Pengzhenrongella frigida sp. nov., isolated from a glacier.

A Gram-stain-positive, rod-shaped bacterium, designated as HLT2-17T, was isolated from soil sample taken from the Hailuogou glacier in Sichuan province, PR China. Strain HLT2-17T was capable of growing at 4-25°C and in NaCl concentrations ranging from 0 to 2% (w/v). The highest level of 16S rRNA gene sequence similarity was observed with Pengzhenrongella phosphoraccumulans M0-14T (98.3 %) and Pengzhenrongella sicca LRZ-2T (98.2 %). The average nucleotide identity and digital DNA-DNA hybridization values between strain HLT2-17T and its closest relatives, P. phosphoraccumulans M0-14T and P. sicca LRZ-2T, were 80.0-84.0 % and 23.3-27.7 %, respectively. Phylogenomic analysis indicated that strain HLT2-17T clustered together with strains P. phosphoraccumulans M0-14T and P. sicca LRZ-2T. Strain HLT2-17T contained C16 : 0 and anteiso-C15 : 0 as the major fatty acids, and MK-9(H4) as the menaquinone. Therefore, based on a polyphasic approach, we propose that strain HLT2-17T (=CGMCC 1.11116T= NBRC 110443T) represents a novel species of the genus Pengzhenrongella and suggest the name Pengzhenrongella frigida sp. nov.

Identification of Flavobacterium algoritolerans sp. nov. and Flavobacterium yafengii sp. nov., two novel members of the genus Flavobacterium.

Six psychrotolerant, Gram-stain-negative, aerobic bacterial strains, designated as LB1P51T, LB2P87T, LB2P84, LB3P48, LB3R18 and XS2P67, were isolated from glaciers on the Tibetan Plateau, PR China. The results of 16S rRNA gene analysis confirmed their classification within the genus Flavobacterium. Strain LB2P87T displayed the highest sequence similarity to Flavobacterium sinopsychrotolerans 0533T (98.18 %), while strain LB1P51T exhibited the highest sequence similarity to Flavobacterium glaciei CGMCC 1.5380T (98.15 %). Strains LB2P87T and LB1P51T had genome sizes of 3.8 and 3.9 Mb, respectively, with DNA G+C contents of 34.2 and 34.1 %, respectively. Pairwise average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) calculations revealed that these strains represented two distinct species within the genus Flavobacterium. The results of phylogenomic analysis using 606 core genes indicated that the six strains formed a distinct clade and were most closely related to F. glaciei CGMCC 1.5380T. The ANI and dDDH values between the two species and other members of the genus Flavobacterium were below 90.3 and 40.1 %, respectively. Genome relatedness, the results of phylogenomic analysis and phenotypic characteristics collectively support the proposal of two novel species of the genus Flavobacterium: Flavobacterium algoritolerans sp. nov. (LB1P51T = CGMCC 1.11237T = NBRC 114813T) and Flavobacterium yafengii sp. nov. (LB2P87T = CGMCC 1.11249T = NBRC 114814T).

Diversity of the genus Cryobacterium and proposal of 19 novel species isolated from glaciers.

The bacterial genus Cryobacterium includes at present 14 species that live in cryospheric environments. In this study, we analyzed 101 genomes of Cryobacterium with pure cultures obtained from GenBank. They could be classified into 44 species based on average nucleotide identity (ANI) analysis, showing the diversity of Cryobacterium. Among these, 19 strains in our laboratory were isolated from the glacier samples in China. The pairwise ANI values of these 19 strains and known species were <95%, indicating that they represented 19 novel species. The comparative genomic analysis showed significant differences in gene content between the two groups with a maximum growth temperature (T max) of ≤ 20°C and a T max of >20°C. A comprehensive and robust phylogenetic tree, including 14 known species and 19 novel species, was constructed and showed five phylogenetic branches based on 265 concatenated single-copy gene sequences. The T max parameter had a strong phylogenetic signal, indicating that the temperature adaptation of Cryobacterium was largely through vertical transfer rather than horizontal gene transfer and was affected by selection. Furthermore, using polyphasic taxonomy combined with phylogenomic analysis, we proposed 19 novel species of the genus Cryobacterium by the following 19 names: Cryobacterium serini sp. nov., Cryobacterium lactosi sp. nov., Cryobacterium gelidum sp. nov., Cryobacterium suzukii sp. nov., Cryobacterium fucosi sp. nov., Cryobacterium frigoriphilum sp. nov., Cryobacterium cryoconiti sp. nov., Cryobacterium lyxosi sp. nov., Cryobacterium sinapicolor sp. nov., Cryobacterium sandaracinum sp. nov., Cryobacterium cheniae sp. nov., Cryobacterium shii sp. nov., Cryobacterium glucosi sp. nov., Cryobacterium algoritolerans sp. nov., Cryobacterium mannosilyticum sp. nov., Cryobacterium adonitolivorans sp. nov., Cryobacterium algoricola sp. nov., Cryobacterium tagatosivorans sp. nov., and Cryobacterium glaciale sp. nov. Overall, the taxonomy and genomic analysis can improve our knowledge of phenotypic diversity, genetic diversity, and evolutionary characteristics of Cryobacterium.

Flavobacterium frigoritolerans sp. nov. and Flavobacterium shii sp. nov., isolated from glaciers on the Tibetan Plateau.

The genus Flavobacterium belongs to the family Flavobacteriaceae and its members are widely distributed in the environment. Taxonomic descriptions of strains LS1R47T and LS1R49T isolated from the Laigu glacier on the Tibetan Plateau, China, are presented in this study. Both strains were psychrotolerant, Gram-stain-negative, aerobic and rod-shaped. The comparative analysis of 16S rRNA gene sequences showed that strain LS1R47T was closest to Flavobacterium bizetiae CIP 105534T (98.90 %) and strain LS1R49T was closest to Flavobacterium collinsii 983-08T (98.73 %). The 16S rRNA gene sequence similarity, average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between the two novel isolates were 99.4, 86.0 and 30.9 %, respectively. The ANI and dDDH values between strains LS1R47T and LS1R49T and their closely relatives were below 87.6 and 33.3 %, respectively. Phylogenomic analysis showed that the two strains cluster together with Flavobacterium hydatis ATCC 29551T. Both strains contained MK-6 as sole quinone, phosphatidylethanolamine as the principal polar lipid, and summed feature 3 (iso-C15 : 0 2-OH and/or C16 : 1 ω7c), iso-C15 : 0 3-OH, C15 : 0 3-OH and iso-C17 : 0 3-OH as the main fatty acids. These results indicated that strains LS1R47T and LS1R49T represented two novel species within the genus Flavobacterium. Therefore, we propose two novel species, Flavobacterium frigoritolerans sp. nov. (LS1R47T=CGMCC 1.11577T=NBRC 113654T) and Flavobacterium shii sp. nov. (LS1R49T=CGMCC 1.11581T=NBRC 113652T).

Mucilaginibacter aurantiaciroseus sp. nov. and Mucilaginibacter flavidus sp. nov., isolated from Renlongba glacier on the Tibetan Plateau.

Strains RB4R14T and RT5R15T, two Gram-stain-negative, aerobic, rod-shaped, non-motile bacteria, were isolated from ice and cryoconite of Renlongba glacier, respectively, on the Tibetan Plateau, PR China. The results of phylogenetic analysis based on 16S rRNA genes indicated that strains RB4R14T and RT5R15T belonged to the genus Mucilaginibacter with the highest similarities to Mucilaginibacter rigui WPCB133T (98.78 %) and Mucilaginibacter xinganensis BJC16-A31T (97.64 %), respectively. The genomic DNA G+C contents of strains RB4R14T and RT5R15T were 42.8 and 43.1 mol%, respectively. The digital DNA-DNA hybridization values between strains RB4R14T, RT5R15T and their close relatives were below 31.9 and 17.4 %, respectively. The average nucleotide identity values between the two novel strains and their close relatives were 79.5-82.0 and 77.9-79.3 % respectively, indicating the novelty of the two isolates at a species level. The two novel strains contained MK-7 as the major menaquinone, and summed feature 3 (comprising C16 : 1 ω7c and/or C16 : 1 ω6c), iso-C15 : 0 and iso-C17 : 0-3OH as the major fatty acids. The major polar lipid of the two novel strains were phosphatidylethanolamine. Based on phenotypic, chemotaxonomic and phylogenetic results, we propose two novel species, Mucilaginibacter aurantiaciroseus sp. nov. (RB4R14T=CGMCC 1.11911T=NBRC 114020T) and Mucilaginibacter flavidus sp. nov. (RT5R15T=CGMCC 1.23117T=NBRC 113930T).

Arthrobacter cheniae and Arthrobacter frigidicola sp. nov., isolated from a glacier.

Two Gram-stain-positive, aerobic, rod-shaped, pink and light pink colony-forming bacteria, designated as Hz2T and MDT2-14T, respectively, were isolated from glacier cryoconite samples. Comparisons based on 16S rRNA gene sequences showed that strains Hz2T and MDT2-14T take Arthrobacter bussei KR32T and Arthrobacter zhaoguopingii J391T as their closest neighbours, respectively. The average nucleotide identity values between the two novel strains and their closest relatives were 83.56 and 93.06 %, respectively. The two strains contain MK-9(H2) as their predominant menaquinone. The polar lipids of both strains were phosphatidylglycerol, diphosphatidylglycerol, phosphatidylinositol and an unidentified glycolipid. The major fatty acids of strain Hz2T were anteiso-C15 : 0, summed feature 3 (comprising C16 : 1 ω7c and/or C16 : 1 ω6c) and iso-C15 : 0, while the major fatty acids of strain MDT2-14T were anteiso-C15 : 0 and anteiso-C17 : 0. Based on these data, we propose two novel species, Arthrobacter cheniae sp. nov. (Hz2T = CGMCC 1.9262T=NBRC 113086T) and Arthrobacter frigidicola sp. nov. (MDT2-14T=CGMCC 1.9882T=NBRC 113089T).

Glacieibacterium frigidum gen. nov., sp. nov., a novel member of the family Sphingosinicellaceae isolated from a glacier.

A novel Gram-stain-negative, rod-shaped, yellow bacterium, designated as LB1R16T, was isolated from the Laigu glacier on the Tibetan Plateau, PR China. Strain LB1R16T was catalase-positive, oxidase-negative and grew at 0-28 °C, pH 6.0-8.0 and in the absence of NaCl. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain LB1R16T belongs to the family Sphingosinicellaceae but formed an independent lineage. The highest level of 16S rRNA gene sequence similarities were found to Polymorphobacter arshaanensis DJ1R-1T (95.24 %), Sphingoaurantiacus capsulatus YLT33T (94.78 %) and Sandarakinorhabdus limnophila DSM 17366T (94.67 %). The genomic DNA G+C content was 68.8 mol%. The main cellular fatty acids were summed feature 8 (C18 : 1 ω7c/C18 : 1 ω6c), summed feature 3 (C16 : 1 ω7c/C16 : 1 ω6c), C16 : 0 and C12 : 0-OH. The respiratory quinone was ubiquinone-10. The polar lipids were phosphatidylethanolamine, phosphatidylglycerol, one sphingoglycolipid, one unidentified aminolipid, one unidentified phospholipid and two unidentified polar lipids, which were different from the type strains of Polymorphobacter arshaanensis, Sphingoaurantiacus capsulatus and Sandarakinorhabdus limnophila. Based on a polyphasic approach, a novel species of a new genus, Glacieibacterium frigidum gen. nov., sp. nov., within the family Sphingosinicellaceae is proposed. The type strain is LB1R16T (=CGMCC 1.11941T=NBRC 113873T).

Flavobacterium franklandianum sp. nov. and Flavobacterium gawalongense sp. nov., isolated from glaciers on the Tibetan Plateau.

The genus Flavobacterium (family Flavobacteriaceae) can be found in diverse environments. In this study, seven novel strains were isolated from glaciers in PR China and subjected to taxonomic research. Phylogenetic analyses based on the 16S rRNA gene revealed that the strains belonged to the genus Flavobacterium. None of the seven strains grew at temperatures above 22 °C, indicating that they are psychrophilic. Furthermore, the average nucleotide identity (ANI) values of the seven strains were calculated and indicated that they represented two novel species in Flavobacterium. Strain LB3P56T was most closely related to Flavobacterium soyangense IMCC26223T (97.70 %) and strain GSP16T was most closely related to Flavobacterium sinopsychrotolerans 0533T (98.03 %). The ANI values between the two Flavobacterium strains and their closest relatives were less than 83.47 %, which was much lower than the threshold for species delineation of 95-96 %. Therefore, we propose two novel species, Flavobacterium franklandianum sp. nov. (LB3P56T=CGMCC 1.11934T=NBRC 113651T) and Flavobacterium gawalongense sp. nov. (GSP16T=CGMCC 1.24642T=NBRC 113664T).

Enlightening the taxonomy darkness of human gut microbiomes with a cultured biobank.

BACKGROUND: In gut microbiome studies, the cultured gut microbial resource plays essential roles, such as helping to unravel gut microbial functions and host-microbe interactions. Although several major studies have been performed to elucidate the cultured human gut microbiota, up to 70% of the Unified Human Gastrointestinal Genome species have not been cultured to date. Large-scale gut microbial isolation and identification as well as availability to the public are imperative for gut microbial studies and further characterizing human gut microbial functions. RESULTS: In this study, we constructed a human Gut Microbial Biobank (hGMB; homepage: hgmb.nmdc.cn ) through the cultivation of 10,558 isolates from 31 sample mixtures of 239 fresh fecal samples from healthy Chinese volunteers, and deposited 1170 strains representing 400 different species in culture collections of the International Depository Authority for long-term preservation and public access worldwide. Following the rules of the International Code of Nomenclature of Prokaryotes, 102 new species were characterized and denominated, while 28 new genera and 3 new families were proposed. hGMB represented over 80% of the common and dominant human gut microbial genera and species characterized from global human gut 16S rRNA gene amplicon data (n = 11,647) and cultured 24 "most-wanted" and "medium priority" taxa proposed by the Human Microbiome Project. We in total sequenced 115 genomes representing 102 novel taxa and 13 previously known species. Further in silico analysis revealed that the newly sequenced hGMB genomes represented 22 previously uncultured species in the Unified Human Gastrointestinal Genome (UHGG) and contributed 24 representatives of potentially "dark taxa" that had not been discovered by UHGG. The nonredundant gene catalogs generated from the hGMB genomes covered over 50% of the functionally known genes (KEGG orthologs) in the largest global human gut gene catalogs and approximately 10% of the "most wanted" functionally unknown proteins in the FUnkFams database. CONCLUSIONS: A publicly accessible human Gut Microbial Biobank (hGMB) was established that contained 1170 strains and represents 400 human gut microbial species. hGMB expands the gut microbial resources and genomic repository by adding 102 novel species, 28 new genera, 3 new families, and 115 new genomes of human gut microbes. Video abstract.

Mucilaginibacter glaciei sp. nov. and Mucilaginibacter pankratovii sp. nov., isolated from a glacier on the Tibetan Plateau.

Two Gram-stain-negative, aerobic, rod-shaped, non-motile, bacterial strains, designated as ZB1P21T and ZT4R22T, were isolated from ice and cryoconite samples collected from Zepu glacier on the Tibetan Plateau, PR China. The phylogenetic analysis of 16S rRNA gene showed that the two strains belong to the genus Mucilaginibacter. Strain ZB1P21T showed the highest similarity to Mucilaginibacter rigui WPCB133T (97.35 %), while strain ZT4R22T showed the highest similarity to Mucilaginibacter gilvus F01003T (99.11 %). The average nucleotide identity values between the two novel strains and their closest relatives were 79.42 and 85.72 % respectively. The two novel strains contained MK-7 as the major menaquinone, and summed feature 3 (comprising C16 : 1 ω7c and/or C16 : 1 ω6c), iso-C15:0, iso-C17 : 03-OH and C16 : 1 ω5c as the major fatty acids. The major polar lipid of the two novel strains were phosphatidylethanolamine. Based on these data, we propose two novel species, Mucilaginibacter glaciei sp. nov. (ZB1P21T=CGMCC 1.23981T=NBRC 113932T) and Mucilaginibacter pankratovii sp. nov. (ZT4R22T=CGMCC 1.23487T=NBRC 113931T).

Flavobacterium restrictum sp. nov., Flavobacterium rhamnosiphilum sp. nov., and Flavobacterium zepuense sp. nov. isolated from glaciers.

Three strains, designated as LB1R34T, LB3P52T and ZT4R6T, were isolated from glaciers located on the Tibetan Plateau, PR China. The strains were Gram-stain-negative, aerobic, rod-shaped, non-motile and yellow. Phylogenetic analysis based on the 16S rRNA gene and genomic sequences indicated that they were related to the members of the genus Flavobacterium. The 16S rRNA gene sequences similarities between the three strains were 92.31-96.93 %. The average nucleotide identity values and digital DNA-DNA hybridization values between these three strains and their closest relatives were 76.80-91.33 % and 21.3-44.2 %, respectively. LB1R34T, LB3P52T and ZT4R6T contained MK-6 as the major menaquinone, summed feature 3 (comprising C16 : 1ω7c and/or C16 : 1ω6c) and iso-C15 : 0 as the major fatty acids. Phosphatidylethanolamine was present in their polar lipids profiles. On the basis of the phenotypic characteristics, he results of phylogenetic analysis and genotypic data, three novel species, Flavobacterium restrictum sp. nov. (type strain=LB1 R34T=CGMCC 1.11493T=NBRC 113650T), Flavobacterium rhamnosiphilum sp. nov. (type strain=LB3 P52T=CGMCC 1.11446T=NBRC 113776T) and Flavobacterium zepuense sp. nov. (type strain=ZT4 R6T=CGMCC 1.11919T=NBRC 113653T) are proposed.

Cryobacterium ruanii sp. nov. and Cryobacterium breve sp. nov., isolated from glaciers.

Strains Sr36T and TMT4-23T were isolated from No. 1 glacier in Xinjiang Uygur Autonomous Region and Toumingmengke glacier in Gansu Province, PR China, respectively. They were Gram-stain-positive and rod-shaped micro-organisms. The optimum growth temperature of the two strains was 10-14 °C. Phylogenetic analysis showed that the two strains were related to members of the genus Cryobacterium. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between strain Sr36T and its close relatives Cryobacterium luteum Hh15T, Cryobacterium aureum Hh31T, Cryobacterium levicorallinum Hh34T and Cryobacterium flavum Hh8T were 81.16-87.24 and 28.0-32.5 %, respectively. The ANI and dDDH values between strain TMT4-23T and its close relative Cryobacterium psychrotolerans 0549T were 81.16 and 22.3 %. The polar lipids of strain Sr36T were diphosphatidylglycerol, phosphatidylglycerol, one unidentified glycolipid and three unidentified lipids. The polar lipids of strain TMT4-23T were diphosphatidylglycerol, phosphatidylglycerol, one unidentified glycolipid, one unidentified phospholipid and six unidentified lipids. The major fatty acids of strain Sr36T were anteiso-C15 : 0, iso-C16 : 0, anteiso-C17 : 0 and anteiso-C15 : 1. The major fatty acids of strain TMT4-23T were anteiso-C15 : 0, anteiso-C17 : 0, iso-C16 : 0, anteiso-C15 : 1 and iso-C15 : 1. Both strains contained 2,4-diaminobutyric acid and their predominant menaquinone was MK-10. On the basis of the phenotypic, phylogenetic and genotypic data, two novel species Cryobacterium ruanii sp. nov. (type strain = Sr36T=CGMCC 1.9275T=NBRC 113797T) and Cryobacterium breve sp. nov. (type strain =TMT4-23T=CGMCC 1.9556T=NBRC 113800T) are proposed.

Phenotypic divergence of thermotolerance: Molecular basis and cold adaptive evolution related to intrinsic DNA flexibility of glacier-inhabiting Cryobacterium strains.

The link between guanine-cytosine (GC) content and thermal adaptation is controversial. Here, we compared maximum growth temperature (TMGT ) and genomics of 78 Cryobacterium strains to avoid unreliable conclusions resulting from distantly phylogenetic groups. Phylogenomic analysis revealed this taxon had much higher diversification than we knew. Interestingly, these strains showed thermotolerance divergence with phylogenetic cohesion. A significant difference was found between TMGT ≤ 20°C strains and TMGT > 20°C strains in genomic GC content which mainly caused by variation of GC3. TMGT ≤ 20°C strains tended to use synonymous codons ended with A/U, but TMGT > 20°C strains tended to use G/C. Lower GC content at synonymous sites (≈GC3) of TMGT ≤ 20°C strains could provide lower intrinsic DNA flexibility which strongly associated with optimal molecular dynamics, and then guarantee DNA function at lower growth temperatures. This analysis of codon bias revealed close relationships for thermal adaptation, GC content at synonymous sites (≈GC3), intrinsic DNA flexibility and optimal DNA dynamics. Natural selection was main force driving this codon bias; strains with lower TMGT endured stronger natural selection. Therefore, this study provided molecular basis for bacterial adaptive evolution from moderate temperature to low temperature.

Flavobacterium psychrotolerans sp. nov., a psychrotolerant bacterium isolated from Renlongba glacier on the Tibetan Plateau.

One aerobic, Gram-stain-negative, rod-shaped bacterium, designated strain RB1R5T, was isolated from Renlongba glacier in Tibet Autonomous Region, China. Growth was observed at 4-25 °C and pH 7.0-8.0. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain RB1R5T belongs to the genus Flavobacterium, and its closest relatives are Flavobacterium luteum IMCC26026T and Flavobacterium psychroterrae CCM 8827T with 97.75 and 97.42% gene sequence similarities, respectively. The average nucleotide identity and DNA-DNA hybridization values between strain RB1R5T and its closest relative Flavobacterium luteum IMCC26026T were 78.02 and 21.8 %, respectively. The major cellular fatty acids were anteiso-C15 : 0 and summed feature 3 (comprising C16:1ω7c/C16:1ω6c). The sole menaquinone was MK-6. The major phospholipid was phosphatidylethanolamine. On the basis of phenotypic, phylogenetic and chemotaxonomic characterizations, strain RB1R5T represents a novel species of the genus Flavobacterium, for which the name Flavobacterium psychrotolerans sp. nov. is proposed. The type strain is RB1R5T (=CGMCC 1.23024T=NBRC 113060T).

Flavobacterium ranwuense sp. nov., isolated from glacier.

A Gram-stain-negative, aerobic, rod-shaped, devoid of flagella, orange-yellow-coloured bacterium (strain LB2P22T) was isolated from an ice sample collected from the Laigu glacier on the Tibetan Plateau, PR China. The results of 16S rRNA gene sequence analysis indicated that strain LB2P22T belongs to the genus Flavobacterium with highest similarity to Flavobacterium laiguense LB2P30T (98.14 %), Flavobacterium glaciei CGMCC 1.5380T (98.02 %), Flavobacterium psychroterrae CCM 8827T (97.94 %) and Flavobacterium granuli CGMCC 1.10125T (97.91 %). Strain LB2P22T had 77.99-84.99 % average nucleotide identity and 22.3-29.3 % digital DNA-DNA hybridization values with its closest relatives, indicating that it represents a novel species of the genus Flavobacterium. Strain LB2P22T grew at 0-25 °C, pH 7.0-8.0 and up to 1.0 % (w/v) NaCl. The main cellular fatty acids were iso-C15 : 0, summed feature 3 (comprising C16 : 1ω7c/C16 : 1ω6c) and anteiso-C15 : 0. The genomic DNA G+C content is 34.3 mol%. The predominant menaquinone was MK-6 and the polar lipids were phosphatidylethanolamine, one unidentified aminolipid and four unidentified lipids. Based on these data, a novel species, Flavobacteriumranwuense sp. nov., is proposed, with LB2P22T (=CGMCC 1.11361T=NBRC 113777T) as the type strain.

Cryobacterium melibiosiphilum sp. nov., a psychrophilic bacterium isolated from glacier ice.

A psychrophilic, Gram-stain-positive, rod-shaped bacterium, designated Hh39T, was isolated from Xinjiang No. 1 glacier in PR China. Strain Hh39T was catalase-positive, oxidase-negative and could grow at 0-18 °C, pH 6.0-11.0 and in the presence of 0-2.5 % (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain Hh39T belonged to the genus Cryobacterium. The highest level of 16S rRNA gene sequence similarities were found to the type strains of Cryobacterium levicorallinum (99.01 %), Cryobacterium luteum (98.90 %), Cryobacterium aureum (98.90 %) and Cryobacterium roopkundense (98.75 %). However, the low average nucleotide identity (80.65-81.89 %) and digital DNA-DNA hybridization values (22.1-23.8 %) between strain Hh39T and its four closest relatives indicated that it represents a novel species of the genus Cryobacterium. The predominant fatty acids were anteiso-C15:0, anteiso-C15:1, iso-C16:0 and anteiso-C17:0. The major menaquinone was MK-10. The polar lipids were diphosphatidylglycerol, phosphatidylglycerol, one unidentified lipid and one unidentified glycolipid. On the basis of results of phenotypic, genotypic and phylogenetic analyses, a novel species, Cryobacterium melibiosiphilum sp. nov., is proposed, with Hh39T (=NBRC 107884T=CGMCC 1.11212T) as the type strain.

Microevolution and Adaptive Strategy of Psychrophilic Species Flavobacterium bomense sp. nov. Isolated From Glaciers.

Numerous mountain glaciers located on the Tibetan Plateau are inhabited by abundant microorganisms. The microorganisms on the glacier surface are exposed to the cold, barren, and high-ultraviolet radiation environments. Although the microbial community composition on glaciers has been revealed by high-throughput sequencing, little is known about the microevolution and adaptive strategy of certain bacterial populations. In this study, we used a polyphasic approach to determine the taxonomic status of 11 psychrophilic Flavobacterium strains isolated from glaciers on the Tibetan Plateau and performed a comparative genomic analysis. The phylogenetic tree based on the concatenated single-copy gene sequences showed the 11 strains clustered together, forming a distinct and novel clade in the genus Flavobacterium. The average nucleotide identity (ANI) values among these strains were higher than 96%. However, the values much lower than 90% between them and related species indicated that they represent a novel species and the name Flavobacterium bomense sp. nov. is proposed. The core and accessory genomes of strains in this new Flavobacterium species showed diverse distinct patterns of gene content and metabolism pathway. In order to infer the driving evolutionary forces of the core genomes, homologous recombination was found to contribute twice as much to nucleotide substitutions as mutations. A series of genes encoding proteins with known or predicted roles in cold adaptation were found in their genomes, for example, cold-shock protein, proteorhodopsin, osmoprotection, and membrane-related proteins. A comparative analysis of the group with optimal growth temperature (OGT) ≤ 20°C and the group with OGT > 20°C of the 32 Flavobacterium type strains and 11 new strains revealed multiple amino acid substitutions, including the decrease of the proline and glutamine content and the increase of the methionine and isoleucine content in the group with OGT ≤ 20°C, which may contribute to increased protein flexibility at low temperatures. Thus, this study discovered a novel Flavobacterium species in glaciers, which has high intraspecific diversity and multiple adaptation mechanisms that enable them to cope and thrive in extreme habitats.

Nocardioides zhouii sp. nov., isolated from the Hailuogou Glacier in China.

A novel Gram-stain-positive, non-motile, rod-shaped, non-spore-forming bacterium, designated HLT2-9T, was isolated from the ice tongue surface of Hailuogou Glacier in Sichuan Province, PR China. Colonies of cells were cream yellow, convex and round. Growth occurred at 0-27 °C (optimum, 20 °C), pH 7.0-10.0 (pH 7.0) and in the presence of 0-2.0 % (w/v) NaCl (0 %). Phylogenetic analysis based on 16S rRNA gene sequences showed that strain HLT2-9T belonged to the genus Nocardioides. The highest level of sequence similarities were found to Nocardioides glacieisoli CGMCC1.11097T (99.24 %), Nocardioides oleivorans CGMCC 4.6882T (98.54 %) and Nocardioides ganghwensis CGMCC 4.6875T (98.54 %). However, the low average nucleotide identity (85.6-87.9 %) and digital DNA-DNA hybridization values (26.4-30.2 %) of strain HLT2-9T to its three closest relatives demonstrated that it represents a novel species of the genus Nocardioides. The major cellular fatty acids of strain HLT2-9T were C17 : 1ω8c and iso-C16 : 0. Strain HLT2-9T contained ll-2,6-diaminopimelic acid as the diamino acid in the cell-wall peptidoglycan. The predominant menaquinone is MK-8(H4). The G+C content of the genomic DNA was 70.65 mol%. Based on evidence collected from the phenotypic, genotypic and phylogenetic analyses, a novel species Nocardioideszhouii sp. nov. is proposed, with HLT2-9T (=CGMCC 1.11084T=NBRC 109783T) as the type strain.

Flavobacterium laiguense sp. nov., a psychrophilic bacterium isolated from Laigu glacier on the Tibetan Plateau.

One bacterial strain, denoted as LB2P30T, was isolated from Laigu glacier located on the Tibetan Plateau, PR China. Strain LB2P30T was an aerobic, non-motile, rod-shaped, yellow-pigmented and Gram-stain-negative bacterium. The temperature range for growth was 4-20 °C (optimum, 14 °C). The phylogenetic analysis based on partial 16S rRNA gene sequences showed that strain LB2P30T belonged to the genus Flavobacterium and was most similar to Flavobacterium fluvii (98.12 %) and Flavobacterium limicola (97.91 %). The average nucleotide identity values between strain LB2P30T and its closest relatives, F. fluvii DSM 19978T and F. limicola DSM 15094T, were 76.73 % and 77.40 %, respectively. Cells of strain LB2P30T contained summed feature 3 (comprising C16:1ω7c and/or C16:1ω6c) and iso-C15 : 0 as the predominant fatty acids and menaquinone-6 as the sole menaquinone. The genomic DNA G+C content was 34.74 mol%. Based on the phenotypic, chemotaxonomic, genotypic and phylogenetic characteristics, we propose strain LB2P30T as a novel species of the genus Flavobacterium with the nomenclature of Flavobacteriumlaiguense sp. nov. The type strain is LB2P30T (=CGMCC 1.11271T=NBRC 113059T).