
==== Front
Sci Rep
Sci Rep
Scientific Reports
2045-2322
Nature Publishing Group UK London

39256493
71969
10.1038/s41598-024-71969-0
Article
Social wasp-associated Tsukamurella sp. strains showed promising biosynthetic and bioactive potential for discovery of novel compounds
Rojas-Villalta Dorian 1
Núñez-Montero Kattia kattia.nunez@uautonoma.cl

2
Chavarría-Pizarro Laura laura.chavarria@itcr.ac.cr

1
1 https://ror.org/04zhrfn38 grid.441034.6 0000 0004 0485 9920 Department of Biology, Biotechnology Research Center, Instituto Tecnológico de Costa Rica, Cartago, Costa Rica
2 https://ror.org/010r9dy59 grid.441837.d 0000 0001 0765 9762 Facultad de Ciencias de la Salud, Instituto de Ciencias Aplicadas, Universidad Autónoma de Chile, Temuco, Chile
10 9 2024
10 9 2024
2024
14 2111811 7 2024
2 9 2024
© The Author(s) 2024
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
In the face of escalating antibiotic resistance, the quest for novel antimicrobial compounds is critical. Actinobacteria is known for producing a substantial fraction of bioactive molecules from microorganisms, nonetheless there is the challenge of metabolic redundancy in bioprospecting. New sources of natural products are needed to overcome these current challenges. Our present work proposes an unexplored potential of Neotropical social wasp-associated microbes as reservoirs of novel bioactive compounds. Using social wasp-associated Tsukamurella sp. strains 8F and 8J, we aimed to determine their biosynthetic potential for producing novel antibiotics and evaluated phylogenetic and genomic traits related to environmental and ecological factors that might be associated with promising bioactivity and evolutionary specialization. These strains were isolated from the cuticle of social wasps and subjected to comprehensive genome sequencing. Our genome mining efforts, employing antiSMASH and ARTS, highlight the presence of BGCs with minimal similarity to known compounds, suggesting the novelty of the molecules they may produce. Previous, bioactivity assays of these strains against bacterial species which harbor known human pathogens, revealed inhibitory potential. Further, our study focuses into the phylogenetic and functional landscape of the Tsukamurella genus, employing a throughout phylogenetic analysis that situates strains 8F and 8J within a distinct evolutionary pathway, matching with the environmental and ecological context of the strains reported for this genus. Our findings emphasize the importance of bioprospecting in uncharted biological territories, such as insect-associated microbes as reservoirs of novel bioactive compounds. As such, we posit that Tsukamurella sp. strains 8F and 8J represent promising candidates for the development of new antimicrobials.

Keywords

Genome mining
Ecological relationships
Antibiotics discovery
Natural products
Bioprospecting
Evolutionary specialization
Subject terms

Biological techniques
Ecology
Microbiology
Instituto Tecnológico de Costa RicaVIE-1510118 [2019-2020] VIE-1510143 [2021] VIE-1510170 [2022] VIE-1510183 [2023] projects issue-copyright-statement© Springer Nature Limited 2024
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pmcIntroduction

Actinobacteria is considered one of the most important phyla regarding their extensive biotechnological applications. They are known to produce a wide range of natural products involved in antibacterial, antifungals, antivirals, and antitumorigenic activities, among others1–3. This group of bacteria is reported to produce at least more than one third of the total bioactive molecules isolated from microorganisms4, therefore representing intensively studied and promising microorganisms.

Regardless, the bioprospecting for natural products fell into metabolic redundancy, meaning that those compounds found in isolated species are the same or highly similar to other previously reported5. This led to a need for more specific and specialized area of research, such as microorganisms from unexplored environments, vaguely studied species, and bacteria adapted to hazardous conditions (extremophiles)6–8. In this sense, insect-associated microbes have been promoted as a hot-spot for novel bioactive molecules discovery9.

The insect’s microbiome presents a diverse bacterial composition, which plays a relevant role in the host’s biology and physiology10. These microorganisms might take part in different aspects, including development, defense, detoxification, food digestion, nutrition, and immunity of the host11. The dynamics involved in their ecological relationship are thought to have a strong impact on the evolutionary trajectory of associated bacteria, especially those related to defensive functions12–14.

The exploration of insect-associated bacteria has resulted in promising results of antibiosis assays15, even against multidrug-resistant human pathogens16. Moreover, common actinobacteria species have been reported with novel bioactive compounds, possibly due to ecological specialization17. Social wasps are considered among the insects with promising bacterial symbionts for antimicrobial molecules bioprospection18. These insects are known to coevolve with bacteria as a defense strategy to prevent pathogens growth18. The established symbiotic relationship and bacteria specialization propose the hypothesis of a wide diversity of undiscovered bioactive metabolites in associated microorganism19.

Nowadays, the discovery of biological molecules is favored by the development of sequencing techniques. Genomics has allowed in silico predictions of genes related to production of bioactive compounds called biosynthetic gene clusters (BGCs)20. Several software tools have been developed for this regard. The antibiotic and Secondary Metabolite Analysis SHell (antiSMASH)21 and Antibiotic Resistance Target Seeker (ARTS)22 are among the most popular, as they perform accurate and precise ‘genome mining’ for BGCs in prokaryotic genomes. This facilitated the anticipation of novel molecules and chemical structure prediction, fastening the discovery of antibiotic compounds23,24.

We have previously sequenced the genome of two rare actinomycete strains, Tsukamurella sp. 8F and 8J, isolated from the cuticle of social wasps25, which are not classified among the known Tsukamurella species. On the other hand, the Natural Products Atlas shows seven metabolites isolated from Tsukamurella species (accessed August 8th, 2024)26. These include compounds with antibacterial (harundomycin), cytotoxic (tsukalipids), and antioxidant (lipocarbazoles) properties27–29. Our group has also confirmed the antibiotic activity of strains 8J and 8F30. Both of our strains inhibited the growth of the insect pathogen Bacillus thuringensis and human pathogen Escherichia coli, while Tsukamurella sp. 8J repressed Pseudomonas aeruginosa30.

Given the demonstrated antimicrobial activity of this previously unexplored microbial resource and the importance of utilizing novel approaches for antibiotic discovery, this study aimed to assess its biosynthetic potential of Tsukamurella sp. 8F and 8J. We also evaluated their phylogenetic and genomic characteristics, examining how environmental and ecological factors might be linked to their promising bioactivity and evolutionary specialization.

Materials and methods

Genome mining for biosynthetic gene clusters

Two bacterial strains (8F and 8J) identified as part of the Tsukamurella genus were previously isolated from social wasps (Vespidae; Polistinae: Epiponini) obtained from Refugio Nacional de Fauna Silvestre Golfito (Costa Rica, 8°39′15.5″ N, 83°10′45.1″ W)25. Further details on strain isolation, DNA sequencing, and genome assembly and annotation are reported in25. Draft genomes are available at National Center for Biotechnological Information (NCBI) under the accession numbers ASM2916737v1 and ASM2916740v1, respectively. The genome sequences of Tsukamurella sp. 8J and 8F strains were annotated looking for biosynthetic gene clusters (BGCs) using antiSMASH v7 beta21 with the detection strictness set to ‘relaxed’. We selected those BGCs with core biosynthetic genes located in the middle of the cluster, excluding those in contig edges, ensuring BGCs completeness. To identify antibiotic resistance genes (ARGs) that could incur in the bioactivity of the strain, the resistome of both strains was predicted through Resistance Gene Identifier (RGI) v.6.0.0 web portal31 (https://card.mcmaster.ca/analyze/rgi). Here, the Open Reading Frames are predicted by Prodigal v2.6.332 and sequences are aligned using DIAMOND software v2.1.033 against the Comprehensive Antibiotic Resistance Database (CARD) v3.2.531 under the Perfect and Strict paradigm of criteria. For a target-directed genome mining, we annotated both strains with the Antibiotic Resistance Target Seeker v2 (ARTS, https://arts.ziemertlab.com/index)22. This tool predicts the mode of action of compounds encoded by uncharacterized BGCs, based on the presence of resistance genes in close proximity, phylogenetic evidence of HGT events and duplication of housekeeping genes withing the cluster by automated target directed genome mining of BGCs with potential antibiotic activity22. To achieve this, ARTS detects possible resistance housekeeping genes with TIGRfam Equivologs based on: (a) rare duplication (all essential genes over the median and standard thresholds for the sum identified in the bacterial family), (b) proximity to a BGC (based on antiSMASH annotation), and (c) evidence for horizontal gene transfer (by comparing the topology of core gene phylogeny), therefore predicting the mode of action of the encoded compounds in uncharacterized BGCs prioritized by ARGs22. To improve the bioprospecting of clusters encoded with potential new antibiotic metabolites, we manually selected the TIGRfam annotated genes which presented two or three of the previously mentioned characteristics.

Phylogenetic and functional analysis of the Tsukamurella genus

To understand more about the evolutionary relationships of our Tsukamurella sp. strains, we carried out a phylogenetic analysis based on multi-locus sequences using the Automated Multi-Locus Sequence Analysis (autoMLSA) web server (https://automlst.ziemertlab.com/)34. We extracted the representative Tsukamurella species available at the NCBI (accessed 01/23/2023). To evaluate the relationship to sister groups, four complete representative genomes of each Gordonia and Rhodococcus genera were randomly downloaded from the NCBI (accessed 08/11/2024). Previous studies have demonstrated that Tsukamurella species share common features to these bacterial groups35, hence representing a compelling choice for this analysis. The reference genome of Escherichia coli K-12 (accession ASM584v2) was used for indicating outgroup in the phylogenetic tree. autoMLSA calculates Average Nucleotide Identity (ANI) values for all query genomes against its database, selecting a total of 50 genomes, half with the nearest values to entire set and other half to individual queries34. Gene homologs are searched among selected genomes and added to a list of pre-identified homologs, to finally identify a maximum of 100 single copy homologs. Finally all selected genes are aligned and used to infer a maximum-likelihood tree34. Resulting tree was visualized using iTOL v6.636.

Name, source of isolation and genome size were obtained from the representative genomes. Files in .fasta format for each species were analyzed on antiSMASH v7 beta21 to predict their BGCs. The results were organized with the number of clusters per type of secondary metabolite. We also calculated the percentage of genome dedicated for secondary metabolism by the summary of total base pairs lengths of BGCs divided by genome size. All data was classified and analyzed according to the source of isolation of each sample (Insect, waste waters, human and plastic).

Results and discussion

Tsukamurella sp. 8F and 8J present BGCs with low to no similarity to known molecules, including antibiotics specific clusters

The Tsukamurella sp. 8J and 8F showed 19 and 17 identified biosynthetic gene clusters (BGCs), respectively. Both strains presented the greatest similarity in BGCs encoding for Non-Alpha PolyAmino group Acids (NAPAA, 100% similarity with ε-Poly-l-lysine) and aminopolycarboxylic acid (100% similarity with [S,S]-EDDS) (Table 1). Notably, only two clusters for each strain were over the threshold for confident similarity (> 75%), an ectoine and a hybrid (ectoine/nocobactin NA) (Table 1). The rest of the identified BGCs had low to no similarity (0–36%) with known clusters, mainly related with genes involved in the biosynthesis of antibiotic compounds (e.g., sisomicin, arginomycin, ambruticin). Both strains presented similar BGCs except for one ectoine and one Type 1 Polyketide Synthase (T1PKS) cluster only present in 8J strain.Table 1 Genomic annotated BGCs in 8J and 8F strains and most similar known cluster when identified.

Strain	Region	Type	From	To	Most similar known cluster	Similarity (%)	
8J	Region 1.1	Terpene	223,597	244,433	Sisomicin	Saccharide (5%)	
Region 2.1	Terpene	81,824	102,861	Lipopolysaccharide	Saccharide:lipopolysaccharide (5%)	
Region 4.1	T3PKS	117,106	158,347	Asukamycin	Polyketide:type II	
Region 5.1	redox-cofactor	60,005	82,858	Griseusin	Polyketide (4%)	
Region 6.1	NRP-metallophore, betalactone, NRPS	1	43,626	Nocobactin NA, nocobactin NA 10152B*†	NRP + polyketide (75%)	
Region 7.1	hglE-KS	33,797	79,991	–	–	
Region 7.2	NAPAA	90,787	124,668	e-Poly-l-lysine*†	NRP (100%)	
Region 12.1	RiPP-like	65,661	76,458	–	–	
Region 13.1	T1PKS	1	45,868	Arginomycin	Other (13%)	
Region 15.1	T1PKS	1	27,311	–	–	
Region 18.1	NRPS, T1PKS	28	69,451	Foxicins A–D	NRP + polyketide (4%)	
Region 23.1	T3PKS, butyrolactone	1	29,451	–	–	
Region 24.1	NRP-metallophore, NRPS	1	64,996	Heterobactin B, heterobactin S2	NRP (36%)	
Region 25.1	Ectoine	1	5420	Ectoine*†	Other (75%)	
Region 34.1	T1PKS	15,421	43,138	–	–	
Region 36.1	Ectoine	34,384	40,265	–	–	
Region 41.1	Aminopolycarboxylic-acid, ectoine	1	11,963	[S,S]-EDDS*†	Other (100%)	
	Region 55.1	Arylpolyene	1	21,197	Dactylocycline A	Polyketide (5%)	
	Region 67.1	T1PKS	1	12,977	Ambruticin*	Polyketide (17%)	
8F	Region 1.1	Redox-cofactor	230,144	252,997	Griseusin	Polyketide (5%)	
Region 2.1	Terpene	149,464	170,501	Lipopolysaccharide	Saccharide:lipopolysaccharide (5%)	
Region 3.1	NAPAA	45,581	79,462	e-Poly-l-lysine*†	NRP (100%)	
Region 3.2	hglE-KS	90,258	136,452	–	–	
Region 3.3	NRP-metallophore, NRPS	157,232	246,720	Heterobactin B/heterobactin S2*	NRP (36%)	
Region 5.1	Terpene	212,504	233,340	Sisomicin	Saccharide (5%)	
Region 6.1	RiPP-like	193,358	204,155	–	–	
Region 8.1	T1PKS	75,640	140,538	Ambruticin*	Polyketide (11%)	
Region 10.1	NRP-metallophore, betalactone, NRPS	104,064	151,811	Nocobactin NA, nocobactin NA 10152B*†	NRP + polyketide (75%)	
Region 14.1	T1PKS	1	45,874	Arginomycin	Other (13%)	
Region 18.1	Aminopolycarboxylic-acid	34,572	52,435	[S,S]-EDDS*†	Other (100%)	
Region 22.1	T1PKS, NRPS	33	66,427	Foxicins A–D	NRP + polyketide (4%)	
Region 23.1	T3PKS, butyrolactone	1	29,457	–	–	
Region 25.1	Ectoine	1	5423	Ectoine*†	Other (75%)	
Region 34.1	T1PKS	15,459	43,176	–	–	
Region 36.1	T3PKS	7841	40,513	Asukamycin	Polyketide: type II (4%)	
Region 52.1	Arylpolyene	1	16,517	–	–	
“Region” refers to the contig in which BGC is encoded. –, not found.

T#PKS type # polyketide synthases, NAPAA non-alpha polyamino group acids, hglE-KS heterocyst glycolipid synthase-like PKS, NRP non-ribosomal peptide, NRPS non-ribosomal peptide synthases, RiPP ribosomally synthesized and post-translationally modified peptide.

*Shares one or more core biosynthetic genes with our cluster.

†Similarity to known cluster surpasses > 75% threshold for confident match.

Recent advances in genomic research facilitates the data mining of bioactive secondary metabolites, and it is considered a valuable tool to uncover novel molecules37. Previous studies have found a wide range of biosynthetic gene clusters encoded in the genome of insect-isolated bacteria, including Non-Ribosomal Peptide Synthases (NRPS), bacteriocins, siderophores, terpenes19,38,39. These molecules with antimicrobial activity might have a role for the control and communication among the microbiome present in their host, as for the protection from potential entomopathogens18,40. Additionally, actinomycetes-derived compounds, similar to those here annotated, are considered bioactive against a wide spectrum of microorganisms. Some NRPS and T1PKS reported a promising antimicrobial effect against the human pathogens: Bacillus subtilis, Staphylococcus aureus, Candida albicans and Streptomyces sp. K1-0141. In bacterial species, the presence of terpenes, siderophores, lanthipeptides, and butyrolactones can be associated with the capacity of inhibiting bacterial growth and antibiotic production42,43. Therefore, the great amount and diversity of annotated BGCs encoding for antimicrobial compounds and other molecules with previously stated bioactivity suggests the idea of the Tsukamurella sp. 8F and 8J are favorable strains for bioactive compounds production.

Although low similarities are found between the annotated BGCs and known clusters, some BGCs (marked with *) share core biosynthetic genes within the reference and query clusters (Table 1). This is an indicative of these BGCs encoding for possible novel antimicrobial molecules with new targets or different molecular structure. The investigation of compounds with these characteristics is considered a promising strategy to fight the increasing antibiotic resistance44. Several new natural products have been isolated from insect-associated actinomycetes, demonstrating an antagonistic effect in the development of other microorganisms9,19,45. Bacterial strains isolated from ants and termites exudated new compounds identified as porphyrins and natalenamides with antimicrobial and insecticidal properties46,47. Regarding wasps, chemical analysis of the antifungal and antibacterial potential of 15 Streptomyces strains provided insights of insect-associated bacteria as valuable sources of novel molecules48. Other studies have contributed to stablish this idea by investigating bacterial extract with encouraging inhibitory potential against common human pathogens17. The wide variety of bioactive compounds and activities presented in bacteria isolated from insects and our results promote our Tsukamurella sp. strains as relevant for bioprospecting for new natural products.

Regarding the resistome prediction, Tsukamurella sp. 8F and 8J showed the same antibiotic resistance genes (ARGs), between our strains. Genes similar to vanX, vanH, vanO and vanW (from the CARD database) were detected in a strict protein homolog model indicating resistance against vancomycin and teicoplanin (ARO accessions: 3002954, 3002948, 3002913 and 3003724, respectively). Tsukamurella species are considered rare opportunistic human pathogens and the presence of ARGs is expected49,50. However, contrary to our results, most human isolates are resistant to penicillins and cephalosporins49.

On the other hand, antimicrobial resistance genes (ARGs) are commonly found in bacteria that produce antibiotics, serving as a mechanism to prevent self-toxicity51. Consequently, the presence of ARGs within a particular BGC is used as an indicator that the cluster may encode antibacterial molecules. In this study, a target-directed genome mining for antibiotic compounds through ARTS software identified some of the annotated cluster as possible producers of antibiotic molecules, based on the identification of known ARG, and essential genes duplication and HGT that might be related to ARG (Table 2). Particularly, TIGRfam genes with known and unknown functions are present and duplicated in both strains, on a BGCs related to the production of Terpene, PKS, ectoine and aminopolycarboxylic acid.Table 2 Tsukamurella sp. 8J and 8F strains’ BGCs and antibiotic auto-resistance-related genes identified through ARTS prediction.

Strain	Region	Type: most similar known cluster (similarity)	Genes: description	Function	
8J	1.1	Terpene: sisomicin (5%)	GrpE: GrpE	Unclassified	
		TIGR02350: chaperone protein DnaK	Protein fate	
4.1	T3PKS: asukamycin (4%)	TIGR01311: glycerol kinase	Energy metabolism	
13.1	T1PKS: arginomycin (13%)	TIGR00105: ribosomal protein bL31	Protein synthesis	
		TIGR01131: ATP synthase F0, A subunit	Energy metabolism	
		TIGR00220: large conductance mechanosensitive channel protein	Cellular processes	
36.1	Ectoine: not found	TIGR00494: protein CrcB	Unknown function	
8F	5.1	Terpene: sisomicin (5%)	GrpE: GrpE	Unclassified	
		TIGR02350: chaperone protein DnaK	Protein fate	
14.1	T1PKS: arginomycin (13%)	TIGR00105: ribosomal protein bL31	Protein synthesis	
		TIGR01260: ATP synthase F0, C subunit	Energy metabolism	
		TIGR01131: ATP synthase F0, A subunit	Energy metabolism	
18.1	Aminopolycarboxylic-acid: [S,S]-EDDS (100%)	TIGR00494: protein CrcB	Unknown function	
“Region” refers to the contig in which BGC is encoded.

The gene prediction due to duplication and Horizontal Gene Transfer (HGT) events specifies for discovering new antibiotic drugs, while unknown function genes incur in the idea of bioactive molecules with new targets22. As stated before, this is considered relevant for antibiotic bioprospecting against the global resistance crisis44. Primary metabolism is one of the main targets for the antibiotics’ mode of action. Duplication of the essential genes is a strategy to develop resistance, and duplicated genes seem to be strongly associated with plasmids, mobilizing by HGT52,53. Our results exhibited the duplication and HGT events, as predicted by the ARTS software, of genes associated to protein fate, energy metabolism, protein synthesis and cellular processes (Table 2). These functions fall among the categories previously proven to be duplicated in order to confer antibiotic resistance52. The presence of these resistance genes within the BGCs supports the idea of the clusters encoding antimicrobial compounds, as they might represent a mechanism to avoid self-toxicity.

Moreover, the majority of identified BGC are correspondent to polyketides-synthases-encoding (PKS) with low similarity to known compounds (4% to 13%), which might indicate new or known molecules with variable substituents54. Some of these BGCs share biosynthetic genes with reference cluster encoding for compounds with common antimicrobial activity (e.g. ambruticin). The annotation of resistance genes and BGCs of known antimicrobials raises speculations about these clusters coding for novel or modified bioactive compounds. A similar hypothesis has been stablished for other PKS clusters with low similarity affiliated with Actinobacteria phylum55. The overall results support the proposed bioactive potential of our strains for antimicrobials bioprospection (48). It should be noted that we have conducted a predictive approach based on genome mining, hence, further research regarding chemical analysis should be conducted to confirm the nature, novelty and structure of the secondary metabolites resulting from our Tsukamurella strains.

The production of natural products with new targets andsimilarity to cluster annotated compounds have been largely associated with other actinobacteria, especially Streptomyces strains56–58. Based on our results, we annotated three BGCs with low similarity to known antimicrobial molecules (asukamycin, sisomicin and arginomycin). Besides presenting a high antibacterial activity59, the engineering of asukamycin and sisomicin led to an increased yield production, antibiotic activity, and possible repurposing to amplify their spectrum of action56,57. In this sense, our Tsukamurella strains must be considered as study subjects for possibly new and similar bioactive metabolites of genetic engineering interest, yet another strategy against the undergoing global antibiotic crisis44.

There is limited knowledge regarding the antimicrobial activity of Tsukamurella species. However, previous analysis showed low to no inhibition against indicator microorganisms, including Escherichia coli and Staphylococcus aureus60. A similar result was reported previously by our group regarding the in vitro antimicrobial potential of strains included in this study. Strains 8J and 8F showed inhibition against E. coli and the insect pathogen Bacillus thuringensis, while 8J showed also activity against the growth of S. aureus,30. Insect-associated bacteria have reported a large evolution history of horizontal gene transfer, as a mechanism to establish a symbiotic relationship with their host organism61. Within these acquired genetic elements, BGCs encoding for antimicrobial compounds are common, possibly to potentiate the defensive role of some bacteria against host-antagonist microbes62,63. Other authors have isolated Tsukamurella strains from agricultural crops and, while unknowing their specific function, it is thought that these bacteria can inhibit the growth of phytopathogenic fungi64. This hypothesis indicates that our strains might take part of the beneficial microbiome of the wasp cuticle65, a function perhaps acquired for specialization in a symbiotic relationship. This idea is also supported by the high bioactivity obtained by one of our strains (8F) against the insect-pathogen B. thuringensis, despite the lack of bioactivity by Tsukamurella strains reported in literature30,60.

Environmental and ecological factors correlated with the diversification and specialization of the genomes of Tsukamurella genus

Considering our strains previous results, we carried out a phylogenetic analysis with other Tsukamurella species in order to evaluate if the possible environment specialization case is evidenced in the genomic composition. The proteome core based phylogenetic tree showed our strains in a completely different clade from those previously reported Tsukamurella species (Fig. 1). As mentioned in the genome announcement, both 8F and 8J might belong to a novel Tsukamurella species25. This is supported by an Average Nucleotide Identity (ANI) analysis, where our strains had values above the threshold (> 95%) that defines species (99.98% similarity), while other species were below25. The insect-isolated bacterium Tsukamurella paurometabola had the closest relationship with 8J and 8F strains. This result is consistent with Average Nucleotide Identity (ANI) analysis, where this species presented the highest similarity with our strains (79.4%)25.Fig. 1 Rooted maximum-likelihood phylogenetic tree based on multi-locus sequences analysis of in the available representative genomes of the Tsukamurella genus and four randomly selected complete representative genomes from Gordonia and Rhodococcus genera. The reference genome of Escherichia coli K-12 was used for indicating outgroup. Phylogenetic tree was generated using autoMLSA66. NCBI accession is specified next to each species name. Color code represents the source of the only Tsukamurella species. For those without color, the source is unknown The branches’ length represents the number of substitutions per site (scale: 0.1 substitution per site) and percentage of 1,000 replications bootstraps is shown. QS  query sequence, TS  type strain, OG  outgroup. Species starting with TS, OG or without acronyms represent those automatically selected from tool’s database based on nearest ANI values to query genomes.

Moreover, common human pathogen species (T. tyroinosolvens, T. asaccharolytica, T. ocularis, T. conjunctivitidis, T. pulmonis and T. sputi) were clustered together away from 8J and 8F. This might indicate a high specialization regarding their pathogenesis67. Additionally, T. spumae and T. pseudospumae, both isolated from waste waters samples, were located in the same clade apart from human pathogens and insect-isolated species.

Tsukamurella species seem to be relevant part of the microbiota of insects, although its function remains unknown68. A previous study supports our idea of high specialization of Tsukamurella genera, regarding their environments and ecology69. The findings evidenced Tsukamurella as the second most abundant bacterial community in Atta sexdens ants (Hymenoptera), through a metagenomic approach69. Similarity percentage (SIMPER) analyses of beta diversity revealed that Tsukamurella had a significant impact on the differences in ants’ microbiota between habitats and geographic location69. In this case, Tsukamurella species were thought to be highly specialized and influential on the microbiota composition due to antimicrobial properties of the bacteria, advantageous in those specific habitats69.

Furthermore, other phylogenetic analysis of arthropod microbiome presented the same pattern of symbionts clustered together away from other species70. Remarkably, a deep analysis from the ecological perspective indicated that microbial composition is related to bacterial interactions and their functional role in the host71. These studies demonstrated a strong associations of insect species to specific bacteria with environmental and ecological factors72, also depending on the development stage and host phylogeny72,73. Note that this fundament, regarding environment and ecology, supports the hypothesis proposed for the specialization of Tsukamurella species with A. sexdens ants69. Hence, we consider diversification and evolutionary specialization towards a defensive role against social wasp antagonists, might be responsible for the phylogenic distance found between our strains and reported genomes.

To evaluate this functional aspect, we explored the biosynthetic gene cluster diversity and genome size of the members of this genus (Table 3). The 11 genomes analyzed presented an average of 15 BGCs. These included clusters encoding for terpenes, ectoines, various types of polyketides synthases, non-ribosomally synthetized peptides, among others (Fig. 2). Most of the predicted BGCs encoded for similar known clusters of antimicrobial compounds such as those mentioned above. Tsukamurella sp. 8F and 8J had the highest quantity of BGCs among the genus (17 and 19, respectively). As stated in previous sections, most of those encoding for polyketides and hybrids involving NRPS, similar to other actinobacteria74.Table 3 Genomic and functional characteristics of representative genomes of Tsukamurella genus.

Accession	Species	Isolation material	Quantity of BGCs	Genome size	% of genome for secondary metabolism	
ASM2916740v1	Tsukamurella sp. 8 J	Insect	19	4,598,787	11	
ASM2916737v1	Tsukamurella sp. 8F	Insect	17	4,680,862	13	
ASM2480761v1	T. ocularis	Insect	14	4,306,542	12	
ASM9222v1	T. paurametabola	Insect	14	4,479,724	11	
ASM785843v1	T. asaccharolytica	Human	14	4,123,067	11	
ASM785847v1	T. conjuntivitidis	Human	16	5,076,401	10	
ASM785844v1	T. sputi	Human	13	4,810,120	9	
52700_E01	T. pulmonis	Human	13	4,773,464	11	
ASM157519v1	T. pseudospumae	Waste water	14	5,206,330	10	
ASM1239601v1	T. spumae	Waste water	13	4,461,018	9	
ASM1691946v1	T. tyrosinosolvens	Plastic	14	4,840,603	10	
All genomes and metadata were retrieved from the NCBI database on 01/23/2023. BGCs biosynthetic gene clusters. “Isolation material” refers to a generalized source of isolation. For example, our strains isolated from wasp cuticle correspond to the “insect” classification.

Fig. 2 Biosynthetic Gene Clusters (BGC) characterization of the Tsukamurella genus. (A) Maximum number of BGCs per type per source of isolation. (B) Average of genetic content for BGCs per source of isolation (insect = 4 species, plastic = 1 species, human = 4, species, and waste water = 2 species). T#PKS type # polyketide synthases, NAPAA non-alpha polyamino group acids, hglE-KS heterocyst glycolipid synthase-like PKS, NRP non-ribosomal peptide, NRPS non-ribosomal peptide synthases, RiPP ribosomally synthesized and post-translationally modified peptide.

Notably, lanthipeptides, heterocyst glycolipid synthase-like PKS and arylpolyenes cluster were only present in our strains. Extensive research has evaluated the antibacterial and antifungal activity of lanthipeptides and arylpolyenes against a wide spectrum of microorganisms75–77. The unique presence of these clusters could represent evidence of specialization of Tsukamurella sp. 8F and 8J to the social wasp association. Moreover, hglE-KS are responsible for promoting plant growth in plant-associated bacteria78. To our knowledge, this is the first time a hglE-KS cluster has been reported for insect-associated bacteria (as well as for the Tsukamurella genus), hence, its function in the insect microbiome remains unknown.

Additionally, the insect-isolated species also presented the greatest number of clusters encoding for PKS and ectoines. As we discussed before, these might represent in our strain some novel or variations of known molecules with possible new targets and structures. Remarkably, a wide variety of polyketides are found in bacterial symbionts of insects, as they are common part of the machinery involved in a defense response79,80. Some studies suggest the idea of these peptides being associated with symbionts by being absent or in low quantities in other environments isolated bacteria81. This same pattern is found in the Tsukamurella genus, supporting this hypothesis.

Moreover, the insect-isolated species presented the highest average percentage of genomic data dedicated for secondary metabolism (11.75%) and one of the smallest average genome sizes, only behind human pathogenic Tsukamurella strains. Evolutionary specialization can lead to the reduction of genome sizes, as a response to accomplish more specific functions in the host82. Although, pathogenicity is the clearest example of genome degradation83,84, symbiont bacteria are also a subject of this phenomenon85,86. Insect-associated bacteria can commonly have reduced genomes, especially in those related to a defensive role87. In this sense, it can be observed that the strains isolated from humans and insects had the smallest genomes. Nonetheless, a high secondary metabolism genomic percentage and reduced genome size could indicate a specialization tendency towards the production of antimicrobial compounds against host-specific antagonists63. In this case, even though the insect has the smallest genomes, the percentage of the genome devoted to secondary metabolisms (BGCs) was the highest among all strains. Previous studies proved that this pattern could be also associated to environmental factors, leading to promising biosynthetic potential88. Then, our strains could represent a preliminary example of symbiont-host coevolution, which positively impacted their biosynthetic potential to produce specialized compounds that might provide a new source for natural products.

Conclusion

Environmental and ecological factors play a key role in metabolic specialization of microorganisms. Wasp cuticle isolated bacteria Tsukamurella sp. 8F and 8J encoded for a wide range of natural products with putative antimicrobial activity, most of which presented low to no similarity with known clusters, anticipating a possible novelty of the compounds. In this sense, the presence of genes encoding for proteins of unknown functions which might represent antibiotics of new mechanisms of actions, a relevant bioprospecting in the antibiotic-resistance crisis era. In vitro antibiosis assays showed promising inhibitory potential against bacterial species which harbor known human and insect pathogens, indicating a possible ecological specialization. Moreover, phylogenetic distance analysis, functional and genomic characterization proved a divergence of the genus related to environmental adaptation, supporting the hypothesis. Therefore, we consider Tsukamurella sp. 8F and 8J as relevant subjects of study due to a promising biosynthetic and bioactive potential for the discovery of possible novel antimicrobial compounds. Additionally, we think these strains might represent an example of preliminary specialization of insect symbionts, a co-evolution phenomenon that might have positive impacted the biosynthetic machinery, therefore promoting insect-associated bacteria for the bioprospecting of bioactive natural products. Overall, Tsukamurella strains 8F and 8J isolated from social wasps seem to present a relevant biosynthetic potential for antibiotic production. Some of which might be novel bioactive molecules due to specialization as insect symbionts. Hence, we consider our strains should be further study with metabolomics analysis to untapped the novelty of these compounds and evaluate its biotechnology potential.

Abbreviations

ANI Average nucleotide identity

ARGs Antibiotic resistance genes

ARO Antibiotic resistance ontology

ARTS Antibiotic resistance target seeker

ATP Adenosine triphosphate

autoMLSA Automated multi-locus sequence analysis

BGCs Biosynthetic gene clusters

CARD Comprehensive Antibiotic Resistance Database

CIB Center of Biotechnology Research (Spanish acronym)

hglE-KS Heterocyst glycolipid synthase-like PKS

HGT Horizontal gene transfer

ITCR Instituto Tecnológico de Costa Rica

NAPAA Non-alpha polyamino group acids

NCBI National Center for Biotechnology Information

NRP Non-ribosomal peptide

NRPS Non-ribosomal peptide synthase

RGI Resistance gene identifier

RiPP Ribosomally synthesized and post-translationally modified peptide

SIMPER Similarity percentage analysis

T#PKS Type # polyketide synthase

Acknowledgements

To Dr. Javier Pizarro Torres from Institut Pasteur Paris (Yersinia Research Unit) for genome sequencing. To Dr. Carlos Chacón Díaz from the Microbiology Department of the Universidad de Costa Rica for ATCC strains donation. To Dr. Sergio Jansen González for his fieldwork assistance to Dr. Laura Chavarría-Pizarro. We thank CONAGEBIO for the collection permits (R-CM-ITCR- 001–2019-OT and R-CM-ITCR-002–2019-OT).

Author contributions

DRV, KNM and LCP performed the conceptualization, designed methodology, validated the research, and carried out data acquisition, curation, formal analysis, interpretation, and visualization. DRV wrote and prepared the original draft. DRV, KNM and LCP wrote, reviewed, and edited the final manuscript. KNM and LCP oversaw project administration and funding acquisition. All authors read and approved the final manuscript.

Funding

Research was funded by the Instituto Tecnológico de Costa Rica (VIE-1510118 [2019–2020], VIE-1510143 [2021], VIE-1510170 [2022], and VIE-1510183 [2023] projects).

Data availability

The datasets analyzed during the current study are available in the National Center for Biotechnology Information (NCBI) repository, under the BioProject PRJNA764377.

Competing interests

The authors declare no competing interests.

Publisher's note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

These authors contributed equally: Dorian Rojas-Villalta and Kattia Núñez-Montero.
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