Phenotypic and Genomic Characterization of Streptomyces pakalii sp. nov., a Novel Species with Anti-Biofilm and Anti-Quorum Sensing Activity in ESKAPE Bacteria

The increasing number of infections caused by antimicrobial multi-resistant microorganisms has led to the search for new microorganisms capable of producing novel antibiotics. This work proposes Streptomyces pakalii sp. nov. as a new member of the Streptomycetaceae family. The strain ENCB-J15 was isolated from the jungle soil in Palenque National Park, Chiapas, Mexico. The strain formed pale brown, dry, tough, and buried colonies in the agar with no diffusible pigment in GAE (glucose–asparagine–yeast extract) medium. Scanning electron micrographs showed typical mycelium with long chains of smooth and oval-shaped spores (3–10 m). The strain grew in all of the International Streptomyces Project (ISP)’s media at 28–37 °C with a pH of 6–9 and 0–10% NaCl. S. pakalii ENCB-J15 assimilated diverse carbon as well as organic and inorganic nitrogen sources. The strain also exhibited significant inhibitory activity against the prodigiosin synthesis of Serratia marcescens and the inhibition of the formation and destruction of biofilms of ESKAPE strains of Acinetobacter baumannii and Klebsiella pneumoniae. The draft genome sequencing of ENCB-J15 revealed a 7.6 Mb genome with a high G + C content (71.6%), 6833 total genes, and 6746 genes encoding putative proteins. A total of 26 accessory clusters of proteins associated with carbon sources and amino acid catabolism, DNA modification, and the antibiotic biosynthetic process were annotated. The 16S rRNA gene phylogeny, core-proteome phylogenomic tree, and virtual genome fingerprints support that S. pakalii ENCB-J15 is a new species related to Streptomyces badius and Streptomyces globisporus. Similarly, its average nucleotide identity (ANI) (96.4%), average amino acid identity (AAI) (96.06%), and virtual DNA–DNA hybridization (67.3%) provide evidence to recognize it as a new species. Comparative genomics revealed that S. pakalli and its closest related species maintain a well-conserved genomic synteny. This work proposes Streptomyces pakalii sp. nov. as a novel species that expresses anti-biofilm and anti-quorum sensing activities.


Introduction
Streptomyces is a Gram-positive, mycelium-forming, sporulating bacteria with a high Guanine-Cytosine (G + C) content (57-75%).The genus belongs to the phylum Actinobacteria, the order Streptomycetales, and the family Streptomycetaceae.The life cycle of Streptomyces begins with the germination of their unigenomic dormant spores, the development of multigenomic filamentous hyphae or the mycelium substrate stage, and its subsequent autolysis for supporting morphological differentiations into aerial mycelium and sporulation [1,2].
The Streptomyces genus is abundant in terrestrial and marine habitats of the biosphere and may establish mainly mutualistic and pathogenic associations with plants, animals, and other microorganisms [3][4][5][6].More than 700 Streptomyces species have been formally described [7], and 416 papers proposing new species have been deposited in the PubMed database since 1965.Although phylogenomic studies have re-clustered taxonomically many Streptomyces species [8], the bona fide genus maintains a comprehensive set of species.Streptomyces species excrete secondary metabolites of diverse chemical families with diverse biological activities and applications in industry and agriculture [9][10][11].
The increased isolation of microorganisms multi-resistant to antimicrobials has led to the search for new Actinobacteria strains capable of producing novel bioactive molecules [12].As a result, the World Health Organization urgently called on public and private institutions to search for and develop new compounds to manage infections caused by antibiotic-resistant microorganisms (ARMs) [13].Among the multiple targets for the control of bacterial infections is quorum sensing (QS) which is a cell-to-cell communication process that arranges the expression of virulence factors of many pathogenic bacteria [14].Biofilm formation, mobility, secretion of extracellular enzymes, and pigment production are regulated by QS, among other cellular processes [15][16][17].For this reason, the search for QS inhibitors has become relevant [18].An example of QS mediated via N-acyl-L-homoserine lactone (AHL) is the synthesis of the red pigment prodigiosin from Serratia marcescens, which may be employed as a model to evaluate the activity of QS inhibitors [19].
The availability of genome sequences and bioinformatics tools provides a new approach to taxonomy and contributes towards the discovery of expressed and cryptic biosynthetic gene clusters encoding bioactive molecules, which could significantly reduce the time and costs for the discovery of new drugs [24][25][26].
The current increase in antimicrobial resistance has led to the search for novel microorganisms with the potential for producing antibiotics.This work isolated a novel Streptomyces species capable of inhibiting biofilm formation and quorum-sensing activity in two ESKAPE bacteria and prodigiosin pigment biosynthesis in S. marcescens.This work aimed to analyze the phenotypic features, antimicrobial activities, and genomic features of the new species Streptomyces pakalii.

Sampling Area
The sampling area was in the Parque Nacional Palenque located in Chiapas, Mexico (17 • 30 33 N, 91 • 58 56 W).A total of 3 rhizospheric soil samples were obtained at a depth of 20 cm.The samples were transported to the laboratory at 5-10 • C.

Isolation and Purification of Actinobacteria
Serial decimal dilutions of the rhizospheric soil samples were performed using distilled water as a diluent in a final volume of 10 mL.The first 4 dilutions were spread onto glucose-asparagine-yeast extract (GAE) solid medium (20 g/L glucose, 1.0 g/L asparagine, 0.5 g/L yeast extract, 0.5 g/L K 2 HPO 4 , 0.01 g/L FeSO 4 , 0.5 g/L MgSO 4 , and 15.0 g/L bacteriological agar).After 5 days of incubation at 28 • C, typical colonies with morphology of actinobacteria (hard, dry, and buried colonies in agar) were reisolated in GAE medium by streak plate method until axenic cultures.
The strain ENCB-J15 was selected from among 15 different colonial morphotypes for further studies due to its remarkable capabilities to inhibit bacterial quorum sensing, biofilm formation, and biofilm destruction of two ESKAPE bacteria analyzed.Additionally, the strain was recognized early as a new species of Streptomyces by the preliminary 16S rRNA gene-based identification.

Growth and Maintenance of the ENCB-J15 Strain
Spores were obtained in GAE medium after 10 days of incubation at 28 • C, preserved with 30% glycerol in cryotubes at −70 • C, and lyophilized with 20% skim milk and 2% glycerol as cryoprotectants.The spores of the strain germinated, and mycelia grew on GAE solid medium at 28 • C for 5 days.Colonial and microscopic morphologies verified the purity of the strain.The strain ENCB-J15 was deposited in the collection of the Escuela Nacional de Ciencias Biológicas (ENCB) of Instituto Politécnico Nacional (IPN) and the Colección de Microorganismos of Centro Nacional de Recursos Genéticos, Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias, Mexico, which is a member of the World Federation of Culture Collection (https://wfcc.info/membership/memberlist,accessed on 15 May 2023).

Growth in Liquid Medium and Lyophilization of Supernatant
A suspension of spores from the strain ENCB-J15 was inoculated and incubated at 28 • C with orbital agitation at 150 rpm in an Erlenmeyer flask with GAE broth for 20 days.The microbial culture was centrifugated at 13,000 rpm for 15 min, and the supernatant was previously filtered through 0.45 and 0.22 µm sterile membranes for lyophilization.The products were rehydrated and concentrated at 10X (100 mg/mL) for further analysis.

Description of Microscopic Morphology by Scanning Electron Microscopy (SEM)
Petri dish microcultures of the strain ENCB-J15 were inoculated, grown in squares of GAE solid medium covered by a circular coverslip, and incubated at 28 • C for 10 days.The coverslip with a culture sample was placed on a 12-well microplate and fixed with 2% glutaraldehyde for 2 h, dehydrated with 10, 20, 30, 40, 50, 60, 70, 80, and 90% ethanol each for 10 min for each dissolution, and finally dehydrated with absolute alcohol for 20 min [27].The sample was critically dried with CO 2 using the Emitech K850 Critical Point Dryer to remove all moisture and subsequently mounted on aluminum sample holders with adhesive carbon tape.The sample was coated with a layer of gold at 20 mA for 2 min using Quorum Q15OR-ES equipment.Finally, the sample was observed in a Hitachi SU1510 SEM at 10-15 kW [28].

Phenotypic Characterization
The GAE solid medium was used as a basal medium to test the assimilation of carbon and nitrogen sources.The glucose was replaced by 1% saccharose, mannitol, lactose, glucose, fructose, galactose, xylose, starch, sorbitol, glycerol, maltose, galactose, or mannose.The asparagine was replaced by 0.5% NH 4 NO 3 , (NH 4 ) 2 SO 4 , lysine, threonine, tyrosine, meat extract, meat peptone, casein peptone, or casamino acids.Additionally, the growth of the strain was evaluated in ISP 2, 3, 4, 5, and 7 media [29].The growth of the strain was also evaluated in GAE solid medium adjusted to a pH range between 5 and 14, and NaCl concentrations ranged between 0 and 20%.The incubation temperatures were tested between 28 • C and 45 • C. All culture media were incubated at 28 • C for 7 days, except for the plates used for the temperature test.

Genomic DNA Extraction
The biomass of the strain ENCB-J15 was obtained from a culture of 5 days of growth in GAE solid medium.The DNA was extracted with the Soil Microbe DNA MiniPrep kit (Zymo Research, Irvine, CA, USA) following the manufacturer's instructions.DNA integrity was verified by electrophoresis in 1% agarose gel, 1X TAE buffer, and staining with 0.5 µg/mL ethidium bromide solution.The DNA yield was 253 ng/µL.

Preliminary Molecular Identification Using 16S rRNA
The amplification of partial sequence belonging to 16S rRNA gene was performed via endpoint PCR using the universal primers 27F (5 -AGAGTTTGATCMTGGCTCAG-3 ) and 1492R (5 -TACGGYTACCTTGTTACGACTT-3 ) as previously reported [30].The mastermix for 25 µL of PCR reaction contained 10X buffer, 25 mM of MgCl 2 , 10 mM of each dNTP, 10 pM of each primer, and 0.5 units of the Taq polymerase.The PCR reaction was carried out with the following thermal cycler conditions: one cycle of initial denaturalization at 94 • C for 5 min, 35 cycles of denaturalization at 94 • C for 1 min, alignment at 54 • C for 1 min, polymerization at 72 • C for 2 min, and a final polymerization at 72 • C for 10 min.Subsequently, 1% agarose gel electrophoresis was performed to corroborate the amplified product.The PCR products (approximately 1.4 kb) were purified with the Zymoclean TM Gel DNA Recovery Kit (Zymo Research, Orange, CA, USA).The Sanger sequencing of the amplicons was performed at Macrogen ® laboratories in South Korea.BLAST compared the sequences with type strains of species of the genomic database bank NCBI and the List of Prokaryotic names with Standing in Nomenclature (LPSN), except when no type strains of the species were available.
The most closely related sequences were manually edited with the Seaview program [31].A multiple alignment was performed by using CLUSTAL X [32].The phylogenetic tree was constructed using the maximum likelihood method (ML) with the JC69 evolutionary model estimated with the MEGA v. 10 software [33].The bootstrap method evaluated the tree topology's robustness, computing 1000 repetitions [34].

Genome Sequencing and Annotation
The Whole Genome Sequencing (WGS) was performed at the Centre for Comparative Genomics and Evolutionary Bioinformatics (CGEB) at Dalhousie University, Canada, using the Illumina sequencing platform.The genomic libraries were prepared with the Nexter-aTM XT Library Preparation Kit from Illumina, and the sequencing reaction was carried out with the Illumina MiSeq equipment.The quality metrics from raw reads were analyzed using FastQC v. 0.11.8 (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/, accessed on 25 August 2022).Raw reads were then trimmed using Trimmomatic v. 0.38 [35].The de novo genome assembling was performed with the SPAdes v. 3.13.0program [36].The quality metrics of the assembly were determined with QUAST v. 5.0.2 [37].The genome annotation was performed with Prokka v. 1.12 [38] and RAST v. 2.0 [39].Prophage prediction was made with PHASTER [40].Insertion elements (IE) were detected and annotated using ISEScan [41].

Phylogenomic Analyses
Phylogenomic reconstruction was performed using three methods: whole genome phylogeny by identifying the Virtual Genome Fingerprints (VGF), a core genome phylogeny (CGP), and a core-proteome-based phylogenomic analysis (CPBP).The VGF method was performed with the VAMPhyRE program [42].CPBP was performed to obtain the clusters of orthologous groups of proteins (COGs) from the proteome of each organism with OrthoFinder v4.0 [43].A maximum likelihood (ML) phylogenomic tree was constructed with the concatenated alignment of the COGs and LG + F + R7 model with IQ-TREE program [44].The confidence level of the tree was estimated via ultrafast bootstrap with 1000 replicates.The CGP was built with 92 genes using the UBCG program [45].The JC69 evolutionary test was used for phylogenomic reconstruction.
Genome synteny analysis among the strain ENCB-J15 and the most related Streptomyces species (Streptomyces badius and Streptomyces bacilliaris) was performed with the progressive MAUVE tool included in the MAUVE software [52,53] using default parameters.Synteny comparisons were plotted with the genoPlot package of R [54].

Pathogenic Bacteria from the ESKAPE Group and Pigmented S. marcescens
The pathogenic strains used in this study were clinical isolates of Acinetobacter baumannii A15 and A22 and Klebsiella pneumoniae KpINP19, HP614, and KpINP8.They were donated by Dra.Graciela Castro-Escarpulli of Laboratorio de Investigación Clínica y Ambiental (ENCB, IPN).S. marcescens ENCB-MG01 belongs to the collection of the ENCB, IPN.
The strains were grown in Luria-Bertani (LB) solid medium (5 g/L yeast extract, 10 g/L casein peptone, and 5 g/L NaCl) at 37 • C for 24 h.After verifying their purity, the strains were preserved in 40% glycerol at −70 • C.

Inhibition of the Production of the Prodigiosin Pigment of S. marcescens by ENCB-J15 Supernatants
The production and inhibition of prodigiosin pigment of S. marcescens ENCB-MG01 were determined by mixing 1 mL of 1 × 10 8 bacteria/mL with 1 mL of the 10X supernatant of the strain ENCB-J15 and incubated at 37 • C for 24 h.For the extraction of the pigment, the method described by Ramanathan et al. was used [55].In brief, 100 mL of the bacterial growth obtained with or without the Streptomyces ENCB-J15 supernatant was placed in 1.5 mL microtubes and mixed with 100 µL of a solution of 2% HCL-absolute ethanol.The mixture was centrifuged at 5000 rpm for 5 min, and the supernatant was read at 535 nm for quantification in a Multiskan FC 357 Microplate Photometer (Thermo Scientific TM, , San Jose, CA, USA).The analyses obtained were evaluated via t-test and considered a significant difference as having a value of p < 0.0001.

Inhibition of the Formation and Destruction of Biofilms Formed by A. baumannii and K. pneumoniae
Multidrug-resistant clinical isolates of A. baumannii A15 and A21 and K. pneumoniae KpINP19, HP614, and KpINP8 were used.Each isolate was grown in 5 mL of medium LB at 37 • C for 24 h.The growth obtained was centrifuged at 13,000 rpm for 5 min, then the supernatant was removed, and the cellular button was washed with PBS 1x buffer 2 times.The inoculum was adjusted to 1 × 10 8 bacteria/mL in RPMI 1640 medium (2 g/L D-glucose, 5 g/L phenol red, 6 g/L NaCl, 2 g/L NaHCO 3 , 1.512 g/L Na 2 HPO 4 , 0.4 g/L KCl, 0.1 g/L MgSO 4 , and 0.1 g/L CaNO 3 at a pH of 7.2).
For the formation of bacterial biofilms, 200 µL of A. baumannii and K. pneumoniae suspensions and 100 µL of the actinobacteria supernatant ENCB-J15 were placed in polystyrene microplates of 96 sterile flat-bottomed wells and incubated for 24 h at 37 • C. For the destruction of preformed biofilms, the biofilms of each pathogenic strain were previously formed by placing 200 µL of bacterial inoculum in polystyrene microplates of 96 sterile flat-bottomed wells and incubating them at 37 • C for 24 h.After 24 h of incubation of biofilm formation, 100 µL of Streptomyces ENCB-J15 supernatant was placed and incubated for another 24 h.Biofilms were quantified with the methodology described by Christensen et al., 1985 [56].
The analyses obtained were evaluated by a two-way ANOVA analysis of variance with a significance level of p < 0.05.

Morphological and Phenotypic Characterization of Strain ENCB-J15
The strain ENCB-J15 cultured in GAE solid medium after 4 days at 28 • C developed buried colonies with a pale brown color, dry appearance, and tough consistency.The 7-day-old colonies acquired a whitish appearance on their surface due to aerial mycelium formation and spore maturation, but no diffusible pigments were observed (Figure 1A).SEM allowed us to observe typical mycelium and long chains of smooth and oval-shaped spores (Figure 1B,C).These morphological features are consistent with the typical features described for the genus Streptomyces [4,7].
Jose, CA, USA).The analyses obtained were evaluated via t-test and considered a significant difference as having a value of p < 0.0001.

Inhibition of the Formation and Destruction of Biofilms Formed by A. baumannii and K. pneumoniae
Multidrug-resistant clinical isolates of A. baumannii A15 and A21 and K. pneumoniae KpINP19, HP614, and KpINP8 were used.Each isolate was grown in 5 mL of medium LB at 37 °C for 24 h.The growth obtained was centrifuged at 13,000 rpm for 5 min, then the supernatant was removed, and the cellular button was washed with PBS 1x buffer 2 times.The inoculum was adjusted to 1 × 10 8 bacteria/mL in RPMI 1640 medium (2 g/L D-glucose, 5 g/L phenol red, 6 g/L NaCl, 2 g/L NaHCO3, 1.512 g/L Na2HPO4, 0.4 g/L KCl, 0.1 g/L MgSO4, and 0.1 g/L CaNO3 at a pH of 7.2).
For the formation of bacterial biofilms, 200 µL of A. baumannii and K. pneumoniae suspensions and 100 µL of the actinobacteria supernatant ENCB-J15 were placed in polystyrene microplates of 96 sterile flat-bottomed wells and incubated for 24 h at 37 °C.For the destruction of preformed biofilms, the biofilms of each pathogenic strain were previously formed by placing 200 µL of bacterial inoculum in polystyrene microplates of 96 sterile flat-bottomed wells and incubating them at 37 °C for 24 h.After 24 h of incubation of biofilm formation, 100 µL of Streptomyces ENCB-J15 supernatant was placed and incubated for another 24 h.Biofilms were quantified with the methodology described by Christensen et al., 1985 [56].
The analyses obtained were evaluated by a two-way ANOVA analysis of variance with a significance level of p < 0.05.

Morphological and Phenotypic Characterization of Strain ENCB-J15
The strain ENCB-J15 cultured in GAE solid medium after 4 days at 28 °C developed buried colonies with a pale brown color, dry appearance, and tough consistency.The 7day-old colonies acquired a whitish appearance on their surface due to aerial mycelium formation and spore maturation, but no diffusible pigments were observed (Figure 1A).SEM allowed us to observe typical mycelium and long chains of smooth and oval-shaped spores (Figure 1B,C).These morphological features are consistent with the typical features described for the genus Streptomyces [4,7].The strain ENCB-J15 grew abundantly in the GAE solid medium and ISP media.However, its growth in the ISP3 medium was weak, and non-sporulation was observed after 5 days of incubation at 28 °C (Figure S1).Except for xylose, the strain grew in a GAE solid medium amended with different carbon sources tested, including monosaccharides, The strain ENCB-J15 grew abundantly in the GAE solid medium and ISP media.However, its growth in the ISP3 medium was weak, and non-sporulation was observed after 5 days of incubation at 28 • C (Figure S1).Except for xylose, the strain grew in a GAE solid medium amended with different carbon sources tested, including monosaccharides, disaccharides, polysaccharides, and polyalcohol.Nonetheless, the growth of the strain in GAE + sorbitol was weak, and only vegetative mycelia were observed (Figure S2).The versatility of the strain ENCB-J15 reflects the high diversity but low availability of assimilable carbon sources in soil.Each carbon source displays a specific regulation of the biosynthesis of secondary metabolites determining the competence, defense, chemical communication, and survival of the microorganism in soil [57,58].
The characteristic pale brown pigment of the colonies was not observed in GAE amended with galactose and glycerol (Figure S2).All organic nitrogen sources that were used instead of asparagine in the GAE medium supported the growth and sporulation of the strain.However, inorganic nitrogen sources generated poor growth and sporulation (Figure S3).A nitrogen source is vital to the life cycle of Streptomyces as a component of proteins, nucleic acids, and many secondary metabolites [58].
The strain ENCB-J15 had a range of temperatures and pH growth typical of most Streptomycetaceae species (Figures S4 and S5) [7,59].At the maximum growth temperature of 37 • C, the strain ENCB-J15 synthesized a non-diffusible melanin-like black pigment.Bacterial pigments are often synthesized in non-optimal or stress culture conditions, with various temperatures and pH values.Melanins generally protect microorganisms from environmental stress conditions, such as ultraviolet radiation and oxidative stress by heavy metals [60].Several Streptomyces species secrete tyrosinases that produce soluble melanins in media [61], but the black pigment of the strain ENCB-J15 is attached to biomass.

Phylogenetic, Phylogenomic, and Pairwise Comparison
The strain ENCB-J15 was located in an independent branch of the 16S rRNA phylogenetic tree, but no clear differentiation among related species was observed (Figure 2).The percentage of nucleotide similarity between the strain ENCB-J15 and its closest phylogenetic relatives ranged from 93.5 to 94.1% (Table 1).The widely accepted cutoff for delimiting new bacterial species using 16S rRNA gene sequences is between 97 and 98.65% [64][65][66][67][68][69][70].Under this criterion, the ENCB-J15 strain could be considered a new species.Currently, it is assumed that the 16S rRNA gene sequence is not sufficient for species-level identification, differentiation between related species, and the definition of new bacteria species [68,[71][72][73], and the Streptomyces spp.are no exception [8,[74][75][76].The identification of Streptomyces species based on a multilocus sequence analysis (MLSA) of atpD, recA, trpB, rpoB, and gyrB genes is a more robust molecular tool for species-level identification [76][77][78][79].Currently, whole-genome sequencing methods and bioinformatic analyses of the G + C content, ANI, AAI, in silico GGH percentages, and phylogenomic reconstructions make it possible to derive useful information about taxonomy and phylogeny.The G + C content measured by chemical analyses allowed a maximum variation within bacterial species of 3-5 mol% [64,80]; meanwhile, the G + C content calculated from the genome sequence proposes a maximum variation value into bacterial species of 1% [48].The ENCB-J15 strain has a G + C content of 71.63%, which is in the range of the Streptomyces genus (69.7-74.5%)[8].
In addition, more and more often, ANI, AAI, and in silico GGH percentages are used as essential criteria to define cutoff values of species and assign new species.The following cutoff values of ANI (95-96%), AAI (<95%), and in silico GGH (<70%) are frequently used to define species limits in bacteria [48,49,81].Nonetheless, the ANI cutoff value of 96.5% is also accepted since this value offers better resolution for bacterial species delimitation [68,82].The ANI cutoff value of ≥96.5% has been proposed for delineating species belonging to Streptomyces and members from Actinobacteria, such as Salinospora [68,83].
The features of the ENCB-J15 genome are outlined in Table 2.According to ANI and AAI, S. badius SP6C4 and S. globisporus TFH56 type species displayed very high values for ANI and AAI between them (99.99 and 99.98%, respectively), but a lower ANI of 96.43% and AAI of 96.06-96.01%with the ENCB-J15 strain.Both indexes were even lower among the strain ENCB-J15 and other Streptomyces species (Figure 3).An in silico GGH analysis between the ENCB-J15 strain and its closest relatives S. badius and S. globisporus showed a value of 67.3% (Table 1).Recently, in silico GGH has been recognized as a valuable criterion of relatedness and has replaced the experimentally complex and variable DNA-DNA hybridization assays [48,84].
The sequencing of whole genomes and the phylogenomic approach have become valuable tools to define and recognize new bacteria species for science [8,[85][86][87][88].CGP, CPBP, and VGF were used to perform the phylogenomic approaches of the strain ENCB-J15, but the most robust maximum likelihood phylogenetic tree was obtained based on CPBP.ENCB-J15 was closely clustered in this tree with S. badius and S. globisporus, but S. filamentosus and S. parvus were more distant (Figure 4).In all the phylogenomic trees, S. badius and S. globisporus formed a clade and shared a close common ancestor; meanwhile, the speciation event of the strain ENCB-J15 was earlier (Figure 4 and Figures S7 and S8).Currently, phylogenomic reconstruction is the most robust tool for species classification in bacteria in general and the Actinobacteria phylum in particular [8,86].Thus, the topology of both phylogenomic trees is consistent with the proposal of ENCB-J15 as a new species.There are non-official criteria for categorizing bacterial species based on phylogenomic metrics.A CGP based on at least 100 single core genes could resolve taxonomical discrepancies among closely related strains [85,89].Even for the Streptomyces genus, CPBP based on approximately 700 COGs allows for clusters and defines the species into a conscious and well-resolved phylogenetic tree [68].Furthermore, confidence trees with well-supported nodes with bootstrap values ≥70% have been suggested [85].Although the phylogenomic metrics for bacterial species designation are in the normalization process, this work fulfills the species identification of ENCB-15 based on phylogenomic reconstruction with the metrics described above.
S. badius and S. globisporus, formally recognized as independent species, have genomic content, indexes, and percentages that suggest they are two strains of the same species.Nonetheless, the ENCB-J15 strain has G + C content, ANI, AAI, in silico GGH percentages, and phylogenomic trees that are consistent with the proposed metrics to assign a strain as a new species which we suggest can be named Streptomyces pakalii sp.nov.ANI and AAI between them (99.99 and 99.98%, respectively), but a lower ANI of 96.43% and AAI of 96.06-96.01%with the ENCB-J15 strain.Both indexes were even lower among the strain ENCB-J15 and other Streptomyces species (Figure 3).An in silico GGH analysis between the ENCB-J15 strain and its closest relatives S. badius and S. globisporus showed a value of 67.3% (Table 1).Recently, in silico GGH has been recognized as a valuable criterion of relatedness and has replaced the experimentally complex and variable DNA-DNA hybridization assays [48,84].The sequencing of whole genomes and the phylogenomic approach have become valuable tools to define and recognize new bacteria species for science [8,[85][86][87][88].CGP, CPBP, and VGF were used to perform the phylogenomic approaches of the strain ENCB-J15, but the most robust maximum likelihood phylogenetic tree was obtained based on CPBP.ENCB-J15 was closely clustered in this tree with S. badius and S. globisporus, but S. filamentosus and S. parvus were more distant (Figure 4).In all the phylogenomic trees, S. badius among closely related strains [85,89].Even for the Streptomyces genus, CPBP based on approximately 700 COGs allows for clusters and defines the species into a conscious and well-resolved phylogenetic tree [68].Furthermore, confidence trees with well-supported nodes with bootstrap values ≥70% have been suggested [85].Although the phylogenomic metrics for bacterial species designation are in the normalization process, this work fulfills the species identification of ENCB-15 based on phylogenomic reconstruction with the metrics described above.

Analysis of Genomic Features among Streptomyces pakalii sp. nov. and Closely Related Species
The genome of S. pakalii sp.nov.has a size of 7.6 Mb, a 71% G + C content, 6833 genes, and 6746 proteins annotated (Figure 5 and Table 2).Additionally, four ribosomal genes, 82 tRNA genes, four sequences corresponding to putative prophages, and five insertion elements (IE) were detected (Table 2 and S1). S. pakalii sp.nov.showed a similar genome size and higher protein content than S. badius and S. globisporus, its most proximal relatives.Nevertheless, these species present more rRNA and tRNA gene copies than S. pakalii sp.nov.ENCB-J15 [90].The results suggest that S. pakalii sp.nov.probably suffered encodinggene expansion into its genome, as well as a putative ribosomal and tRNA depuration.The encoding-gene gain is a common phenomenon in the genus Streptomyces that helps it to adapt to the environment [91].Although plasmids were not evidenced, other mobile elements were detected, such as prophages or insertion elements (Table 2).size and higher protein content than S. badius and S. globisporus, its most proximal relatives.Nevertheless, these species present more rRNA and tRNA gene copies than S. pakalii sp.nov.ENCB-J15 [90].The results suggest that S. pakalii sp.nov.probably suffered encoding-gene expansion into its genome, as well as a putative ribosomal and tRNA depuration.The encoding-gene gain is a common phenomenon in the genus Streptomyces that helps it to adapt to the environment [91].Although plasmids were not evidenced, other mobile elements were detected, such as prophages or insertion elements (Table 2).The synteny analysis of the entire genome reflected a similar genome structure among S. pakalii sp.nov., S. badius, and S. bacilliaris.However, S. pakalii sp.nov.displayed events of reordering and gene insertions (Figure 6).As previously observed, the chromosome structure in the species analyzed was highly conserved at the central region generally associated with the core genome.However, the insertions were enriched at the terminal regions of the chromosome, a region corresponding to the accessory genome that includes metabolite biosynthetic genes [92].The synteny analysis of the entire genome reflected a similar genome structure among S. pakalii sp.nov., S. badius, and S. bacilliaris.However, S. pakalii sp.nov.displayed events of reordering and gene insertions (Figure 6).As previously observed, the chromosome structure in the species analyzed was highly conserved at the central region generally associated with the core genome.However, the insertions were enriched at the terminal regions of the chromosome, a region corresponding to the accessory genome that includes metabolite biosynthetic genes [92].

Comparative Analysis of Functional Annotation
S. pakalii sp.nov.ENCB-J15 showed 6337 proteins with known functions and 409 with non-determined functions.Most of the annotated genes of S. pakalii sp.nov.were assigned roles in transcription, amino acids, carbohydrate transport, and metabolism (684, 458, and 456 genes, respectively) (Table S2).COGs with large repertories were related to lipid transport and metabolism, energy production and conversion, translation and biogenesis,

Comparative Analysis of Functional Annotation
S. pakalii sp.nov.ENCB-J15 showed 6337 proteins with known functions and 409 with non-determined functions.Most of the annotated genes of S. pakalii sp.nov.were assigned roles in transcription, amino acids, carbohydrate transport, and metabolism (684, 458, and 456 genes, respectively) (Table S2).COGs with large repertories were related to lipid transport and metabolism, energy production and conversion, translation and biogenesis, cell wall development or biogenesis, signal translation mechanisms, and inorganic ion and transport.All genomes showed an extensive repertory of COGs related to secondary metabolite biosynthesis (SMB) with a content of 218-277 COGs (Figure 7A).The analysis with OrthoVenn indicated that 4812 core COGs are shared among the Streptomyces species included in this analysis (Figure 7B).S. pakalii sp.nov.exhibited 26 accessory COGs, mainly involved in carbon source and amino acid catabolism, DNA modification, and the antibiotic biosynthetic process (Figure 7B and Table S3).Core genomes are frequently involved in primary metabolism and DNA processing functions, whereas genes associated with SMB are increased in the Streptomyces pangenome [91,93].This suggests that acquiring accessory genes is a central phenomenon associated with speciation in Streptomyces [94].The complexity of forest soil microhabitats may maintain selective pressures that explain the vast repertoire of accessory genes and the phenotypic versatility of S. pakalii sp.nov.

Inhibition of Prodigiosin Biosynthesis of S. marcescens by S. pakalii sp. nov. ENCB-J15 Supernatants
The expression of diverse bacterial virulence factors, such as biofilm formation in pathogenic bacteria and the biosynthesis of pigments such as prodigiosin in S. marcescens, depends on QS [15].Therefore, QS has been pointed out as a possible target for controlling pathogenic bacterial infections [95,96].In this work, the supernatant of S. pakalii sp.nov.was inhibited until 50% of the biosynthesis of prodigiosin of S. marcescens, which was achieved with no decrease in bacterial growth (Figure 8).Nonetheless, more research should be conducted to determine if the target of the inhibitory activity is at the level of pigment biosynthesis or QS.Hence, assays of the inhibition of prodigiosin biosynthesis are a tool to recognize potential QS inhibitors.

Inhibition of Prodigiosin Biosynthesis of S. marcescens by S. pakalii sp. nov. ENCB-J15 Supernatants
The expression of diverse bacterial virulence factors, such as biofilm formation in pathogenic bacteria and the biosynthesis of pigments such as prodigiosin in S. marcescens, depends on QS [15].Therefore, QS has been pointed out as a possible target for controlling pathogenic bacterial infections [95,96].In this work, the supernatant of S. pakalii sp.nov.was inhibited until 50% of the biosynthesis of prodigiosin of S. marcescens, which was achieved with no decrease in bacterial growth (Figure 8).Nonetheless, more research should be conducted to determine if the target of the inhibitory activity is at the level of pigment biosynthesis or QS.Hence, assays of the inhibition of prodigiosin biosynthesis are a tool to recognize potential QS inhibitors.

Inhibition of the Formation and Destruction of Biofilms Formed by A. baumannii and K. pneumoniae
One of the most important virulence factors today is the formation of biofilms, which a critical role in human pathogenic bacteria such as A. baumannii and K. pneumoniae, since they act as a structure that increases resistance to different agents, such as antimicrobial compounds, detergents, and disinfectants, among others, and prevents their action, causing the microorganisms that form these biofilms to become resistant [55].The supernatant of Streptomyces pakalii sp.nov.displayed anti-biofilm activity because it was able to inhibit more than 50% of the formation of these (Figure 9A), as well as destroy the biofilms already formed (Figure 9B) by the ESKAPE isolates of K. pneumoniae and A. baumannii.It showed activity similar to that reported by Sangkanu et al. in 2017 [97], whose results showed a decrease in the formation and destruction of biofilms formed by Staphylococcus epidermidis ATCC35984 in the presence of the supernatants of different actinobacteria isolated from a mangrove swamp in Thailand.These results suggest that a molecule, hitherto unknown, in this supernatant may be used as an anti-biofilm agent.One of the most important virulence factors today is the formation of biofilms, which play a critical role in human pathogenic bacteria such as A. baumannii and K. pneumoniae, since they act as a structure that increases resistance to different agents, such as antimicrobial compounds, detergents, and disinfectants, among others, and prevents their action, causing the microorganisms that form these biofilms to become resistant [55].The supernatant of Streptomyces pakalii sp.nov.displayed anti-biofilm activity because it was able to inhibit more than 50% of the formation of these (Figure 9A), as well as destroy the biofilms already formed (Figure 9B) by the ESKAPE isolates of K. pneumoniae and A. baumannii.It showed activity similar to that reported by Sangkanu et al. in 2017 [97], whose results showed a decrease in the formation and destruction of biofilms formed by Staphylococcus epidermidis ATCC35984 in the presence of the supernatants of different actinobacteria isolated from a mangrove swamp in Thailand.These results suggest that a molecule, hitherto unknown, in this supernatant may be used as an anti-biofilm agent.

Figure 2 .
Figure 2. Maximum likelihood phylogenetic tree of strain ENCB-J15 and closely related species based on 16S rRNA gene sequences (1500 nt).Streptomyces pakalii sp.nov.ENCB-J15 is highlighted in the green box.Numbers over the branches are the bootstrap values >50% computed by 1000 replicates.The scale bar represents the number of substitutions per base.Currently, whole-genome sequencing methods and bioinformatic analyses of the G + C content, ANI, AAI, in silico GGH percentages, and phylogenomic reconstructions make it possible to derive useful information about taxonomy and phylogeny.The G + C content measured by chemical analyses allowed a maximum variation within bacterial species of 3-5 mol% [64,80]; meanwhile, the G + C content calculated from the genome

Figure 2 .
Figure 2. Maximum likelihood phylogenetic tree of strain ENCB-J15 and closely related species based on 16S rRNA gene sequences (1500 nt).Streptomyces pakalii sp.nov.ENCB-J15 is highlighted in the green box.Numbers over the branches are the bootstrap values >50% computed by 1000 replicates.The scale bar represents the number of substitutions per base.

Figure 3 .
Figure 3. Heatmaps of Average Nucleotide Identity (ANI) (A) and Average Amino Acid Identity (AAI) (B) calculated in pairwise comparisons of related Streptomyces species.The color gradient bar at the x-axis represents the ANI and AAI values.Streptomyces pakalii sp.nov.ENCB-J15 is highlighted in blue.

Figure 3 .
Figure 3. Heatmaps of Average Nucleotide Identity (ANI) (A) and Average Amino Acid Identity (AAI) (B) calculated in pairwise comparisons of related Streptomyces species.The color gradient bar at the x-axis represents the ANI and AAI values.Streptomyces pakalii sp.nov.ENCB-J15 is highlighted in blue.

Figure 4 .
Figure 4. Maximum likelihood phylogenomic tree of Streptomyces pakalii sp.nov.ENCB-J15 and other related species of the genus Streptomyces.The phylogeny was based on the core-proteome of 1218 Clusters of Orthologous Groups of Proteins (COGs) and reconstructed with Orthofinder and IQ-TREE.Numbers over the nodes represent the ultrafast bootstrap values >50% computed by 1000 replicates.The scale bar represents the number of substitutions per base.Streptomyces pakalii sp.nov.ENCB-J15 is highlighted in the purple box.

Figure 4 .
Figure 4. Maximum likelihood phylogenomic tree of Streptomyces pakalii sp.nov.ENCB-J15 and other related species of the genus Streptomyces.The phylogeny was based on the core-proteome of 1218 Clusters of Orthologous Groups of Proteins (COGs) and reconstructed with Orthofinder and IQ-TREE.Numbers over the nodes represent the ultrafast bootstrap values >50% computed by 1000 replicates.The scale bar represents the number of substitutions per base.Streptomyces pakalii sp.nov.ENCB-J15 is highlighted in the purple box.

Figure 6 .
Figure 6.Genomic synteny and comparison among Streptomyces pakalii sp.nov.ENCB-J15 and related species.Color code: conserved regions (rainbow shades), variable regions (white gaps), conservation of the direction or position of the regions (red lines), and inversions or translocations (blue lines).

Figure 6 .
Figure 6.Genomic synteny and comparison among Streptomyces pakalii sp.nov.ENCB-J15 and related species.Color code: conserved regions (rainbow shades), variable regions (white gaps), conservation of the direction or position of the regions (red lines), and inversions or translocations (blue lines).

Figure 7 .
Figure 7. Predicted protein content comparison among S. pakalii sp.nov.ENCB-J15 and closely related species.(A) Heatmap of clusters of orthologous groups of proteins (COGs)' abundance sorted into different functional categories.The gradient color bar represents the relative content of COGs.Numbers in the boxes represent the absolute content of COGs.Names on the x-axis represent the organisms analyzed.The functional categorization is represented on the y-axis.(B) Venn diagram of shared and accessory COGs among Streptomyces pakalii sp.nov.ENCB-J15 and close species.Numbers in the diagram represent the shared and accessory content of COGs.The bar plot represents the overall content of COGs in each Streptomyces species included in the analysis.

Figure 7 .
Figure 7. Predicted protein content comparison among S. pakalii sp.nov.ENCB-J15 and closely related species.(A) Heatmap of clusters of orthologous groups of proteins (COGs)' abundance sorted into different functional categories.The gradient color bar represents the relative content of COGs.Numbers in the boxes represent the absolute content of COGs.Names on the x-axis represent the organisms analyzed.The functional categorization is represented on the y-axis.(B) Venn diagram of shared and accessory COGs among Streptomyces pakalii sp.nov.ENCB-J15 and close species.Numbers in the diagram represent the shared and accessory content of COGs.The bar plot represents the overall content of COGs in each Streptomyces species included in the analysis.

Figure 8 .
Figure 8. Inhibition of the production of the prodigiosin pigment of S. marcescens by Streptom-ces pakalii sp.nov.ENCB-J15 supernatants.The symbol on the bar (*) represents a statistically significant difference by t-tests.p < 0.0001.

Figure 8 .
Figure 8. Inhibition of the production of the prodigiosin pigment of S. marcescens by Streptom-ces pakalii sp.nov.ENCB-J15 supernatants.The symbol on the bar (*) represents a statistically significant difference by t-tests.p < 0.0001.

3. 6 .
Inhibition of the Formation and Destruction of Biofilms Formed by A. baumannii and K. pneumoniae

3. 7 .
Description of Streptomyces pakalii sp.nov.Streptomyces pakalii (Etimology: pak.al'i.iN.L. gen.masc.n. pakalii; the name is derived from the Mayan King Kʼinich Janaab Pakal, who was buried in a Mayan pyramid) is a Gram-positive filamentous bacterium that shows the formation of smooth spore chains.The front and back of the colony have a pale brown color without the presence of diffusible pigments in culture media.Its growth was observed at temperatures of 28-37 °C with the optimal growth at a temperature of 28 °C.When it grows at 37 °C, it produces a non-diffusible melanin-like pigment in GAE medium.The strain grew in NaCl concentrations up to 10% and pH values of 5-13.It uses glucose, starch, maltose, lactose, sucrose,

Table 1 .
Comparison of nucleotide similitude percentages for 16S rRNA gene, genomic G + C content, and in silico genome-genome hybridization (GGH) of Streptomyces pakalii sp.nov ENCB-J15 with related species.