Amycolatopsis camponoti sp. nov., new tetracenomycin-producing actinomycete isolated from carpenter ant Camponotus vagus

An actinobacterial strain A23T, isolated from adult ant Camponotus vagus collected in Ryazan region (Russia) and established as tetracenomycin X producer, was subjected to a polyphasic taxonomic study. Morphological characteristics of this strain included well-branched substrate mycelium and aerial hyphae fragmented into rod-shaped elements. Phylogenetic analyses based on 16S rRNA gene and genome sequences showed that strain A23T was most closely related to Amycolatopsis pretoriensis DSM 44654T. Average nucleotide identity and digital DNA–DNA hybridization values between the genome sequences of isolate A23T and its closest relative, Amycolatopsis pretoriensis DSM 44654T, were 39.5% and 88.6%, which were below the 70% and 95–96% cut-off point recommended for bacterial species demarcation, respectively. The genome size of the isolate A23T was 10,560,374 bp with a DNA G + C content of 71.2%. The whole-cell hydrolysate contained meso-diaminopimelic acid and arabinose and galactose as main diagnostic sugars as well as ribose and rhamnose. It contained MK-9(H4) as the predominant menaquinone and iso-C16:0, iso-C15:0, anteiso-C17:0 and C16:0 as the major cellular fatty acids. Diphosphatidylglycerol and phosphatidylethanolamine prevailed among phospholipids. Mycolic acids were not detected. Based on the phenotypic, genomic and phylogenetic data, isolate A23T represents a novel species of the genus Amycolatopsis, for which the name Amycolatopsis camponoti sp. nov. is proposed, and the type strain is A23T (= DSM 111725T = VKM 2882T). Supplementary Information The online version contains supplementary material available at 10.1007/s10482-022-01716-w.


Introduction
The genus Amycolatopsis Lechevalier et al. 1986 belonged to the family Pseudonocardiaceae Embley et al. 1989(order Pseudonocardiales Labeda and Goodfellow 2015, class Actinomycetia Salam et al. 2020(Salam et al. 2020, phylum Actinomycetota corrig. Goodfellow 2021 (Oren and Garrity 2021)), and encompasses 85 validly published species names with Amycolatopsis orientalis as its type species (https:// lpsn. dsmz. de/ genus/ amyco latop sis). Amycolatopsis is aerobic to facultatively anaerobic and characterized by branched vegetative hyphae that undergo fragmentation into rod-like and squarish elements. Whole-cell hydrolysates are rich in meso-2,6-diaminopimelic acid, along with arabinose and galactose as whole-cell sugars. The peptidoglycan is of the A1g type. Muramic acid moieties are N-acetylated. Does not contain mycolic acids. The diagnostic phospholipid is phosphatidylethanolamine and/or phosphatidylmethylethanolamine while the occurrence of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol and phosphatidylinositol mannosides is variable (Tan and Goodfellow 2015). Amycolatopsis have been described from diverse environments such as soil, vegetation, human and animal clinical sources, fresh water, rock and subterranean sites (Lee 2009).
The 16S rRNA gene phylogeny provides a useful framework for inferring the relationships between genera in the Pseudonocardiaceae family, but did not seem to have enough taxonomic resolution to distinguish species. The use of multi-locus sequence analysis (MLSA) is able to clarify the taxonomic positions of Amycolatopsis species (Glaeser and Kämpfer 2015). The most reliable way to identify actinobacteria strains is the comparison of genomes from the point of view of DNA-DNA relatedness and construction whole-genome phylogenetic trees .
The genus Amycolatopsis comprises a large group of commercially and medically important actinobacteria capable of producing two major types of antibiotics: glycopeptides and polyketides (Kisil et al. 2021). In this paper, we describe strain A23 T , isolated from body of ant Camponotus vagus collected in Ryazan region, Russia. This strain is able to produce the aromatic polyketide antibiotic tetracenomycin X (TcmX) and its new congener 6-hydroxytetracenomycin X (6-OH-Tcm X) which possess antimicrobial and cytotoxic activity (Osterman et al. 2020;Alferova et al. 2021). As recently demonstrated TcmX and 6-OH-Tcm X are potent inhibitors of protein synthesis due to the fact that they bind to the large subunits of prokaryotic or eukaryotic ribosomes, within the polypeptide exit tunnel (Osterman et al. 2020).
Polyphasic study was used to clarify the taxonomic position of novel strain A23 T . We propose to establish this strain as a representative for the novel species of the genus Amycolatopsis, with the name Amycolatopsis camponoti sp. nov.

Collection and microbial isolation
Strain A23 T was isolated from bodies of adult workers of Camponotus vagus collected in Kasimovsky District, Ryazan region, Russia (55.01138 N, 41.73078 E) (Zakalyukina et al. 2021). Five individuals were washed three times in sterile distilled water and then crushed by tissue microhomogenizer with sterile saline solution. Aliquots of this mixtures were spread over M490 medium (HiMediaLab) supplemented with nystatin and nalidixic acid at final concentrations of 250 μg/mL and 10 μg/mL, respectively, and incubated for 14 days at 28 °C (Zakalyukina et al. 2019). The strain was purified and maintained on Organic medium 79 (Prauser and Falta 1968), and preserved as suspension of mycelial fragments and spores in 20% (v/v) glycerol at − 20 °C.

16S rRNA phylogeny
The full-length 16S rRNA gene sequences of strain A23 T was extracted from the whole genome sequence (CABVGP010000001.1) and was compared to sequences of type strains in the EzBioCloud database (www. ezbio cloud. net).
Evolutionary trees based on 16S rRNA gene sequences were inferred with the neighbour-joining, maximum-parsimony and maximum-likelihood treemaking algorithms after CLUSTAL W alignment by using MEGA software version X (Kumar et al. 2018) (https:// www. megas oftwa re. net). These evolutionary analyzes involved 24 nucleotide sequences. All positions with less than 95% site coverage were eliminated, i.e., fewer than 5% alignment gaps, missing data, and ambiguous bases were allowed at any position (partial deletion option). There were a total of 1414 positions in the final dataset.

Phenotypic characterization
Cultural characteristics of strain A23 T were observed on the range of ISP 2-ISP 7 media (Shirling and Gottlieb 1966), Organic medium 79 and modified Bennett's agar (Tan et al. 2006b) after cultivation up to 14 days at 28 °C. Cell morphology of strain A23 T on Organic medium 79 after cultivation at 28 °C for 10 days was studied using scanning electron microscopy (JSM-6380LA, JEOL). Motility test was carried out using the "hanging drop" method by light microscope (Fisherbrand AX-502, Thermo Fisher Scientific).
Carbon source utilization was assessed on basal medium ISP 9 (Shirling and Gottlieb 1966) with addition of 0.04% solution of bromocresol purple at 28 °C for 14 days. Enzyme activities were estimated using paper indicator system (NPO Microgen, Russia) according to the manufacturer's recommendations at 28 °C for 7 days. The degradation of casein, starch and cellulose was estimated on clearing of the insoluble compounds around areas of growth (Williams et al. 1983). The ability of strain to grow at a different pH (pH 5.0, pH 6.0, pH 7.0, pH 8.0, pH 9.0) was tested on a modified ISP 9 medium (g/L: (NH 4 ) 2 SO 4 -2.64, MgSO 4 × 7H 2 O-1, glucose-10, agar-20), buffered with phosphate solutions according to Williams et al. (Williams et al. 1971), at 28 °C for 14 days. The growth at different range of temperature (5 °C, 10 °C, 20 °C, 30 °C, 40 °C) and salinity (1%, 5%, 8%) were assessed on Organic medium 79 after 14 days of incubation.

Chemotaxonomy
Cell biomass of strains A23 T and A. pretoriensis DSM 44654 T were obtained from cultures grown in DSMZ 554 broth medium and on a rotary shaker (180 r.p.m) at 28 °C. After 120-h growth cells were centrifuged, washed three times in sterile distilled water and freeze-dried. Whole-cell sugars (Lechevalier and Lechevalier 1970;Staneck and Roberts 1974), menaquinone (Collins et al. 1985) and diaminopimelic acids analyses (Schleifer and Kandler 1972) were carried out. Standard chromatographic procedures were used to determine polar lipid pattern for strain A23 T and A. pretoriensis DSM 44654 T following the protocol of Minnikin et al. (1984). Cellular fatty acids extracts were prepared using minor modifications of the protocol of Miller (Miller 1982) and Kuykendall et al. (Kuykendall et al. 1988). Gas chromatography (Agilent 6890N instrument) was used to analyse the fatty acid methyl esters which were identified using Sherlock Microbial Identification system (MIDI, Microbial ID, Newark, DE 19711 U.S.A.) and the Actin6 database (Sasser 1990). The analysis was supplemented by a GC-MS run on an Agilent GC-MS 7000D for identity confirmation. Mycolic acids extraction was performed according to Vilchéze and Jacobs (Vilcheze and Jacobs 2007). Cell lysis was carried out in KOH/MeOH solution at 95 °C overnight and extracted with chloroform. Dried extracts were recovered in chloroform:MeOH (9:1) and analyzed in negative ion mode on an Agilent QTof mass spectrometer by direct infusion into the ESI source (300 µL/min). Mycolic acids were identified by comparison of the exact masses of known mycolic acid structures to the measured ones (Bouam et al. 2018).
For predicting the pathogenicity of A23 T and closely related strains we used PathogenFinder (http:// cge. cbs. dtu. dk/ servi ces/ Patho genFi nder/), a web-server for the prediction of bacterial pathogenicity by analysing the input genome (Cosentino et al. 2013).

Results and discussion
Amycolatopsis strains are well known as producers for the commercially used antibiotics, e.g. vancomycin, rifamycin, eremomycin (Kisil et al. 2021). Other compounds with antibacterial, antifungal or antiviral properties that have been derived from Amycolatopsis strains are quartromycin, octacosamicin, chelocardin, kigamicin and the macrotermycins A-D (Chen et al. 2016;Kumari et al. 2016;Beemelmanns et al. 2017). It has been shown that there is a relationship between the distribution of biosynthetic gene clusters (BGCs) and phylogenetic lineages in Amycolatopsis genomes. Amycolatopsis strains that produce, or have the potential to produce, a particular class of antibiotic are phylogenetically related (Adamek et al. 2018). Strain A23 T was previously known to produce bioactive compounds (Osterman et al. 2020;Alferova et al. 2021) was subjected to a polyphasic taxonomic approach to clarify its taxonomic position.
Phylogenomic analysis based on whole-genome sequences showed that strain A23 T formed a wellsupported monophyletic clade with A. pretoriensis DSM 44654 T with 100% bootstrap value (Fig. 1). The neighbor-joining (Fig. S1), maximum-likelihood ( Fig. S2)  Previous phylogenomic analyses revealed that the Amycolatopsis clade is divided into four major phylogenomic subclades (Adamek et al. 2018;Sangal et al. 2018). This study showed that strain A23 T is closely related to type strains within the group B subclades sensu Adamek et al. 2018 andSangal et al. 2018. The study of Sánchez-Hidalgo with colleagues presented in this study, based on GBDP distances calculated from genome sequences, showed consistency with previous studies (Fig. 1).
The complete genome size of strain A23 T was 10,560,374 bp with DNA G + C content of 71.2%, which was consistent with the G + C content of the genus Amycolatopsis (Teo et al. 2021). Similar genome features were observed for the closest neighbor A. pretoriensis DSM 44654 T (genome size 10,299,026 bp and G + C content 71.2%). Representatives of the group B are characterized by the presence of a high number of biosynthetic gene clusters ranged from 28 to 41 per genome (Adamek et al. 2018). That fact explains the ability of majority of this clade to produce antibiotics and/or bioactive molecules (Tan and Goodfellow 2015). The genome properties of strain A23 T and other type strains within group B sensu Adamek et al. 2018 andSangal et al. 2018 are summarized in Table 1.
The ANI and in silico DDH values between strain A23 T and strain DSM 44654 T were 39.5% and 88.6%, respectively. The ANI and in silico DDH values between strain A23 T and other related species of the genus Amycolatopsis were below the recommended thresholds of 95-96% and 70% for species demarcation (Ciufo et al. 2018; Meier-Kolthoff and Göker 2019) ( Table S1).
Cells of strain A23 T were aerobic, Gram-positive, non-motile. This strain showed a good growth on ISP 2-ISP 5 and moderate on both ISP 6 and ISP 7. The colours of substrate mycelia varied from light ivory to sulfur yellow while aerial hyphae usually were white or cream ( Table 2). The branching mycelia (diameter 0.42 µm) fragmented into different lengths rod-shaped elements (Fig. 2), which is typically observed for Amycolatopsis (Franco and Labeda 2014 (Table 3). The majority of considered strains including strain A23 T were able to utilize arabinose, fructose, galactose, sucrose and decompose of urea. They grew at 30 °C and in the presence of NaCl 1% w/v, but none produced amylases. The optimum growth temperature and pH of strain A23 T were 28-30 °C and pH 7, accordingly, but it was able to grow between pH 6.0-9.0 and up to NaCl 5.0% (w/v).
Online tool antiSMASH predicted 32 secondary metabolite gene clusters in the genome of strain A23 T (Table S5) and 30 ones in A. pretoriensis (Table S6). In total, from A23 T genome it was identified 11 PKS clusters, 10 NRPS and gene NRPS-like clusters, 2 RiPPs and 4 terpenes, while from DSM 44654 T there were 10, 10, 2 and 4 accordingly.
The genome mining of strain A23 T revealed that this strain has the potential to produce wide range of secondary metabolites including limazepines A, C-F and macrotermycins A-D (Table S5). The limazepines belong to the growing group of the pyrrolo[1,4]benzodiazepine antitumor antibiotics. Their inherent antitumor and antibacterial activities are due to their ability to regulate gene expression by recognizing and binding with DNA GC base pairs (Fotso et al. 2009). These limazepines' clusters were identical to that in strain Streptomyces sp. ICBB 8177, and showed also 82% of genes similarity with the sequence of tomaymycin in strain Streptomyces achromogenes (Li et al. 2009) (Fig. 3). Macrotermycin biosynthetic gene clusters with 96% similarity to that of Amycolatopsis  . 2 Scanning electron micrograph of strain A23 T , showing aerial mycelium that fragmented into rod-shaped elements after incubation on Organic medium 79 at 28 °C for 10 days strain sp. M39 (Beemelmanns et al. 2017) were found in the genome of strains A23 T (Fig. S8). Macrotermycins A-D are 20-membered glycosylated polyketide macrolactams's had antibacterial and also selective antifungal activity and were isolated from a termiteassociated actinomycete, Amycolatopsis sp. M39 (Beemelmanns et al. 2017). The DSM 44654 T genome contained the same clusters encoded limazepines and macrotermycins, supporting the view that phylogenetic similarity implies the presence of closely related biosynthetic pathways (Adamek et al. 2018).
Previously described tetracenomycin biosynthesis gene cluster and its paralogus Tcm2 cluster (Osterman et al. 2020) were detected in 1.7 and 2.5 regions of genome A23 T accordingly. Among all related strains A. rifamycinica DSM 46095 T and A. balhimycina DSM 44591 T have orthologous clusters in their  (Labeda et al. 2003); 7, A. mediterranei ATCC 13685 T (Lechevalier et al. 1986); 9, A. rifamycinica DSM 46095 T (Bala et al. 2004 (Osterman et al. 2020). Despite the relatively closeness, no Tcm clusters were detected in the genome sequence of strain DSM 44654 T and other related strains. The vast majority of Amycolatopsis from B-group were predicted as non human pathogen, the values of probability of being a human pathogen were below one (Table S7). Nevertheless, the protein families associated with pathogenicity were detected, so ABCtype transport system protein of Saccharomonospora viridis DSM 43017 was present in the genome of A23 T and all the strains examined (Table S7).
Based on phenotypic, phylogenetic and genomics analyses, strain A23 T , producer of a valuable substance tetracenomycin X, is considered as a type strain of a novel species with the proposed name, Amycolatopsis camponoti.
Aerobic, Gram-strain-positive, non-motile and filamentous actinobacteria. The aerial mycelia fragments into rod-shaped fragments (0.42 µm in diameter). Well-developed substrate mycelium varies from light ivory to sulfur yellow, and the colour of aerial mycelium usually is white on ISP 2-ISP 4, ISP 6, MBA and Organic 79 media. When growing for three weeks in liquid Organic medium 79, it produces soluble pigments that ranges from faintly brown to red.
The type strain is A23 T (= DSM 111725 T = VKM Ac-2882 T ), isolated from bodies of ants Camponotus vagus in Ryazan region, Russia. The genome size of the isolate A23 T is 10,560,374 bp with a DNA G + C content of 71.2%. The GenBank accession number for the 16S rRNA gene sequence and the genome Fig. 3 The BGCs in genome of Amycolatopsis A23 T A the structure of region 1.7 (location: 2,798,054-2,907,649 nt, total: 109,596 nt); B limazepines cluster of Streptomyces sp. ICBB 8177; C tetracenomycin X biosynthesis cluster assembly of strain A23 T are KY952635.2 and GCA_902497555, respectively.

Data availability statement
The GenBank accession number for the 16S rRNA gene sequence and the genome assembly of strain A23 T are KY952635.2 and GCA_902497555, respectively. Detailed data on genome analysis A23 T are presented IMG/M DataBase (https:// gold. jgi. doe. gov/).

Conflict of interest
The authors declare no conflict of interest.
Ethical approval This article does not contain any studies with human participants and/or animals performed by any of the authors. The formal consent is not required in this study.
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