Streptomyces pyxinae sp. nov. and Streptomyces pyxinicus sp. nov. isolated from lichen Pyxine cocoes (Sw.) Nyl.

Two novel actinobacteria, designated as LP05-1T and LP11T, were isolated from the lichen Pyxine cocoes (Sw.) Nyl. collected in Bangkok, Thailand. Genotypic and phenotypic analyses revealed that both strains represented members of the genus Streptomyces. The 16S rRNA gene of LP05-1T showed the highest similarity to the genome of Streptomyces gelaticus (98.41 %), while the 16S rRNA gene of LP11T was most similar to that of Streptomyces cinerochromogenes (98.93 %). The major menaquinones in LP05-1T were MK-9(H8), MK-9(H6), MK-9(H4) and MK-9(H2), and in LP11T, they were MK-9(H8) and MK-9(H6). Both strains exhibited the major fatty acids iso-C15 : 0, anteiso-C15 : 0, iso-C16 : 0 and anteiso-C17 : 0, with LP05-1T also possessing iso-C17 : 0. The polar lipids of LP05-1T included phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylinositol, phosphatidylinositol mannoside and an unidentified lipid, while those of LP11T consisted of phosphatidylethanolamine, lyso-phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylinositol, phosphatidylinositol mannoside, an unidentified aminolipid and an unidentified glycolipid. The digital DNA-DNA hybridisation (dDDH) and average nucleotide identity (ANI) values indicated that both strains are distinct from each other with values below 70 and 95 %, respectively. dDDH, ANI by blast (ANIb) and ANI by MUMmer (ANIm) values between LP05-1T and its closely related type strains were 26.07-26.80 %, 81.24-82.01 % and 86.82-86.96 %, respectively, while those for LP11T and its closely related type strains were 30.70-31.70 %, 84.09-85.31 % and 88.02-88.39 %, respectively. The results of the taxonomic investigation, including dDDH and ANI values, indicate that LP05-1T and LP11T are novel type strains of two novel species within the genus Streptomyces. The names proposed are Streptomyces pyxinae sp. nov. for strain LP05-1T (=TBRC 15494T, =NBRC 115434T) and Streptomyces pyxinicus sp. nov. for strain LP11T (=TBRC 15493T, =NBRC 115421T).

Actinoplanes pyxinae sp. nov., a new lichen-derived rare actinobacterium exhibiting antimicrobial and anticancer activity.

A novel lichen-derived actinobacterium, designated Pm04-4T, was isolated from Pyxine cocoes (Sw.) Nyl. lichen collected from Chaiyaphum, Thailand. A polyphasic approach was used to describe the taxonomic position of the strain. The strain had morphological and chemotaxonomic properties similar to members of the genus Actinoplanes. It produced sporangia on the substrate mycelia. Meso-diaminopimelic acid, galactose, glucose and mannose were detected in the whole-cell hydrolysate of the strain. The major menaquinone was MK-9(H4). The polar lipids were phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylinositol and phosphatidylinositol mannoside. The predominant cellular fatty acids were iso-C15 : 0, anteiso-C15 : 0, iso-C16 : 0 and anteiso-C17 : 0. Strain Pm04-4T showed the highest 16S rRNA gene sequence similarity to Actinoplanes akusuensis TRM 8003T (99.0 %). In the phylogenomic tree, strain Pm04-4T was positioned close to A. aksuensis TRM88003T, A. maris M416T, A. polyasparticus TRM66264T, A. hotanensis TRM88002T, A. abujensis DSM 45518T, A. bogorensis NBRC 110975T, A. brasiliensis DSM 43805T, A. lichenicola LDG1-01T and A. ovalisporus LDG1-06T. The average nucleotide identity and digital DNA-DNA hybridization values between strain Pm04-4T and its closely related neighbours were below the threshold values for describing new species. Moreover, the strain could be distinguished from its closely related type strains by phenotypic properties. Based on genotypic and phenotypic evidence, it can be concluded that strain Pm04-4T is a representative of a new Actinoplanes species for which the name Actinoplanes pyxinae sp. nov. is proposed. The type strain is Pm04-4T (=TBRC 16207T=NBRC 115836T). The type strain exhibited activity against Staphylococcus aureus ATCC 25923 as well as four yeast strains, namely Candida albicans TISTR 5554, Candida glabrata TISTR 5006, Candida krusei TISTR 5351 and Candida parapsilosis TISTR 5007. It also showed cytotoxicity against Caco-2, MNT-1 and MCF-7 cancer cells.

Genome sequences and functional analysis of Levilactobacillus brevis LSF9-1 and Pediococcus acidilactici LSF1-1 from fermented fish cake (Som-fak) with gamma-aminobutyric acid (GABA) production.

Gamma-aminobutyric acid (GABA) is a crucial inhibitory neurotransmitter in the sympathetic nervous system that exerts regulatory effects on the blood, immune, and nervous systems. GABA production in som-fak, a traditional fermented fish of Thailand, has been attributed to the activity of lactic acid bacteria (LAB). The present study aims to characterize the LAB isolates and compare the genomes and GABA synthesis genes of selected isolates capable of GABA production. Thirteen isolates demonstrating GABA synthesis capability were identified based on their phenotypic and genotypic characteristics. Seven isolates (group I: LSF3-3, LSF8-3, LSF9-1, LSF9-3, LSF9-6, LSF9-7, and LSF10-14) were identified as Levilactobacillus brevis with 99.78-100% similarity. LSF2-1, LSF3-2, LSF5-4, and LSF6-5 (group II) were identified as Lactiplantibacillus pentosus with 99.86-100% similarity. Strain LSF1-1 (group III) was identified as Pediococcus acidilactici (99.47%), and LSF10-4 (group IV) was identified as Pediococcus pentosaceus with 99.93% similarity. The GABA production of isolates ranged from 0.087 to 16.935 g/L. The maximum production of 16.935 g/L from 3% monosodium glutamate was obtained from strain LSF9-1. Gene and genome analysis revealed that L. brevis LSF9-1 has multiple gad genes in the genome, such as gadB1, gadB2, gadC1, and gadC2, making it the potential strain for GABA production. Additionally, the genome analysis of P. acidilactici LSF1-1 consists of gadA, gadB, and gadC, which respond to controlling GABA production and export. Furthermore, strain LSF1-1 was considered safe, containing no virulence factors. Thus, Levilactobacillus brevis LSF9-1 and Pediococcus acidilactici LSF1-1 have the potential for GABA production and probiotic use in future studies.

Streptomyces macrolidinus sp. nov., a novel soil actinobacterium with potential anticancer and antimalarial activity.

A novel actinomycete, strain RY43-2T, belonging to the genus Streptomyces, was isolated from a peat swamp forest soil collected from Rayong Province, Thailand. The strain was characterized by using a polyphasic approach. The cell-wall peptidoglycan contained ll-diaminopimelic. Ribose and glucose were detected in its whole-cell hydrolysates. The strain contained anteiso-C15:0, iso-C14:0 and iso-C16:0 as the predominant fatty acids, and MK-9(H4), MK-9(H6) and MK-9(H8) as the major menaquinones. The phospholipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, phosphatidylinositol mannoside, three unidentified ninhydrin-positive phospholipids and two unidentified phospholipids. Strain RY43-2T showed the highest 16S rRNA gene similarity to Streptomyces misionensis JCM 4497T (98.9 %) and Streptomyces lichenis LCR6-01T (98.9 %). The draft genome of RY43-2T was 6.7 Mb with 6078 coding sequences with an average G+C content of 70.8 mol%. Genomic analysis revealed that the average nucleotide identity (ANI) values based on blast (ANIb) and MUMmer (ANIm) between strain RY43-2T and S. misionensis JCM 4497T were 80.1 and 86.1%, respectively. The ANIb and ANIm values between strain RY43-2T and S. lichenis LCR6-01T were 77.0 and 85.5%, respectively. The digital DNA-DNA hybridization values were 25.2 and 23.0% in comparison with the draft genomes of S. misionensis JCM 4497T and S. lichenis LCR6-01T, respectively. The results of taxonomic analysis suggested that strain RY43-2T represented a novel species of the genus Streptomyces for which the name Streptomyces macrolidinus sp. nov. is proposed. The type strain is RY43-2T (=TBRC 7286T=NBRC 115640T). Strain RY43-2T exhibited antimicrobial activity against Enterococcus faecium ATCC 51559, Colletotrichum capsici BMGC 106 and Colletotrichum gloeosporioides BMGC 107 with the minimum inhibitory concentration values of 25.0, 12.5, and 6.25 µg ml-1. It also exhibited potent antimalarial activity against Plasmodium falciparum K1 with IC50 of 0.0031 µg ml-1. In addition, it showed cytotoxicity against Vero, KB, MCF-7 and NCI-H187 with IC50 values of 0.0347, 6.15, 3.36 and 0.0352 µg ml-1, respectively.

Actinomadura parmotrematis sp. nov., isolated from the foliose lichen, Parmotrema praesorediosum (Nyl.) Hale.

A novel actinomycete strain PM05-2T was isolated from the lichen Parmotrema praesorediosum (Nyl.) Hale collected from Chaiyaphum Province, Thailand. The taxonomic position of the strain was studied using the polyphasic approach. Based on the morphology and chemotaxonomic properties, strain PM05-2T was identified as a member of the genus Actinomadura. The whole-cell hydrolysate contained meso-diaminopimelic acid, rhamnose, ribose, xylose, madurose, glucose and galactose. The polar lipids were phosphatidylglycerol, diphosphatidylglycerol, phosphatidylinositol, phosphatidylinositol mannoside, one unidentified phosphoglycolipid, four unidentified phospholipids and one unidentified lipid. The menaquinones were MK-9(H6), MK-9(H4), MK-9(H2), MK-9(H8) and MK-9(H0). The major cellular fatty acids were C16:0 and C18:1 ω9c. Strain PM05-2T showed the highest 16S rRNA gene similarity to Actinomadura hibisca NBRC 15177T (98.58%), Actinomadura kijaniata NBRC 14229T (98.29 %) and Actinomadura namibiensis DSM 44197T (98.14 %). The phylogenetic tree analysis revealed that strain PM05-2T was related to A. hibisca NBRC 15177T, A. kijaniata NBRC 14229T, A. namibiensis DSM 44197T and Actinomadura macrotermitis RB68T. The genomic analysis revealed that average nucleotide identity values based on both blast and MUMmer between strain PM05-2T and the relative type strains ranged from 77.6 to 86.4%. The digital DNA-DNA hybridization values among the strains were lower than the threshold for assigning to the same species. The taxonomic results suggested that strain PM05-2T represented a novel species of the genus Actinomadura for which the name Actinomadura parmotrematis is proposed. The type strain is PM05-2T (=TBRC 15492T=NBRC 115416T).

Diversity of the culturable lichen-derived actinobacteria and the taxonomy of Streptomyces parmotrematis sp. nov.

A total of 37 actinobacteria were isolated from eighteen lichen samples collected in Thailand. Based on the 16S rRNA gene sequences, they were identified into five genera including Actinoplanes (1 strain), Actinomadura (1 strain), Pseudosporangium (1 strain), Wangella (1 strain) and Streptomyces (33 strains). Among these isolates, strain Ptm05T, Ptm01 and Ptm12 showed low 16S rRNA gene similarity and was selected for the further taxonomic study using the polyphasic approach. These strains showed the highest 16S rRNA gene sequence similarity with Streptomyces sparsogenes ATCC 25498T (97.44-97.72%). Strain Ptm05T was selected for the type strain. The chemical cell composition of the strain was similar to the members of Streptomyces genus. LL-diaminopimelic acids were detected in the peptidoglycan. Menaquinones were MK-9(H8) and MK-9(H6). Phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylinositol, phosphatidylinositol mannoside, one unidentified phospholipid, one unidentified glycolipid and one unidentified lipid were detected as the polar lipids. The predominant cellular fatty acids are anteiso-C15:0, iso-C15:0, iso-C16:0, iso-C17:0 and C16:0. The dDNA-DNA hybridization values among strain Ptm05T and its closely related Streptomyces type strains were 17.2-18.0%. In addition, the ANIb and ANIm between strain Ptm05T and related Streptomyces type strains were ranged from 75.69 to 76.13% and 85.21 to 85.35%, respectively. Based on phenotypic and genomic evidence, strain Ptm05T (= TBRC 14546T = NBRC 115203T) represents the novel species of the genus Streptomyces for which the name Streptomyces parmotrematis sp. nov. is proposed. This study showed that the lichens are the promising source of the novel actinobacterial taxa.

Bacterial microbiome in tropical lichens and the effect of the isolation method on culturable lichen-derived actinobacteria.

Ten samples of tropical lichens collected from Doi Inthanon, Thailand, were explored for the diversity of their bacterial microbiomes through 16S rRNA-based metagenomics analysis. The five predominant lichen-associated bacteria belonged to the phyla Proteobacteria (31.84%), Planctomycetota (17.08%), Actinobacteriota (15.37%), Verrucomicrobiota (12.17%), and Acidobacteriota (7.87%). The diversity analysis metric showed that Heterodermia contained the highest bacterial species richness. Within the lichens, Ramalina conduplicans and Cladonia rappii showed a distinct bacterial community from the other lichen species. The community of lichen-associated actinobacteria was investigated as a potential source of synthesized biologically active compounds. From the total Operational Taxonomic Units (OTUs) found across the ten different lichen samples, 13.21% were identified as actinobacteria, including the rare actinobacterial genera that are not commonly found, such as Pseudonocardia, Kineosporia, Dactylosporangium, Amycolatopsis, Actinoplanes, and Streptosporangium. Evaluation of the pretreatment method (heat, air-drying, phenol, and flooding) and isolation media used for the culture-dependent actinobacterial isolation revealed that the different pretreatments combined with different isolation media were effective in obtaining several species of actinobacteria. However, metagenomics analyses revealed that there were still several strains, including rare actinobacterial species, that were not isolated. This research strongly suggests that lichens appear to be a promising source for obtaining actinobacteria.