Preliminary Application Of Sandwich Plate Technology In Marine Bacterial Resources Research And Polyphasic Taxonomy Analysis Of Three Novel Bacterial Strains

Both metagenomics and single-cell sequencing can detect unknown genes from uncultured microbial strains in environments,and either method may find the significant potential metabolites and roles of these strains.However,such gene/genome-based techniques do not allow detailed investigations that are possible with cultures.To solve this problem,various approaches for cultivation of uncultured bacteria have been developed,but there are still difficulties in maintaining pure cultures by subculture.For example,the separation efficiency of new species is reduced,the influence of dominant flora on other species,the impact of information exchange on growth,and the use of high-end instruments involved in bacterial isolation.In nature,the interaction between individual bacteria has an important impact on their growth.Based on this phenomenon,we have established a sandwich plate technology.This technique consists of medium commonly used in laboratories and culturable bacteria.The method was initially verified by studying marine bacteria.The medium used in the sandwich plate technology consists of three parts:upper,middle and lower.The lower medium is the 2216E medium commonly used in the laboratory.The upper medium was a modified 1/10 2216E medium.The culturable bacteria were placed between two layers of medium as sandwich strain.Finally,we determined the coated area of the sandwich strain on the surface of the lower medium is about 70 mm in diameter,the agar concentration of the upper medium is 4%?w/v?.Studies on bacterial diversity in marine sediments showed that the experimental groups S255 S63,S69 and S70 could increase the diversity of bacteria under the action of sandwich strains.The experimental groups S08,S09,S12,S38 and S39 could reduce the diversity of bacteria under the action of sandwich strains.At the genus level,Psychrobacter,Ruegeria,Microbulbifer,Aequorivita and Bacillus were the dominant groups with obvious changes in abundance in each experimental group.In terms of potential new species,each group can increase the proportion of new bacteria under the action of sandwich strains,especially the experimental groups S08,S25,S38,S39,S69 and S70.Moreover,the potential new species of the family Flavobacteriaceae,Moraxellaceae,Rhodobacteraceae,Halomonadaceae and Phyllobacteriaceae are more distinct in each group.Through pure culture,a total of 481 strains were obtained,which were distributed in four phylums of Actinobacteria,Bacteroidetes,Firmicutes and Proteobacteria.There are 149 potential new species,124 new species,24 new genus,and 1 new family.Furthermore,17 strains with growth-dependent strains were obtained by screening and verifying the pure culture strains.In addition,this paper describes the multiple classification and identification of three new strains.A Gram-stain-negative,rod-shaped bacterium?designated strain M1309^T?with slow gliding motility was isolated from marine sediment obtained off the coast of Weihai,China.The growth of strain M1309^T was observed at 16-42 ??optimum,37 ??and pH 6.5-8.0?optimum,7.0-7.5?in the presence of 2.0-6.0%?w/v?NaCl?optimum,2.0-3.0%?.Phylogenetic analyses based on the 16S rRNA gene sequence revealed that the strain belonged to the genus Winogradskyella.Strain M1309^T exhibited the highest 16S rRNA gene sequence similarity of 95.5%to Winogradskyella poriferorum JCM 12885^T.Chemotaxonomic analysis showed that the sole respiratory quinone was menaquinone 6?MK-6?and the major fatty acids included iso-C_15:0,iso-C_15:1 G and iso-C_17?0-30H.The major polar lipids included phosphatidylethanolamine,two aminolipids,and three unidentified lipids.The DNA G+C content of the strain was 36.1 mol%.On the basis of phenotypic distinctiveness and phylogenetic divergence,strain M1309^T is considered to represent a novel species of the genus Winogradskyella?for which the name Winogradskyella tangerina sp.nov.is proposed.The type strain is M1309^T?=KCTC 52896"=MCCC 1K03310^T?.A Gram-stain-negative,facultatively aerobic,rod-shaped,motile by gliding and pink-pigmented bacterial strain,designated ELS1360^T,was isolated from lake sediment collected from Inner Mongolia,China?40°34'52.74"N,106°49'57.24"E?.Strain ELS1360-T grew optimally at 33 ??at pH 6.5-7.0 and without NaCl.Strain ELS1360^T exhibited 96.9 and 96.8%16S rRNA gene sequence similarities with H.luteus JCM 30328^T and H.latericoloratus JCM 30327^T,respectively,and 90.3-96.7%with other members of the genus Hymenobacter.Phylogenetic analysis based on 16S rRNA gene sequences showed that strain EL:L360-V belonged to the genus Hymenobacter and clustered with Hymenobacter luteus JCM 30328-T and Hymenobacter latericoloratus JCM 30327N.The predominant cellular fatty acids were iso-C_15:0,Summed feature 3 and Ci6:1?5c.Strain ELS1360^T contained MK-7 as the sole menaquinone.The major polar lipid profile contained phosphatidylethanolamine,one aminophospholipid and one unidentified lipid.The genomic DNA G+C content of strain ELS1360^T was 57.1 mol%.Based on the phylogenetic,phenotypic,genotypic and chemotaxonolic analyses,it is concluded that strain ELJ13601 represents a novel species within the genus Hymenobacter,for which the name Hymenobacter sediminis sp.nov.is proposed.The type strain is ELS1360^T?=KCTC 62449^T=MCCC 1H00319^T?.A Gram-stain-negative,rod-shaped bacterium?designated strain N24^T?with gliding motility was isolated from thermal spring water at Xi'an,China.Cells were rods,0.4-0.8?m wide and 1.8-7.8?m long.Optimal growth occurred at 33 ? and pH 7.0 on R2A agar.Strain N24^T can produce exopolysaccharide?EPS?on R2A at 33 ?.Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain N24^T formed a distinct phyletic lineage within the family Chitrnophagaceae and was most closely related to members of the genera Flavisolibacter,Cnuella,Niveitalea,Flavitalea,Flaviaesturariibacter and Niastella with 91.7-93.9%16S rRNA gene sequence similarities.The major fatty acids of strain N24^T were iso-C_15:0?31.8%?,iso-C_17:0 3-OH?16.1%?and iso-C_15:1 G?12.9%?.The polar lipid profile consisted of phosphatidylethanolamine,two aminolipids,and six unknown lipids;the quinone system consisted of menaquinone-7?MK-7?.The genomic G?C content was 49.3 mol%.On the basis of the evidence presented in this study,strain N24-t represents a novel species of a new genus in the family Chitinophagaceae,for which the name Paracnuella aquatica gen.nov.,sp.nov.is proposed.The type strain of the type species is N24^T?=KCTC 62083^T=MCCC 1H00301^T?.