Polyphasic classification of Nonomuraea strains isolated from the Karakum Desert and description of Nonomuraea deserti sp. nov., Nonomuraea diastatica sp. nov., Nonomuraea longispora sp. nov. and Nonomuraea mesophila sp. nov.

636 Polyphasic classification of Nonomuraea strains isolated from the Karakum Desert and description of Nonomuraea deserti sp. nov., Nonomuraea diastatica sp. nov., Nonomuraea longispora sp. nov. and Nonomuraea mesophila sp.nov. HayrettinSaygin1, ImenNouioui2, HilalAy3, KiymetGuven4, DemetCetin5, HansPeterKlenk2, MichaelGoodfellow2 and NevzatSahin3,* TAXONOMIC DESCRIPTION Saygin etal., Int. J. Syst. Evol. Microbiol. 2020;70:636–647 DOI 10.1099/ijsem.0.003808 Author affiliations: 1Department of Biology, Faculty of Science and Arts, Ondokuz Mayis University, 55139, Samsun, Turkey; 2School of Natural and Environmental Sciences, Newcastle University, Ridley Building 2, Newcastle upon Tyne NE1 7RU, UK; 3Department of Molecular Biology and Genetics, Faculty of Sciences and Arts, Ondokuz Mayis University, 55139, Samsun, Turkey; 4Department of Biology, Faculty of Science, Eskisehir Technical University, 26555, Eskisehir, Turkey; 5Division of Science Education, Department of Mathematics and Science Education, Gazi University, 06500, Ankara, Turkey. *Correspondence: Nevzat Sahin, nsahin@ omu. edu. tr Keywords: Actinobacteria; genomics; Nonomuraea; phylogenomic analyses; Karakum Desert. Abbreviations: ANI, Average Nucleotide Identity; dDDH, digital DNADNA Hybridization; GGDC, Genome to Genome Distance Calculator; TYGS, Type Strain Genome Server. The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene of KC201T, KC401, KC310T, KC712T and 6K102T are MG770626, MG770651, MG770639, MG770679 and MG770753, respectively. The GenBank/EMBL/DDBJ accession numbers for the draft genome sequences of KC201T, KC401, KC310T, KC712T and 6K102T are SMJZ00000000, VBUN00000000, SMKO00000000, SMKP00000000 and SMLD00000000, respectively. One supplementary figure and two supplementary tables are available with the online version of this article. 003808 © 2020 The Authors Abstract Five actinobacteria isolates, KC201T, KC401, KC310T, KC712T and 6K102T, were recovered from the Karakum Desert during an investigation of novel actinobacteria with biotechnological potential. A polyphasic approach confirmed the affiliation of the strains to the genus Nonomuraea. The strains showed chemotaxonomic and morphological properties consistent with their classification in the genus Nonomuraea. Furthermore, these strains clearly distinguished and formed well supperted clades in phylogenetic and phylogenomic trees. Low ANI and dDDH values and distinguishing phenotypic properties between isolates KC201T, KC310T, KC712T and 6K102T showed that these strains belonged to novel Nonomuraea species, the names proposed for these taxa are Nonomuraea deserti sp. nov., Nonomuraea diastatica sp. nov., Nonomuraea longispora sp. nov. and Nonomuraea mesophila sp. nov., with the type strains KC310T (=CGMCC 4.7331T =DSM 102919T =KCTC 39774T), KC712T (=CGMCC 4.7334T =DSM 102925T =KCTC 39776T), KC201T (=CGMCC 4.7339T =DSM 102917T =KCTC 39781T) and 6K102T (=CGMCC 4.7541T =JCM 32916T), respectively. The genus Nonomuraea was first proposed by Zhang et al. [1] and is classified in the family Streptosporangiaceae. The original genus name ‘Nonomuria’ was corrected to ‘Nonomuraea’ by Chiba et al. [2] and the genus has recently been emended by Cao et al. [3]. The genus is characterized by extensively branched substrate and aerial mycelia. Aerial hyphae may differentiate into hooked, spiral, or straight chains of spores with folded, irregular, smooth, or warty spore surfaces and may also produce single spores or spherical sporangia [3–6]. At the time of writing, the genus comprises 50 species and two subspecies with validly published names ( www. bacterio. net/ nonomuraea. html). Members of the genus are characterized by mesodiaminopimelic acid in the cell wall peptidoglycan, madurose in wholecell hydrolysates as the diagnostic sugar, diphosphatidylglycerol, phosphatidylethanolamine, hydroxylated phosphatidylethanolamine as predominant phospholipids, MK-9(H4), MK-9(H2) and MK-9(H0) as major menaquinones, C17 : 010methyl and isoC16 : 0 as predominant fatty acids [4]. Nonomuraea strains like other members of the family Streptosporangiaceae are an increasingly rich source of commercial products, notably antibiotics and enzymes [7]. Nonomuraea sp. ATCC 55076 has the largest genome in Actinobacteria and this strain has 32 gene clusters including vancomycin, teicoplanin and kistamicin biosynthetic gene clusters [8]. In addition, it is known that Nonomuraea rosea, Nonomuraea roseoviolacea, 637 Saygin etal., Int. J. Syst. Evol. Microbiol. 2020;70:636–647 Nonomuraea rubra, Nonomuraea pusilla, Nonomuraea spiralis and Nonomuraea gerenzanensis produce deoxycephalomycin B, carminomicins, maduromycin, actinotiocin, pyralomicin and glycopeptide antibiotic A40926 [9–15]. Members of the genus have been isolated from diverse natural habitats such as rhizosphere [16, 17], leaf [18], cave [19], limestone mine [20], root [5, 21], coastal sediment [22], mangrove sediment [23] and mushroom compost [24], while many members were isolated from different types of soil including cave soil [25], forest soil [26, 27], arid soil [28], acidic soil [29], rhizosphere soil [3, 30–32] and mangrove soil [33]. In the current study, we determined the taxonomic positions of strains KC201T, KC310T, KC401, KC712T and 6K102T recovered from the Karakum Desert soil. The strains were compared with one another and with related type strains of the genus Nonomuraea using polyphasic approach and complementary data drawn from whole genome sequences. The resultant data show that four of the strains represent novel species of the genus Nonomuraea. HAbITAT AND ISOlATION Slightly alkaline soil samples with low organic matter content were collected from the Karakum Desert soil at Darvaz (40° 15′ 38.13″ N and 58° 26′ 20.39″ E) and Mary (38° 12′ 31.41″ N and 62° 53′ 37.29″ E) in Turkmenistan. After airdrying at room temperature for 14 days, soil samples (1 g) were suspended in ¼ strength Ringer’s (Oxoid) solutions (9 ml) and the resultant suspensions incubated at 60 °C for 20 min. 200 µl samples of the serial dilutions suspensions were spread onto different media, including Stevenson’s medium no. 3 (SM3) [34], humic acid vitamin agar (HV) [35], marine agar [36], Reasoner's 2A (R2A) agar [37] and M1 agar [38] media supplemented with cycloheximide (50 µg ml−1) and nalidixic acid (10 µg ml−1) and the isolation plates were incubated in 28 °C for 28 days. Strains KC201T, KC401, KC310T, KC712T and 6K102 T isolated from SM3, HV, marine, R2A and M1 agar plates, respectively. Strain 6K102T was isolated from the soil sample collected from Mary while the other strains were isolated from the soil collected from Darvaz. The strains were purified and maintained on yeast extractmalt extract agar (International Streptomyces Project medium 2; ISP 2) [39] and stored in glycerol stock solutions (25%, v/v) at −80 °C. PHylOgENy AND gENOME fEATuRES For the extraction of genomic DNA, strains KC201T, KC401, KC310T, KC712T and 6K102T were grown in ISP 2 broth [39] at 28 °C for 14 days. The genomic DNA of strains were extracted from the isolates using a DNA extraction kit (Invitrogen) according to the manufacturer’s instructions. PCR amplification of 16S rRNA gene was carried out using the universal primers 27F (5′-AGAGTTTGATC(AC)TGGCTCAG-3′) and 1492R (5′-ACGG(CT)TACCTTGTTACGACTT-3′) [40] and PCR products purified and sequenced using an abi prism 3730 XL automatic sequencer. The obtained sequences (1479–1484 bp) were aligned with corresponding sequences of closely related Nonomuraea type strains retrieved from the EzBioCloud server [41]. Phylogenetic trees were reconstructed using the software package mega X [42] following multiple alignment of the sequence data by clustal_w [43]. 16S rRNA phylogenetic trees were constructed using the algorithms: neighbourjoining [44], maximumlikelihood [45] and maximumparsimony [46]. Topologies of the resultant trees were assessed by bootstrap resampling with 1000 replicates [47]. The genomes of the strains were sequenced externally (MicrobesNG, Birmingham, UK) using the Illumina HiSeq 2500 next generation sequencing platform and a ×250 bp pairedend protocol. Assemblies of raw data were performed using the full Spades assemble strategy on the patric web server (https:// patricbrc. org/) [48]. The draft genome sequences were deposited in the National Centre for Biotechnology Information (ncbi) database under accession numbers SMJZ00000000, VBUN00000000, SMKO00000000, SMKP00000000 and SMLD00000000, and then, annotated using the rast annotation server [49]. The genome sequences were uploaded to the Type Strain Genome Server (tygs), a free bioinformatics platform available at https:// tygs. dsmz. de, for comprehensive whole genomebased taxonomic analyses [50]. The results were provided by the tygs. The resulting intergenomic distances were used to infer a balanced minimumevolution tree with branch support via fastme 2.1.4 including SPR postprocessing [51] and branch support inferred from 100 pseudobootstrap replicates. The resultant trees were rooted at the midpoint [52] and visualized with PhyD3 [53]. Typebased species clustering using a 70 % digital DNA–DNA hybridization (dDDH) radius around each of the 27 type strains was done as described previously [54] and subspecies clustering achieved using a 79 % dDDH threshold after MeierKolthof et al. [55]. The dDDH similarities between strains KC201 T , KC401, KC310 T , KC712 T and 6K102 T were determined using formula 2 of the GGDC web server [56]. Similarly, average nucleotide identity (ANI) and ANIblast (ANIb) values between the strains were calculated using JSpeciesWS [57]. Secondary metabolite biosynthetic gene clusters in the genomes of the strains were predicted using antiSMASH web server [58]. It can be seen from Fig.1 that the isolates formed a distinct and well supported clade in the Nonomuraea 16S rRNA gene tree. Strains KC201T, KC401 and 6K102T are most closely related to Nonomuraea salmonea DSM 43678T sharing 16S rRNA gene sequence similarities with the later of 98.0, 98.1 and 98.3 %, respectively. In turn, isolates KC310T and KC712T are most closely related to Nonomuraea candida HMC10T sharing 98.1 % gene sequence identity with this strain. All of these sequence identity values are below the threshold of 98.65 % for delineation of novel species [59, 60]. The 16S rRNA gene identities between the strains ranged from 99.2 to 99.7 %. The total size of the draft genome sequences of strains KC201T, KC401, KC310T, KC712T and 6K102T are 9.16, 9.79, 10.69, 10.86 and 10.14 Mb with G+C contents of 70.6, 70.6, 638 Saygin etal., Int. J. Syst. Evol. Microbiol. 2020;70:636–647 Fig. 1. Neighbourjoining phylogenetic tree based on 16S rRNA gene sequences showing relationships between strains KC201T, KC310T, KC401, KC712T, 6K102T and between them and type strains of closely related Nonomuraea species. Evolutionary distances were computed using the Jukes–Cantor method [76] and are in the units of the number of base substitutions per site. The analysis was based on 39 nucleotide sequences. Positions containing gaps and missing data were eliminated from the data set. There was a total of 1316 positions in the final dataset. Asterisks indicate corresponding nodes recovered in the maximumlikelihood and maximumparsimony trees. Numbers at the nodes indicate percentage levels of bootstrap support, only values over 50 % are shown. Bar, 0.005 substitutions per nucleotide position. 639 Saygin etal., Int. J. Syst. Evol. Microbiol. 2020;70:636–647 70.9, 70.3 and 70.8 %, respectively; general features of the genomes are listed in Table1. In the whole genomebased tree, all strains clustered together and the average branch support value was 97.3 % in this tree (Fig.2). The dDDH values between the isolates ranged from 45.60 % to 88.20 %. In addition, the isolates shared relatively low average nucleotide identities ranging from 90.7 and 98.6 % calculated by using the ANIb and ANIm algorithms. Thus, the dDDH and ANI values are well below 70 and 95 % for DNA–DNA hybridization and average nucleotide identity, respectively, which are wellestablished thresholds for species delineation, except for strains KC201T and KC401, having 88.20 % dDDH and 98.6 % ANI values (Table2). Consequently, both dDDH and ANI values confirmed that strains KC201T, KC310T, KC712T and 6K102T represent novel species within the genus Nonomuraea while strains KC201T and KC401 are members of the same species. CHEMOTAXONOMy The isolates were examined for chemotaxonomic properties found to be of value in Streptosporangiaceae systematics [7]. Biomass for chemotaxonomic studies was obtained by growing isolates in ISP 2 broth at 160 r.p.m. at 28 °C for 14 days, then; cells were harvested by centrifugation, washed in distilled water, recentrifuged and freezedried. Wholecell hydrolysates were examined for isomers of diaminopimelic acid (A2pm) [61] and cellwall sugars [62]. Cellular fatty acids from the isolates and related strains, namely Nonomuraea antimicrobica JCM 16904T, Nonomuraea candida DSM 45086T, Nonomuraea jiangxiensis JCM 18570T, Nonomuraea salmonea DSM 43678T and Nonomuraea turkmeniaca DSM 43926T were extracted, methylated and analysed by gas chromatography using an Agilent Technologies 6890 N instrument, fitted with a G2614A autosampler and a 6783 injector, according to the standard protocol of the Microbial Identification (midi) System and analysed using Sherlock software version 6.1, the resultant peaks were identified using the tsba5 database [63, 64]. Polar lipids were extracted and analysed by twodimensional TLC following Minnikin et al. [65] and isoprenoid quinones after Collins [66]. The results of chemotaxonomic analyses were consistent with the assignment of the isolates to the genus Nonomuraea [4]. All of the isolates produced whole cell hydrolysates containing mesodiaminopimelic acid, glucose, mannose, madurose and ribose showing that they had a sugar pattern type B sensu Lechevalier and Lechevalier [62]; galactose was found in strains KC310T, KC712T, KC201T and KC401 though only in trace amounts in the latter two strains. All of the isolates contained major amounts of menaquinone was found to be MK-9(H4) (range, 66.2–74.0 %) for all strains. Isolates KC201T, KC401 and 6K102T contained minor amounts of MK-9(H2) (9.4, 13.7 and 15.0 %, respectively) and isolates KC310T and KC712T contained MK-9(H6) (19.0 and 12.5 %, respectively). The polar lipid profiles of all of the isolates contained diphosphatidylglycerol, phosphatidylmonomethylethanolamine, phosphatidylethanolamine, hydroxyphosphatidylethanolamine, dihydroxyphosphatidylethanolamine, phosphatidylglycerol and phosphatidylinositol (Fig. S1, available in the online version of this article). Although the polar lipid patterns of most Nonomuraea strains contain glucosamine, this component was absent from isolates KC201T, KC401, KC310T, KC712T and 6K102T; this is also the case with Nonomuraea gerenzanensis [9], Nonomuraea lactucae [3] and Nonomuraea zeae [17]. The fatty acid profiles of the isolates and those of the type strains of closely related Nonomuraea species are shown in Table S1. All of the isolates contained major amounts of C17 : 010methyl and isoC16:0 as did most of the reference strains. Similarly, isolates KC201T, KC712T and 6K102T contained major amounts of C16:0, isolate KC310T also contained isoC16:0 2OH as a major component. In general, the fatty acid profiles distinguished the isolates from one another and from the reference strains. CulTuRAl, MORPHOlOgICAl AND PHySIOlOgICAl PROPERTIES The isolates were examined for a broad range of phenotypic characteristics, known to be of value in the classification of filamentous, sporeforming actinobacteria. Cultural properties were recorded after incubation at 28 °C for 14 days on ISP 2–7 agar [39], modified Bennett’s agar [67], Czapek’s agar [68], nutrient agar [69] and tryptic soy agar (Difco) media by comparison against. Aerial and substrate mycelia colours and those of diffusible pigments were determined using the ISCCNBS Colour Charts [70]. Spore arrangement and spore surface ornamentation were observed following growth either Table 1. General features of the genomes of the isolates using the rast annotation server [49] KC201TKC401 KC310TKC712T6K102T Number of contigs 465 494 470 488 427 Genome size (bp) 9164341 9792882 10694046 10861993 10138478 Proteincoding genes 9216 9862 10 741 11 200 9989 RNA genes 70 74 77 70 68 G+C content (mol%) 70.6 70.6 70.9 70.3 70.8 N50 length (bp) 34 207 36 947 42 533 44 791 44 189 L50 83 79 75 78 66 640 Saygin etal., Int. J. Syst. Evol. Microbiol. 2020;70:636–647 Fig. 2. Phylogenomic tree based on whole genome sequence data of strains KC201T, KC310T, KC401, KC712T, 6K102T and closely related type strains of the family Streptosporangiaceae reconstructed on the Type (Strain) Genome Server (TYGS). The tree was inferred with FastME 2.1.6.1 [51] from GBDP distances calculated from genome sequences. The branch lengths are scaled in terms of GBDP distance formula d5. The numbers above branches are GBDP pseudobootstrap support values from 100 replications, with an average branch support of 97.3 %. The tree was rooted at the midpoint [52]. 641 Saygin etal., Int. J. Syst. Evol. Microbiol. 2020;70:636–647 on ISP 2 or ISP 4 media at 28 °C for up to 21 days by scanning electron microscopy (JSM 6060, jeol) instrument. Growth at different temperatures (4, 10, 20, 28, 37, 45 and 55 °C) and different pH values (pH 4.0–10.0, at intervals of 1.0 pH unit) and tolerance to several NaCl concentrations (0–15 %, at intervals of 1.0 %, w/v) were determined on ISP 2 agar (pH 7.0) after 28 °C for up to 14 days. The pH range for growth was evaluated after Saygin et al. [71]. The degradation of Tweens 20, 40 and 80 was tested as described by Nash and Krent [72]. The isolates were examined for their ability to degrade adenine, casein, chitin, gelatin, guanine, hypoxanthine, starch, xanthine and xylan using media and methods described by Williams et al. [67]. Aesculin, allantoin, arbutin and urea hydrolysis, nitrate reduction and H2S production were examined using established methods [67, 73–75]. In turn, carbon source utilization was recorded on ISP 9 agar [39] supplemented with either at 1 %, w/v or in the case of sodium succinate at 0.1 %, w/v. Similarly, the ability of the isolates was examined using media and method recommended by Williams et al. [67] supplemented with a final concentration of 0.1 % of the tested nitrogen sources. The closely related type strains, N. candida DSM 45086T, N. salmonea DSM 43678T, N. antimicrobica JCM 16904T, N. jiangxiensis JCM 18570 T and N. turkmeniaca DSM 43926 T , which were chosen by evaluating sequence similarities and phylogenetic trees formed on the basis of 16S rRNA gene, were also included for comparison in all tests. The isolates formed extensively branched substrate and aerial mycelia. The aerial hyphae was differentiated into spore chains or single spores with smooth surfaces (Fig.3). In general, the isolates and reference strains grew well on all of the growth media, notably on modified Bennett’s and ISP 7 and 4 agar (Table S2). A range of substrate mycelial pigments were detected ranging from cream to brown and reddish black. All but two of the strains formed white aerial mycelia on ISP 4 agar, aerial hyphae were not formed on modified Bennett’s agar or on ISP 5, ISP 6 or ISP 7 agar. It can be seen from Table3 that a broad range of phenotypic properties can be given weight to distinguish between the isolates and between them and the reference strains. In contrast, all of the isolates hydrolyzed aesculin, though none used Larabinose, myoinositol, Lrhamnose, Dsorbitol or Lsorbose as sole carbon sources. Similarly, all of the isolates grow from 28 to 37 °C, from pH 6 to 8 and in the presence of up to 3%, w/v sodium chloride. characteristics of strains are given in the species descriptions. It can be concluded that the isolates not only form distinct clades in phylogenetic and phylogenomic trees but also have chemotaxonomic and morphological properties consistent with their classification in the genus Nonomuraea [4]. It is also apparent from the ANI, dDDH and associated phenotypic data that isolates KC201 T , KC310 T , KC712 T and 6K102T merit recognition as Nonomuraea species, the names proposed for these taxa are Nonomuraea deserti sp. nov., Nonomuraea diastatica sp. nov., Nonomuraea longispora sp. nov. and Nonomuraea mesophila sp. nov., respectively. It is clear from the ANI and DDH data that isolate KC401 is a bona fide member of N. longispora. DESCRIPTION Of NoNomuraea deserti S P. NOv. Nonomuraea deserti sp. nov. ( de. ser′ti. L. gen. n. deserti of a desert, referring to the source of the type strain). Aerobic, Gramstainpositive, nonmotile actinobacterium that forms extensively branched substrate and aerial mycelia. Aerial hyphae differentiated to hooked chains of spores with a smooth surfaces. Good growth occurs on ISP 2, 3, modified Bennett’s and nutrient agar and moderate growth on ISP Table 2. 16S rRNA gene sequence and genome sequence comparisons between the isolates based on digital DNA–DNA hybridization (dDDH) and average nucleotide identity (ANI) analyses Strain 1 Strain 2 16S rRNA gene identity (%) dDDH ANIb ANIm KC201TKC401 99.73 88.20±2.5 98.04 98.67 KC310T99.19 47.50±2.4 90.70 92.66 KC712T99.19 46.50±2.4 91.04 92.43 6K102T99.39 61.00±2.9 94.58 95.28 KC401 KC310T99.32 47.30±2.5 91.22 92.61 KC712T99.19 46.30±2.5 90.67 92.39 6K102T99.53 60.60±2.8 94.17 95.22 KC310TKC712T99.73 61.50±2.8 93.85 95.40 6K102T99.26 46.50±2.5 90.72 92.44 KC712T6K102T99.25 45.60±2.6 90.54 92.19 642 Saygin etal., Int. J. Syst. Evol. Microbiol. 2020;70:636–647 4–7, Czapek’s and tryptic soy agar, substrate mycelia may be brown, cream or yellow. Does not produce diffusible or melanoid pigments. Grows from 28–37 °C (optimum, 28 °C), from pH 6.0–8.0 (optimum, pH 7.0) and in the presence of up to 3 % (w/v) NaCl. Hydrolyses aesculin and arbutin, reduces nitrate but does not form H2S and does not hydrolyse allantoin or urea. Gelatin, hypoxanthine and xylan are degraded, but not adenine, casein, chitin, guanine, starch, Tweens 40 or 80 or xanthine. DArabinose, cellobiose, dextran, Dfructose, Dglucose, lactose, Dmannitol and raffinose are utilized as sole carbon and energy sources, but not adonitol, Larabinose, dextrin, Dgalactose, Lglutamine, maltose, myoinositol, Dmannose, melezitose, melibiose, inulin, Lrhamnose, Dsorbitol, Lsorbose, sodium succinate, sucrose, xylitol or xylose. LArginine, Lasparagine, Lcysteine, glycine, Lhydroxyproline, Lmethionine, Lphenylalanine, Lproline, Fig. 3. Scanning electron microscopy images of isolates (a) KC201T grown on ISP 2 medium at 28 °C for 21 days, (b) KC401 grown on ISP 4 medium at 28 °C for 21 days, (c) KC310T, (d) KC712T and (e) 6K102T grown on ISP 2 medium at 28 °C for 14 days. Bars, 1 µm. 643 Saygin etal., Int. J. Syst. Evol. Microbiol. 2020;70:636–647 Table 3. Differential properties that distinguish between the isolates and between them and reference strains closely related type strains of Nonomuraea species. Strains: 1, KC201T; 2, KC401; 3, KC310T; 4, KC712T; 5, 6K102T; 6, Nonomuraea candida DSM 45086T; 7, Nonomuraea salmonea DSM 43678T; 8, Nonomuraea antimicrobica JCM 16904T; 9, Nonomuraea jiangxiensis JCM 18570T; 10, Nonomuraea turkmeniaca DSM 43926T. All data were obtained from this study unless indicated. nr, Not reported. 1 2 3 4 5 6 7 8 9 10 Isolation source Desert Desert Desert Desert Desert Soil* nr Leaf†Acidic soil‡nr NaCl range (%, w/v) 0–3 0–4 0–3 0–4 0–5 0–3 0–3 0–4 0–5 0–4 pH tolerance for growth 6–9 6–9 6–8 6–8 6–8 6–9 6–9 6–10 5–10 6–11 Temperature range for growth (°C) 20–37 20–37 28–37 28–37 20–37 28–45 28–37 20–37 28–37 20–45 Degradation of (%, w/v): Gelatin + + + + − + + − + + Starch + + − + + + + − − − Tween 40 − − − − + + − + − + Tween 80 − − − − + + − + − + Xylan + + + + − − + + + + Nitrogen source utilization (0.1 %, w/v): α-Isoleucine − + − + + + − − − + lAlanine − + − + + + + + − + lHistidine − + − − − + − − − + Biochemical tests: Arbutin hydrolysis + + + + − + + + + + Aesculin hydrolysis + + + + + + + − + + Nitrate reduction − + + + + + + − + + Carbon source utilization (1 %, w/v): Adonitol + − − − + + + − − + dArabinose − + + − + + + − − + dFructose − − + − + + + − + + dSorbitol − − − − − + − − − + dGalactose − − − − + − + + + + dMannose + + − − − − − − − − Melezitose − − − − + + − − − + Melibiose + + − − + − + − − + Dextrin − + − − − + − + + + Inulin − − − − + − + − − + lSorbose − − − − − + + − − + lArabinose − − − − − + + − + + lRhamnose − − − − − + + − − + Lactose − − + − + + + − − − Maltose + − − − + + + − + + Mannitol + + + + + + − − + − Continued 644 Saygin etal., Int. J. Syst. Evol. Microbiol. 2020;70:636–647 Lserine, Ltyrosine and Lvaline are utilized as sole nitrogen sources, but not Lalanine, Lhistidine or α-isoleucine. The predominant menaquinones are MK-9(H 4 ) and MK-9(H 6 ). The polar lipid profile includes diphosphatidylglycerol, phosphatidylmonomethylethanolamine, phosphatidylethanolamine, hydroxyphosphatidylethanolamine, dihydroxyphosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, three unidentified glycophospholipids, an unidentified phospholipid, an unidentified glycolipid and four unidentified lipids. Wholecell hydrolysates contain mesoA2pm, glucose, mannose, madurose, ribose and galactose. The major fatty acids are isoC16:0, isoC16:02OH and C17:010methyl. The DNA G+C content of strain KC310T is 70.9 mol% and the genome size 9.69 Mbp. The type strain, KC310T (=CGMCC 4.7331T=DSM 102919T=KCTC 39774T), was isolated from desert soil sample collected from the Karakum Desert, Turkmenistan. The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain KC310T is MG770639 and draft genome sequence accession number SMKO00000000. DESCRIPTION Of NoNomuraea diastatica SP. NOv. Nonomuraea diastatica ( di. a. sta′ ti. ca. N.L. fem. adj. diastatica from eng. n. diastase, a starchdegrading enzyme, derived from Gr. fem. n. diastasis a parting, a separation). Aerobic, Gramstainpositive, nonmotile actinobacter’um that forms extensively branched substrate and aerial mycelia. Aerial hyphae bear clusters of oval spores. Good growth occurs on ISP 2, modified Bennett’s, Czapek’s, tryptic soy and nutrient agar, moderate growth on ISP 4, 5 and 7 agar and weak growth on ISP 6 agar substrate mycelia may be brown or cream, not produce diffusible or melanoid pigments. Grows from 28–37 °C (optimum, 28 °C), from pH 6.0–8.0 (optimum, pH 7.0) and in the presence of up to 4 % (w/v) NaCl. Hydrolyses aesculin and arbutin, reduces nitrate but does not produce H 2 S and does not hydrolyse allantoin or urea. Gelatin, hypoxanthine, starch and xylan are degraded, but not adenine, casein, chitin, guanine, Tweens 40 or 80 or xanthine. Cellobiose, dextran, Dglucose, Dmannitol, raffinose and sodium succinate are utilized as sole carbon and energy sources, but not adonitol, Darabinose, Larabinose, dextrin, Dfructose, Dgalactose, Lglutamine, myoinositol, inulin, lactose, maltose, Dmannose, melezitose, melibiose, Lrhamnose, Dsorbitol, Lsorbose, sucrose, xylitol or Dxylose. LAlanine, Larginine, Lasparagine, Lcysteine, glycine, Lhydroxyproline, α-isoleucine, Lmethionine, Lphenylalanine, Lproline, Lserine, Ltyrosine and Lvaline are used as sole nitrogen sources, but not lhistidine. The predominant menaquinones are MK-9(H 4 ) and MK-9(H 6 ). The polar lipid profile includes diphosphatidylglycerol, phosphatidylmonomethylethanolamine, phosphatidylethanolamine, hydroxyphosphatidylethanolamine, dihydroxyphosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, three unidentified glycophospholipids, an unidentified phospholipid and three unidentified lipids. Wholecell hydrolysates contain mesoA2pm, glucose, mannose, madurose, ribose and galactose. The major fatty acids are C17 : 010methyl, isoC16:0 and C16:0. The DNA G+C content of strain KC712T is 70.3 mol% and the genome size 10.6 Mbp. The type strain, KC712T (=CGMCC 4.7334T=DSM 102925T=KCTC 39776T), was isolated from desert soil sample collected from the Karakum Desert, Turkmenistan. The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain KC712T is MG770679 and draft genome sequence accession number SMKP00000000. DESCRIPTION Of NoNomuraea loNgispora SP. NOv. Nonomuraea longispora ( lon. gi. spo′ra. L. adj. longus long; Gr. fem. n. spora spore; N.L. fem. n. longispora long spore). Aerobic, Gramstainpositive, nonmotile actinobacteria that form extensively branched substrate and aerial mycelia. Aerial hyphae differentiated into long straight spore chains with a smooth surfaces. Good growth occurs on ISP 2, ISP 4 agar, modified Bennett’s agar, tryptic soy agar and nutrient agar and moderately well on Czapek’s, ISP 3, 5, 6 and 7 agar. Substrate mycelia produce a range of pigments (cream, brown, reddish black or claret red). Melanoid pigments are not produced on ISP6 agar. Red or light pink diffusible pigments are produced on ISP 2, ISP 3 and modified Bennett’s agar. Grow from 20–37 °C (optimum, 28 °C), from pH 6.0–9.0 (optimum, pH 7.0), in the presence of NaCl 1 2 3 4 5 6 7 8 9 10 myoinositol − − − − − + + − − + Succinic acid − − − + − + − − + + Sucrose − − − − + + + − − + Xylose − + − − + − + − − + *Data taken from Le Roes and Meyers [77]. †Data taken from Qin et al. [18]. ‡Data taken from Li et al. [29]. Table 3. Continued