolated from a marine solar saltern

Halophilic archaeal strain TGN-42-S1^T was isolated from the Tanggu marine solar saltern, China. Cells from strain TGN-42-S1^T were observed to be pleomorphic rods, stained Gram-negative, and formed red-pigmented colonies on solid media. Strain TGN-42-S1^T was found to be able to grow at 20–50 °C (optimum 35–37 °C), at 1.7–4.8 M NaCl (optimum 3.1 M), at 0–1.0 M MgCl₂ (optimum 0.1 M), and at pH 5.0–9.0 (optimum pH 7.0–7.5). The cells lysed in distilled water, and the minimal NaCl concentration to prevent cell-lysis was found to be 10 % (w/v). The major polar lipids of the strain were phosphatidic acid, phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, phosphatidylglycerol sulfate, galactosyl mannosyl glucosyl diether (TGD-1), sulfated galactosyl mannosyl glucosyl diether (S-TGD-1), sulfated galactosyl mannosyl galactofuranosyl glucosyl diether (S-TeGD), and three unidentified glycolipids which were chromatographically identical to those of the Halobacterium species. The 16S rRNA gene and rpoB′ gene of strain TGN-42-S1^T were phylogenetically related to the corresponding genes of Halobacterium jilantaiense CGMCC 1.5337^T (98.8 and 93.5 % nucleotide identity, respectively), Halobacterium salinarum CGMCC 1.1958^T (98.4 and 91.9 %), and Halobacterium noricense JCM 15102^T (96.9 and 91.1 %). The DNA G + C content of strain TGN-42-S1^T was determined to be 69.2 mol %. Strain TGN-42-S1^T showed low DNA–DNA relatedness with Hbt. jilantaiense CGMCC 1.5337^T and Hbt. salinarum CGMCC 1.1958^T, the most closely related members of the genus Halobacterium. The phenotypic, chemotaxonomic, and phylogenetic properties suggested that strain TGN-42-S1^T (=CGMCC 1.12575^T =JCM 19908^T) represents a new species of Halobacterium, for which the name Halobacterium rubrum sp. nov. is proposed.     

Gramella jeungdoensis sp. nov., isolated from a solar saltern in Korea

A non-motile, Gram-stain-negative, yellow pigmented, rod-shaped bacterium, strain HMD3159^T, was isolated from a solar saltern in Korea. The major fatty acids were iso-C_15:0 (26.3%), iso-C_17:0 3OH (12.1%), iso-C_16:0 (12.0%), summed feature 3 (comprising C_16:1 ω7c and/or C_16:1 ω6c; 11.0%) and summed feature 9 (iso-C17:1 ω9c and/or 10-methyl C_16:0; 10.0%). The major respiratory quinone was MK-6. The DNA G+C content was 40.9 mol%. The phylogenetic tree based on 16S rRNA gene sequences showed that strain HMD3159^T formed a lineage within the genus Gramella and closely related to Gramella gaetbulicola (95.5% sequence similarity), Gramella portivictoriae (94.9%), Gramella echinicola (94.6%), and Gramella marina (93.6%). On the basis of the evidence presented in this study, strain HMD3159^T represents a novel species of the genus Gramella, for which the name Gramella jeungdoensis sp. nov., is proposed. The type strain is HMD3159^T (=KCTC 32123^T =CECT 7683^T).     

江苏盐城盐场中度嗜盐菌的分离与鉴定

采用高盐的牛肉膏蛋白胨培养基(盐浓度为8%NaCl),研究江苏省盐城市盐场土壤里中度嗜盐菌的分布情况及种群特征。从盐城市的射阳、新滩、灌东三处盐场土壤中共采集和分离得到13株中度嗜盐菌。通过形态观察、生理生化分析、16S rRNA序列分析和系统进化分析等方法进行初步鉴定,结果表明:分离到的中度嗜盐菌分属3个属,Virgibacillus属4株、Halomonas属7株和Marinobacter属2株。研究结果揭示盐城市的盐场存在较为丰富的中度嗜盐菌,具有较高的研究和利用价值。     

Identification of Streptomyces sp. nov. WH26 producing cytotoxic compounds isolated from marine solar saltern in China

A moderately halophilic actinomycetes strain, designated as WH26, was isolated from Weihai Solar Saltern in China. The identification of the strain WH26 was performed by its morphological characteristics, physiological and biochemical tests as well as phylogenetic analysis based on 16S rRNA sequence comparison. The results showed that the nucleotide sequence of the 16S rRNA gene (1,677 bp) of the strain WH26 exhibited close similarity (97–99 %) with other Streptomyces 16S rRNA genes and the strain WH26 was identified to belong to the genus Streptomyces. An ethyl acetate extraction of Streptomyces sp. nov. WH26 demonstrated significant cellular toxicity. Two compounds, 8-O-methyltetrangulol and naphthomycin A were isolated from the extract via silica gel column chromatography and HPLC. These two compounds showed potent cytotoxic activity against several human tumor cell lines including A549, HeLa, BEL-7402 and HT-29. The present studies suggest that moderately halophilic actinomycetes may be a novel biological source for the discovery of anticancer agents.     

Halorubellus salinus gen. nov., sp. nov. and Halorubellus litoreus sp. nov., novel halophilic archaea isolated from a marine solar saltern

Two extremely halophilic archaeal strains GX3(T) and GX26(T) were isolated from the Gangxi marine solar saltern near the Weihai city of Shandong Province, China. Cells from the two strains were pleomorphic and stained Gram-negative, colonies were red-pigmented. Strains GX3(T) and GX26(T) were able to grow at 25-50 °C (optimum 37 °C), at 1.4-5.1M NaCl (optimum 3.1M), at pH 5.5-9.5 (optimum pH 7.0) and neither strain required Mg(2+) for growth. Cells lyse in distilled water and the minimal NaCl concentration to prevent cell-lysis was 8% (w/v). The major polar lipids of the two strains were PA (phosphatidic acid), PG (phosphatidylglycerol), PGP-Me (phosphatidylglycerol phosphate methyl ester) and three major glycolipids (GL1, GL2 & GL3) chromatographically identical to S-TGD-1 (sulfated galactosyl mannosy glucosyl diether), S-DGD-1 (sulfated mannosyl glucosyl diether), and DGD-1 (mannosyl glucosyl diether) respectively, an unidentified lipid (GL4) was also detected in strain GX26(T). Phylogenetic analysis based on 16S rRNA gene revealed that strain GX3(T) and strain GX26(T) formed a distinct clade with the closest relative, Haladaptatus paucihalophilus (89.9-92.4% and 90.4-92.7, respectively). The rpoB' gene similarities between strains GX3(T) and GX26(T), and between the two strains and the closest relative, Halorussus rarus TBN4(T) are 96.5%, 84.3% and 83.9%, respectively. The DNA G+C contents of strain GX3(T) and strain GX26(T) are 67.3 mol% and 67.2 mol%, respectively. The DNA-DNA hybridization value between strain GX3(T) and strain GX26(T) was 44%. The phenotypic, chemotaxonomic and phylogenetic properties suggest that strain GX3(T) and strain GX26(T) represent two novel species in a new genus within the family Halobacteriaceae, Halorubellus salinus gen. nov., sp. nov. (type strain GX3(T)=CGMCC 1.10384(T)=JCM 17115(T)) and Halorubellus litoreus sp. nov. (type strain GX26(T)=CGMCC 1.10386(T)=JCM 17117(T)).     

Molecular community analysis of magnesium-rich bittern brine recovered from a Tunisian solar saltern

The microbial community of a magnesium-rich bittern brine saturated with NaCl (380-400 g/L) from a Tunisian solar saltern was investigated using a molecular approach based on 16S rRNA gene analysis and viability tests. The results revealed the existence of microbial flora. Viability test assessment showed that 46.4% of this flora was viable but not detectable by culturability tests. 16S rRNA genes from 49 bacterial clones and 38 archaeal clones were sequenced and phylogenetically analyzed. Eleven operational taxonomic units (OTUs) determined by the DOTUR program with 97% sequence similarity were generated for Bacteria. These OTUs were affiliated with Bacteroidetes and Gammaproteobacteria. The archaeal community composition exhibited more diversity with 38 clones, resulting in 13 OTUs affiliated with the Euryarchaeota phylum. Diversity measurement showed a more diverse archaeal than bacterial community at the saturated pond.     

Use of a mixed culture strategy to isolate halophilic bacteria with antibacterial and cytotoxic activity from the Manaure solar saltern in Colombia

Background

Water evaporation in solar salterns creates salinity gradients that promote the adaptation of microbial species to different salinities. This competitive habitat challenges the metabolic capabilities of microorganisms and promotes alterations in their production of secondary metabolites. Thus, solar salterns are a potentially important source of new natural products. In Colombia, the most important and representative solar saltern is located in Manaure (La Guajira) in the north of Colombia. The aim of this study was to develop an alternative screening strategy to select halophilic bacteria as producers of bioactive compounds from mixed microbial cultures rather than individual environmental isolates. Brine and sediment samples from different ponds (across a salinity gradient) were inoculated in seven different culture media to grow bacteria and archaea, allowing for a total of 40 different mixed cultures. An organic extract from each mixed culture was obtained and tested against multidrug resistant pathogens, including Klebsiella pneumoniae, vancomycin-resistant Enterococcus faecium, methicillin-resistant Staphylococcus aureus and Bacillus subtilis. In addition, the extracts were tested against two human cancer cell lines, cervical adenocarcinoma (SiHa) and lung carcinoma (A-549).

Results

Twenty-four of the forty extracts from mixed cultures obtained from brine and sediment samples from the Manaure solar saltern showed antibacterial activity against Bacillus subtilis. Two extracts, referred to as A1SM3–29 and A1SM3–36, were also active against a methicillin-resistant Staphylococcus aureus, with the latter extract also showing slight cytotoxic activity against the assayed human lung cancer cell line. From this mixed culture, nine isolates were cultivated, and their extracts were tested against the same pathogens, resulting in the identification of a Vibrio sp. strain (A1SM3–36-8) with antimicrobial activity that was similar to that observed for the mixed culture extract. The extract of this strain was subjected to a bioautography assay, and 3 different fractions exhibited antibacterial activity against methicillin-resistant Staphylococcus aureus. Based on the amount obtained for each fraction, F3 was selected to isolate and identify its metabolites. The major compound was identified by NMR and HRMS as 13-cis-docosenamide, an amide that has been previously reported to be an antimicrobial and cytotoxic compound.

Conclusions

Our results shows the utility of our strategy in detecting bioactive molecules in initial mixed cultures by biological assays, resulting in the isolation and characterization of Vibrio sp. A1SM3–36-8, a halophilic strain with great antibacterial and cytotoxic potential.