Multilocus Sequence Analysis Evolutionary Distance Research Articles

Streptomyces koelreuteriae sp. nov., isolated from the rhizosphere soil of Koelreuteria paniculata and healthy leaves of Xanthium sibiricum.

Two actinomycete strains, designated MG62T and CRLD-Y-1, were isolated from rhizosphere soil of Koelreuteria paniculata and healthy leaves of Xanthium sibiricum, respectively, in Hunan province, PR China. They could produce abundant aerial mycelia that generated rod-shaped spores with spiny surfaces. Morphological features of the two strains are typical of the genus Streptomyces. Strains MG62T and CRLD-Y-1 exhibited 99.93 % 16S rRNA gene sequence similarity. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between them were 99.99 and 100 %, respectively, suggesting that they belonged to the same species. 16S rRNA gene sequences analysis revealed that the two strains belonged to the genus Streptomyces and showed highest similarities to Streptomyces violarus NBRC 13104T (99.07-99.29 %) and Streptomyces arenae ISP 5293T (99.21-99.35 %). Phylogenetic analysis based on 16S rRNA gene sequences indicated that strains MG62T and CRLD-Y-1 were closely related to S. violarus NBRC 13104T and S. arenae ISP 5293T. However, the ANI, dDDH and multilocus sequence analysis evolutionary distance values between the two strains and their relatives provide a robust basis upon which to verify strains MG62T and CRLD-Y-1 as representing a novel species. Moreover, a comprehensive comparison of phenotypic and chemotaxonomic characteristics further confirmed that the two strains were distinct from their relatives. Based on all these data above, strains MG62T and CRLD-Y-1 should represent a novel Streptomyces species, for which the name Streptomyces koelreuteriae sp. nov. is proposed. The type strain is MG62T (=JCM 34747T=MCCC 1K06175T).

Taxonomic Positions of a Nyuzenamide-Producer and Its Closely Related Strains.

Streptomyces sp. N11-34 is a producer of bicyclic peptides named nyuzenamides A and B. We elucidated its taxonomic position and surveyed its nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) gene clusters by whole genome analysis. Streptomyces sp. N11-34 showed 16S rRNA gene sequence similarities of 99.9% and 99.8% to Streptomyces hygroscopicus NBRC 13472T and Streptomyces demainii NRRL B-1478T, respectively. Although these members formed a clade in a phylogenetic tree based on 16S rRNA gene sequences, the clade split into two closely related subclades in multilocus sequence analysis (MLSA). One included Streptomyces sp. N11-34, S. demainii NRRL B-1478T, S. hygroscopicus NBRC 100766, S. hygroscopicus NBRC 16556 and S. hygroscopicus TP-A0867 and the other comprised S. hygroscopicus NBRC 13472T and S. hygroscopicus NBRC 12859. These phylogenetic relationships were supported by phylogenomic analysis. Although Streptomyces sp. N11-34 was classified to S. hygroscopicus at the species level based on MLSA evolutionary distances and DNA–DNA relatedness, these distances and relatedness of members between the two subclades were comparatively far (0.004–0.006) and low (75.4–76.4%), respectively. Streptomyces sp. N11-34 possessed six NRPS, seven PKS and four hybrid PKS/NRPS gene clusters in the genome. Among the seventeen, ten were identified to be biosynthetic gene clusters (BGCs) of nyuzenamide, echoside, coelichelin, geldanamycin, mediomycin, nigericin, azalomycin, spore pigment, alchivemycin and totopotensamide, whereas the remaining seven were orphan in our bioinformatic analysis. All seventeen are conserved in S. hygroscopicus NBRC 100766, S. hygroscopicus NBRC 16556 and S. hygroscopicus TP-A0867. In contrast, S. hygroscopicus NBRC 13472T and S. hygroscopicus NBRC 12859 lacked the BGCs of alchivemycin, totopotensamide, a nonribosomal peptide and a hybrid polyketide/nonribosomal peptide compound. This difference was in a good accordance with the abovementioned phylogenetic relationship. Based on phenotypic differences in addition to phylogenetic relationship, DNA–DNA relatedness and BGCs, strains of S. hygroscopicus should be reclassified to two subspecies: S. hygroscopicus subsp. hygroscopicus and a new subspecies, for which we proposed S. hygroscopicus subsp. sporocinereus subsp. nov. The type strain is NBRC 100766T (=ATCC 43692T = DSM 41460T = INMI 32T = JCM 9093T = NRRL B-16376T = VKM Ac-312T). S. demainii was classified in this subspecies.

Streptomyces typhae sp. nov., a novel endophytic actinomycete with antifungal activity isolated the root of cattail (Typha angustifolia L.).

A novel endophytic actinomycete with antagonistic activity against various phytopathogenic fungi, designated strain p1417T, was isolated from the root of cattail (Typha angustifolia L.) collected from Yunnan Province, Southwest China. A polyphasic taxonomic study was carried out to establish the taxonomic status of this strain. Strain p1417T was found to have morphological and chemotaxonomic characteristics typical of the genus Streptomyces. The diamino acid present in the cell wall was LL-diaminopimelic acid. Xylose and arabinose occurred in whole cell hydrolysates. The menaquinones were identified as MK-9(H8), MK-9(H6) and MK-9(H4). The polar lipid profile was found to contain diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol and phosphatidylinositol mannoside. The major fatty acids were found to be iso-C16:0, anteiso-C15:0, iso-C15:0 and C16:0. The genomic DNA G + C content of strain p1417T based on the genome sequence was 72.0mol%. Based on 16S rRNA gene, five housekeeping genes and whole genome sequences analysis, strain p1417T was most closely related to Streptomyces flavofungini JCM 4753T (99.4% 16S rRNA gene sequence similarity), Streptomyces alboflavus JCM 4615T (98.8%) and Streptomyces aureoverticillatus JCM 4347T (98.2%). However, the average nucleotide identity values, the digital DNA-DNA hybridization values and the multilocus sequence analysis evolutionary distances between this strain and its closely related strains showed that it belonged to one distinct species. In addition, these results were also supported by differences in the phenotypic and chemotaxonomic characteristics between strain p1417T and three closely related type strains. Therefore, it is concluded that strain p1417T represents a novel species of the genus of Streptomyces, for which the name Streptomyces typhae sp. nov. is proposed. The type strain is p1417T (= CCTCC AA 2019091T = DSM 110636T).

Streptomyces scabichelini sp. nov., isolated from soil

A novel actinomycete, designated strain HC44T, was isolated from a soil sample collected from Hacibektaş, Turkey, and characterized using a polyphasic approach. The strain had morphological characteristics and chemotaxonomic properties identical to those of members of the genus Streptomyces. Phylogenetic analyses based on 16S rRNA gene sequence comparisons revealed that HC44T clustered with members of the genus Streptomyces and the highest 16S rRNA gene sequence similarity values were obtained with Streptomyces vastus NBRC 13094T (97.6 %) and Streptomyces kalpinensis TRM 46509T (96.9 %). Multi-locus sequence analysis (MLSA) based on five housekeeping genes (atpD, gyrB, recA, rpoB and trpB) showed that the MLSA evolutionary distance value was 0.043 between strain HC44T and S. vastus NBRC 13094T. Whole-cell hydrolysates contained ll-diaminopimelic acid, glucose, mannose and ribose. The predominant menaquinones were MK-9(H6) and MK-9(H8). The major polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol and phosphatidylinositol mannoside. The DNA G+C content of the draft genome sequence, consisting of 11.2 Mbp, was 69.8 mol%. On the basis of polyphasic taxonomic evidence, strain HC44T represents a novel species of the genus Streptomyces, for which the name Streptomyces scabichelini sp. nov. is proposed. The type strain is HC44T (=DSM 106874T=KCTC 39872T).

Steptomyces fagopyri sp. nov., a novel actinomycete isolated from rhizospheric soil of Fagopyrum dibotrys.

A novel actinomycete, designated strain QMT-28T, was isolated from rhizosphere soil of Fagopyrum dibotrys collected from Shuangfeng, Hunan Province, PR China. Strain QMT-28T grew well on International Streptomyces Project series media and formed well-developed, branched substrate hyphae and aerial mycelium that differentiated into loose spiral spore chains consisting of cylindrical spores with smooth surfaces. The diagnostic diamino acid was ll-diaminopimelic acid and the whole-cell sugars were galactose and glucose. The predominant fatty acids were C18 : 1 cis9, summed feature 6 (C18 : 2 cis 9,12/C18 : 0 a) and C16 : 0. The polar lipids included diphosphatidylglycerol, hydroxy phospatidylethanolamine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylinositol mannosides, phospholipids of unknown structure containing glucosamine and several unidentified phospholipids. The major menaquinones were MK-9, MK-9(H2), MK-9(H4), MK-9(H6) and MK-9(H8). The genome size of strain QMT-28T was about 8.7 Mbp with a G+C content of 71.2 mol%. Phylogenetic analysis showed that the novel strain was closely related to Streptomyces olivochromogenes DSM 40451T (99.5 % similarity), Streptomyces mirabilis NBRC 13450T (98.9 %), Streptomyces kanamyceticus NBRC 13414T (98.9 %), Streptomyces kaempferi I37T (98.9 %) and Streptomyces arcticus ZLN234T (98.8 %). However, the average nucleotide identity values, the digital DNA-DNA hybridization values and the multilocus sequence analysis evolutionary distances between this strain and closely related strains showed that it belonged to a distinct species. In addition, these results were also supported by differences in the phenotypic characteristics between QMT-28T and five closely related type strains. Consequently, strain QMT-28T should represent a novel species of the genus Streptomyces, with the suggested name Streptomyces fagopyri sp. nov. The type strain is QMT-28T (=CICC 24808T=JCM 33796T).

Streptomyces phaeolivaceus sp. nov. and Streptomyces broussonetiae sp. nov., isolated from the leaves and rhizosphere soil of Broussonetia papyrifera.

Two novel actinobacteria, designated strains GY16T and T44T, were isolated from the leaves and rhizosphere soil of Broussonetia papyrifera, respectively. A polyphasic approach was used for determining their taxonomic position. Results of 16S rRNA gene sequence analysis indicated that strain GY16T exhibited highest similarities to Streptomyces cinereoruber subsp. fructofermentans CGMCC 4.1593T (98.82 %), Streptomyces deccanensis KCTC 19241T (98.76 %), Streptomyces scabiei NRRL B-16523T (98.69 %), Streptomyces europaeiscabiei KACC 20186T (98.69 %) and Streptomyces rishiriensis NBRC 13407T (98.69 %), and strain T44T showed 99.2, 99.1, 99.1 and <98.7 % sequence similarities to Streptomyces filipinensis CGMCC 4.1452T, Streptomyces achromogenes subsp. achromogenes DSM 40028T, Streptomyces durhamensis DSM 40539T and other Streptomyces species, respectively. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain GY16T formed an independent subclade, which indicated that strain GY16T should belong to a potential novel species; and strain T44T was closely related to S. filipinensis CGMCC 4.1452T, S. achromogenes subsp. achromogenes DSM 40028T, S. durhamensis DSM 40539T and S. yokosukanensis DSM 40224T. However, the multilocus sequence analysis evolutionary distance, average nucleotide identity and DNA-DNA hybridization values between closely related relatives were far from the species-level thresholds. In addition, phenotypic and chemotaxonomic characteristics further confirmed that strains GY16T and T44T belonged to two distinct species. Based on these results, it is concluded that the isolated strains represent novel species within the genus Streptomyces, for which the names Streptomyces phaeolivaceus sp. nov. (type strain GY16T=CICC 24807T=KCTC 49326T) and Streptomyces broussonetiae sp. nov. (type strain T44T=CICC 24819T=JCM 33918T) are proposed.

Streptomyces paludis sp. nov., isolated from an alpine wetland soil.

A Gram-stain-positive actinobacterium, designated strain GSSD-12T, was isolated from an alpine wetland soil. blast search of the full-length 16S rRNA gene sequence of GSSD-12T indicated it represented a member of the genus Streptomyces, and displayed highest similarity with Streptomyces scopuliridis NRRL B-24574T (99.2 %) and less than 98.6 % similarity with other species of the genus Streptomyces with validly published names. Phylogenetic analysis based on 16S rRNA gene sequences indicated that GSSD-12T was closely related to S. scopuliridis NRRL B-24574T, Streptomyces odonnellii NRRL B-24891T and Streptomyces lushanensis NRRL B-24994T. However, the digital DNA-DNA hybridization value, the average nucleotide identity value and the multilocus sequence analysis evolutionary distance between this strain and its closest relatives indicated that it represented a distinct species. Furthermore, GSSD-12T was also distinctly differentiated from them by morphological, physiological and biochemical characteristics. Therefore, strain GSSD-12T(=CICC 11051T=JCM 33019T) represents a novel species of the genus Streptomyces, for which the name Streptomyces paludis sp. nov. is proposed.

Streptomyces tibetensis sp. nov., an actinomycete isolated from the Tibetan Plateau.

A novel actinomycete, designated strain XZ 46T, was isolated from acid sandy soil collected from the Tibetan Plateau, China. Its taxonomic position was determined using a polyphasic approach. Strain XZ 46T shows the typical morphological and chemotaxonomic features of members of the genus Streptomyces: slightly yellow to brown substrate mycelia and grayish white to slightly yellow aerial hyphae forming cylindrical and spiny spores; meso-diaminopimelic acid in the cell wall peptidoglycan; MK-9(H8), MK-9(H4) and MK-9(H2) as predominant menaquinones; diphosphatidylglycerol, phospatidylethanolamine, phosphatidylglycerol and phosphatidylinositol as main polar lipids; and iso-C15:0, iso-C16:0 and anteiso-C15:0 as major cellular fatty acids. The G+C content of the draft genome sequence, consisting of 8,995,813bp, is 71.23%. The16S rRNA gene sequence analysis indicated that strain XZ 46T shows high sequence similarity to Streptomyces luteogriseus NBRC 13402T as well as forming an independent lineage clade with it in phylogenetic trees. Multilocus sequence analysis (MLSA) of five housekeeping genes (atpD, gyrB, recA, rpoB and trpB) illustrated that Streptomyces hawaiiensis is also a very closely related taxon. However, DNA-DNA hybridization, MLSA evolutionary distance and phenotypic properties demonstrate that strain XZ 46T can be distinguished from these phylogenetically related Streptomyces species. Therefore, it is concluded that strain XZ 46T represents a novel species of the genus Streptomyces, for which the name Streptomyces tibetensis is sp. nov. proposed. The type strain is XZ 46T (= CGMCC 4.7579T = KCTC 49221T).

Streptomyces reniochalinae sp. nov. and Streptomyces diacarni sp. nov., from marine sponges.

Two marine actinomycete strains, LHW50302T and LHW51701T, were isolated from marine sponges collected in Sansha, Hainan Province, China. The morphological, chemotaxonomic and phylogenetic characteristics were consistent with their classification in the genus Streptomyces. The strains formed hooked and looped chains of arthrospores with smooth surfaces. The cell-wall hydrolysates of the strains contained ll-diaminopimelic acid as the diagnostic diamino acid. MK-9(H8) was the predominant menaquinone. The major polar lipids were diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylinositol. Major fatty acids of the strains were iso-C16 : 0, anteiso-C15 : 0 and anteiso-C17 : 0. The 16S rRNA gene sequences indicated that the strains clustered together with Streptomyces albus CGMCC 4.1640T and Streptomyces qinglanensis CGMCC 4.6825T. Multilocus sequence analysis (MLSA) confirmed their relationship. Genome relatedness in forms of average nucleotide identity, digital DNA-DNA hybridization value and MLSA evolutionary distance between each of the strains and its closest relatives showed that they belonged to distinct species. On the basis of these results, strains LHW50302T and LHW51701T belong to two novel species in the genus Streptomyces, for which the names Streptomyces reniochalinae sp. nov. (type strain LHW50302T=CCTCC AA 2018013T=DSM 106194T) and Streptomyces diacarni sp. nov. (type strain LHW51701T=CCTCC AA 2018017T=DSM 106126T) are proposed, respectively.

Taxonomic evaluation of Streptomyces albus and related species using multilocus sequence analysis and proposals to emend the description of Streptomyces albus and describe Streptomyces pathocidini sp. nov.

In phylogenetic analyses of the genus Streptomyces using 16S rRNA gene sequences, Streptomyces albus subsp. albus NRRL B-1811(T) forms a cluster with five other species having identical or nearly identical 16S rRNA gene sequences. Moreover, the morphological and physiological characteristics of these other species, including Streptomyces almquistii NRRL B-1685(T), Streptomyces flocculus NRRL B-2465(T), Streptomyces gibsonii NRRL B-1335(T) and Streptomyces rangoonensis NRRL B-12378(T) are quite similar. This cluster is of particular taxonomic interest because Streptomyces albus is the type species of the genus Streptomyces. The related strains were subjected to multilocus sequence analysis (MLSA) utilizing partial sequences of the housekeeping genes atpD, gyrB, recA, rpoB and trpB and confirmation of previously reported phenotypic characteristics. The five strains formed a coherent cluster supported by a 100 % bootstrap value in phylogenetic trees generated from sequence alignments prepared by concatenating the sequences of the housekeeping genes, and identical tree topology was observed using various different tree-making algorithms. Moreover, all but one strain, S. flocculus NRRL B-2465(T), exhibited identical sequences for all of the five housekeeping gene loci sequenced, but NRRL B-2465(T) still exhibited an MLSA evolutionary distance of 0.005 from the other strains, a value that is lower than the 0.007 MLSA evolutionary distance threshold proposed for species-level relatedness. These data support a proposal to reclassify S. almquistii, S. flocculus, S. gibsonii and S. rangoonensis as later heterotypic synonyms of S. albus with NRRL B-1811(T) as the type strain. The MLSA sequence database also demonstrated utility for quickly and conclusively confirming that numerous strains within the ARS Culture Collection had been previously misidentified as subspecies of S. albus and that Streptomyces albus subsp. pathocidicus should be redescribed as a novel species, Streptomyces pathocidini sp. nov., with the type strain NRRL B-24287(T).

Taxonomic evaluation of the Streptomyces hygroscopicus clade using multilocus sequence analysis and DNA–DNA hybridization, validating the MLSA scheme for systematics of the whole genus

Streptomyces hygroscopicus and related species are the most well known candidate producers of antibiotics and many other industrially and agronomically important secondary metabolites in the genus Streptomyces. Multilocus sequence analysis (MLSA) has shown to be a powerful and pragmatic molecular method for unraveling streptomycete diversities. In this investigation, a multilocus phylogeny of 58 representatives of the S. hygroscopicus 16S rRNA gene clade including S. violaceusniger and related species was examined. The result demonstrated that the MLSA data were helpful in defining members of the S. hygroscopicus clade, providing further evidence that the MLSA scheme of five housekeeping genes ( atpD, gyrB, recA, rpoB and trpB) is a valuable alternative for creating and maintaining operational protocols for the Streptomyces species assignment. DNA–DNA hybridization (DDH) between strains with representative MLSA evolutionary distances, combined with previous data from S. griseus and S. albidoflavus clades, revealed a high correlation between MLSA and DDH, and sustains that the five-gene nucleotide sequence distance of 0.007 could be considered as the species cut-off for the whole genus. This significant correlation thus makes the MLSA scheme applicable to construction of a theory-based taxonomy for both ecology and bioprospecting of streptomycetes. Based on the MLSA and DDH data, as well as phenotypic characteristics, 10 species and three subspecies of the S. hygroscopicus clade are considered to be later heterotypic synonyms of eight genomic species, and Streptomyces glebosus sp. nov., comb. nov. (type strain CGMCC 4.1873 T = LMG 19950 T = DSM 40823 T) and Streptomyces ossamyceticus sp. nov., comb. nov. (type strain CGMCC 4.1866 T = LMG 19951 T = DSM 40824 T) are also proposed.

Proposal to reclassify the Streptomyces albidoflavus clade on the basis of multilocus sequence analysis and DNA–DNA hybridization, and taxonomic elucidation of Streptomyces griseus subsp. solvifaciens

The Streptomyces albidoflavus 16S rRNA gene clade contains 10 species and subspecies with identical 16S rRNA gene sequences and very similar numerical taxonomic data, including Streptomyces griseus subsp. solvifaciens. Type strains of this clade, as well as three CGMCC strains which were received as Streptomyces galilaeus, Streptomyces sioyaensis and Streptomyces vinaceus, respectively, that shared the same 16S rRNA gene sequences with the clade, were subjected to multilocus sequence analysis (MLSA), DNA–DNA hybridization (DDH) and phenotypic characterization for a comprehensive reevaluation. The 13 strains still formed a distinct, albeit loosely related, clade in the phylogenetic tree based on concatenated sequences of aptD, gyrB, recA, rpoB and trpB genes, supported by a high bootstrap value and different tree-making algorithms, with MLSA evolutionary distances ranging from 0 to 0.003. DDH values among these strains were well above the 70% cut-off point for species delineation. Based on the genotypic data of MLSA and DDH, combined with key phenotypic properties in common, it is proposed that the 10 species and subspecies of the S. albidoflavus clade, namely S. albidoflavus, S. canescens, S. champavatii, S. coelicolor, S. felleus, S. globisporus subsp. caucasicus, S. griseus subsp. solvifaciens, S. limosus, S. odorifer and S. sampsonii, should be merged into a single genomic species, for which the name S. albidoflavus is retained, and that the three strains S. galilaeus CGMCC 4.1320, S. sioyaensis CGMCC 4.1306 and S. vinaceus CGMCC 4.1305 should be assigned to S. albidoflavus as well. The results also indicated that MLSA could be the procedure of choice for distinguishing between species within Streptomyces 16S rRNA gene clades.