Biosynthetic gene clusters with biotechnological applications in novel Antarctic isolates from Actinomycetota.

Actinomycetota have been widely described as valuable sources for the acquisition of secondary metabolites. Most microbial metabolites are produced via metabolic pathways encoded by biosynthetic gene clusters (BGCs). Although many secondary metabolites are not essential for the survival of bacteria, they play an important role in their adaptation and interactions within microbial communities. This is how bacteria isolated from extreme environments such as Antarctica could facilitate the discovery of new BGCs with biotechnological potential. This study aimed to isolate rare Actinomycetota strains from Antarctic soil and sediment samples and identify their metabolic potential based on genome mining and exploration of biosynthetic gene clusters. To this end, the strains were sequenced using Illumina and Oxford Nanopore Technologies platforms. The assemblies were annotated and subjected to phylogenetic analysis. Finally, the BGCs present in each genome were identified using the antiSMASH tool, and the biosynthetic diversity of the Micrococcaceae family was evaluated. Taxonomic annotation revealed that seven strains were new and two were previously reported in the NCBI database. Additionally, BGCs encoding type III polyketide synthases (T3PKS), beta-lactones, siderophores, and non-ribosomal peptide synthetases (NRPS) have been identified, among others. In addition, the sequence similarity network showed a predominant type of BGCs in the family Micrococcaceae, and some genera were distinctly grouped. The BGCs identified in the isolated strains could be associated with applications such as antimicrobials, anticancer agents, and plant growth promoters, among others, positioning them as excellent candidates for future biotechnological applications and innovations. KEY POINTS: • Novel Antarctic rare Actinomycetota strains were isolated from soil and sediments • Genome-based taxonomic affiliation revealed seven potentially novel species • Genome mining showed metabolic potential for novel natural products.

Biotechnological potential of actinomycetes in the 21st century: a brief review.

This brief review aims to draw attention to the biotechnological potential of actinomycetes. Their main uses as sources of antibiotics and in agriculture would be enough not to neglect them; however, as we will see, their biotechnological application is much broader. Far from intending to exhaust this issue, we present a short survey of the research involving actinomycetes and their applications published in the last 23 years. We highlight a perspective for the discovery of new active ingredients or new applications for the known metabolites of these microorganisms that, for approximately 80 years, since the discovery of streptomycin, have been the main source of antibiotics. Based on the collected data, we organize the text to show how the cosmopolitanism of actinomycetes and the evolutionary biotic and abiotic ecological relationships of actinomycetes translate into the expression of metabolites in the environment and the richness of biosynthetic gene clusters, many of which remain silenced in traditional laboratory cultures. We also present the main strategies used in the twenty-first century to promote the expression of these silenced genes and obtain new secondary metabolites from known or new strains. Many of these metabolites have biological activities relevant to medicine, agriculture, and biotechnology industries, including candidates for new drugs or drug models against infectious and non-infectious diseases. Below, we present significant examples of the antimicrobial spectrum of actinomycetes, which is the most commonly investigated and best known, as well as their non-antimicrobial spectrum, which is becoming better known and increasingly explored.

Description of an anaerobic actinobacterium, Kribbibacterium absianum gen. nov., sp. nov., a new member of the novel family Kribbibacteriaceae fam. nov., and reclassification of the genera Granulimonas and Leptogranulimonas.

Two rod-shaped, obligate anaerobic, Gram-stain-positive bacteria isolated from the pig faeces were designated YH-ols2216 and YH-ols2217T. Analysis of 16S rRNA gene sequences revealed that these isolates were most related to the members of the family Atopobiaceae, within the order Coriobacteriales, and Granulimonas faecalis KCTC 25474T with 92.0 and 92.5% similarities, respectively. The 16S rRNA gene sequence similarity within isolates was 99.9 %; and those between isolates YH-ols2216 and YH-ols2217T, and Atopobium minutum DSM 20586T, the type species of the type genus Atopobium within the family Atopobiaceae, were 88.5 and 88.7 %, respectively. Those between isolates and Coriobacterium glomerans PW2T, the type species of the type genus Coriobacterium within the family Coriobacteriaceae, were 88.7 and 89.1 %, respectively. The multi-locus sequence tree revealed that the isolates, alongside the genera Granulimonas and Leptogranulimonas, formed a distinct cluster between the families Atopobiaceae and Coriobacteriaceae. The average nucleotide identities and digital DNA-DNA hybridization values for the isolates and their most closely related strains ranged from 67.7 to 76.2 % and from 18.4 to 23.3 %, respectively. The main cellular fatty acids of the isolates were C18 : 0 DMA, C18 : 1 ω9c, C18 : 0 12OH, C18 : 0, and C16 : 0. The cell wall contained the peptidoglycan meso-diaminopimelic acid. Lactate was the main end-product of the isolates. The major polar lipids of isolate YH-ols2217T were aminophospholipid, aminolipids, and lipids. Menaquinones were not identified in the cells of the isolates. The DNA G+C contents of isolates YH-ols2216 and YH-ols2217T were 67.5 and 67.6 mol%, respectively. Considering these chemotaxonomic, phenotypic, and phylogenetic properties, Kribbibacteriaceae fam. nov. is proposed within the order Coriobacteriales. YH-ols2216 (=KCTC 25708=NBRC 116429) and YH-ols2217T (=KCTC 25709T=NBRC 116430T) represent a novel taxon within this new family and the name Kribbibacterium absianum gen. nov., sp. nov. is proposed. In addition, the genera Granulimonas and Leptogranulimonas are transferred to the family Kribbibacteriaceae fam. nov.

Biodegradation and antimicrobial capability-induced heavy metal resistance of the marine-derived actinomycetes Nocardia harenae JJB5 and Amycolatopsis marina JJB11.

Currently, heavy metal-resistant (HMR) marine actinomycetes have attracted much attention worldwide due to their unique capabilities. In this study, 27 marine-derived actinomycetes were isolated from coastal beaches in the Arabian Gulf of Al-Jubail in Saudi Arabia and screened for resistance to 100 mg/L of the heavy metals Cd2+, Cr6+, Cu2+, Fe2+, Pb2+, and Ni2+ using different assay techniques. Six isolates were selected as HMRs, of which two isolates, JJB5 and JJB11, exhibited the highest maximum tolerance concentrations (200- > 300 mg/L). Both isolates were the highest among six-HMR screened for their biodegradation potential of plastics low-density polyethylene, polystyrene, and polyvinyl chloride, recording the highest weight loss (15 ± 1.22 - 65 ± 1.2%) in their thin films. They also showed the highest biodegradability of the pesticides acetamiprid, chlordane, hexachlorocyclohexane, indoxacarb and lindane, indicating promising removal capacities (95.70-100%) for acetamiprid and indoxacarb using HPLC analysis. Additionally, the cell-free filtrate (CFF) of both isolates displayed the highest antimicrobial activity among the six-HMR screened against a variety of microbial test strains, recording the highest inhibition zone diameters (13.76 ± 0.66 - 26.0 ± 1.13 mm). GC‒MS analyses of the ethyl acetate extract of their CFFs revealed the presence of diverse chemical compounds with a multitude of remarkable biological activities. Based on their spore morphology and wall-chemotype, they were assigned to the nocardioform-actinomycetes. Furthermore, their phenotypic characteristics, together with 16S rRNA gene sequencing (OR121525-OR121526), revealed them as Nocardia harenae JJB5 and Amycolatopsis marina JJB11. Our results suggest that marine HMR actinomycetes are promising candidates for various biotechnological applications.

Stomatohabitans albus gen. nov., sp. nov., an oral living facultative anaerobic actinobacteria isolated form Steller sea lion, and proposal of Stomatohabitantaceae fam. nov. and Stomatohabitantales ord. nov.

Two novel actinobacteria, designated as SYSU M7M538T and SYSU M7M531, were isolated from oral of Eumetopias jubatus in Zhuhai Chimelong Ocean Kingdom, China. The cells of these microorganisms stained Gram-positive and were rod shaped. These strains were facultative anaerobic, and catalase-positive. Optimal growth occurred at 37 °C and pH 7.0 over 7 days of cultivation. Both strains possessed diphosphatidylglycerol, phosphatidylglycerol and phosphocholine as the major polar lipids. The main menaquinone was MK-9(H4). The major fatty acids were C16:0, C17:1w8c, C17:0, C18:1w9c and C18:0. Analyses of genome sequences revealed that the genome size of SYSU M7M538T was 2.1 Mbp with G + C content of 52.5 %, while the genome size of SYSU M7M531 was 2.3 Mbp with G + C content of 52.7 %. The ANI and 16S rRNA gene analysis results showed that the pairwise similarities between the two strains and other recognized Nitriliruptoria species were less than 64.9 % and 89.0 %, respectively. Phylogenetic analysis of the 16S rRNA gene sequences indicated that strains SYSU M7M538T and SYSU M7M531 formed a well-separated phylogenetic branch distinct from other orders of Nitriliruptoria. Based on the data presented here, these two strains are considered to represent a novel species of a novel genus, for which the name Stomatohabitans albus gen. nov., sp. nov., with the type strain SYSU M7M538T (=KCTC 59113T = GDMCC 1.4286T), are proposed. We also propose that these organisms represent a novel family named Stomatohabitantaceae fam. nov. of a novel order Stomatohabitantales ord. nov.

Actinomycetospora termitidis sp. nov., an insect-derived actinomycete isolated from termite (Odontotermes formosanus).

Strain Odt1-22T, an insect-derived actinomycete was isolated from a termite (Odontotermes formosanus) that was collected from Chanthaburi province, Thailand. Strain Odt1-22T was aerobic, Gram-stain-positive, and produced bud-like spore chain on the substrate hypha. According to chemotaxonomic analysis, strain Odt1-22T contained meso-diaminopimelic acid in peptidoglycan and the whole-cell hydrolysates contained arabinose, galactose, glucose, and ribose. The major menaquinone was MK-8(H4). The diagnostic phospholipids were diphosphatidylglycerol, hydroxyphosphatidylethanolamine, phosphatidylethanolamine and phosphatidylglycerol. Phylogenetic analysis based on 16 S rRNA gene sequence revealed that strain Odt1-22T was identified to the genus Actinomycetospora and showed high similarity values with A. chiangmaiensis DSM 45062 T (99.24%), A. soli SF1T (99.24%) and A. corticicola 014-5 T (98.17%). The genomic size of strain Odt1-22T was 6.6 Mbp with 73.8% G + C content and 6355 coding sequences (CDSs). The genomic analysis, strain Odt1-22T and closely related species A. chiangmaiensis DSM 45062 T, A. soli SF1T and A. corticicola DSM 45772 T displayed the values of average nucleotide identity-blast (ANIb) at 83.7-84.1% and MUMmer (ANIm) at 86.6-87.0%. Moreover, the results of digital DNA-DNA hybridization values between strain Odt1-22T and related Actinomycetospora species were 45.8-50.5% that lower than the threshold value of commonly used to delineate separated species level. On the basis of phenotypic, chemotaxonomic, and genotypic data, strain Odt1-22T represented a novel species within the genus Actinomycetospora, for which the name Actinomycetospora termitidis sp. nov. is proposed. The type strain of the species is Odt1-22T (= TBRC 16192 T = NBRC 115965 T).

Streptomyces corallincola and Kineosporia corallincola sp. nov., two new coral-derived marine actinobacteria.

Two new marine actinobacteria, designated as J2-1T and J2-2T, were isolated from a coral, Favites pentagona, collected from Rayong Province, Thailand. The taxonomic positions of the two strains were identified based on polyphasic taxonomy. Based on morphological characteristics and chemotaxonomy, strains J2-1T and J2-2T were identified as members of the genus Streptomyces and Kineosporia, respectively. Strains J2-1T and J2-2T showed the highest 16S rRNA gene sequence similarity to Streptomyces broussonetiae T44T (98.62 %) and Kineosporia babensis VN05A0415T (98.08 %), respectively. Strain J2-1T had chemotaxonomic properties resembling members of the genus Streptomyces. ll-Diaminopimelic acid, glucose and ribose were detected in the whole-cell hydrolysate. Diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylinositolmannoside, unidentified aminolipid and five unidentified phospholipids were detected as the polar lipids. The major cellular fatty acids were C16 : 0 iso, C15 : 0 anteiso, C15 : 0 iso, C16 : 0, C17 : 0 anteiso, C14 : 0 iso and C17 : 0 iso. Strain J2-2T a showed similar cell composition to members of the genus Kineosporia. Both isomers of ll- and meso-diaminopimelic acid were detected in the peptidoglycan. Arabinose, galactose, madurose and xylose were observed in the whole-cell hydrolysate. The polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol, phosphatidylinositol mannoside, phosphatidylcholine, an unidentified phospholipid and an unidentified glycolipid. The major cellular fatty acids were C16 : 0, C18 : 1 ω9c, C18 : 0 10-methyl, tuberculostearic acid, C18 : 0 and C17 : 0. Both strains could be distinguished from their closely related type strains according to their phenotypic characteristics. Comparative genome analysis indicated the delineation of two novel species based on digital DNA-DNA hybridization and average nucleotide identity values, which were below 70 and 95 %, respectively. The names proposed are Streptomyces corallincola sp. nov. (J2-1T=TBRC 13503T=NBRC 115066T) and Kineosporia corallincola sp. nov. (J2-2T=TBRC 13504T=NBRC 114885T).

Solihabitans fulvus gen. nov., sp. nov., a member of the family Pseudonocardiaceae isolated from soil.

A polyphasic taxonomic study was carried out on an actinobacterial strain (AN110305T) isolated from soil sampled in the Republic of Korea. Cells of the strain were Gram-stain-positive, aerobic, non-motile and rod-shaped. Comparative 16S rRNA gene sequence studies showed a clear affiliation of strain AN110305T with Actinomycetia, with highest pairwise sequence similarities to Goodfellowiella coeruleoviolacea DSM 43935T (97.6%), Umezawaea tangerina MK27-91F2T (97.0%), Kutzneria chonburiensis NBRC 110610T (96.9%), Kutzneria buriramensis A-T 1846T (96.8%), Umezawaea endophytica YIM 2047XT (96.8%), Kutzneria albida NRRL B-24060T (96.7%) and Saccharothrix coeruleofusca NRRL B-16115T (96.6%). Cells of strain AN110305T formed pale-yellow colonies on Reasoner's 2A agar. MK-9 (H4) (68%) and MK-10 (H4) (32%) were the predominant menaquinones. Diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylmethyl ethanolamine, hydroxy-phosphatidylethanolamine, an unidentified aminolipid and an unidentified aminophospholipid were major polar lipids. Iso-C16:0 (24.5%), anteiso-C15:0 (19.3%), anteiso-C17:0 (15.7%) and iso-C15:0 (15.2%) were the major fatty acids and meso-diaminopimelic acid was the pepdidoglycan. The cell-wall sugars were composed of galactose, glucose, mannose and ribose. The genomic DNA G+C content was 70.7 mol%. Based on genotypic and phenotypic data, strain AN110305T could be distinguished from all genera within the family Pseudonocardiaceae and represents a novel genus and species named Solihabitans fulvus gen. nov., sp nov. The type strain is AN110305T (=KCTC 39307T =DSM 103572T).

Ornithinimicrobium laminariae sp. nov., isolated from the kelp Laminaria japonica.

A Gram-stain-positive, aerobic, non-sporulating, yellow-pigmented and rod or cocci-shaped bacterium, designated Arc0846-15T, was isolated from the kelp Laminaria japonica. Strain Arc0846-15T was found to grow at 16-35 °C (optimum, 28 °C), at pH 6.0-9.5 (optimum, 7.0) and in the presence of 0-6 % (w/v) NaCl (optimum, 2 %). Cells were positive for catalase and negative for oxidase activity. Phylogenetic analyses, based on 16S rRNA gene sequence comparisons, revealed that the nearest phylogenetic neighbour strains of strain Arc0846-15T were Ornithinimicrobium murale 01 Gi-040T (96.2 %), Ornithinimicrobium kibberense K22-20T (96.1 %) and Ornithinimicrobium humiphilum HKI 0124T (95.2 %). Based on phylogenomic analysis, the average nucleotide identity values between strain Arc0846-15T and the neighbour strains were 69.8, 69.7 and 69.8 %, respectively; the digital DNA-DNA hybridization values between strain Arc0846-15T and its three closest neighbour strains were 18.8, 19.1 and 19.3 %, respectively. The predominant menaquinone was MK-8 (H4). The dominant cellular fatty acids were C17 : 1 ω8c, iso-C15 : 0, iso-C16 : 0 and C17 : 0. The polar lipids contained diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol, glycolipid, one unidentified aminolipid and four unidentified lipids. The DNA G+C content of strain Arc0846-15T was 61.6 mol% based on the whole genome sequence. Based on the phylogenetic and phenotypic characteristics, strain Arc0846-15T is considered to represent a novel species of the genus Ornithinimicrobium, for which the name Ornithinimicrobium laminariae sp. nov. is proposed, with Arc0846-15T (=KCTC 49655T=MCCC 1K06093T) as the type strain.

Distribution and Evolutionary History of Sialic Acid Catabolism in the Phylum Actinobacteria.

Sialic acids are present in humans and other metazoans, playing essential roles in physiological and pathological processes. Commensal and pathogenic bacteria have evolved the capacity to utilize sialic acids as nutrient and energy sources. However, in some actinobacteria, sialic acid catabolism (SAC) is associated with free-living populations. To unravel the distribution and evolutionary history of SAC in the phylum Actinobacteria, we analyzed the presence and diversity of the putative SAC gene cluster (nan) in 7,180 high-quality, nonredundant actinobacterial genomes that covered 1,969 species. The results showed that ∼13% of actinobacterial species had the potential to utilize sialic acids, with 45 species capable of anhydro-SAC, all except two of them through the canonical pathway. These species belonged to 20 orders and 81 genera, with ∼36% of them from four genera, Actinomyces, Bifidobacterium, Corynebacterium, and Streptomyces. Moreover, ∼40% of the nan-positive species are free living. Phylogenetic analysis of the key nan genes, nanA, nanK, and nanE, revealed a strong signal of horizontal gene transfer (HGT), accompanied with vertical inheritance and gene loss. This evolutionary pattern led to high diversity and differential distribution of nan among actinobacterial taxa and might cause the cluster to spread to some free-living species while losing in some host-associated species. The evolution of SAC in actinobacteria probably represents the evolution of certain kinds of noncore bacterial functions for environmental adaptation and lifestyle switch, in which HGT plays a dominant role. IMPORTANCE Sialic acids play essential roles in the physiology of humans and other metazoan animals, and microbial sialic acid catabolism (SAC) is one of the processes critical for pathogenesis. To date, microbial SAC is studied mainly in commensals and pathogens, while its distribution in free-living microbes and evolutionary pathway remain largely unexplored. Here, by examining all actinobacterial genomes available, we demonstrate that putative SAC is present in a small proportion of actinobacterial species, of which, however, ∼40% are free-living species. We also reveal remarkable difference in the distribution of SAC among actinobacterial taxa and high diversity of the putative SAC gene clusters. HGT plays a significant role in the evolution of SAC, accompanied with vertical inheritance and gene loss. Our results provide a comprehensive and systematic picture of the distribution and evolutionary history of SAC in actinobacteria, expanding the current knowledge on bacterial adaptation and diversification.

Agromyces agglutinans sp. nov., isolated from saline lake sediment.

A novel actinobacterium, designated strain CFH 90414T, was isolated from sediment sampled at a saline lake in Yuncheng, Shanxi, PR China. The taxonomic position of the strain was investigated by using a polyphasic approach. Cells of strain CFH 90414T were Gram-reaction-positive, aerobic and non-motile. Growth occured at 4-40 °C, pH 5.0-9.0 and in the presence of up to 0-3.0 % (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain CFH 90414T was a member of the genus Agromyces. The 16S rRNA gene sequence similarity analysis indicated that strain CFH 90414T was most closely related to Agromyces italicus JCM 14320T (98.07 %) and Agromyces lapidis JCM 14321T (97.18 %). The whole genome of CFH 90414T was 3.64 Mb, and showed a G+C content of 71.5 mol%. The average nucleotide identity (ANI) values and digital DNA-DNA hybridization (dDDH) values between CFH 90414T and the other species of the genus Agromyces were found to be low (ANI <78.99 % and dDDH <22.9 %). The whole-cell sugars were rhamnose, mannose, ribose, glucose and galactose. The isolate contained l-2,4-diaminobutyric acid, d-alanine, d-glutamic acid and glycine in the cell-wall peptidoglycan. The predominant menaquinone was MK-12. The major cellular fatty acids were anteiso-C15 : 0, anteiso-C17 : 0 and iso-C16 : 0. The polar lipid profile contained diphosphatidylglycerol, phosphatidylglycerol and an unidentified glycolipid. On the basis of phenotypic, genotypic and phylogenetic data, strain CFH 90414T is considered to represent a novel species of the genus Agromyces, for which the name Agromyces agglutinans sp. nov. is proposed. The type strain is CFH 90414T (=DSM 105966T=KCTC 49062T).

Intestinal gluconeogenesis shapes gut microbiota, fecal and urine metabolome in mice with gastric bypass surgery.

Intestinal gluconeogenesis (IGN), gastric bypass (GBP) and gut microbiota positively regulate glucose homeostasis and diet-induced dysmetabolism. GBP modulates gut microbiota, whether IGN could shape it has not been investigated. We studied gut microbiota and microbiome in wild type and IGN-deficient mice, undergoing GBP or not, and fed on either a normal chow (NC) or a high-fat/high-sucrose (HFHS) diet. We also studied fecal and urine metabolome in NC-fed mice. IGN and GBP had a different effect on the gut microbiota of mice fed with NC and HFHS diet. IGN inactivation increased abundance of Deltaproteobacteria on NC and of Proteobacteria such as Helicobacter on HFHS diet. GBP increased abundance of Firmicutes and Proteobacteria on NC-fed WT mice and of Firmicutes, Bacteroidetes and Proteobacteria on HFHS-fed WT mice. The combined effect of IGN inactivation and GBP increased abundance of Actinobacteria on NC and the abundance of Enterococcaceae and Enterobacteriaceae on HFHS diet. A reduction was observed in the amounf of short-chain fatty acids in fecal (by GBP) and in both fecal and urine (by IGN inactivation) metabolome. IGN and GBP, separately or combined, shape gut microbiota and microbiome on NC- and HFHS-fed mice, and modify fecal and urine metabolome.

Changpingibacter yushuensis gen. nov., sp. nov., isolated from fluvial sediment in Qinghai Tibet Plateau of China.

Two facultatively anaerobic, short rod-shaped, non-motile, Gram-stain-positive, unknown bacterial strains (JY-X040T and JY-X174) were isolated from fluvial sediments of Tongtian River in Yushu Tibetan Autonomous Prefecture, Qinghai province, China. Cells formed translucent, gray, round and convex colonies, with a diameter of less than 0.5 mm after 5 days of incubation at 30°C on brain heart infusion-5% sheep blood agar. The 16S rRNA gene sequence similarity between strain JY-X040T and Fudania jinshanensis 313T is 93.87%. In the four phylogenetic trees constructed based on the 16S rRNA gene and 423 core genes, the two isolates form an independent branch, phylogenetically closest to F. jinshanensis 313T, but could not be classified as a member of the genus Fudania or any other genus of the family Arcanobacteriaceae. The DNA G + C content of strain JY-X040T was 57.8%. Calculation results of average nucleotide identity, digital DNA-DNA hybridization value and amino acid identity between strain JY-X040T and F. jinshanensis 313T are 69.9%, 22.9%, and 64.1%. The major cellular fatty acids were C16:0 (23%) and C18:1ω9c (22%). The cell-wall peptidoglycan type was A5α (L-Lys-L-Ala-L-Lys-D-Glu). The polar lipids comprised diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol, phosphatidylinositol mannoside and four unidentified components. The whole-cell sugars contained rhamnose and ribose. MK-10(H4) was the sole respiratory quinone. The minimum inhibitory concentration of streptomycin was 32 µg/ml. All physiological, biochemical, chemotaxonomic and genomic characteristics support that strains JY-X040T and JY-X174 represent members of a novel species in a new genus, Changpingibacter yushuensis gen. nov., sp. nov. The type strain is JY-X040T (GDMCC 1.1996T = KCTC 49514T).

Bending is required for activation of dsz operon by the TetR family protein (DszGR).

The dsz operon responsible for the biodesulfurization of organosulfurs is under the control of a 385 bp long promoter. Recently, a TetR family protein was identified which served as an activator of operon. Here we report that the TetR family protein (WP_058249973.1), named DszGR can specifically activate the dsz operon. Direct binding of the DszGR to DNA was observed at single molecule level by AFM. It was found that the binding of DszGR to the promoter DNA induces a bend by about âˆ¼40-50° degrees which may not be enough for the activation of the promoter. Thus, bendability in the promoter sequence was analyzed. The results show that the promoter has a curvature at around -235 and -200 bp with respect to dszA start codon. On mutating this region, a decrease in activity of the promoter was observed. Our results suggest that the DszGR protein binds to the upstream sequences and induces a bend, which is facilitated by further bending of the DNA which is required for dsz promoter activity. IHF binding site present in the promoter, and a significant reduction in desulphurization activity in the absence of either IHF subunits, suggested role of IHF in regulation of the dsz operon.

Paraconexibacter antarcticus sp. nov., a novel actinobacterium isolated from Antarctic tundra soil.

A Gram-negative, non-motile, aerobic bacterium, named 02-257T, was isolated from Antarctic soil. The cells are surrounded by relatively thin capsules and were catalase-positive and oxidase-negative cocci. Growth of strain 02-257T was observed at 4-35 °C (optimum, 28-30 °C), pH 6.0-8.0 (optimum, pH 6.0) and with 0-1.5% NaCl (optimum, 0 %). Strain 02-257 showed the highest 16S rRNA gene sequence similarity to Paraconexibacter algicola Seoho-28T (95.06 %). Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain 02-257T is a member of a novel species belonging to the clade formed by members of the genus Paraconexibacter in the family Paraconexibacteraceae. The DNA G+C content was 72.9 mol%. Strain 02-257T contained C16 : 0-iso (23.0 %), C18  :  1 ω9c (13.8 %), C16 : 0 (12.5 %) and C17 : 1 ω9c-iso (10.8 %) as major cellular fatty acids and menaquinone MK-7(H4) was detected as the only isoprenoid quinone. Diphosphatidylglycerol, phosphatidylinositol, phosphatidylinositole mannoside, phosphatidylinositole dimannoside, unidentified phosphoglycolipid, unidentified aminophospholipid, two unidentified phospholipids, three unidentified aminolipids and six unidentified lipids were the major polar lipids. meso-Diaminopimelic acids were the diagnostic diamino acids in the cell-wall peptidoglycan. On the basis of phenotypic, chemotaxonomic and phylogenetic data, strain 02-257T is considered to represent a novel species of the genus Paraconexibacter, for which the name Paraconexibacter antarcticus sp. nov. is proposed. The type strain is 02-257T (=CCTCC AB 2021030T=KCTC 49619T).

Reclassification of Olsenella gallinarum as Thermophilibacter gallinarum comb. nov. and description of Thermophilibacter immobilis sp. nov., isolated from the mud in a fermentation cellar used for the production of Chinese Luzhou-flavour Baijiu.

A novel Gram-stain-positive, strictly anaerobic, elliptical, non-motile and non-flagellated bacterium, designed LZLJ-2T, was isolated from the mud in a fermentation cellar used for the production of Chinese Luzhou-flavour Baijiu. Growth occurred at 28-45 °C (optimum, 37 °C), at pH 6.0-7.0 (optimum, pH 6.0) and with concentrations of NaCl up to 2 % (w/v; optimum, 0 %). On the basis of 16S rRNA gene sequence similarity, strain LZLJ-2T belonged to the genus Thermophilibacter and was most closely related to Thermophilibacter mediterraneus Marseille-P3256T (similarity 96.9 %), Olsenella gallinarum ClaCZ62T (similarity 96.6 %) and Thermophilibacter provencensis Marseille-P2912T (similarity 96.4 %). In addition, strain LZLJ-2T had high similarity to the genus Olsenella, including Olsenella profusa DSM 13989T (similarity 94.9 %), Olsenella umbonata DSM 22620T (similarity 94.9 %), Olsenella uli ATCC 49627T (similarity 94.22 %), Tractidigestivibacter scatoligenes DSM 28304T (similarity 93.9 %) and Paratractidigestivibacter faecalis KCTC 15699T (similarity 93.25 %). Comparative genome analysis showed that orthoANI values between strain LZLJ-2T and Thermophilibacter mediterraneus Marseille-P3256T, Olsenella gallinarum ClaCZ62T, Thermophilibacter provencensis Marseille-P2912T, Olsenella profusa DSM 13989T, Olsenella umbonata DSM 22620T, Olsenella uli ATCC 49627T, Tractidigestivibacter scatoligenes DSM 28304T and Paratractidigestivibacter faecalis KCTC 15699T were 78.68, 78.99, 78.29, 73.40, 74.00, 74.30, 75.08 and 77.23 %, and the genome-to-genome distance values were respectively 22.3, 22.5, 22.4, 19.6, 20.5, 19.7, 20.5 and 21.5 %. The genomic DNA G+C content of strain LZLJ-2T was 65.21 mol%. The predominant cellular fatty acids (>10 %) of strain LZLJ-2T were C18 : 1 cis 9 (33.7 %), C14 : 0 (22.0 %) and C18 : 1 cis 9 DMA (13.5 %). d-Glucose, sucrose, mannose, maltose, lactose (weak), salicin, glycerol (weak), cellobiose and trehalose (weak) could be used by strain LZLJ-2T as sole carbon sources. Enzyme activity results showed positive reactions with valine arylamidase, leucine arylamidase, crystine arylamidase, acid phosphatase, alkaline phosphatase, esterase (C4) (weakly positive), naphthol-AS-BI-phosphohydrolase, α-glucosidase and ß-glucosidase. The major end products of glucose fermentation were lactic acid and acetic acid. It produced skatole from indole acetic acid, and produced p-cresol from modified peptone-yeast extract medium with glucose. Based on the 16S rRNA gene trees as well as the genome core gene tree, it is suggested that Olsenella gallinarum are transferred to genus Thermophilibacter as Thermophilibacter gallinarum comb. nov. Based on phenotypic, genotypic and phylogenetic data, strain LZLJ-2T is considered to represent a novel species of the genus Thermophilibacter, for which the name Thermophilibacter immobilis sp. nov. is proposed. The type strain is LZLJ-2T (=KCTC 25162T=JCM 34224T).

Leucobacter soli sp. nov., from soil amended with humic acid.

A Gram-positive, non-spore-forming actinobacterium (IMT-300T) was isolated from soil amended with humic acid in Malvern, AL, USA. This soil has been used for 50+years for the cultivation of earthworms for use as fish bait. Based on 16S rRNA gene sequence similarity studies, strain IMT-300T was shown to belong to the genus Leucobacter and was closely related to the type strain of 'Leucobacter margaritiformis' L1T (97.8%). Similarity to all other type strains of Leucobacter species was lower than 97.2 %. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between the IMT-300T genome assembly and those of the closest relative Leucobacter type strain were 81.4 and 23.3 % (Leucobacter chironomi), respectively. The peptidoglycan of strain IMT-300T contained l-2,4-diaminobutyric acid as the diagnostic diamino acid. In addition, glycine, d- and l-alanine and d-glutamic acid were found. The peptidoglycan type represents a variant of B2δ (B11). The major quinones were menaquinones MK-10 and MK-11. The polar lipid profile consisted of the major lipids diphosphatidylglycerol, phosphatidylglycerol and moderate to minor amounts of two unidentified phospholipids, two unidentified glycolipids and an unidentified aminophospholipid. The polyamine pattern contained major amounts of spermidine and spermine. Strain IMT-300T contained the major fatty acids C15 : 0 anteiso, C16 : 0 iso and C17 : 0 anteiso, like other members of the genus Leucobacter. The results of ANI and dDDH analyses and physiological and biochemical tests allowed a genotypic and phenotypic differentiation of strain IMT-300T from the most closely related Leucobacter species. Strain IMT-300T represents a novel Leucobacter species, for which we propose the name Leucobacter soli sp. nov., with the type strain IMT-300T (CIP 111803T=DSM 110505T=CCM 9020T=LMG 31600T).

Antagonistic activity of endophytic actinobacteria from native potatoes (Solanum tuberosum subsp. tuberosum L.) against Pectobacterium carotovorum subsp. carotovorum and Pectobacterium atrosepticum.

BACKGROUND: The native potatoes (Solanum tuberosum subsp. tuberosum L.) grown in Chile (Chiloé) represent a new, unexplored source of endophytes to find potential biological control agents for the prevention of bacterial diseases, like blackleg and soft rot, in potato crops. RESULT: The objective of this study was the selection of endophytic actinobacteria from native potatoes for antagonistic activity against Pectobacterium carotovorum subsp. carotovorum and Pectobacterium atrosepticum, and their potential to suppress tissue maceration symptoms in potato tubers. This potential was determined through the quorum quenching activity using a Chromobacterium violaceaum ATCC 12472 Wild type (WT) bioassay and its colonization behavior of the potato plant root system (S. tuberosum) by means of the Double labeling of oligonucleotide probes for fluorescence in situ hybridization (DOPE-FISH) targeting technique. The results showed that although Streptomyces sp. TP199 and Streptomyces sp. A2R31 were able to inhibit the growth of the pathogens, only the Streptomyces sp. TP199 isolate inhibited Pectobacterium sp. growth and diminished tissue maceration in tubers (p ≤ 0.05). Streptomyces sp. TP199 had metal-dependent acyl homoserine lactones (AHL) quorum quenching activity in vitro and was able to colonize the root endosphere 10 days after inoculation. CONCLUSIONS: We concluded that native potatoes from southern Chile possess endophyte actinobacteria that are potential agents for the disease management of soft rot and blackleg.

Adlercreutzia hattorii sp. nov., an equol non-producing bacterium isolated from human faeces.

Two obligately anaerobic, Gram-stain-positive, rod-shaped bacteria were isolated from faecal samples of healthy humans in Japan. 16S rRNA gene sequence analysis indicated that these two strains (8CFCBH1T and 9CBH6) belonged to the genus Adlercreutzia, which is known as an equol-producing bacterium. The closest neighbours of strain 8CFCBH1T were Adlercreutzia equolifaciens subsp. equolifaciens DSM 19450T (98.6%), Adlercreutzia equolifaciens subsp. celatus do03T (98.4%), Adlercreutzia muris WCA-131-CoC-2T (96.6%), Parvibacter caecicola NR06T (96.4%), Adlercreutzia caecimuris B7T (95.3%) and Adlercreutzia mucosicola Mt1B8T (95.3%). The closest relatives to strain 9CBH6 were A. equolifaciens subsp. equolifaciens DSM 19450T (99.8%), A. equolifaciens subsp. celatus do03T (99.6%) and A. muris WCA-131-CoC-2T (96.8%). Strain 8CFCBH1T showed 22.3-53.5% digital DNA-DNA hybridization (dDDH) values with its related species. In addition, the average nucleotide identity (ANI) values between strain 8CFCBH1T and its related species ranged from 75.4 to 93.3%. On the other hand, strain 9CBH6 was considered as A. equolifaciens based on the dDDH and ANI values (>70% dDDH and >95-96% ANI). Strain 9CBH6 showed daidzein-converting activity, as expected from the result of genome analysis. The genome of strain 8CFCBH1T lacked four genes involved in equol production. Growing cells of strain 8CFCBH1T were not capable of converting daidzein. Based on the collected data, strain 8CFCBH1T represents a novel species in the genus Adlercreutzia, for which the name Adlercreutzia hattorii sp. nov. is proposed. The type strain of A. hattorii is 8CFCBH1T (=JCM 34083T=DSM 112284T).