ГЕНОМ БАКТЕРИАЛЬНОГО ШТАММА NONOMURAEA SP., ВЫДЕЛЕННОГО ИЗ ПЛОДОВОГО ТЕЛА ЧЕРНОГО ТРЮФЕЛЯ TUBER AESTIVUM

Genus Nonomuraea includes Gram-positive aerobic bacteria inhabiting diverse environments

Summer or black truffle Tuber aestivum is a fungus of the Tuber genus. This species can be found in Europe, including Russia, and is widely known for its black fruit bodies located underground. Fruit bodies are of a size 2-8 cm and are covered by black plaques which gave name to this truffle. T. aestivum grows in symbiosis with the roots of trees and also has many bacteria, fungi and protists inhabiting its fruitbody. All these living organisms, in relation to their presence and amount, can influence the condition of truffle, leading it to prosperity and oppression. As the truffles are promising organisms for different industries including biotechnology, information about organisms inhabiting them is necessary, for instance, for the comprehensive analysis and interpretation and prognosis of metabolite production. Therefore, detailed analysis of each of such organisms can improve the knowledge of T. aestivum. 

We sequenced, assembled and analyzed the genome of the novel Nonomuraea species isolated from the truffle Tuber aestivum. 16S rRNA sequence retrieved from the assembled contigs using ContEst16S tool

Strain of Nonomuraea sp. LPB2021202275-12-8 was isolated from wild ascocarp of Tuber aestivum collected in coniferous forest in Krasnodar, Russia (GPS 43.61824381815124, 39.84066203618042). The ascocarp surface was cleaned with a toothbrush. Then the ascocarp was split and the central part of the tuber gleba was dissected, homogenized in Tryptic Soy Broth (TSB, Kat. T8907, Merck, Germany) and plated on Potato Dextrose Agar nutrient medium (PDA, Kat. 1.10130, Merck, Germany). The spread plate method followed by the streak plate method was used to isolate a single colony of the strain. Ten passages were performed to ensure the purity of clones. Then confirmation of culture purity was checked during PCR identification of strain with universal eubacterial primers (27F 5'-AGAGTTTGATYMTGGCTCAG-3' and 1510R 5'-TACGGYTACCTTGTTACGACTT-3') used for 16S rRNA gene amplification. Then analysis of sequence chromatogram quality was performed as in

For DNA isolation, the strain was cultured for 36 hours in liquid TSB nutrient medium in Erlenmeyer flasks with deflectors at 28°C and shaking rate 180 rpm. DNA was extracted using MP Biomedicals™ Gnome™ DNA Isolation Kit (USA), for library preparation we used KAPA HyperPrep kit (Roche Sequencing Solutions, USA). The genome was sequenced on Illumina MiSeq using 250 bp paired-end reads.

As the next step, we performed genome assembly. The Uncicycler (v.0.5.0) tool performs raw reads quality control during assembly, so we performed genome de novo assembly using this tool with default parameters

Then we annotated the genome with PGAP (online program v.6.7 on the NCBI website, accessed on 08.03.2024) and predicted 9876 genes located in this genome. Annotation of secondary metabolite biosynthesis gene clusters was retrieved from antiSMASH (v.7.0, 19). In total, 30 genetic clusters were detected. Twenty-seven clusters were presented as novel and unknown. Among detected informative clusters we found clusters which were predicted to be associated with polyketides (alkylresorcinol, 100% similarity), terpenes (geosmin, 100% similarity) and NRP (coelichelin, 72% similarity). In theoretical and experimental studies it was shown that alkylresorcinols being natural chemical compounds that have anticancer activity, can be potential regulators of different processes including immune, antimicrobial and metabolic. For geosmin there are no observations about antimicrobial activity, but it may have warning chemical activity. It gives a smell similar to the soil smell and can attract or repel organisms. Coelichelin is less studied than two previous compounds and is involved in interactions with metals in cells. Taken together, found clusters have a potential for different medical and biotechnological purposes and can be considered in detail in further studies.

Overall, our study presents a novel genome sequence of Nonomuraea sp. isolated from T. aestivum and describes it for possible valuable secondary metabolites. This information can be used in further studies in a search for different drugs, including antibiotics, and extends the knowledge of genomes and their functions for this rare genus.

3.1. Data availability

Genome assembly project is available in NCBI BioProject database with accession number PRJNA1075312, assembly is available in NCBI Genome with accession number GCF_037044575.1, raw reads are available in NCBI SRA database with accession number SRX24330061, genome annotation is available in NCBI Gene database with accession number GCF_037044575.1. antiSMASH report is available on FigShare 10.6084/m9.figshare.26303158.

The truffle fungi is a well known source of microorganisms. They includes both bacteria, and truffle associated fungi. Our previous study revealed the symbiotic microorganisms related to such genera as Rahnella, Enterobacter, Stenotrophomonas, Acinetobacter, Citrobacter, Comamonas, Enterohabdus, Klebsiella, Lactobacillus, Massilia, Nitrobacter, Nocardioides, Pedobacter, Ralstonia, Raoultella, Rahnella, Rhizobium, Sphingobacterium, and Staphylococcus. Nonomurea sp., isolated in these study, was first time detected from the truffles.

Thus, the symbiotic organisms of truffle T. aestivum can be interesting and perspective object for biomedical and genetic engineering research.