Introduction

Bark beetles (Coleoptera, Curculionidae, Scolytinae) are a group of insects that comprises thousands of species [1]. They have gained notoriety in recent years due to the decimation of conifer forests and have become one of the most destructive insects in forest ecosystems, where they cause huge economic losses [2]. The expansion of bark beetle populations has been enhanced by climate change [3] and the increase of international trade, contributing to the introduction of new exotic bark beetles species into foreign forest ecosystems [4].

One bark beetle is Ips acuminatus Gyll, which is a secondary pest of the pine engraver beetle. This species infests the thin bark of Scots pine (Pinus sylvestris L.) throughout Europe [5] and overwinters inside its host tree branches. Scots pine is among the most common and important tree species with a wide distribution in hemiboreal forests in Europe. For many years, I. acuminatus was not considered a serious problem; however, during the last decades, tree mortality and damages caused by this bark beetle in young plantations and stands of Scots pine seem to have increased in central and southern Europe [6, 7]. In the first decade of the century, a review of bark- and wood-boring insects rated I. acuminatus as one of the ten most important bark-boring pest species in Europe, and it was scored as particularly damaging in Germany, Slovakia, Switzerland, Romania and Spain [8].

As most of the bark beetle species, I. acuminatus cohabits with several assemblages of fungi and bacteria under the bark. Interactions between bark beetles and fungi have been researched intensively for many years [9, 10]; some fungal species establish a mutualistic relationship with the beetles, whereas others act as antagonists, either directly by causing disease or competing with the insect for nutrients [10]. However, the role of bacteria in the bark beetles’ ecology has only recently been appreciated [1]. As Popa et al. reported in 2012 [11], bacteria and fungi establish a tripartite interaction with the bark beetle in the subcortical environment, forming a symbiotic complex. This complex is considered as an entity known as “bark beetle holobiont” and may be influencing the host’s fitness [1]. In addition, some bacterial strains involved in this scenery are associated to the bark beetle playing a role in the protection of the beetle holobiont, using a broad array of strategies [1, 11]. A key strategy, widespread among insects, could be the use of molecular defences from associated bacteria, which produce substances that inhibit the development of pathogens and other antagonists [1]. Better understandings of these complex interactions are needed to realize the ecology of I. acuminatus and might be useful in integrated pest management programmes, to target the inhibition of microbial allies or empowering natural enemies [1].

The bacterial genus Arthrobacter (family Micrococcaceae) was proposed by Conn and Dimmick in 1947 [12] with the description of Arthrobacter globiformis (NBRC 12137T) as the type species of the genus. Different strains of the genus Arthrobacter have been found worldwide in a variety of environments, including water [13], glacier [14], sewage [15], sediments [16], soil environment [17] and human clinical specimens [18]. Recently, Saati-Santamaría et al. [19] reported the isolation of strain IA7T from adult specimens of I. acuminatus. A comparative 16S rRNA gene sequence analysis indicated that the strain IA7T is most closely affiliated to the genus Arthrobacter; however, it seems that this strain constitutes a new species within the genus.

The analysis of the possible role of microorganisms in certain environments is important to understand the huge importance and influence of microbial associates on the ecology of macroorganisms’ populations. Thus, identification and characterization of strain IA7T related to I. acuminatus would help us to describe a putative role about its interaction with the bark beetle and better understand the ecology of the insect. The genome sequence analysis of a microbial strain provides new insights into its role within the bark beetle holobiont [20, 21], which can be further experimentally tested. Moreover, pan-genome analyses and comparison of the genome sequence of a bacterium with those of closely related taxa provide a better understanding of its specificities, allowing the correlation of those differences with possible roles in its specific niche [22]. Therefore, we describe the novel bacterial species, designated as Arthrobacter ipsi sp. nov. IA7T in this study, which was previously isolated from adult bark beetles of the species I. acuminatus collected in Stará Boleslav, Czech Republic [19]. In addition, we provide the genome sequence of the strain and the analysis of its genetic potential. In parallel, we present a pan-genome analysis of 21 type strains of the genus Arthrobacter, which conveys the functional capabilities of this genus, and the genomic comparison of strain IA7T with other genomes of the genus, describing its unique proteins, not present in those strains isolated from other niches. Finally, since our analysis suggested the capability of A. ipsi to synthesize antimicrobial compounds, we proved the capability of this strain to inhibit Ips entomopathogenic fungi, suggesting its potential role in the protection of the bark beetle holobiont.

Experimental Procedures

Isolation and Identification of Strain

The isolation of strain IA7T from I. acuminatus had been previously described by Saati-Santamaría et al. in 2018 [19]. Briefly, a pool of 3 adult specimens of I. acuminatus was crushed. Serial dilutions were inoculated onto Petri dishes containing different media and strain IA7T was recovered from Nutrient Agar (NA).

The comparison of the almost complete 16S rRNA gene sequence of the IA7T strain with other publicly available ones was done as described previously [19, 23].

For phylogenetic tree construction, complete gene sequences of the 16S rRNA gene and housekeeping genes tuf, secY, rpoB, recA, fusA and atpD of strain IA7T were obtained from the genome sequence and sequences of related type strains were obtained from the NCBI database and processed as described in [19, 23]. The type strain Cellulomonas composti TR7-06T (AB166887.1) was used as an outgroup in the phylogenetic analysis of the 16S rRNA gene.

The mol% G + C content of DNA of IA7T was determined from the complete genome sequence, together with its length.

All genomes belonging to type strains of the genus Arthrobacter available in the NCBI genome were retrieved. The average nucleotide identity was calculated with ANIb [24] through the Python package PYANI (v0.2.1) [25].

Finally, the digital DNA-DNA hybridization (dDDH) with A. globiformis (NBRC 12137T) was estimated by using genome-to-genome distance calculator (GGDC) version 2.1 online service [26] with the formula recommended by Auch et al. [27].

Genome Sequencing and Functional Annotation

Total DNA was obtained from cells from a fresh culture of A. ipsi IA7T grown for 1 day in NA at 28 °C using Quick DNATM ZR Fungal/Bacterial DNA MiniPrep (Zymo Research®).

DNA sequencing was carried out in on Illumina MiSeq platform via a paired-end run (2 × 250 bp). The sequence reads were assembled with Velvet 1.2.10 [28]. Contigs were oriented and gaps were filled with MeDuSa [29], using all genomic sequences listed in Table 1 as reference.

Table 1 Strains and data of the genomes used in this study. Unique, accessory and core genes of each strain of the pan-genome are also displayed
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Gene calling and functional annotations of genes were made with RAST (v2.0) [30]. KofamKOALA [31] was employed in order to assign more functions to predicted genes.

Pan-genome Analysis

Pan-genome analysis was performed with all genomes belonging to type strains of the genus Arthrobacter available in the NCBI genome database at the moment of carrying out this study—a total of 22—including the genome sequence from A. ipsi IA7T strain obtained in this work. Data of each genome appear in Table 1.

Core genome and pan-genome analyses of all these genomes were performed with the bacterial pan genome analysis (BPGA) pipeline (v1.3) [32]. The USEARCH (v11.0.667) algorithm was used with a threshold of 0.7 for the identification of orthologous proteins. Pan-core plots were calculated over 500 iterations. The assignation of an open or closed pan-genome was made according to the formula of a power law regression f(x) = a.x^b (Heap’s law), considering as open pan-genome when 0 < b < 1 [33]. A flower-plot of core and unique genes was drawn with plotrix package of R-project [34].

Functional analysis of core and unique proteins was performed with KofamKOALA [31]. Blast2Go [35] software was used in order to compare unique proteins of the A. ipsi IA7T strain with the RefSeq-Protein database (threshold = 1*E-05) through BLASTp method. The taxonomy of all reference top-hits was retrieved with the same program. InterPro annotation and BLAST-based mapping were performed for Gene Ontology (GO) assignment. Summary of GO functions was also made within this software.

All these genomes were processed with antiSMASH (v5.1.1) [36] for specific functional annotation over the secondary metabolism.

Clustered heatmaps were constructed with R-project by calling heatmap.2 from gplots package [37].

Cultural and Physiological Characterization

For the phenotypical characterization, strain IA7T was routinely grown aerobically on tryptone soy agar (TSA) or tryptone soy broth (TSB) for 2 days at 28 °C, except where indicated otherwise. Growth at 4, 12, 28 and 37 °C was assessed on TSA medium after the incubation period. To evaluate the strain IA7T growth capability at different pHs, we used TSB medium with an adjusted final pH of 6.0, 6.5, 7.5, 8.5, 9.5 and 10.0, by using citric acid-sodium citrate buffer for pH 6.0; phosphate buffer for pH 6.5, 7.5 and 8.5; and carbonate-bicarbonate buffer for pH 9.5 and 10.0; the same medium supplemented with 0, 1.0, 2.0, 3.0, 4.0, 5.0, 7.5 and 10.0 (w/v) NaCl (%), respectively, was used to asses salt tolerance. Gram staining and catalase and oxidase test were performed as previously detailed [19]. Bacterial cells’ size was measured under an optical microscope. To study carbon source utilization and enzyme activities, we used API-20E, API-20NE and API-ZYM test kits (bioMerieux) following the instructions given by the manufacturer. In all cases, results were checked after up to 1 week.

Antifungal Compounds’ Production Assay

To assess the inhibition of fungal pathogens’ capability, 7 fungal pathogen strains of Ips beetles were grown on PDA medium (Metarhizium anisopliae CCF0966, Lecanicillium muscarium CCF6041, L. muscarium CCF3297, Isaria fumosorosea CCF4401, I. farinosa CCF4808, Beauveria bassiana CCF4422 and B. brongniartii CCF1547 [38,39,40]) at 25 °C during 3 days. A fresh culture of the strain IA7T was used to seed a single streak in the centre of Potato Dextrose Agar (PDA), NA and TSA plates. These plates were incubated at 28 °C for 10 days in order to ensure the metabolites’ diffusion. After this pre-incubation period, each fungal species was transferred to plates where strain IA7T had been growing. Mycelia plugs (0.5 cm) were deposited 0.5 cm far from the bacterial biomass. After additional incubation under suitable conditions for the tested fungal strains’ growth (25 °C), the antifungal capability of strain IA7T was evaluated at 5 and 10 days by comparison of the fungal growth in these test plates with the one obtained in control plates (fungal plugs cultivated in the same conditions with no presence of the strain IA7T). Absence of fungal mycelia development was considered as positive antifungal capability of the strain IA7T.

Results

Bacterial Isolation and identification

The isolation of strain IA7T from Ips acuminatus and its identification within the genus Arthrobacter were previously described by Saati-Santamaría et al. [19]. A more in-depth analysis of the strain identity based on the comparison of its 16S rRNA gene sequence (1446 bp) revealed that Arthrobacter sp. IA7T could belong to a new species within the genus Arthrobacter. Strain IA7T showed the highest identity of 16S rRNA sequence with A. globiformis NBRC 12137T (99.52%), followed by A. pascens DSM 20545T (98.82%), A. nitrophenolicus SJConT (98.69%), A. humicola KV-653T (98.40%) and A. oryzae KV-651T (98.20%) and low identities with other type strains of the genus Arthrobacter (96.65–97.81%).

Genomic Features

We obtained the genome sequence of strain IA7T to go deeper in the analysis of both its phylogenetic classification and its potential role within the bark beetle holobiont. Genome sequencing and assembly yielded 18 scaffolds built from 58 original contigs. The estimated genome size is 4,509,596 bp, with a G + C content of 66.0%. Values for L50 and N50 are 13 and 151,583, respectively. The number of coding sequences predicted is 4236, while 57 RNAs were found by RAST.

Phylogenetic Analyses

ML and NJ trees based on 16S rRNA sequence including all related species within the genus Arthrobacter presented a similar topology. In both cases, IA7T clustered with A. globiformis NBRC 12137T in a broader cluster that includes A. pascens DSM 20545T, A. humicola KV-653T and A. oryzae KV-651T, among others (Fig. 1). The results clearly indicated that strain IA7T was phylogenetically affiliated to the genus Arthrobacter. As regards ML and NJ trees constructed on the nucleotide sequences of six concatenated housekeeping genes (tuf, secY, rpoB, recA, fusA and atpD) (Fig. 2) both showed a similar phylogenetic clustering. The trees revealed that the strain IA7T clustered with A. enclensis NIO-1008T and A. silvisoli NEAU-SA1T.

Fig. 1
figure 1

Maximum likelihood phylogenetic tree based on almost complete 16S rRNA gene sequences showing the relationships between strain IA7T and the type strains of related Arthrobacter species. Dots indicate that the corresponding nodes were also recovered in the trees generated with neighbour-joining algorithm. The numbers at the nodes indicate levels of bootstrap support based on a maximum-likelihood of 1000 resampled datasets. Scale bar = 0.01 substitutions per nucleotide position. Accession numbers of the sequences are indicated in parentheses. Cellulomonas composti TR7-06T (AB166887) is used as outgroup

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Fig. 2
figure 2

Maximum likelihood phylogenetic tree based on concatenated tuf, SecY, rpoB, recA, fusA and atpD gene sequences showing the relationships between strain IA7T and the type strains of some related Arthrobacter species. Dots indicate that the corresponding nodes were also recovered in the trees generated with neighbour-joining algorithm. The numbers at the nodes indicate levels of bootstrap support based on a maximum likelihood of 1000 resampled datasets. Scale bar = 0.1 substitutions per nucleotide position

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ANIb values between the genomes of all type strains of species of the genus Arthrobacter are available on the public database and that of strain IA7T were under the 95% limit, proposed as barrier for species definition [41] (Fig. 3). This supports the classification of strain IA7T as a new species of the genus Arthrobacter. Clustering based on ANIb values reveals that the closest related strain to strain IA7T is A. globiformis NBRC 12137T (88.9% identity) and confirms their classification as different species. Digital DNA-DNA hybridization (dDDH) between the genome sequences of these two strains offered a value of 37.10% [34.6–39.6%], which is far below the widely accepted threshold of 70% dDDH for bacterial species delineation [27, 42], thus confirming that strain IA7T belongs to a new species within the genus Arthrobacter.

Fig. 3
figure 3

Clustered heatmap of ANIb values shared among type Arthrobacter strains

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Phenotypic Characterization

Phenotypic characteristics of strain IA7T are presented in the species description and compared with those of the type strains of closely related Arthrobacter species in Table 2.

Table 2 Phenotypic characteristics of strain IA7T and the most closely related Arthrobacter species: A. globiformis NBRC 12137T [66, 67], A. enclensis NIO-1008T [16], A. silvisoli NEAU-SA1T [68]. +, positive; -, negative; ND, no data
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Genome and Pan-genome Analyses Towards the Host-Bacteria Interaction

None of the previously described species within the genus Arthrobacter has been isolated from a bark beetle. Thus, and based on the hypothesis that unique genes for A. ipsi IA7T might reveal some clue in this sense, we compared the genomic potential of this new species with that of other described species of the genus through a pan-genome analysis to aid in the description of the potential role of A. ipsi IA7T within the bark beetle holobiont.

Genomic sizes of type strains belonging to the genus Arthrobacter range between 3,176,728 and 5,042,614 bp, with a median of 4,159,105 bp. The pan-genome is composed of a core (conserved) genome of 252 genes, an accessory (dispensable) genome of 12,347 genes and 25,562 unique (strain specific) genes (Table 1 and Online Resource 1a). The genome sequence with more unique genes, corresponding to A. castelli DSM 16402T, contains 3035 unique genes, whereas A. cupressi CGMCC 1.10783T, with 464 unique genes, is the genome with the lowest number of unique genes. A. ipsi IA7T gives 800 unique genes to the pan-genome. Core-pan plot shows a constant core genome; therefore, the incorporation of more genomes to the analysis will probably keep the number of gene families in the core (Online Resource 1b). The parameter 'b' of the power fit pan-genome formula is 0.708178, so the pan-genome is still open; more gene families will be probably added if more Arthrobacter genomes were incorporated into the pan-genome.

KEGG analysis of the pan-genome shows some differences among core, accessory and unique genes between different fractions of the metabolism (Fig. 4). Strain specificity is represented by functions assigned into diverse KEGG categories: cell motility, biosynthesis of secondary metabolites, membrane transport, drug resistance and carbohydrate, glycan, lipid, amino acid, xenobiotics, terpenoid and other polyketide metabolism, all of them with a reduced core genome in comparison with accessory or unique genes falling in these groups. Nevertheless, the core genome is well represented in energy metabolism, translation, cell growth and death, folding, sorting and degradation, metabolism of cofactors and vitamins, nucleotide metabolism and replication and repair.

Fig. 4.
figure 4

Bars representing KEGG categories of core, accessory and unique genes

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Biosynthetic gene clusters (BGCs) found with antiSMASH and clustering based on BGC profiles is represented in Online Resource 2. We can see that, in agreement with the ANIb analysis, A. ipsi IA7T is placed close to the type species for A. globiformis and A. oryzae. There are 14 BGC types detected, being 5 of them broadly distributed among these 21 genomes, such as BGCs encoding for betalactones, terpenes, siderophores, as well as type III polyketide synthases (PKS) and NRPS-like clusters of genes. In addition, there are some others in lower amount: ectoine, butyrolactone, non-ribosomal peptide synthetases (NRPS), bacteriocins, furan, linear azol(in)e-containing peptides and pheganomycin-style protein ligase-containing clusters.

Unique proteins for A. ipsi IA7T were analysed in detail in order to reveal exclusive functions of this strain against other Arthrobacter strains isolated from different sources. Blast2Go analyses let us to detect the most similar proteins to IA7T unique sequences and extract the taxonomy of the top-hit BLASTp results for them (Online Resource 3a); the taxon with the higher number of BLASTp hits is A. globiformis, followed by many other Arthrobacter strains not included in this pan-genome analysis and 176 out of 800 sequences did not give any similarity with any other protein under the chosen threshold. The main fraction of the similarity distribution of the BLASTp hits is over 70% (Online Resource 3b).

Specific annotation for these unique proteins yielded 95 sequences with assigned function by KofamKOALA; Blast2Go gives Gene Ontology (GO) terms for 190 sequences, distributed in diverse functions as detailed in Online Resource 4. Significantly, we found that many of the predicted functions are related with hydrolase and oxidoreductase activities, carbohydrate derivative binding and protein binding, among others. In addition, almost half of the GO terms represent proteins placed as integral components of the membrane (Online Resource 5).

Not only IA7T unique genes are responsible of the strain function within its niche, so we performed a genome mining of A. ipsi IA7T genome sequence, which revealed genes related to its possible roles within nutrient cycling of its holobiont (Online Resource 4). A. ipsi IA7T genome harbours complete pathways for the synthesis of thiamine, riboflavin, biotin, nicotinate and nicotinamide vitamins, all vitamins belonging to the B group. Regarding nitrogen and sulphur metabolism, based on genes’ annotation information, this strain could assimilate nitrates and nitrites, as well as oxidize H2S. A. ipsi IA7T has also many genes related to the production and acquisition of siderophores, molecules involved in the chelation of iron and used in the transport of this element across cell membranes.

Aiming a deeper analysis on the secondary metabolism genetic potential, antiSMASH tool was used in order to improve IA7T genome annotations regarding secondary metabolism. This process showed 7 BGCs related with the secondary metabolism within the genome, a type III polyketide synthase (PKS), a betalactone, a siderophore (closely related to the BGC described to produce deferoxamine E) and 4 NRPS-like BGCs. Since all these BGCs may be related with the production of antimicrobial compounds, we hypothesised that this strain could have some role in the protection of the bark beetle holobiont against microbial pathogens.

Fungal Development Inhibition

Because of the above-mentioned results regarding the genetic potential of the strain IA7T to synthesize antimicrobial compounds, an antagonism assay was carried out against entomopathogenic fungi of this insect group. In all tested media (PDA, NA and TSA), growth of all entomopathogenic fungal strains used in this assay was totally inhibited, both at 5 and 10 days, whereas the development of mycelia in control plates presented a clearly larger diameter than that of the inoculation plug, which supports the idea of a protective role of A. ipsi in its bark beetle host.

Description of Arthrobacter ipsi sp. nov.

Cells of strain IA7T form cream, smooth and circular with entire margins colonies when grown for 3 days at 28 °C on TSA medium. Strain IA7T was found to be able to grow at 12–37 °C with optimum at 28 °C, at pH 6.5–8.5 with optimum at 6.5–7.5 and in the presence of 0–4% but not at 5% (w/v) NaCl, with optimum at 0–1% (w/v) NaCl. Cells are short Gram-negative rods with 0.4 μm length and 0.1 μm width. IA7T is catalase positive and oxidase negative. In the API20E system, glucose fermentation/oxidation test was negative. Strain IA7T was found to be positive for aesculin hydrolysis, production of urease, gelatinase and β-galactosidase and for assimilation of D-mannose, D-mannitol, N-acetyl glucosamine and D-maltose; but negative for reduction of nitrates, glucose fermentation, production of indole and arginine dihydrolase and assimilation of D-glucose, L-arabinose, potassium gluconate, caprate, adipate, malate, trisodium citrate and phenylacetate. Enzyme activities were observed to be positive for alkaline phosphatase, esterase, esterase lipase, acid phosphatase, naphthol-AS-BI-phosphohydrolase, α-galactosidase, β-galactosidase, α-glucosidase, β-glucosidase, N-acetyl-β-glucosaminidase and α-mannosidase, but negative for lipase, leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin, α-chymotrypsin and a-fucosidase.

The type strain IA7T (=CECT 30100T = LMG 31782T) was isolated from a bark beetle from the species Ips acuminatus in the Czech Republic. The G + C base composition was 66.0 mol%.

Discussion

In a previous work on the bacterial diversity associated to bark beetles, we isolated the strain IA7T; a comparison of the 16S rRNA gene sequence of strain IA7T with those available on public databases revealed that it belongs to the genus Arthrobacter, with A. globiformis NBRC 12137T, the type species of the genus, its most closely related type strain [12].

A previous taxonomic study with the genus Arthrobacter showed how a MLSA based on the concatenated sequence of the six housekeeping genes tuf-secY-rpoB-recA-fusA-atpD is quite resolutive to differentiate different species within the genus [43]. In the case of strain IA7T, the MLSA based on the above-mentioned housekeeping genes showed how it clusters with A. enclensis NIO-1008T and A. silvisoli NEAU-SA1T, but the phylogenetic distances between those three strains clearly support that IA7T belongs to a different species. However, it is currently accepted that the gold standard for the prokaryotic species definition is average nucleotide identity (ANI) [24, 41]. ANI represents a mean of identity values between homologous genomic regions shared by two genomes and a value of 95–96 % constitutes the generally accepted threshold for bacterial species delineation [41]. The higher ANIb value for strain IA7T, obtained with A. globiformis NBRC 12137T, was quite below the species definition limit. Moreover, the dDDH value between these two strains was also under the species delineation threshold of 70% DDH [27, 42]. Thus, all our analyses indicated that strain IA7T represents a novel species of the genus Arthrobacter and we propose the description of A. ipsi sp. nov., isolated from Ips acuminatus.

Since IA7T was isolated from the bark beetle Ips accuminatus [19] and it has been broadly described how microbial associates may have essential roles for its host fitness, we wanted to explore if this newly described bacterial species has a potentially relevant role for its host. For that purpose, we performed a pan-genome analysis of the genus Arthrobacter, to find unique genetic features of IA7T, which might be related to its role in the niche where it was isolated: the bark beetle. Interestingly, the pan-genome analysis showed that only 252 genes—out of an average of 3585 genes per genome—form the core genome of the Arthrobacter genus. This number of core genes covers an average of only about 7% of the genes of each genome, a relatively low amount for strains belonging to the same genus [44, 45] that underlines the genetic diversity of this pan-genome. These results are in consonance with the ANIb values between the different species of the genus obtained in this study, which are around 75%, a value used for genus delimitation in other genera [46, 47]. A possible explanation of this broad genomic variability within species classified within the same genus based on 16S gene sequence analysis could be the large repertory of bacteriophages found in Arthrobacter strains, which are key drivers of microbial genetic evolution [48].

Regarding the annotations of core, accessory and unique genes, we show that, as expected, important processes such as cell division and energy metabolism are carried out fundamentally by core genes, whereas the most diverse genes encode proteins involved in the metabolism of secondary metabolites or in the transport or the metabolism of lipids or amino acids, which are primary blocks for many secondary metabolites> [49]; this can be explained by the diversity of niches from which the type strains of Arthrobacter species were isolated. Some other core genes are implicated in the nitrogen (N) and sulphur (S) metabolisms. In this sense, all genomes have enzymes responsible of the assimilation of H2S and NH3, so they could help in the incorporation of S and N into the amino acid metabolism, suggesting that these type strains could play relevant roles in N and S cycles in their specific niches.

Based on these data of core genes and supported by a deeper genome analysis, A. ipsi IA7T could have a possible contribution to the N and S cycles within its host. The capability of this strain to metabolize diverse forms of N might be making this element available for the beetle [50], and a higher N content has been related to a more efficient conversion of plant material into insect body tissues [51]. Regarding S, H2S is usually produced in animal guts by the bacterial population and high concentrations of this compound may become toxic to the host [52]. Thus, assimilation of H2S by A. ipsi IA7T could aid the beetle in this regard. On the other hand, animals usually require more nutrients than those acquired with the diet, being most of these metabolites produced by their gut microbial symbionts [53]. In this case, strain A. ipsi IA7T has the genetic machinery to synthesize B-group vitamins and supply iron by the production of siderophores. Further -omics approaches or isotope-labelled experiments might help in deciphering if this strain’s genetic potential is benefiting its host [1].

Apart from the core genes, A. ipsi IA7T also possesses 800 unique genes. The acquisition of these genes could have diverse origins, being horizontal gene transference from other genera a likely explanation [54]. However, more than 20% of the unique proteins of this genome present no closely related discovered proteins, so the origin or evolution of their encoding genes remains uncertain. Indeed, KO or GO terms were assigned only to a small percentage of these unique proteins found in IA7T, so their functions are probably uncharacterized. Interestingly, some of those unique proteins with predicted functions are annotated as enzymes implicated in aromatic compounds’ breakdown, such as a benzoate 1,2-dioxygenase involved in the degradation of methyl-, fluoro- or unsubstituted benzoates. It has been described that methyl benzoate is a plant metabolite with insecticidal properties [55] and how the presence of this compound is increased in pines when beetles are present [56]; thus, a role of A. ipsi IA7T in the protection of the beetle against this tree defence seems quite feasible. Apart from that, many other unique genes are classified under functional categories related with more general and vast processes.

In regards of the secondary metabolism of this pan-genome, some types of BGCs are present in many species. All these genomes possess a BGC encoding for a β-lactone. All but 2 strains harbour type III polyketide synthases (PKS). Terpene, non-ribosomal peptide synthetase (NRPS)-like and siderophore biosynthetic machinery are also broadly represented in the genus. The average of BGCs per genome detected by antiSMASH is 5, a relatively high amount for a genus with a small genome (average of 4,159,105 bp). Moreover, most of the annotated BGCs of this pan-genome have low similarity to already described BGCs. This highlights the promising potential of strains belonging to the genus Arthrobacter for the discovery of new secondary metabolites.

Regarding the specific annotation of the secondary metabolism encoded in the genome sequence of strain IA7T, the predicted BCGs—4 NRPS-like, a type III PKS, a β-lactone and a siderophore—have been described as gene clusters related to the synthesis of molecules with antimicrobial activity [57,57,58,59,61].Indeed, the siderophore BGC is highly similar to the gene cluster implicated in the synthesis of deferoxamine E, a compound with described antifungal activity [62].

Following the clues obtained from the secondary metabolism genome mining and the previous available information on the capability of A. ipsi IA7T to inhibit development of two yeast-like fungi—Candida humilis and Pichia fermentans—and weakly reduce the growth of a filamentous fungus, Aspergillus sp. [19], we thought that strain IA7T could play a role in the protection of the engraver beetle I. acuminatus against entomopathogenic fungi. Thus, we tested the capability of this bacterium to inhibit some antagonist fungal strains, which resulted in a total inhibition of the development in all fungal strains tested. These data supports the possibility of a potential role of this new species in the protection of the bark beetle holobiont. Nonetheless, further studies to test this protective role in the beetle should be performed.

Pathogens and parasites of bark beetles have been intensively investigated, and fungi occupy an important place in the list of bark beetle antagonists [63, 64]. From an environmental point of view, studies of diversity and occurrence of bark beetle pathogens are related to the development of environmentally friendly methods to control the mass outbreaks of this pest. Contemporary forest protection requires the advancement of integrated methods for pest insect and disease control through developing methods of forecasting forest dangers, the use of natural enemies and agro-technical methods for regulation of pests [1, 65]. For this reason, a better knowledge of the biology and natural protection mechanisms of bark beetles is necessary to a more effective management of some pests with economic impact, as I. acuminatus. In this work, we describe how Arthrobacter ipsi IA7T may protect I. acuminatus from its natural pathogens; thus, a strategy focused on the inhibition of this bacterium might aid in the control of this forest pest.