Isolation and Characterization of Antimicrobial Peptides Isolated from Brevibacillus halotolerans 7WMA2 for the Activity Against Multidrug-Resistant Pathogens.

Multidrug resistance to pathogens has posed a severe threat to public health. The threat could be addressed by antimicrobial peptides (AMPs) with broad-spectrum suppression. In this study, Brevibacillus halotolerans 7WMA2, isolated from marine sediment, produced AMPs against Gram-positive and Gram-negative bacteria. The AMPs were precipitated by ammonium sulfate 30% (w/v) from culture broth and dialyzed by a 1 kDa membrane. Tryptone Soy Agar (TSA) was used for the cultivation and resulted in the largest bacteria-inhibiting zones under aerobic conditions at 25 °C, 48 h. An SDS-PAGE gel overlay test revealed that strain 7WMA2 could produce AMPs of 5-10 kDa and showed no degradation when held at 121 °C for 30 min at a wide pH 2-12 range. The AMPs did not cause toxicity to HeLa cells with concentrations up to 500 µg/mL while increasing the arbitrary unit up to eight times. The study showed that the AMPs produced were unique, with broad-spectrum antimicrobial ability.

Whole-genome analysis and secondary metabolites production of a new strain Brevibacillus halotolerans 7WMA2: A potential biocontrol agent against fungal pathogens.

The extensive usage of synthetic fungicides against fungal diseases has caused adverse impacts on both human and agricultural crops. Therefore, the current study aims to establish a new bacterium 7WMA2, as a biocontrol agent to achieve better antifungal results. The strain 7WMA2 was isolated from marine sediment, displayed a broad spectrum of several fungi that includes Alternaria alternata, Cladosporium sp., Candida albicans, Fusarium oxysporum, Trichosporon pullulans, and Trichophyton rubrum. The 16S rRNA phylogeny inferred that strain 7WMA2 was a member of Brevibacillus. The phylogenetic and biochemical analyses revealed that the strain 7WMA2 belongs to the species of Brevibacillus halotolerans. The complete genome sequence of Brevibacillus halotolerans 7WMA2 consists of a circular chromosome of 5,351,077 bp length with a GC content of 41.39 mol %, including 4433 CDS, 111 tRNA genes, and 36 rRNA genes. The genomic analysis showed 23 putative biosynthetic secondary metabolite gene clusters responsible for non-ribosomal peptides, polyketides and siderophores. The antifungal compounds concentrated from cell-free fermentation broth demonstrated strong inhibition of fungi, and the compounds are considerably thermal stable and adaptable to pH range 2-12. This complete genome sequence has provided insight for further exploration of antagonistic ability and its secondary metabolite compounds indicated feasibility as biological control agents against fungal infections.

Flavobacterium litorale sp. nov., isolated from red alga.

A Gram-stain-negative and rod-shaped bacterial strain (WSW3-B6T) was isolated from red alga collected from the West Sea, Republic of Korea. Cells of strain WSW3-B6T were non-motile, aerobic and produced slightly yellow and mucoid colonies on marine agar. The strain grew optimally at 23-30 °C, with 0.5-4 % NaCl (w/v) and at pH 6.5-8.5. A phylogenetic analysis of the 16S rRNA gene revealed that strain WSW3-B6T belongs to the genus Flavobacterium within the family Flavobacteriaceae, having the highest sequence similarity to Flavobacterium arcticum SM1502T (96.7%), followed by Flavobacterium salilacus subsp. altitudinum LaA7.5T (96.2%) and Flavobacterium salilacus subsp. salilacus SaA2.12T (96.2%). The complete sequence of a circular chromosome of strain WSW3-B6T determined by combination of Oxford Nanopore and Illumina platforms comprised a total 2 725 095 bp with G+C content of 37.1 mol%. A comparative analysis based on the whole genome also showed the distinctiveness of strain WSW3-B6T. The average nucleotide identity (ANI) values between strain WSW3-B6T and the closest strains F. arcticum SM1502T, F. salilacus subsp. altitudinum LaA7.5T and F. salilacus subsp. salilacus SaA2.12T were 78.3, 77.8 and 77.7 %, respectively, while the digital DNA-DNA hybridization (dDDH) values between strain WSW3-B6T and the above closely related strains were 21.0, 20.4 and 20.3 %, respectively. Both the ANI and dDDH values supported the creation of a new species in the genus Flavobacterium. The major fatty acids (>10 %) were iso-C15 : 0 (19.3 %), C16 : 0 (14.0 %), iso-C17 : 0 3-OH (13.1 %) and C18 : 0 (10.7 %). The polar lipids of strain WSW3-B6T included phosphatidylethanolamine, three unidentified aminolipids and three unidentified lipids. Moreover, MK-6 was the only respiratory quinone. A comparison of the phylogenetic distinctiveness and the unique phenotypic and chemotaxonomic characteristics among strain WSW3-B6T and closely related type strains supported that strain WSW3-B6T (=KCTC 82708T=GDMCC 1.2627T) represents a novel species of the genus Flavobacterium, for which the name Flavobacterium litorale sp. nov. is proposed.

Cellulosimicrobium protaetiae sp. nov., isolated from the gut of the larva of Protaetia brevitarsis seulensis.

A Gram-stain-positive, non-spore-forming, yellow-pigmented, non-motile, non-flagellated, facultative anaerobic and rod-shaped bacterial strain, designated BI34T, was isolated from the gut of the larva of Protaetia brevitarsis seulensis. Strain BI34T grew at 15-40 °C (optimum, 37 °C), at pH 6.5-9.0 (optimum, pH 7.5) and in the presence of 0-7 % (w/v) NaCl (optimum, 2 %). Based on the results of 16S rRNA gene sequence analysis, strain BI34T belonged to the phylum Actinobacteria and was closely related to Cellulosimicrobium funkei NBRC 104118T (99.3 %), Cellulosimicrobium cellulans NBRC 15516T (99.1 %), Cellulosimicrobium composti BIT-GX5T (99.0 %), Cellulosimicrobium fucosivorans SE3T (99.0 %), Cellulosimicrobium marinum NBRC 110994T (98.4 %) and Cellulosimicrobium terreum DS-61T (97.0 %). The genome to genome relatedness of the average nucleotide identity (ANI) and the digital DNA-DNA hybridization (dDDH) values calculated by the Genome-to-Genome Distance Calculator between strain BI34T and its related species mentioned above were lower than the threshold of 95 and 70 % for speciation, respectively. The predominant menaquinone of strain BI34T contained MK-9(H4), and the major fatty acids were anteiso-C15 : 0, C16 : 0 and anteiso-C17 : 0. Strain BI34T had diphosphatidylglycerol and phosphatidylglycerol as major polar lipids. The whole-cell sugars were galactose, glucose and ribose, and the cell-wall peptidoglycan contained lysine, alanine, aspartic acid and glutamic acid. The DNA G+C content of strain BI34T was 73.8 mol%. The difference in physiological and biochemical characteristics and the below-threshold values of genome-to-genome relatedness indicate that strain BI34T represents a novel species in the genus Cellulosimicrobium, for which the name Cellulosimicrobium protaetiae sp. nov. is proposed. The type strain is BI34T (=KCTC 49302T=NBRC 114073T).

Flavobacterium ustbae sp. nov., isolated from rhizosphere soil of Alhagi sparsifolia.

A yellow-pigmented bacterial strain, designated T13T, was isolated from the rhizosphere soil of Alhagi sparsifolia collected from Xinjiang, PR China. Cells were rod-shaped, Gram-stain-negative, aerobic and gliding. Strain T13T grew optimally at 25-30 °C and pH 7.0-8.0 with a NaCl tolerance of 0-2 % on Reasoner's 2A agar. Phylogenetic analysis based on the 16S rRNA gene sequence showed that strain T13T belonged to the genus Flavobacterium within the family Flavobacteriaceae and was closely related to Flavobacterium nitrogenifigens KCTC 42884T with a similarity value of 97.4 %. The major polar lipid was phosphatidylethanolamine; the only respiratory quinone was MK-6, and the polyamine profile contained sym-homospermidine as the major polyamine and a trace amount of spermidine. The major fatty acids were iso-C15 : 0, C16 :  1ω7c and summed feature 3 (comprising C16 : 1ω7c or C16 : 1ω6c). The G+C content of the genomic DNA was 34.1 mol%. It is concluded from the phenotypic and genotypic data that strain T13T represents a novel species of the genus Flavobacterium, for which the name Flavobacteriumustbae sp. nov. with the type strain T13T (=KCTC 62874T=ACCC 60126T) is proposed.

Lacinutrix chionocetis sp. nov., isolated from gut of a red snow crab.

A Gram-negative, aerobic, non-motile, rod-shaped bacterial strain, designated MAB-07T, was isolated from the gut of a red snow crab. The novel strain grew optimally at 20 °C, pH 7.0-8.0, and in the presence of 3% (w/v) NaCl. A phylogenetic analysis based on the 16S rRNA gene sequence indicated that the strain MAB-07T belongs to the type strains of species of the genus Lacinutrix. Strain MAB-07T exhibited 16S rRNA gene sequence similarity values of 95.5-97.8% with the type strains of species of the genus Lacinutrix. The predominant cellular fatty acids of strain MAB-07T were iso-C15:1 G (27.5%) and iso-C15:0 (21.7%). The major respiratory quinine was identified as MK-6. The polar lipids consisted of phosphatidylethanolamine, four unidentified aminolipids, and two unidentified lipids. The genomic DNA G + C content was determined to be 33.3%, and its DNA-DNA relatedness values with the type strains of L. venerupis, L. mariniflava, L. jangbogonensis, L. algicola, and Olleya aquimaris were 28-32%. Based on the data from this polyphasic taxonomic study, strain MAB-07T is considered to represent a novel species of the genus Lacinutrix, for which the name L. chionocetis sp. nov. is proposed. The type strain is MAB-07T (=KCTC 42767T = JCM 30988T).