Nocardia noduli sp. nov., a novel actinobacterium with biotechnological potential

A genome-based polyphasic study was undertaken to establish the taxonomic status of an actinobacterium strain isolated from an actinorhizal root nodule. Strain ncl1T was found to have chemotaxonomic, cultural and morphological properties characteristic of members of the genus Nocardia. The strain was closely related to Nocardia aurea in the phylogenetic trees based on 16S rRNA gene and genome sequences. The draft genome of the strain is 8.9 Mbp in size, has a genomic DNA G + C content of 67.0% and was predicted to contain at least 19 biosynthetic gene clusters encoding for specialized metabolites. Strain ncl1T was distinguished from its closest neighbour, N. aurea DSM 103986T, by a broad range of phenotypic properties and by low average nucleotide identity and digital DNA-DNA hybridization scores. Consequently, the strain represents a novel Nocardia species for which the name Nocardia noduli sp. nov. is proposed. The type strain is ncl1T (CECT 30123T = DSM 110878T). The present study provides further evidence that actinorhizal nodules are a source of novel species of Nocardia. Supplementary Information The online version contains supplementary material available at 10.1007/s00203-022-02878-x.


Introduction
Data acquired from polyphasic taxonomic and genome-based studies led to marked improvements in the classification of the genus Nocardia and related mycolic acid-containing bacteria (Nouioui et al. 2018) which had previously been classified using a few subjectively weighted features. The genus forms a monophyletic clade within the family Nocardiaceae (Goodfellow and Maldonaldo 2012) and encompasses 120 validly published species (https:// lpsn. dsmz. de/ genus/ nocar dia) most of which have been recognized using combinations of genotypic and phenotypic properties (Nouioui et al. 2020) though the steady flow of new species shows that the genus remains underspeciated (Goodfellow and Maldonaldo 2012;Nouioui et al. 2020).
Nocardia are well known as serious causal agents of progressive invasive infections of humans and animals, notably mycetoma and nocardiosis (Conville et al. 2018), but are also seen to be successful saprophytes as they degrade complex organic compounds (Luo et al. 2014), and as attractive sources of new drug leads (Männle et al. 2020). Nocardia strains have been isolated from diverse ecological settings (Goodfellow and Maldonaldo 2012), notably from soil, sediments, sponges and wastewater treatment plants (Goodfellow and Maldonado 2012), as well as from coastal marine foams (Wright et al. 2021) and healthy plant tissues (Kaewkla and Franco 2010), including nodules of actinorhizal plants (Ghodhbane-Gtari et al. 2014) thereby suggesting they may be beneficial for plant growth (Nouioui et al. 2022).
The improved classification of the genus provides an invaluable framework for the detection of new Nocardia species. The present study was designed to establish the taxonomic status of a Nocardia strain isolated from a sterilized root nodule of Alnus glutinosa and to gain an insight into its biotechnological potential. The isolate ncl1 T was the focus of a genomic-based polyphasic study which showed that it represented a new species of the genus Nocardia for which the name Nocardia noduli sp. nov. is proposed. Genome mining Communicated by Erko Stackebrandt. showed that the type strain of the species has the potential to synthesize novel natural products, notably antibiotics and anticancer compounds.

Isolation, maintenance and cultivation
Strain ncl1 T was isolated from a surfaced sterilized root nodule of an Alnus glutinosa tree growing in Leazes Park, Newcastle upon Tyne, UK, as described by Nouioui et al. (2022). An active culture of the strain was obtained following growth on yeast extract-malt extract agar (International Streptomyces project [ISP] medium 2 (Shirling and Gottlieb 1966)). The strain was checked for purity and maintained in 35% (w/v) glycerol at − 80 °C, as was the type strain of N. aurea DSM 103986 T , which was obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ). Biomass for the chemotaxonomic studies carried out on the strains was harvested from ISP2 broths shaken at 200 rpm in baffled flasks for 7 days at 28 °C. The harvested cells were washed in distilled water and freeze dried.

Cultural and morphological properties of strain ncl1 T
Acid-alcohol-fast staining was carried out following growth on ISP2 agar for 10 days at 28 °C using the Ziehl-Neelsen method (Gordon 1967). Hanging drop preparations were examined to check for motility. The micromorphological properties of the strain were observed by light microscopy of the Gram stained smear. The ability of the strain to grow at a range of temperatures (4 °C, 10 °C, 15 °C, 25 °C, 28 °C, 37 °C, 42 °C and 45 °C) and pH (pH4.0-9.0 at intervals of 0.50 unit) was recorded from ISP2 agar plates. The pH levels were achieved using KH 2 PO 4 /HCl, KH 2 PO 4 /K 2 HPO 4 and K 2 HPO 4 /NaOH buffer systems. Growth and cultural properties were determined after incubation for 7 days at 28 °C on GYM agar (DSMZ medium 65), nutrient agar (NA, (Mac-Faddin 1986)), peptone-meat extract-glucose agar (DSMZ medium 250), tryptic soy agar (TSA, (MacFaddin 1986)) and ISP media 2, 3, 4 (Shirling and Gottlieb 1966). The ability of the strain to grow under anaerobic conditions was determined by inoculating it onto ISP2 agar and incubating at 28 °C for 7 days using anaerobic bag system (Sigma-Aldrich 68,061).

Molecular identification and genome sequencing
DNA was extracted from strain ncl1 T prior to PCR-mediated amplification of a 16S rRNA gene (Weisburg et al. 1991) and sequencing using the Sanger method (Sanger and Coulson 1975;Sanger et al. 1977). Genomic DNA of the strain was extracted, purified and quantified following the protocol provided by MicrobesNG (Birmingham, UK, https:// micro besng. com/ micro besng-faq/), then sequenced on an Illumina HiSeq 250 next generation sequencing platform using the 250 bp paired end sequencing protocol at MicrobesNG. Assembly of the reads and associated procedures were also carried out at MicrobesNG. The draft genome sequence of the strain was annotated using the RAST-SEED webserver (Aziz et al. 2008). The completeness of the genome sequence of the strain was estimated using BUSCO v.5.1.2 (Seppey et al. 2019).

Genome comparison
The draft genome of strain ncl1 T was compared with that of N. aurea SYSU K10002 T (accession number NZ_ QCYJ00000000.

Phylogeny
An almost full length 16S rRNA gene sequence extracted directly from the draft genome of strain ncl1 T (1523 pb) was deposited in the GenBank under accession number OK597210. This sequence was identical to the 16S rRNA gene sequence obtained using the Sanger method. The gene sequence was compared with corresponding gene sequences of the type strains of closely related Nocardia species retrieved from the EzBioCloud database (Yoon et al. 2017). 16S rRNA gene and whole-genome phylogenetic trees were inferred using the Type Strain Genome Server (TYG), available on the GGDC webpage (Meier-Kolthoff and Göker 2019).

Phenotypic and chemotaxonomic properties
Strain ncl1 T and N. aurea DSMZ 103986 T were examined for a range of phenotypic features; notably for their ability to use sole carbon and nitrogen sources; to grow at various pH values and in the presence of antibiotics and inorganic inhibitory compounds using GENIII microplates and an Omnilog device (Biolog Inc., Hayward, CA, USA), as described by Nouioui et al. (2020). The resultant data were analysed using opm package version 1.3.36 (Vaas et al. 2013). Enzymatic profiles of the strains were established using API-ZYM kits (BioMerieux, Lyon, France) by following the manufacturer's instructions. Catalase and oxidase tests were determined using standard procedures. All of the tests cited above were performed in duplicate. Standard chromatographic procedures were used to establish the polar lipid profile and diaminopimelic acid isomers using the integrated method of Minnikin et al. (1984) and the protocol of Schleifer and Kandler (1972), respectively. Whole-organism sugar patterns were determined after Lechevalier and Lechevalier (1970) and Staneck and Roberts (1974). Mycolic acids were extracted and purified as described by Goodfellow et al. (1976) and identified by gas chromatography (Agilent Technologies 6890 N, Santa Clara, CA, USA).
Cellular fatty acids extracted from the strain and N. aurea DSM 103986 T were methylated following Miller (1982), as modified by Kuykendall et al. (1988) and analysed by gas chromatography using the Agilent instrument. The resultant peaks were identified using the standard Microbial Identification (MIDI) system version 4.5 and the ACTIN6 database (Sasser 1990).

Biosynthetic gene clusters coding for specialized metabolites
The presence of natural product-biosynthetic gene clusters (NP-BGCs) in the genome of the isolate and the N. aurea strain were detected using antiSMASH, version 6.0 (Blin et al. 2019) with the detection relaxed option.

Cultural, chemotaxonomic and biochemical properties
Strain ncl1 T did not grow under anaerobic conditions, but did grow from 28 °C to 37 °C, optimally at 28 °C, and from pH 6-7.5. It formed orange colonies on GYM and ISP2 media and orange yellowish aerial hyphae on DSM 250 medium. It was also found to be catalase positive and oxidase negative.
Excellent congruence was found between the phenotypic data acquired on the duplicated tests. Table 1 shows that the isolate and N. aurea DSM 103986 T can be distinguished using a combination of phenotypic features though they also have many properties in common. Only the isolate was able to metabolise d-arabitol, D-fucose, β-gentiobiose, glucuronamide, N-acetyl-D-glucosamine, d-mannitol, methyl pyruvate and l-rhamnose (sugars), gelatin (polymer), and was resistant to vancomycin. In contrast, N. aurea, unlike the strain, metabolised 3-O-methyl-d-glucose, d-turanose, β-methyl-d-glucoside, and d-salicin (sugars) and was resistant to fusidic acid and minocycline.
Chemotaxonomic, colonial and micromorphological features of the strain were consistent with its classification in the genus Nocardia (Goodfellow and Maldonaldo 2012). The strain was found to be aerobic, nonmotile, Gram-stainpositive, partial acid-alcohol-fast and formed substrate and aerial hyphae that fragmented into rod and coccoid-like elements. It was shown to have meso-A2pm as the wall peptidoglycan, arabinose, galactose and glucose as whole-organism sugars; mycolic acids with 46-64 carbon atoms and a polar lipid profile composed of diphosphatidylglycerol, phosphatidylethanolamine (diagnostic phospholipid) and phosphatidylinositol and spots corresponding to unknown lipid, phosphoglycolipid and glycolipid components (Figure S1). The strain and the N. aurea strain were found to be rich in straight chain, saturated, unsaturated and 10-methyl (tuberculostearic) fatty acids though quantitative differences were found between the various components (Table S1). Both strains feature meso-A2pm as the wall peptidoglycan and arabinose, galactose and glucose as diagnostic sugars, but can be distinguished as the strain also contain glucose and the N. aurea strain ribose (Fang et al. 2019).

Genome comparison
The draft genome sequence of the strain has been deposited in the GenBank database under accession number JAFMPL000000000. The genome of the strain has a BUSCO score of 99.7% complete genes (355/356 from the actinobac-teria_class_odb10). The genome sizes of strain ncl1 T and the type strain of N. aurea were 8.9 Mbp and 8.3 Mbp, respectively, and the corresponding genomic DNA G + C contents were 67.0 and 67.4%. The dDDH similarity between the strains was 61.7% (Table 2), a value much lower than the corresponding threshold of 70% used to assign closely related strains to the same species (Wayne et al. 1987). However, the ANI value between these strains was shown to be 95%, and the AAI 93.7%, which is below the cut-off point of 95-96% for species demarcation (Goris et al. 2007;Richter and Rosselló-Móra 2009;Thompson et al. 2013).

Phylogeny
The maximum-likelihood 16S rRNA Nocardia tree (Fig. 1) showed that the strain formed a well separated branch in a clade that included the type strains of seven Nocardia species. Its closest relative was found to be N. aurea SYSU K10002 T (99.4%) in the phylogenomic tree. Greater confidence can be placed in the topology of phylogenomic trees as they are generated from millions of unit characters not limited to about 1500 as in the case of corresponding 16S rRNA gene trees (Nouioui et al. 2018). It is evident from the phylogenomic tree that the strain and N. aurea SYSU K10002 T formed a well-supported subclade that is most closely related to a second well delineated taxon composed of Nocardia arizonensis NBRC 108935 T and Nocardia takedensis NBRC 100417 T (Fig. 2). The resultant wellsupported clade was seen to be distant from corresponding clades encompassing several Nocardia species, notably Nocardia brasiliensis NBRC 14402 T and Nocardia vulneris NBRC 108936 T , and N. altamirensis NBRC 108246 T and Nocardia tenerifensis DSM 44704 T , respectively.
In contrast, only the N. aurea strain has bioclusters associated with the synthesis of cosmomycin D (5% gene identity), an anthracycline antibiotic which shows antitumor activity (Ando et al. 1985); platencin (6% gene identity), a betalactone which inhibits multi-drug resistant M. tuberculosis strains (Martens and Demain 2011); pepticinnamin E (6% gene identity), a farnesyl transferase inhibitor used in cancer, malaria, and trypanosome therapy (Santa et al. 2019); xantholipin, a polycyclic xanthone antibiotic derived from Streptomyces flavogriseus, which was found to be cytotoxic and to show antibacterial activity against Grampositive bacteria, including methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecalis (Wu et al. 2017). In addition, the aziridine alkaloid antibiotic ficellomycin (3% gene identity) and the type I PKs compounds tetarimycin A and B (3% gene identity) inhibit multi-drug resistant S. aureus strains (Kallifidas et al. 2012;He et al. 2018). The N. aurea strain also has the capacity to produce abyssomicin C/ atrop-abyssomicin C (25% gene similarity), polycyclic macrolactones, first detected in the type strain of Verrucosispora maris (now Micromonospora maris) (Nouioui et al. 2018), which show pronounced activity against multi-drug resistant Gram-positive bacteria (Fiedler 2021). Additional compounds associated with each of the strains are shown in Table 3. It is evident from the results outlined above that strain ncl1 T and N. aurea DSM 103986 T , its closest phylogenomic neighbour, have genomes rich in BGCs predicted to encode for novel polyketides. These results provide further evidence that nocardiae should feature more prominently in natural product discovery campaigns designed to find novel antibiotics of therapeutic value. This prospectus is in line with the view that novel Nocardia species represent a prolific source of natural products, one that rivals that of better characterised genera such as Amycolatopsis and Streptomyces (Männle et al. 2020).

Conclusions
Nodular tissues provide shelter and a plentiful supply of carbohydrates for bacteria, including actinobacteria found to belong to the genera Frankia, Micromonospora and Streptomyces. The present study provides further evidence that novel Nocardia species are associated with actinorhizal nodules though ecophysiological studies are needed to establish their ecological role and potential as inoculants to promote plant growth. Further, their ability to inhibit phytopathogens offers a role as an eco-friendly alternative to the use of pesticides in agriculture.
It can be concluded from the phylogenetic and phylogenomic trees that the strain forms a distinct lineage that is most closely related to the type strain of N. aurea. However, it can be distinguished from the latter using a broad range of phenotypic data and by low ANI and dDDH similarities. It is, therefore, proposed that strain ncl1 T represents a new species within the genus Nocardia for which the name Nocardia noduli sp. nov. is proposed.

Description of Nocardia noduli. sp. nov
Nocardia noduli (no'du.li. L. gen. dim. n. noduli, of a nodule, referring to the isolation of the type strain from a root nodule of Alnus glutinosa).
Aerobic, Gram-stain-positive, non-motile organism which forms substrate and aerial hyphae that fragment into rod-and coccoid-like elements. Orange colonies are formed on GYM and ISP2 agar. Grows from pH 5-7.5, optimally at pH 7.0 and from 28 to 37 °C, optimally at 28 °C, and in presence of up to 4% w/v sodium chloride. It is catalase positive, oxidase negative, able to metabolise D-arabitol, D-fucose, β-gentiobiose, glucuronamide, N-acetyl-d-glucosamine, D-mannitol, methyl pyruvate and L-rhamnose (sugars), gelatin (polymer), is resistant to vancomycin and positive for α-galactosidase, valine aminopeptidase, β-glucosidase, n-acetyl-β-glucosaminidase and naphthol-AS-BI phosphohydrolase. The diamino acid of the peptidoglycan is meso-A2pm, the whole-organism sugars are arabinose, galactose and glucose; the predominant fatty acids (> 10%) are C 16:0 , C 18:1 ω9c and 10-methyl C 18:0 , and the mycolic acids have 46-64 carbon atoms. The polar lipids consist of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, a glycophospholipid and two unidentified components. The genome size of the strain is 8.9 Mbp and its genomic DNA G + C content 67.0%. The genome is rich in biosynthetic gene clusters predicted to encode for specialized metabolites, notably antibiotics.
The type strain, ncl1 T (CECT 30123 T = DSM 110878 T ), was isolated from the root nodule of an Alnus glutinosa plant growing in Leazes Park, Newcastle upon Tyne, UK.
The 16S rRNA gene and genome sequences have been deposited in the DDBJ/ENA/GenBank databases under the accession numbers OK597210 and JAFMPL000000000, respectively. The Whole Genome Shotgun project has been deposited at DDBJ/ENA/GenBank under the accession number JAFMPL000000000. The version described in this paper is version JAFMPL010000000.

Acknowledgements
We are grateful to Dr. Katharina Huber, Dr. Meina Neumann-Schaal and Ms. Caroline Pilke (Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures, 38124 Braunschweig, Germany), for their support with some phenotypic analyses and to Dr. Juan P. Gomez Escribano (DSMZ), for support in estimating completeness of the genome. Genome sequencing was provided by MicrobesNG (http:// www. micro besng. com) which is supported by the BBSRC (grant number BB/L024209/1).
Author contributions IN and MG conceived the study. Material preparation, data collection and analysis were performed by all authors. The first draft of the manuscript was written by IN and MG and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.
Funding Open Access funding enabled and organized by Projekt DEAL. The authors declare that no funds, grants, or other support were received during the preparation of this manuscript.

Conflict of interest
The authors have no relevant financial or non-financial interests to disclose.
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