Metatranscriptomic and comparative genomic insights into resuscitation mechanisms during enrichment culturing.

BACKGROUND: The pure culture of prokaryotes remains essential to elucidating the role of these organisms. Scientists have reasoned that hard to cultivate microorganisms might grow in pure culture if provided with the chemical components of their natural environment. However, most microbial species in the biosphere that would otherwise be "culturable" may fail to grow because of their growth state in nature, such as dormancy. That means even if scientist would provide microorganisms with the natural environment, such dormant microorganisms probably still remain in a dormant state. RESULTS: We constructed an enrichment culture system for high-efficiency isolation of uncultured strains from marine sediment. Degree of enrichment analysis, dormant and active taxa calculation, viable but non-culturable bacteria resuscitation analysis, combined with metatranscriptomic and comparative genomic analyses of the interactions between microbial communications during enrichment culture showed that the so-called enrichment method could culture the "uncultured" not only through enriching the abundance of "uncultured," but also through the resuscitation mechanism. In addition, the enrichment culture was a complicated mixed culture system, which contains the competition, cooperation, or coordination among bacterial communities, compared with pure cultures. CONCLUSIONS: Considering that cultivation techniques must evolve further-from axenic to mixed cultures-for us to fully understand the microbial world, we should redevelop an understanding of the classic enrichment culture method. Enrichment culture methods can be developed and used to construct a model for analyzing mixed cultures and exploring microbial dark matter. This study provides a new train of thought to mining marine microbial dark matter based on mixed cultures.

Reichenbachiella versicolor sp. nov., isolated from red alga.

A Gram-stain-negative, strictly aerobic, non-flagellated, rod-shaped bacterial strain, designated DC003T, was isolated from the alga Gracilariablodgettii of the phylum Rhodophyta collected from the coast of Lingshui county, Hainan, China. The strain grew optimally at 28 °C, pH 7.0-7.5 and in the presence of 2.0-3.0 % (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences revealed strain DC003T to be within the genus Reichenbachiella, and most closely related to Reichenbachiella agariperforans JCM11238 (94.5 %), followed by Reichenbachiella faecimaris JCM 16588T (94.2 %). The major respiratory quinone was menaquinone 7 and the major fatty acids were iso-C15 : 0 and summed feature 3 (C16 : 1ω7c and/or iso-C15 : 0 2-OH). The polar lipids consisted of phosphatidylethanolamine, two unidentified aminophospholipids, three unidentified phospholipids and 10 unidentified lipids. The G+C content of the genomic DNA was 37.1 mol%. On the basis of the phenotypic, genotypic and phylogenetic analysis, strain DC003T is considered to represent a novel species of the genus Reichenbachiella, for which the name Reichenbachiellaversicolor sp. nov. is proposed. The type strain is DC003T (=KCTC 42867T=MCCC 1H00130T).

Halioglobus lutimaris sp. nov., isolated from coastal sediment.

A novel Gram-stain-negative, strictly aerobic, non-flagellated and rod-shaped bacterium, designated HF004T, was isolated from a marine sediment sample collected from the coast of Weihai, China. The strain grew optimally at 28 °C, pH 7.5-8.0 and in the presence of 2.0-3.0 % (w/v) NaCl. Based on the 16S rRNA gene sequence analysis, strain HF004T was a member of the genus Halioglobus, appearing to be closely related to Halioglobus pacificus (96.1 %) and Halioglobus japonicus (95.6 %). The major fatty acids were summed feature 3 (i.e. C16 : 1ω7c and/or iso-C15 : 0 2-OH), C17 : 1ω8c and C18 : 1ω7c. The major polar lipids were diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol. The predominant respiratory quinone was Q-8. The DNA G+C content was 57.2 mol%. Cells of strain HF004T were rod-shaped and formed circular, mucous and beige-pigmented colonies on marine agar after incubation for 72 h at 28 °C. On the basis of phenotypic, genotypic and phylogenetic evidence, strain HF004T is presented as a novel species, for which the name Halioglobus lutimaris sp. nov. is proposed. The type strain is HF004T (=KCTC 42395T=MCCC 1H00127T).

Crocinitomix algicola sp. nov., isolated from Gracilaria blodgettii.

A Gram-stain-negative, strictly aerobic, non-flagellated, yellow-pigmented, rod-shaped bacterial strain, designated 0182T, was isolated from Gracilaria blodgettii, an algae of phylum Rhodophyta collected from coast of Lingshui county, Hainan, China (110° 03' 44.2'' E, 18° 24' 29.8'' N). The strain grew optimally at 30 °C, pH 7.0-7.5 and in the presence of 2.0-3.0 % (w/v) NaCl. Cells of strain 0182T were approximately 0.9-2.5 µm in length and 0.2-0.4 µm in width. The major respiratory quinone was MK-7. The predominant cellular fatty acids were iso-C15 : 0, C15 : 0 and iso-C17 : 0 3-OH. The major polar lipids were phosphoaminolipid, glycolipid, two unknown aminolipids and four unknown lipids. The DNA G+C content was approximately 35.4 mol%. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain 0182T was phylogenetically related to members of the genus Crocinitomix and was closely related to Crocinitomix catalasitica with 94.6 % sequence similarity. On the basis of genotypic and phenotypic characteristics and phylogenetic evidence, strain 0182T is thought to represent a novel species of the genus Crocinitomix, for which the name Crocinitomix algicola sp. nov. is proposed. The type strain is 0182T (=KCTC 42868T =MCCC 1H00128T).

Catenovulum sediminis sp. nov., isolated from coastal sediment.

A novel Gram-stain-negative, strictly aerobic, rod-shaped and agar-hydrolysing bacterium, designated D2T, was isolated from a marine sediment sample collected from the coast of Weihai, China (37° 31' 59″ N 122° 03' 47″ E). The cells were motile by a lateral flagellum. Growth was observed at 10-42 °C, at pH 6.0-9.0 and with 0.5-8 % (w/v) NaCl. Phylogenetic analysis based on the 16S rRNA gene sequence showed that strain D2T belonged to the genus Catenovulum, appearing closely related to Catenovulum agarivorans YM01T (96.3 % 16S rRNA gene sequence similarity) and Catenovulum maritimumQ1T (93.9 %). The dominant fatty acids were summed feature 3 (C16 : 1ω7c and/or iso-C15 : 0 2-OH), C16 : 0 and C10 : 0 3-OH. The polar lipids were phosphatidylethanolamine, phosphatidylglycerol, two phosphoaminolipids, two unknown lipids and three phospholipids. Ubiquinone 8 (Q-8) was found to be the major respiratory quinone. The DNA G+C content was 40.4 mol%. On the basis of genotypic, phenotypic and phylogenetic evidence, strain D2T is presented as a representative of a novel species of the genus Catenovulum, for which the name Catenovulum sediminis sp. nov. is proposed. The type strain is D2T (=KCTC 42869T=MCCC 1H00129T). An emended description of the genus Catenovulum is also provided.

Brumimicrobium aurantiacum sp. nov., isolated from coastal sediment.

A Gram-negative, facultatively anaerobic, non-motile, rod-shaped and orange-coloured bacterium, designated N62T, was isolated from marine sediment of the coast of Weihai, PR China. Strain N62T was found to grow optimally at 28-30 °C, pH 7.0-7.5 and with 2.0-3.0 % (w/v) NaCl. The dominant cellular fatty acids of strain N62T were iso-C15 : 0, iso-C15 : 0 G, iso-C17 : 0 3-OH and iso-C17 : 1 3-OH. The major respiratory quinone was MK-6, and the DNA G+C content was 35.3 mol%. The predominant polar lipids were phosphatidylethanolamine, two unidentified aminolipids, an unidentified glycolipid and three unidentified lipids. Phylogenetic analysis based on 16S rRNA gene sequences revealed that N62T was a member of the family Crocinitomicaceae and had a 16S rRNA gene sequence similarity of 95.8-97.2 % with recognized Brumimicrobium species. On the basis of the phylogenetic and phenotypic evidences, strain N62T represents a novel species of the genus Brumimicrobium, for which the name Brumimicrobium aurantiacum sp. nov. is proposed. The type strain is N62T (=KCTC 42589T=MCCC 1H00117T).

Bizionia sediminis sp. nov., isolated from coastal sediment.

A Gram-stain-negative, aerobic, non-gliding, rod-shaped and orange-coloured bacterium, designated strain P131T, was isolated from marine sediment of the coast of Weihai, China, and subjected to a polyphasic study. Strain P131T was found to grow optimally at 28-30 °C, at pH 7.0-7.5 and in the presence of 2-3 % (w/v) NaCl. In a phylogenetic analysis based on 16S rRNA gene sequences, strain P131T was found to belong to the genus Bizionia and exhibited 94.6-97.0 % 16S rRNA gene sequence similarity with recognized Bizionia species. The dominant cellular fatty acids of strain P131T were identified as iso-C15 : 0, iso-C15 : 0 G, iso-C17 : 0 3-OH and iso-C17 : 1ω9c. The predominant polar lipids were phosphatidylethanolamine, phospholipid, two aminolipids and two unidentified lipids. The predominant respiratory quinone was menaquinone MK-6 and the DNA G+C content was 36.7 mol%. On the basis of the phylogenetic and phenotypic evidence presented, strain P131T represents a novel species of the genus Bizionia, for which the name Bizionia sediminis sp. nov. is proposed. The type strain is P131T (=KCTC 42587T=MCCC 1H00124T).

Hyunsoonleella rubra sp. nov., isolated from coastal sediment.

A novel Gram-stain-negative, aerobic, rod-shaped, non-flagellated and non-gliding bacterium, designated FA042T, was isolated from a marine sediment sample collected from the coast of Weihai, China (37° 32' 02″ N 122° 03' 44″ E). Optimal growth occurred at 33 °C, pH 7.0-7.5 and in the presence of 2-3 % (w/v) NaCl. Phylogenetic analysis based on the 16S rRNA gene sequence showed that strain FA042T belonged to the genus Hyunsoonleella. The closest described neighbour, in terms of 16S rRNA gene sequence identity, was Hyunsoonleella jejuensis (95.0 %). The major fatty acids were iso-C15 : 0, iso-C15 : 1 G, C15 : 0, iso-C17 : 0 3-OH and iso-C15 : 0 3-OH. The major respiratory quinone was MK-6. The major polar lipids of strain FA042T were phosphatidylethanolamine, three unidentified lipids and two unidentified aminolipids. The DNA G+C content was 38.5 mol%. Based on its phylogenetic and phenotypic characteristics, strain FA042T is presented as a representative of a novel species, for which the name Hyunsoonleella rubra sp. nov. is proposed. The type strain of Hyunsoonleella rubra sp. nov. is FA042T (=KCTC 42398T=MCCC 1H00110T).