Genome-resolved analyses show an extensive diversification in key aerobic hydrocarbon-degrading enzymes across bacteria and archaea.

BACKGROUND: Hydrocarbons (HCs) are organic compounds composed solely of carbon and hydrogen that are mainly accumulated in oil reservoirs. As the introduction of all classes of hydrocarbons including crude oil and oil products into the environment has increased significantly, oil pollution has become a global ecological problem. However, our perception of pathways for biotic degradation of major HCs and key enzymes in these bioconversion processes has mainly been based on cultured microbes and is biased by uneven taxonomic representation. Here we used Annotree to provide a gene-centric view of the aerobic degradation ability of aliphatic and aromatic HCs in 23,446 genomes from 123 bacterial and 14 archaeal phyla.  RESULTS: Apart from the widespread genetic potential for HC degradation in Proteobacteria, Actinobacteriota, Bacteroidota, and Firmicutes, genomes from an additional 18 bacterial and 3 archaeal phyla also hosted key HC degrading enzymes. Among these, such degradation potential has not been previously reported for representatives in the phyla UBA8248, Tectomicrobia, SAR324, and Eremiobacterota. Genomes containing whole pathways for complete degradation of HCs were only detected in Proteobacteria and Actinobacteriota. Except for several members of Crenarchaeota, Halobacterota, and Nanoarchaeota that have tmoA, ladA, and alkB/M key genes, respectively, representatives of archaeal genomes made a small contribution to HC degradation. None of the screened archaeal genomes coded for complete HC degradation pathways studied here; however, they contribute significantly to peripheral routes of HC degradation with bacteria. CONCLUSION: Phylogeny reconstruction showed that the reservoir of key aerobic hydrocarbon-degrading enzymes in Bacteria and Archaea undergoes extensive diversification via gene duplication and horizontal gene transfer. This diversification could potentially enable microbes to rapidly adapt to novel and manufactured HCs that reach the environment.

Metagenomes and metatranscriptomes shed new light on the microbial-mediated sulfur cycle in a Siberian soda lake.

BACKGROUND: The planetary sulfur cycle is a complex web of chemical reactions that can be microbial-mediated or can occur spontaneously in the environment, depending on the temperature and pH. Inorganic sulfur compounds can serve as energy sources for specialized prokaryotes and are important substrates for microbial growth in general. Here, we investigate dissimilatory sulfur cycling in the brine and sediments of a southwestern Siberian soda lake characterized by an extremely high pH and salinity, combining meta-omics analyses of its uniquely adapted highly diverse prokaryote communities with biogeochemical profiling to identify key microbial players and expand our understanding of sulfur cycling under haloalkaline conditions. RESULTS: Peak microbial activity was found in the top 4 cm of the sediments, a layer with a steep drop in oxygen concentration and redox potential. The majority of sulfur was present as sulfate or iron sulfide. Thiosulfate was readily oxidized by microbes in the presence of oxygen, but oxidation was partially inhibited by light. We obtained 1032 metagenome-assembled genomes, including novel population genomes of characterized colorless sulfur-oxidizing bacteria (SOB), anoxygenic purple sulfur bacteria, heterotrophic SOB, and highly active lithoautotrophic sulfate reducers. Surprisingly, we discovered the potential for nitrogen fixation in a new genus of colorless SOB, carbon fixation in a new species of phototrophic Gemmatimonadetes, and elemental sulfur/sulfite reduction in the "Candidatus Woesearchaeota." Polysulfide/thiosulfate and tetrathionate reductases were actively transcribed by various (facultative) anaerobes. CONCLUSIONS: The recovery of over 200 genomes that encoded enzymes capable of catalyzing key reactions in the inorganic sulfur cycle indicates complete cycling between sulfate and sulfide at moderately hypersaline and extreme alkaline conditions. Our results suggest that more taxonomic groups are involved in sulfur dissimilation than previously assumed.

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.

Oceanobacillus longus sp. nov., a moderately halophilic bacterium isolated from a salt lake.

A Gram-stain-positive, endospore-forming, long rod-shaped, strictly aerobic, moderately halophilic bacterium, designated strain T9BT, was isolated from a brine sample of the hypersaline lake Aran-Bidgol in Iran. Cells of strain T9BT were motile and produced colonies with a brown pigment. Growth occurred between 1.0 and 20 % (w/v) NaCl and the isolate grew optimally at 5.0 % (v/w) NaCl. The optimum pH and temperature for growth of the strain were pH 7.0 and 35 °C, while it was able to grow over pH and temperature ranges of pH 6.0-9.0 and 25-45 °C. Phylogenetic analysis based on the comparison of 16S rRNA gene sequences revealed that strain T9BT is a member of the genus Oceanobacillus. The closest relative to this strain was Oceanobacillus rekensis PT-11T with a similarity of 97.4 %, followed by Oceanobacillus profundus CL-MP28T and Oceanobacillus polygoni SA9T with 97.3 and 97.1 % similarity, respectively. The major cellular fatty acids of the isolate were anteiso-C15 : 0, iso-C14 : 0 and iso-C16 : 0. The polar lipids of strain T9BT consisted of phosphatidylglycerol, diphosphatidylglycerol, three phospholipids and one aminoglycolipid. It contained MK-7 as the predominant menaquinone and meso-diaminopimelic acid in the cell-wall peptidoglycan. The G+C content of the genomic DNA of this strain was 42.9 mol%. Phylogenetic analysis, DNA-DNA hybridization data and phenotypic characteristics allowed strain T9BT to be differentiated from other members of the genus Oceanobacillus. A novel species, Oceanobacillus longus sp. nov., is therefore proposed to accommodate this strain. The type strain is T9BT (=IBRC-M 10703T=LMG 29250T).

Halosiccatus urmianus gen. nov., sp. nov., a haloarchaeon from a salt lake.

A novel, orange-pigmented, halophilic archaeon, strain DC8T, was isolated from Urmia salt lake in north-west Iran. The cells of strain DC8T were non-motile and pleomorphic, from small rods to triangular or disc shaped. The novel strain needed at least 2.5 M NaCl and 0.02 M MgCl2 for growth. Optimal growth was achieved at 4.0 M NaCl and 0.1 M MgCl2. The optimum pH and temperature for growth were pH 7.5 and 45 °C, respectively, and it was able to grow over a pH range of 7.0 to 8.5 and a temperature range of 25 to 55 °C. Analysis of the 16S rRNA gene sequence showed that strain DC8T was a member of the family Halobacteriaceae; however, its similarity was as low as 90.1 %, 89.3 % and 89.1 % to the most closely related haloarchaeal taxa, including type species of members of the genera Halosimplex, Halobaculum and Halomicrobium, respectively. The G+C content of its DNA was 68.1 mol%. Polar lipid analyses revealed that strain DC8T contained phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, phosphatidylglycerol sulfate and phosphatidic acid. One unknown phospholipid, two major glycolipids and one minor glycolipid were also detected. The only quinone present was MK-8 (II-H2). The physiological, biochemical and phylogenetic differences between strain DC8T and other extremely halophilic archaeal genera with validly published names supported that this strain represents a novel species of a new genus within the family Halobacteriaceae, for which the name Halosiccatus urmianus gen. nov., sp. nov. is proposed. The type strain is strain DC8T ( = IBRC-M 10911T = CECT 8793T).

Halovarius luteus gen. nov., sp. nov., an extremely halophilic archaeon from a salt lake.

An extremely halophilic archaeon, strain DA50T, was isolated from a brine sample of Urmia lake, a hypersaline environment in north-west Iran. Strain DA50T was orange-pigmented, motile, pleomorphic and required at least 2.5 M NaCl but not MgCl2 for growth. Optimal growth was achieved at 4.0 M NaCl and 0.3 M MgCl2. The optimum pH and temperature for growth were pH 7.0 and 45 °C, while it was able to grow over a pH range of 6.5-8.0 and a temperature range of 25-50 °C. Analysis of 16S rRNA gene sequences revealed that strain DA50T is a member of the family Halobacteriaceae, showing a low level of similarity with other members of this family. Highest similarities, 94.4, 94.0 and 93.9 %, were obtained with the 16S rRNA gene sequences of the type strains of Natrialba aegyptia, Halobiforma lacisalsi and Halovivax asiaticus, respectively. Polar lipid analyses revealed that strain DA50T contains phosphatidylglycerol and phosphatidylglycerol phosphate methyl ester. Four unidentified glycolipids and two minor phospholipids were also observed. The only quinone present was MK-8(II-H2). The G+C content of its DNA was 62.3 mol%. On the basis of the data obtained, the new isolate could not be classified in any recognized genus. Strain DA50T is thus considered to represent a novel species of a new genus within the family Halobacteriaceae, order Halobacteriales, for which the name Halovarius luteus gen. nov., sp. nov. is proposed. The type strain of Halovarius luteus is DA50T ( = IBRC-M 10912T = CECT 8510T).

Halovivax cerinus sp. nov., an extremely halophilic archaeon from a hypersaline lake.

An extremely halophilic archaeon, strain IC35(T), was isolated from a mud sample of the Aran-Bidgol salt lake in Iran. The novel strain was cream, non-motile, rod-shaped and required at least 2.5 M NaCl, but not MgCl2, for growth. Optimal growth was achieved with 3.4 M NaCl and 0.1 M MgCl2. The optimum pH and temperature for growth were pH 7.0 (grew over a pH range of 6.5-9.0) and 40 °C (grew over a temperature range of 30-50 °C), respectively. Analysis of 16S rRNA gene sequences revealed that strain IC35(T) clustered with species of the genus Halovivax, with sequence similarities of 97.3%, 96.6% and 96.3%, respectively, to Halovivax limisalsi IC38(T), Halovivax asiaticus EJ-46(T) and Halovivax ruber XH-70(T). The rpoB' gene similarities between the novel strain and Halovivax limisalsi IBRC-M 10022(T), Halovivax ruber JCM 13892(T) and Halovivax asiaticus JCM 14624(T) were 90.2 %, 90.2% and 89.9%, respectively. The polar lipid pattern of strain IC35(T) consisted of phosphatidylglycerol and phosphatidylglycerol phosphate methyl ester; six unknown glycolipids and two minor phospholipids were also observed. The only quinone present was MK-8 (II-H2). The G+C content of the genomic DNA was 63.2 mol%. DNA-DNA hybridization studies (29% hybridization with Halovivax limisalsi IBRC-M 10022(T)), as well as biochemical and physiological characterization, allowed strain IC35(T) to be differentiated from other species of the genus Halovivax. A novel species, Halovivax cerinus sp. nov., is therefore proposed to accommodate this strain. The type strain is IC35(T) ( = IBRC-M 10256(T) = KCTC 4050(T)).

Halovivax limisalsi sp. nov., an extremely halophilic archaeon from hypersaline mud.

A Gram-stain-negative, cream-pigmented, motile, extremely halophilic archaeon, designated strain IC38(T), was isolated from a saline mud sample taken from a hypersaline lake, Aran-Bidgol, in Iran. The strain required at least 2.5 M NaCl for growth. However, MgCl2 was not required. Optimal growth occurred with 4.3 M NaCl and 0.2 M MgCl2. The optimum pH and temperature for growth were pH 7.0 and 35 °C, respectively, and strain IC38(T) was able to grow over a pH range of 6.5-9.0, and a temperature range of 25-45 °C. Analysis of the 16S rRNA gene sequence revealed that strain IC38(T) clustered with the two species of the genus Halovivax, Halovivax asiaticus EJ-46(T) and Halovivax ruber XH-70(T), with sequence similarities of 96.4% and 96.1%, respectively. The similarities between the rpoB' gene of the novel strain and Halovivax asiaticus and Halovivax ruber were 90.7% and 90.3%, respectively. The polar lipid pattern of strain IC38(T) consisted of phosphatidylglycerol and phosphatidylglycerol phosphate methyl ester. Three unidentified glycolipids and two minor phospholipids were also observed. The DNA G+C content of strain IC38(T) was 62.6 mol%. On the basis of the phylogenetic analysis, as well as the biochemical and physiological characteristics, the new isolate is suggested to be a representative of a novel species of the genus Halovivax, for which the name Halovivax limisalsi sp. nov. is proposed. The type strain of Halovivax limisalsi is IC38(T) ( = IBRC-M 10022(T) = KCTC 4051(T)).

Halorientalis persicus sp. nov., an extremely halophilic archaeon isolated from a salt lake and emended description of the genus Halorientalis.

An extremely halophilic archaeon, strain D108(T), was isolated from a brine sample of Aran-Bidgol salt lake in Iran. The novel strain was cream-pigmented, motile, pleomorphic rod-shaped and required at least 2.5 M NaCl but not MgCl2 for growth. Optimal growth was achieved with 4.3 M NaCl and 0.1 M MgCl2. The optimum pH and temperature for growth were pH 7.5 and 40 °C, respectively, and the strain was able to grow over a pH range of 6.5 to 9.0, and a temperature range of 30 to 50 °C. Phylogenetic analysis based on 16S rRNA gene sequence comparisons revealed that strain D108(T) clustered with the type strain of the sole species of the genus Halorientalis, Halorientalis regularis TNN28(T), with a sequence similarity of 98.8 %. The polar lipid pattern of strain D108(T) consisted of phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, one phosphoglycolipid and two glycolipids. The only quinone present was MK-8(II-H2). The DNA G+C content of strain D108(T) was 62.8 mol%. DNA-DNA hybridization studies (45 % with Halorientalis regularis IBRC-M 10760(T)), as well as biochemical and physiological characterization, allowed strain D108(T) to be differentiated from Halorientalis regularis. A novel species of the genus Halorientalis, Halorientalis persicus sp. nov., is therefore proposed to accommodate this strain. The type strain is D108(T) ( = IBRC-M 10043(T) = CECT 8375(T)). An emended description of the genus Halorientalis is also proposed.

Aquibacillus halophilus gen. nov., sp. nov., a moderately halophilic bacterium from a hypersaline lake, and reclassification of Virgibacillus koreensis as Aquibacillus koreensis comb. nov. and Virgibacillus albus as Aquibacillus albus comb. nov.

A novel Gram-stain-positive, moderately halophilic bacterium, designated strain B6B(T), was isolated from the water of an Iranian hypersaline lake, Aran-Bidgol, and characterized taxonomically using a polyphasic approach. Cells of strain B6B(T) were rod-shaped, motile and produced ellipsoidal endospores in terminal positions in non-swollen sporangia. Strain B6B(T) was a strictly aerobic bacterium and catalase- and oxidase-positive. The strain was able to grow at NaCl concentrations of 0.5-20.0% (w/v), with optimum growth occurring at 10.0% (w/v) NaCl. The optimum temperature and pH for growth were 35 °C and pH 7.0. On the basis of 16S rRNA gene sequence analysis, strain B6B(T) was shown to belong to the phylum Firmicutes and its closest phylogenetic similarities were with the species Virgibacillus koreensis BH30097(T) (97.5%), Virgibacillus albus YIM 93624(T) (97.4%), Sediminibacillus halophilus EN8d(T) (96.8%), Sediminibacillus albus NHBX5(T) (96.6%), Virgibacillus carmonensis LMG 20964(T) (96.3%) and Paraliobacillus quinghaiensis YIM-C158(T) (96.0%), respectively. Phylogenetic analysis revealed that strain B6B(T), along with V. koreensis BH30097(T) and V. albus YIM 93624(T), clustered in a separate clade in the family Bacillaceae. The DNA G+C content of the novel isolate was 35.8 mol%. DNA-DNA hybridization experiments revealed low levels of relatedness between strain B6B(T)and V. koreensis BH30097(T) (13%) and V. albus YIM 93624(T) (33%). The major cellular fatty acid of strain B6B(T) was anteiso-C15 : 0 (75.1%) and its polar lipid pattern consisted of phosphatidylglycerol, diphosphatidylglycerol, an unknown phospholipid and an unknown glycolipid. The isoprenoid quinones were MK-7 (90%) and MK-6 (3%). The peptidoglycan contained meso-diaminopimelic acid as the diagnostic diamino acid. All of these features support the placement of isolate B6B(T) within the phylum Firmicutes. It is closely related to V. koreensis and V. albus, but with features that clearly distinguish it from species of the genus Virgibacillus or of other related genera. On the basis of the polyphasic evidence derived in this study, we propose that strain B6B(T) be placed within a new genus, as Aquibacillus halophilus gen. nov., sp. nov., with B6B(T) as the type strain ( =IBRC-M 10775(T) =KCTC 13828(T)). We also propose that V. koreensis and V. albus should be transferred to this new genus and be named Aquibacillus koreensis comb. nov. and Aquibacillus albus comb. nov., respectively. The type strain of Aquibacillus koreensis comb. nov. is BH30097(T) ( =KCTC 3823(T) =IBRC-M 10657(T) =JCM 12387(T)) and the type strain of Aquibacillus albus comb. nov. is YIM 93624(T) ( =DSM 23711(T) =IBRC-M 10798(T) =JCM 17364(T)).

DF-Phos: Prediction of Protein Phosphorylation Sites by Deep Forest.

Phosphorylation is the most important and studied post-translational modification (PTM), which plays a crucial role in protein function studies and experimental design. Many significant studies have been performed to predict phosphorylation sites using various machine-learning methods. Recently, several studies have claimed that deep learning-based methods are the best way to predict the phosphorylation sites because deep learning as an advanced machine learning method can automatically detect complex representations of phosphorylation patterns from raw sequences and thus offers a powerful tool to improve phosphorylation site prediction. In this study, we report DF-Phos, a new phosphosite predictor based on the Deep Forest to predict phosphorylation sites. In DF-Phos, the feature vector taken from the CkSAApair method is as input for a Deep Forest framework for predicting phosphorylation sites. The results of 10-fold cross-validation show that the Deep Forest method has the highest performance among other available methods. We implemented a Python program of DF-Phos, which is freely available for non-commercial use at https://github.com/zahiriz/DF-Phos Moreover, users can use it for various PTM predictions.

Halopenitus malekzadehii sp. nov., an extremely halophilic archaeon isolated from a salt lake.

Strain CC65(T), a novel extremely halophilic archaeon, was isolated from a brine sample of a salt lake in Iran. The novel strain was light yellow-pigmented, non-motile, pleomorphic and required at least 1.7 M NaCl and 0.02 M MgCl2 for growth. Optimal growth was achieved at 3.5 M NaCl and 0.4 M MgCl2. The optimum pH and temperature for growth were pH 7.5 and 40 °C, respectively, while it was able to grow over a pH and a temperature range of pH 6.5-9.0 and 30-50 °C, respectively. Analysis of 16S rRNA gene sequence revealed that strain CC65(T) clustered with the sole member of the genus Halopenitus, Halopenitus persicus DC30(T) with a sequence similarity of 98.0%. The polar lipid profile of strain CC65(T) consisted of phosphatidylglycerol and phosphatidylglycerol phosphate methyl ester. An unidentified glycolipid and two minor phospholipids were also observed. The only quinone present was MK-8(II-H2). The DNA G+C content of strain CC65(T) was 63.8 mol%. On the basis of the biochemical and physiological characteristics, as well as DNA-DNA hybridization (44% with Halopenitus persicus IBRC 10041(T)), strain CC65(T) is classified as a novel species of the genus Halopenitus, for which the name Halopenitus malekzadehii sp. nov. is proposed. The type strain is CC65(T) ( = IBRC-M 10418(T) =KCTC 4045(T)).

Bacillus persicus sp. nov., a halophilic bacterium from a hypersaline lake.

A novel gram-positive, slightly halophilic bacterium, designated strain B48(T), was isolated from soil around the hypersaline lake Aran-Bidgol in Iran and characterized taxonomically using a polyphasic approach. Cells of strain B48(T) were non-motile rods and produced ellipsoidal endospores at a central or subterminal position in swollen sporangia. Strain B48(T) was a strictly aerobic bacterium, catalase- and oxidase-positive. The strain was able to grow at NaCl concentrations of 0.5-10.0 % (w/v), with optimum growth occurring at 2.5 % (w/v) NaCl. The optimum temperature and pH for growth were 35 °C and pH 7.5-8.0, respectively. On the basis of 16S rRNA gene sequence analysis, strain B48(T) was shown to belong to the genus Bacillus within the phylum Firmicutes and showed the closest phylogenetic similarity to the species Bacillus foraminis CV53(T) (97.4 %) and Bacillus purgationiresistens DS22(T) (96.9 %). The DNA G+C content of this new isolate was 40.1 mol%. The major cellular fatty acids of strain B48(T) were iso-C15 : 0 and anteiso-C15 : 0, and its polar lipid pattern consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, an aminophospholipid and two unknown phospholipids. The only quinone present was menaquinone 7 (MK-7). The peptidoglycan contained meso-diaminopimelic acid as the diagnostic diamino acid. All these features confirm the placement of isolate B48(T) within the genus Bacillus. DNA-DNA hybridization experiments revealed a low level of relatedness between strain B48(T) and Bacillus foraminis IBRC-M 10625(T) (8.1 %). On the basis of polyphasic evidence from this study, a new species of the genus Bacillus, Bacillus persicus sp. nov., is proposed, with strain B48(T) ( = IBRC-M 10115(T) = DSM 25386(T) = CECT 8001(T)) as the type strain.

Bacillus halosaccharovorans sp. nov., a moderately halophilic bacterium from a hypersaline lake.

A novel Gram-stain-positive, moderately halophilic bacterium, designated strain E33(T), was isolated from water of the hypersaline lake Aran-Bidgol in Iran and characterized taxonomically using a polyphasic approach. Cells of strain E33(T) were motile rods and produced ellipsoidal endospores at a central or subterminal position in swollen sporangia. Strain E33(T) was a strictly aerobic bacterium, catalase- and oxidase-positive. The strain was able to grow at NaCl concentrations of 0.5-25 % (w/v), with optimum growth occurring at 5-15 % (w/v) NaCl. The optimum temperature and pH for growth were 40 °C and pH 7.5-8.0, respectively. On the basis of 16S rRNA gene sequence analysis, strain E33(T) was shown to belong to the genus Bacillus within the phylum Firmicutes and showed the closest phylogenetic similarity with the species Bacillus niabensis 4T19(T) (99.2 %), Bacillus herbersteinensis D-1-5a(T) (97.3 %) and Bacillus litoralis SW-211(T) (97.2 %). The DNA G+C content of the type strain of the novel species was 42.6 mol%. The major cellular fatty acids of strain E33(T) were anteiso-C15 : 0 and iso-C15 : 0, and the polar lipid pattern consisted of diphosphatidylglycerol, phosphatidylglycerol, two unknown glycolipids, an unknown lipid and an unknown phospholipid. The isoprenoid quinones were MK-7 (97 %), MK-6 (2 %) and MK-8 (0.5 %). The peptidoglycan contained meso-diaminopimelic acid as the diagnostic diamino acid. All these features confirm the placement of isolate E33(T) within the genus Bacillus. DNA-DNA hybridization experiments revealed low levels of relatedness between strain E33(T) and Bacillus niabensis IBRC-M 10590(T) (22 %), Bacillus herbersteinensis CCM 7228(T) (38 %) and Bacillus litoralis DSM 16303(T) (19 %). On the basis of polyphasic evidence from this study, a novel species of the genus Bacillus, Bacillus halosaccharovorans sp. nov. is proposed, with strain E33(T) (= IBRC-M 10095(T) = DSM 25387(T)) as the type strain.

Hypersaline Lake Urmia: a potential hotspot for microbial genomic variation.

Lake Urmia located in Iran is a hypersaline environment with a salinity of about 27% (w/v). Metagenomic analyses of water samples collected from six locations in the lake exhibited a microbial community dominated by representatives of the family Haloferacaceae (69.8%), mainly those affiliated to only two genera, Haloquadratum (59.3%) and Halonotius (9.1%). Similar to other hypersaline lakes, the bacterial community was dominated by Salinibacter ruber (23.3%). Genomic variation analysis by inspecting single nucleotide variations (SNVs) and insertions/deletions (INDELs) exhibited a high level of SNVs and insertions, most likely through transformation for abundant taxa in the Lake Urmia community. We suggest that the extreme conditions of Lake Urmia and specifically its high ionic concentrations could potentially increase the SNVs and insertions, which can consequently hamper the assembly and genome reconstruction from metagenomic reads of Lake Urmia.

Challenges in supplying empirical proof for predictions derived from Species Distribution Models (SDMs): the case of an invasive cyanobacterium.

Species distribution models (SDMs) calibrated with bioclimatic variables revealed a high probability for range expansion of the invasive toxin producing cyanobacterium, Raphidiopsis raciborskii to Sweden, where no reports of its presence have hitherto been recorded. While predictions focused on the importance of climate variables for possible invasion, other barriers to dispersal and successful colonization need to be overcome by the species for successful invasion. In this study, we combine field-based surveys of R. raciborskii (microscopy and molecular analysis using species-specific primers) of 11 Swedish lakes and in-silico screening of environmental DNA using 153 metagenomic datasets from lakes across Europe to validate the SDMs prediction. Field-based studies in lakes with high/low predicted probability of occurrence did not detect the presence of R. raciborskii, and in-silico screening only detected hints of its presence in 5 metagenomes from lakes with probability ranging from 0.059 to 0.825. The inconsistencies between SDMs results and both field-based/in-silico monitoring could be due to either sensitivity of monitoring approaches in detecting early invasions or uncertainties in SDMs that focused solely on climate drivers. However, results highlight the necessity of proactive monitoring with high temporal and spatial frequency.

Large-scale phylogenomics of aquatic bacteria reveal molecular mechanisms for adaptation to salinity.

The crossing of environmental barriers poses major adaptive challenges. Rareness of freshwater-marine transitions separates the bacterial communities, but how these are related to brackish counterparts remains elusive, as do the molecular adaptations facilitating cross-biome transitions. We conducted large-scale phylogenomic analysis of freshwater, brackish, and marine quality-filtered metagenome-assembled genomes (11,248). Average nucleotide identity analyses showed that bacterial species rarely existed in multiple biomes. In contrast, distinct brackish basins cohosted numerous species, but their intraspecific population structures displayed clear signs of geographic separation. We further identified the most recent cross-biome transitions, which were rare, ancient, and most commonly directed toward the brackish biome. Transitions were accompanied by systematic changes in amino acid composition and isoelectric point distributions of inferred proteomes, which evolved over millions of years, as well as convergent gains or losses of specific gene functions. Therefore, adaptive challenges entailing proteome reorganization and specific changes in gene content constrains the cross-biome transitions, resulting in species-level separation between aquatic biomes.

High-resolution metagenomic reconstruction of the freshwater spring bloom.

BACKGROUND: The phytoplankton spring bloom in freshwater habitats is a complex, recurring, and dynamic ecological spectacle that unfolds at multiple biological scales. Although enormous taxonomic shifts in microbial assemblages during and after the bloom have been reported, genomic information on the microbial community of the spring bloom remains scarce. RESULTS: We performed a high-resolution spatio-temporal sampling of the spring bloom in a freshwater reservoir and describe a multitude of previously unknown taxa using metagenome-assembled genomes of eukaryotes, prokaryotes, and viruses in combination with a broad array of methodologies. The recovered genomes reveal multiple distributional dynamics for several bacterial groups with progressively increasing stratification. Analyses of abundances of metagenome-assembled genomes in concert with CARD-FISH revealed remarkably similar in situ doubling time estimates for dominant genome-streamlined microbial lineages. Discordance between quantitations of cryptophytes arising from sequence data and microscopic identification suggested the presence of hidden, yet extremely abundant aplastidic cryptophytes that were confirmed by CARD-FISH analyses. Aplastidic cryptophytes are prevalent throughout the water column but have never been considered in prior models of plankton dynamics. We also recovered the first metagenomic-assembled genomes of freshwater protists (a diatom and a haptophyte) along with thousands of giant viral genomic contigs, some of which appeared similar to viruses infecting haptophytes but owing to lack of known representatives, most remained without any indication of their hosts. The contrasting distribution of giant viruses that are present in the entire water column to that of parasitic perkinsids residing largely in deeper waters allows us to propose giant viruses as the biological agents of top-down control and bloom collapse, likely in combination with bottom-up factors like a nutrient limitation. CONCLUSION: We reconstructed thousands of genomes of microbes and viruses from a freshwater spring bloom and show that such large-scale genome recovery allows tracking of planktonic succession in great detail. However, integration of metagenomic information with other methodologies (e.g., microscopy, CARD-FISH) remains critical to reveal diverse phenomena (e.g., distributional patterns, in situ doubling times) and novel participants (e.g., aplastidic cryptophytes) and to further refine existing ecological models (e.g., factors affecting bloom collapse). This work provides a genomic foundation for future approaches towards a fine-scale characterization of the organisms in relation to the rapidly changing environment during the course of the freshwater spring bloom. Video Abstract.

mOTUpan: a robust Bayesian approach to leverage metagenome-assembled genomes for core-genome estimation.

Recent advances in sequencing and bioinformatics have expanded the tree of life by providing genomes for uncultured environmentally relevant clades, either through metagenome-assembled genomes or through single-cell genomes. While this expanded diversity can provide novel insights into microbial population structure, most tools available for core-genome estimation are sensitive to genome completeness. Consequently, a major portion of the huge phylogenetic diversity uncovered by environmental genomic approaches remains excluded from such analyses. We present mOTUpan, a novel iterative Bayesian method for computing the core genome for sets of genomes of highly diverse completeness range. The likelihood for each gene cluster to belong to core or accessory genome is estimated by computing the probability of its presence/absence pattern in the target genome set. The core-genome prediction is computationally efficient and can be scaled up to thousands of genomes. It has shown comparable estimates to state-of-the-art tools Roary and PPanGGOLiN for high-quality genomes and is capable of using genomes at lower completeness thresholds. mOTUpan wraps a bootstrapping procedure to estimate the quality of a specific core-genome prediction, as the accuracy of each run will depend on the specific completeness distribution and the number of genomes in the dataset under scrutiny. mOTUpan is implemented in the mOTUlizer software package, and available at github.com/moritzbuck/mOTUlizer, under GPL 3.0 license.