An antimicrobial thiopeptide producing novel actinomycetes Streptomyces terrae sp. nov., isolated from subsurface soil of arable land

Abstract An antimicrobial producing Gram-positive, aerobic, nonmotile, and filamentous actinobacterial strain SKN60T was isolated from soil The isolate exhibited 99.3% and 99.0% identity with Streptomyces laurentii ATCC 31255T and S. roseicoloratus TRM 44457T, respectively, in 16S rRNA gene sequence analysis. However, the genome sequence displayed maximum ANI (88.45%) and AAI (85.61%) with S. roseicoloratus TRM 44457T. Similarly, the dDDH showed 33.7% identity with S. roseicoloratus TRM 44457T. It formed a cluster with S. roseicoloratus TRM 44457T and S. laurentii ATCC 31255T in phylogenomic tree. Cell wall analysis revealed the presence of diphosphatidylglycerol, phosphatidylethanolamine, and phosphatidylcholine as major polar lipids and diaminopimelic acid as diagnostic diamino acid. Major fatty acids were iso-C15:0, anteiso-C15:0, and iso-C16:0. The G+C content was found to be 72.3 mol%. Genome sequence analysis using antiSMASH database showed occurrence of a thiopeptide biosynthesis gene cluster with 94% similarity to berninamycin from S. bernensis UC5144. The mass of 1146 Da is identical with berninamycin. But subtle differences observed in leader peptide sequence of thiopeptide and berninamycin. Notably, S. bernensis is not validly reported and thus SKN60T is the only strain containing berninamycin BGC as no other phylogenetic relative had it. Additionally, strain SKN60T differed in phenotypic and genetic characteristics with all phylogenetic relatives of the genus Streptomyces. Therefore, it is proposed as a novel species with the name Streptomyces terrae sp. nov. strain SKN60T (=MTCC 13163T; = JCM 35768T).


Introduction
Actinomycetes constitute a major portion of soil microbial biomass.Streptomyces is one of the important genera of Actinomycetota phylum and contains the largest number of species, whic h differ mostl y in their physiology, bioc hemical, and morphological features.Members of the genus Streptomyces are well known for the antibiotic production (Neha et al. 2013 ).The genus Streptom yces contains Gr am-positiv e filamentous bacterial species with high G + C content (Skerman et al. 1989 ). Curr entl y, it contains more than 800 species isolated from aquatic and terrestrial ecosystems, indicating their ubiquitous natur e. Streptom yces species exhibit a mycelial life cycle with the formation of aerial mycelium containing single spore or spore chains (Zhang et al. 2013 ).The spores are grown as nonseparate mycelium with br anc hing hyphal filaments under optimal environmental conditions.It is best studied genus to display such modifications and a complex life c ycle (Flar dh and Buttner 2009 ).Members of the genus exhibit an extensive secondary metabolism and produce an array of bioactive substances, including antibiotics of medical importance (Challis and Hopwood 2003 ).The production of secondary metabolic bioactive substances is usually growth phase dependent and observed during the shifting of mycelium to the sporulation phase (van Wezel andMcDo w all 2011 , Hw ang et al. 2014 ).Appr oximatel y, tw o-thir ds of all av ailable natur all y deri ved bioacti ve substances including anticancer, anthelmintic, antibacterial, and antifungal compounds wer e r eported to pr o-duce by the various species of the genus Streptomyces (Uyeda et al. 2001, Procopio et al. 2012, Ahmad et al. 2017 ).In fact, some species have been emplo y ed as biocontrol agents in agriculture (Wang et al. 2018 ).Consequently, these bacteria are of major importance to the biotechnology and pharmaceutical industry.Furthermor e, identification of antimicr obial biosynthesis potential str ain discov ery has been incr eased with the adv ances in genomics, particularly with the deposition of whole genome and draft genome sequences in public databases.Genome mining has helped to identify the exact constituents of secondary metabolite producers and revealed the biosynthetic potential of different bacteria (Udwary et al. 2011, Bauman et al. 2021 ).It also provides an opportunity for genome mining combined with metabolic pr ofiling, whic h r esults in the heter ologous expr ession of antimicrobial compounds (Yamada et al. 2015, Chen et al. 2017 ).Berninamycin is an example for genome mining-based heter ologous expr ession (Malcolmson et al. 2013 ).It is identified as pyridine containing thiopeptide that shows post-translational modifications upon synthesis .T he modifications include formation of deh ydroalanine/deh ydrobutyrines and azole/azoline rings (Bycroft and Gowland 1978 ).These antimicrobial peptides are an emerging class of antibiotics against drug-resistant Grampositive bacteria.Since species of the genera Streptomyces and Nocardia are primarily reported to produce antimicrobials (Jiang et al. 2023, Bauman et al. 2021, Bagley et al. 2005 ) in this study, we have made an attempt to isolate and identify such antimicrobial substances including antimicrobial peptide producing strains of these genera.

Soil sample collection and isolation of actinomycetes
Soil sample was collected from subsurface (depth of 20 cm) environment of an arable land in Chandigarh and processed immediatel y to scr een micr oor ganisms exhibiting antimicr obial activity .Briefly , 1 g of soil was serially diluted in 10 ml of sterile saline and 100 μl aliquots of each dilution (10 −2 -10 −4 ) were inoculated on to starch casein agar (Himedia, India) plates .T he plates were incubated at 30ºC for 3-4 days and observed after every 24 h time interval for antimicrobial producing strains.Colonies showing inhibitory zone in their periphery were picked up and str eaked on Streptom yces a gar (Himedia) for further purification, and stored as glycerol stocks in −70ºC deep freezer until further use.A strain designated as SKN60 was selected for detailed studies.

Morphological and physiological char acteriza tion
The morphological features were checked by growing the strain aer obicall y at 30ºC for 2-3 days on various International Streptom yces Pr oject (ISP) media (Shirling and Gottlieb 1966 ) including tryptone yeast extract agar (ISP1), yeast malt agar (ISP2), oat meal agar (ISP3), inorganic salt starch agar (ISP4), glycerol aspar a gine a gar (ISP5), peptone yeast extr act ir on a gar (ISP6), tyr osine agar (ISP7), and Streptomyces agar.The aerial and submerged mycelium was observed for color and pigment production.Strain morphology was observed through a phase contrast microscope and sporulation pattern was observed by allowing strain growth onto coverslips inserted into agar medium.Physiological studies were determined at different conditions like temperature (20-40ºC), pH (2-10), and salt concentrations (0%-8%) (Co w an and Steel 1965 ).Different biochemical tests including the ability to utilize different carbon substrates were studied as per the recommended methods (Smibert and Krieg 1994 ).

Chemotaxonomic char acteriza tion
Lyophilized cell biomass of strain SKN60 T was used for different c hemotaxonomic anal ysis .T he fatty acid methyl esters were anal yzed by Sherloc k Micr obial Identification System using v ersion 6.1 (Sasser 1990 ).Total cellular polar lipids , menaquinone , and peptidogl ycan anal ysis wer e performed by thin layer c hr omatogr a phy (TLC) anal yses (Minnikin et al. 1984, Sc humann 2011 ).The extracted polar lipids were analyzed using two-dimensional TLC.Different staining reagents, including molybdatophosphoric acid, ninhydrin, molybdenum blue, and α-naphthol reagents, were emplo y ed to detect total lipids , aminolipids , phospholipids , and glycolipids, r espectiv el y.

16S rRNA gene sequence analysis
The genomic DNA of SKN60 T was extracted using Zymo DNA isolation kit and used for 16S rRNA gene amplification.Briefly, 16S rRN A gene w as amplified using the primers 27F and 1492R and 5x firepol master mix (SolisBioDyne, Estonia) in a 50 μl reaction volume .T he amplified product was run using 1% a gar ose gel and purified using HiYield™ Gel/PCR DNA mini kit (Real Biotech Corporation).The purified amplicon was sequenced using three internal primers including 27F , 786F , and 1492R (10 μl reaction volume) and the sequencing was performed on an ABI Prism 3700 automatic DNA sequencer (Applied Biosystems).The nucleotide sequence file (.ab1) files wer e anal yzed using Finc hTV v ersion (1.4.0) software and assembled manually.The consensus sequence was used for BLAST sequence similarity search on EzTaxon server (Chun et al. 2007 ).All closel y r elated sequences wer e r etrie v ed in fasta format and aligned with SKN60 T sequence using CLUSTAL_W (Thompson et al. 1994 ).The phylogenetic tree was constructed by neighbor-joining algorithm using MEGA software version 7.0 (Tam ur a et al. 2011 ).Kim ur a two-par ameter was used to calculate evolutionary distances and the tree topologies were evaluated by bootstr a p anal yses with 1000 r eplications.

Genome sequencing and analyses
The genome sequence of SKN60 T was produced using illumina-HiSeqX10 at Agrigenome , India.T he total genome cov er a ge was 150X and library was prepared using Paired-end and Mate pair.The library for genome sequencing was prepared using Nextera DNA XT libr ary pr epar ation Kit (15031942).Fastq quality c hec k of the reads was performed using FastUniq (Xu et al. 2012 ).De-novo assembly was performed using ABySS (1.2.10), SPAdes (3.1.30),and Velvet (1.2.150) ( http:// www.usadellab.org/cms ) version 3.13 (Banke vic h et al. 2012 ).Quality assessment of the genome assemblies was performed using QUAST 4.0 v ersion (Gur e vic h et al. 2013 ), whic h pr ovides better statistics for v elv et assembl y and the presence of assembled genes wer e c hec ked using B USCO V2 (Simao et al. 2015 ).Genome annotation of strain SKN60 T was performed using Rapid annotation using subsystem technology (RAST) serv er ( https://r ast.nmpdr.org/).Pr ediction of pr otein coding genes was performed using BLASTX search against UniProt database and Prokka, v.1.14.6 (Seemann 2014 ).The 16S rRNA gene was extracted using RNAmmer tool (Lagesen et al. 2007 ).The whole genome sequence (WGS) shotgun data of the strain SKN60 T was deposited in GenBank.Further, the WGS of Streptomyces sp.SKN60 T was uploaded to the Type (Strain) Genome Server (TYGS) for conducting a taxonomic based analysis of the whole genome (Meier-Kolthof and Göker 2019 ).Genome blast distance phylogeny (GBDP) was used to compare the SKN60 T genome with the type str ains.Inter genomic distances wer e accur atel y estimated using the 'trimming' algorithm and the d5 distance formula.These intergenomic distances were used to construct a balanced minim um e volution tr ee using FASTME 2.1.4with br anc h support determined through 100 pseudo-bootstrap replicates (Henz et al. 2005 ).Biosynthetic gene cluster (BGC) encoding for the production of secondary metabolites was analyzed using antiSMASH 7.0 database tool (Blin et al. 2019 ).Whole genome-based comparison of SKN60 T was analyzed using bioinformatic tools like CGView Comparison Tool (CCT) ( http:// stothard.afns.ualberta.ca/cgview _ ser ver ) and Proksee ( https:// proksee.ca/ ) for circular map representations.

Pan and core-genome analysis
P an-genome anal ysis of SKN60 T was performed using a method of Vernikos et al. ( 2015 ).The pan-genome analysis was performed with genome sequence of the type strains of various species of the genus Streptomyces downloaded from the NCBI database.The pan-genome studies were performed using bacterial pangenome analysis (BPGA) pipeline (Chaudhari et al. 2016 ).Av er a ge nucleotide identity (ANI) and av er a ge amino acid identity (AAI) with the close r elativ es wer e determined using ANI/AAI matrix service using Kostas lab ( http://enve-omics.ce.ga? 020tech.edu/ g-matrix/ ).Genetic relatedness based on digital DN A-DN A hybridization (dDDH) was calculated using genome-to-genome distance-based calculator (GGDC, www.dsmz.de/services/onlinetools).

Antimicrobial activity determination
Gr owth curv e anal ysis of str ain SKN60 T was performed using Streptom yces br oth to detect antimicr obial pr oduction.Cell-fr ee fermented broth (CFB) collected at different time intervals was c hec ked for activity against different test strains procured from Micr obial Type Cultur e Collection and Gene bank (MTCC), Chandigarh.Briefly, 50 ml of culture medium was inoculated with a loopful of inoculum and incubated at 30 • C, 180 rpm and CFB fractions collected after every 24 h were tested for antimicrobial activity against test strains and cell biomass also determined.Gr am-positiv e bacteria including Bacillus subtilis (MTCC 121), Staphylococcus aureus (MTCC 1430), Micrococcus luteus (MTCC 106), and Gr am-negativ e bacteria including Pseudomonas aeruginosa (MTCC 1934), Vibrio cholera (MTCC 3904), and Esc heric hia coli (MTCC 1610) were used as test strains in this study.Overnight gr own cultur e (50 μl) was spr ead on a nutrient a gar plate.Subsequently, w ells w ere made using a cork-borer and 100 μl of CFB collected at different time intervals was added to the wells.Plates were incubated overnight under optimal growth conditions to measure the zone of inhibition.

Extraction and purification of antimicrobial compound
The production of antimicrobial compound was carried out by inoculating 1% of an overnight grown culture of strain SKN60 T (grown in Streptomyces broth) in 1000 ml of nutrient broth (NB) medium and incubated at 30 • C for a period of 96 h at 180 rpm.The r esulting cultur e medium was centrifuged at 7000 rpm, 4 • C for 30 min.The harvested CFB was sterilized using polyether sulfone (PES) membranes with a pore size of 0.2 μm.The antimicrobial compound was extracted by solvent extraction method using ethyl acetate: CFB in equal volumes .T he organic layer was collected and e v a por ated in a r otatory e v a por ator (Buc hi).The activ e fr action was finall y suspended in 100% methanol.The compound was analyzed on a silica plate using TLC (in duplicates).The organic solvent mobile phase emplo y ed w as c hlor oform: methanol: water (65:45:4).The processed TLC plate was dried inside a fume hood and cut into two sections.One strip was sprayed with ninhydrin r ea gent to detect the peptide.Another strip was used to c hec k the antimicrobial activity against S. aureus MTCC 1430 ( ∼10 6 cells ml −1 ) in a bioautogr a phy assay .Briefly , the strip was placed on a cultured nutrient agar plate and allo w ed to diffuse at 4 • C for 1 h.The TLC strip was r emov ed after diffusion and plates were k e pt at 37 • C to observe its inhibitory activity.The antimicrobial compound was purified using r e v erse phase high-performance liquid c hr omatogr a phy (HPLC) on 1260 infinity instrument (Agilent Technologies, USA) using a semipre parati ve C18 column.The mobile phase consisted of solvent system A [HPLC grade water with 0.12% trifluoroacetic acid (TFA)] and solvent system B (HPLCgrade acetonitrile (ACN) with 0.1% TFA) and was monitored via UV detector at 220 and 280 nm.The purified fraction was collected manually and used for further studies.

Mass spectrometry
T he ESI-Q-T OF mass spectrometry was carried out using Agilent 6550 (LC 1290 infinity) system coupled with 6550 Q-TOF (Agilent Technologies).Mobile phase consisted of 0.1% formic acid in water (solvent A) and acetonitrile with 0.1% formic acid (solvent B).The eluted sample was exposed to positive ionization mode at fr a gmentor volta ge 100 V.The spectrum was acquir ed in low m/z range .T he intact mass of HPLC purified peptide was determined by matrix-assisted laser desorption ionization (MALDI).The pep-tide (0.7 mg ml −1 ) was mixed with α-cyano-4-hydroxycinnamic acid matrix in a ratio of 1:1 and loaded on a stainless steel MALDI target plate .T he sample was then processed after air drying for MALDI analysis (Bruker, Microflex).

MIC and killing kinetic determination assay
The method of Gupta et al. ( 2019 ) w as follo w ed to determine minimum inhibitory concentration (MIC) of the purified peptide using 96-well plate assay.MIC is defined as 90% growth inhibition of the test str ains.Additionall y, killing kinetics of the antimicrobial substance from SKN60 T was determined against various Grampositive test strains.Briefly, cells grown to the logarithmic phase were collected by centrifugation and subsequently washed with phosphate buffer saline (PBS, Gibco).The final cell count was adjusted to an OD 600 0.3-0.4.The cells were exposed to different concentrations of the active compound for various time intervals and aliquots of the suspensions wer e spr eaded on plates.Time-kill kinetics wer e expr essed as CFU counts.Untr eated cells wer e used as a control to check CFU count.

TEM sample prepar a tion
TEM was performed to elucidate the mechanism of antimicrobial activity resulting upon treatment of S. aureus (MTCC 1430) with differ ent concentr ations of antimicr obial substance.Untr eated control also processed in parallel (Gupta et al. 2019 ).Briefly, cells were suspended in 1X PBS to obtain the cell density of 10 8 CFU ml −1 and treated with different concentrations (2X and 5X MIC) of a peptide.Samples wer e subsequentl y subjected to negativ e stain with 0.1% (w/v) Sodium phosphotungstate (PTA, Sigma) for 1 min using carbon-coated copper grid (300 mesh, Polybioscience) and cells were observed using a transmission electron microscope (TEM) at a resolution of 0.1-0.5 μm range (Raje et al. 2006 ).

Haemolysis
Haemolysis assay was performed using blood of New Zealand white r abbit, whic h was collected fr om iCARE-CSIR-IMTec h.Fr eshl y collected blood was centrifuged and cells were reconstituted using 1X PBS.The number of RBCs was counted using a hemocytometer and the final cell number was adjusted to 2 × 10 8 cells ml −1 .About 5% of RBCs were treated with different SKN60 T peptide concentrations and incubated in a CO 2 (5%) incubator for various time intervals at 37 • C. PBS and 1% tritonX 100 were considered as negative and positive controls, respectively.Lysis of RBCs was measured using a spectrophotometer with absorbance at 541 nm for different time intervals to observe lysis of RBCs (Singh et al. 2014 ).

Isolation and characterization
While scr eening rhizospher e soil samples for micr oor ganisms possessing antimicrobial activity, a strain designated as SKN60 with an inhibitory zone was isolated and studied in detail.Strain SKN60 T sho w ed good gro wth on all media including ISP1-ISP7 and Streptom yces a gar.It pr oduced aerial and submer ged mycelium.Aerial mycelium was off-white on ISP1, ISP2 and Streptomyces agar media, and it was cream in color on all other media used for cultivation (Fig. 1 A).Soluble pigment production was observed when it was grown on ISP6 medium but no diffusible pigment production was observed in any other media.Substrate mycelia wer e cr eam-color ed in all media except ISP4, ISP6, and ISP7, which sho w ed bro wnish color.Cream sporulation w as observed on ISP1, It sho w ed a negativ e r eaction for indole, ur ease, citr ate, ar ginine deh ydrolase, meth yl red, and Voges-Proskauer tests.Hydrolysis of esculin, casein and star ch w er e observ ed.Hydr ol ysis of Tween 20 and 40 was negative but weakly positive for Tween 60 and 80.It sho w ed a negativ e r eaction to catalase and gelatine tests.It was able to utilize ar abinose, sucr ose, dulicitol, galactose , lactose , mannose , mannitol, melibiose , d-myo inositol, raffinose , rhamnose , sorbitol, salicin, trehalose , and xylose as carbon sour ce.Gro wth w as observ ed up to 40ºC temper atur e with optim um gr owth at 30ºC and pH tolerance up to 10.It was observed that strain SKN60 T was able to tolerate 8% NaCl concentration.Further, str ain SKN60 T differ ed in bioc hemcial and physiological properties with closet relatives (Table S1 , Supporting Information).The fatty acid profile sho w ed presence of (%) iso-C 10:0 (10.9), iso-C 14:0 (7.2), iso-C 15:0 (10.9), anteiso-C 15:0 (11.2), iso-C 16:0 (8.4), anteiso-C 17:0 (3.0), iso-C 17:1 ω5c (6.1), and iso-C 17:0 3-OH (7.5) as major fatty acids .T he polar lipid analysis using two-dimensional TLC and subsequent differential staining sho w ed presence of diphosphatidylgl ycer ol (DPG) and phosphatidylethanolamine (PE) as major polar lipids .T he pr esence of phosphatidylc holine (PC), phosphatidylinositol (PI), and an unidentified phospholipid was also observed in minor quantities (Figure S1 , Supporting Information).The menaquinone analysis sho w ed presence of MK-9.The peptidogl ycan anal ysis of cell wall l ysate using TLC sho w ed diaminopimelic acid (DAP) as a c har acteristic cell wall diamino acid.

16S rRNA gene phylogeny
Taxonomic affiliation of strain SKN60 T was established using EzBioCloud database blast search of 16S rRNA gene sequence (1404 bp, NCBI accession number MZ642351) and closely related phylogenetic neighbors were identified.Further, 16S rRNA gene sequence r etrie v ed fr om genome sequence (1532 bp) was r elativ el y longer and identical with sequence obtained from Sanger method, and thus, it w as used for similarity sear ch and phylogen y.The maxim um sequence identity was observed with type str ains of Streptom yces laurentii ATCC 31255 T (99.31%), S. roseicoloratus TRM 44457 T (99.03%), S. showdoensis NBRC 13417 T (98.47%), and S. vietnamensis GIMV4.0001T (98.31%).The phylogenetic tree constructed using neighbor-joining phylogenetic analysis sho w ed strain SKN60 T forming a cluster with closest phylogenetic relatives in a distinct clade (Fig. 1 B).Indeed, phylogenetic analysis rev ealed that str ain SKN60 T formed a monophyletic cluster with type strains of S. roseicoloratus TRM 44457 T and S. laurentii ATCC 31255 T .

Genome sequence analyses
The WGS of Streptomyces sp.strain SKN60 T contained 7.8 Mb assembled in 87 contigs (with pegs) with N50 contig size of 312 247 bp.The whole genome was submitted to NCBI under accession number JAHTMW000000000.Sequence based GC content was 72.3 mol%.The draft genome of strain SKN60 T was annotated using the prokaryotic genome annotation pipeline (PGAP, NCBI) and RAST analysis revealed 7396 CDS, 73 tRNA, 3 rRNA, 10 L50, and 312 247 N50.The genome of strain SKN60 T w as emplo y ed for ANI calculation against 30 closely related genomes using Kostas lab server.None of the genomes of type str ains compar ed with SKN60 T sho w ed more than 95% ANI value.Ho w ever, ANI analysis of genome sequences displayed maximum identity with S. roseicoloratus TRM 44457 T (88.45%), S. showdoensis NBRC 13417 T (87.02%), S. laurentii ATCC 31255 T (86.42%), and S. vietnamensis GIMV4.0001T (85.91%) (Figure S2A , Supporting Information).Similarly, dDDH values also shown in descending order 33.7%, 30.7%, 29.2%, and 28.3% with its closest strains S. roseicoloratus TRM 44457 T , S. showdoensis NBRC 13417 T , S. laurentii ATCC 31255 T , and S. vietnamensis GIMV4.0001T , r espectiv el y (Figur e S2B , Supporting Information).The descending order of AAI values with closely related species sho w ed 85.61%, 83.52%, and 80.71% identity with S. roseicoloratus TRM 44457 T , S. showdoensis ATCC 15227 T , and S. laurentii ATCC 31255 T , r espectiv el y (Figur e S2C , Supporting Information).The phylogenomic tree derived from intergenomic distances calculated using GBDP on TYGS server sho w ed a distinct clade containing strains SKN60 T and S. roseicoloratus TRM 44457 T in a monophyletic cluster along with type strain of S. laurentii ATCC 31255 T (Fig. 1 C).
The high number of coding sequences (CDS) is accounted for the large genome size and functionally categorized CDSs have been assigned to cluster of orthologous groups using BASys platform.Gr a phical ma ps wer e gener ated using CCT to display the percentage blast similarities between the genomes of strain SKN60 T and those of S. roseicoloratus TRM 4457 T , S. showdoensis ATCC 15227 T , and S. laurentii ATCC31255 T for comparison purposes .T he sense and antisense strands of genome are represented by the outermost two rings colored in blue hues, while the percentage of sequence similarity based on blast hits is displayed through coloring in the graphical representation (Fig. 2 A).The three inner rings displayed areas of sequence similarity between CDS translation of the strain SKN60 T genome and those of S. roseicoloratus TRM 4457 T , S. showdoensis ATCC 15227 T , and S. laurentii ATCC 31255 T , r espectiv el y, identified thr ough BLAST comparisons.Different colors represent the percentage of blast hit identification, r anging fr om 100% to 0% in the gr a phical displa y.T he genome sequence of strain SKN60 T was visualized in right order using Pr oksee serv er.The anal ysis displayed distinct rings in the outermost region indicating the presence of CDS in both the sense and antisense orientations, follo w ed b y various contigs.One of the contigs, depicted in bro wn, w as identified as the berninamycin encoding BGC region in strain SKN60 T (Fig. 2 B).The pan-genome analysis in terms of protein coding genes of 30 Streptomyces species genomes r e v ealed 46 047 ortholog clusters fr om 162 482 total genes that comprised pan-genome .T he sum of unique genes was clustered into 24 930 gene clusters .T he core-genome contained 1568 gene clusters (Fig. 2 C).The increase in total genomes decr eased cor e-genome size, whic h is stabilized with about 1580 genes after the addition of 27th genome indicating the closure of core-genome .T he core-pan plot revealed increase in pan-genome with the addition of new genomes, and therefore, it is considered as an open pan-genome (Figure S3 , Supporting Information).The core and accessory gene pools wer e mainl y associated with carbohydrate , nucleotide , and amino acid metabolism, with some involved in the biosynthesis of secondary metabolites.

Antimicrobial purification and characterization
The growth of Streptomyces sp.strain SKN60 T was analyzed up to 8 days in Streptomyces broth at 30 • C and shaking condition with biomass determination at 24 h regular time intervals.Simultaneously, the CFB obtained was used to determine antimicrobial substance production by testing inhibitory activity on indicator strain S. aureus MTCC 1430.Cell biomass production of strain SKN60 T displayed a steady increase in cell biomass up to 5 days of incubation and thereafter decrease in the biomass was observed.An agar well diffusion assay showed inhibition of S. aureus MTCC 1430 from day 3 onw ar ds with maximum zone of inhibition (13 mm) at day 5.Among the media used for antimicrobial substance production, Streptom yces br oth yielded good gr owth of str ain SKN60 T and also displayed maximum inhibition zone against S. aureus MTCC 1430.Similar biomass yield and activity were also observed with NB medium.The CFB obtained inhibited Gr am-positiv e bacterial indicator strains but did not inhibit Gr am-negativ e test str ains.The antimicrobial substance was extracted using ethyl acetate solvent and used as a crude extract for further purification.HPLC analysis of crude extract sho w ed multiple peaks (Fig. 3 A) and TLC sho w ed multiple bands (inset A Fig. 3 A), but only the HPLC peak eluted at 8.7 min retention time sho w ed promising inhibitory activity against S. aureus MTCC 1430 (inset B Fig. 3 A).The mass of the peptide was determined by LC-MS (Q-ToF), which showed a predominant peak with mass m/z 1146.3Da (Fig. 3 B).A minor peak observed with mass m/z 573 Da shows presence of precursor ion corresponding to M + H 2 + .The fraction showing activity was r epeatedl y injected on HPLC and collected manually.Mass spectrum (MALDI) analysis of collected fragment sho w ed a predominant peak with mass signal m/z 1169 Da (Fig. 3 C) and another peak at 1146 Da (M + H + ).The 23 Da difference observed between both peaks indicated the formation of sodium adduct.Analysis of the sample on TLC sho w ed a distinct band under UV light (inset A Fig. 3 C) and stained with ninhydrin (inset B Fig. 3 C).It also displayed activity against S. aureus MTCC 1430 in bioautogr a phy assay (inset C Fig. 3 C).

BGC analysis
The bioinformatic analysis of Streptomyces sp.strain SKN60 T genome sequence was carried out to determine genes encoding secondary metabolites using antiSMASH 7.0 software.Results r e v ealed that 23 putative BGCs encode various secondary metabolite synthesis with different similarity index (Table S2 , Supporting Information).One of the BGCs sho w ed the presence of genes involved in the synthesis of a RiPP (ribosomally synthesized post-tr anslationall y modified peptide).The pr edicted cluster contained 17 ORFs (spans ∼30 kb) encoding all genes required for thiopeptide biosynthesis including enzymes involved in post-translational modifications.Bioinformatic analysis of the BGC r e v ealed 94% identity to berninamycin, ho w e v er, tr anslated sequence of thiopeptide encoding berA gene sho w ed minor difference in the amino acid composition of the leader peptide (Fig. 3 D).The structural gene encoding thiopeptide ( berA ) contained a total of 74 amino acids .T he core peptide had 16 amino acids with Serine (Ser) residue at the 10th position and was identical to berninamycin amino acid composition (Fig. 3 D).The remaining clusters comprised the hallmark of thiopeptide synthesis, such as BerB-D encoding lantibiotic dehydratase N-and C-terminus, pyridine forming genes, r espectiv el y.BerE1 and E2 wer e observ ed for installing the lantibiotic dehydrogenase.BerG1 and G2 encompass YcaO-type cyclodehydratases and docking scaffold that control the synthesis of thiazole , oxazole , and methyloxazole .Additionally, BerI encodes for C-terminal amide-forming enzymes.Nevertheless, an additional gene was observed in the biosynthetic cluster of strain SKN60 T encoding l -alanyl peptidase, which is known to carry out cleav a ge of the propeptide during the maturation of acti ve pe ptide.

Antimicrobial activity
In vitro antimicrobial studies sho w ed that the antimicrobial peptide from strain SKN60 T inhibited the growth of Gram-positive bacteria.Details of MIC e v aluation r esults observ ed for v arious str ains ar e pr ovided in Table 1 .MIC of antimicr obial peptide cor-responded to 60, 70, and 90 μg ml −1 against Enterococcus faecium MTCC 789, Enterococcus faecalis MTCC 439, and S. aureus MTCC 1430, r espectiv el y.Further, MIC determination of SKN60 T peptide a gainst v arious species of the genus Bacillus r anges fr om 70 to 90 μg ml −1 .Ne v ertheless, no effect was detected a gainst Gr amnegativ e indicator str ains including P. aeruginosa MTCC 1934 and E. coli MTCC 1610.Results of killing kinetics of the peptide sho w ed a reduction of growth of test strains as 2-log kill was observed within 1 h of incubation with peptide (5X MIC) for all strains (Fig. 4 A).Amongst test strains, E. faceium MTCC 789 sho w ed complete killing within 3 h of incubation with a peptide concentration of 5X MIC.Ov er all, within 4 h of incubation the growth of all strains was inhibited.Electron microscopic observation of untreated cells (control) sho w ed intact cells with a smooth surface.TEM analysis sho w ed the disintegration of cell membrane upon  exposure to the peptide (Fig. 4 B).It also sho w ed complete lysis with the accumulation of cell lysate debris in surroundings at higher peptide concentration.

Haemolysis
The haemolytic activity of the compound was confirmed by testing its effect on New Zealand white rabbit RBCs.It was observed that 400 μg ml −1 concentration of peptide sho w ed about 5% of haemol ysis with r espect to the positiv e contr ol (1% Triton X100) observed at 0 h time point.Ho w e v er, haemol ysis was incr eased to 66% with 400 μg ml −1 concentration upon 3 h of incubation (Fig. 4 C).

Discussion
Members of the genus Streptomyces are ubiquitous and r eceiv ed m uc h attention due to their ability to produce secondary metabo-lites with extensive medicinal value.Ho w ever, most of the Streptom yces str ains wer e not explor ed for antimicr obial metabolites, as r e v ealed by genome sequences.Further, r esearc h is r e viving for the undiscov er ed bioactiv e molecules thr ough genome mining of available sequences (Smanski et al. 2016 ).Ther efor e, aiming to isolate bacterial str ains pr oducing suc h antimicr obial compound, we have plated fertile subsurface soil sample on actinomycetes medium.One of the bioactiv e pr oducing isolates, str ain SKN60 T with actinom ycetes lik e colon y featur es was studied in detail.The phenotypic features including colony morphology, fatty acids composition, polar lipid profile, and G + C mol% contents align with the properties described for the genus Streptomyces .The 16S rRNA gene sequence analysis of SKN60 T showed closest sequence identity (99.31%) to S. laurentii ATCC 31255 T .Subsequent genome sequence analyses of strain SKN60 T confirmed the maximum ANI (88.45%) and dDDH (33.7%), r espectiv el y, with S. roseiocoloratus TRM44457 T .Ho w e v er, these v alues ar e m uc h lo w er than the thr eshold v alues r equir ed for species delineation (Rossello- It is reported that Streptomyces strains are known to contain an enormous repository of BGCs encoding secondary metabolites with a promising ability for the secretion of novel antimicrobials (Nandhini et al. 2018, Pacios-Michelena et al. 2021, Alam et al. 2022 ).Accordingl y, str ain SKN60 T identified as a member of the genus Streptomyces and its genome analysis by antiSMASH sho w ed the potential to secr ete bioactiv e compounds.Detailed analysis sho w ed the presence of a RiPP cluster showing 94% sequence identity with thiope ptide berninam ycin.The unique featur e observ ed in the BGC of berninamycin is the presence of genes encoding cyclodehydratase and dehydr ogenase involv ed in posttr anslational modifications, whic h ar e observ ed in str ain SKN60 T .Further, considering the enzyme activity, the presence of cysteine, serine and threonine residues at a conserved region in propeptide clearly suggests it to be a substrate for the enzymes involved in post-translational modifications.Schneider et al. ( 2018 ) observed these modifications are essentially required in synthesis of geninthiocin, a thiopeptide antibiotic.These modifications were in alignment with observed mass of 1146 Da (M + H + ) for r ecombinant v ersion of berninamycin (Malcolmson et al. 2013 ).BGC of berninamycin sho w ed BerH gene belongs to P450 hydroxylase famil y, whic h is mostl y involv ed in the hydr oxylation of v aline .T hese genes are responsible for hydroxylation at the tailoring stage after the formation of main scaffold (Malcolmson et al. 2013, Schneider et al. 2018 ).A previous study demonstrated the involvement of gene BerI in post-translational modifications that acts on a macr ocyclic substr ate to yield C-terminal amide and generates acti ve berninam ycin (Malcolmson et al. 2013 ).It is pertinent to mention that the presence of l -alanyl peptidase only observed in berninamycin biosynthetic cluster of strain SKN60 T is typically known to carry out the cleav a ge of the peptide at N-terminal alanine r esidue perha ps to yield activ e peptide .T he subtle difference observed in the leader peptide region of berA gene sequence indicates the micr oe volution of BGCs with subsequent effect on secr etion le v els (Smanski et al. 2016 ).
Thiopeptides or thiazolyl peptides reported mainl y fr om genus Streptom yces ar e w ell-kno wn antimicrobial compounds with complex structural configurations .T he research in this class of antibiotics has gained significant attention since it sho w ed promising antimicr obial activity a gainst drug-r esistant pathogens, including penicillin-resistant Streptococcus pneumoniae , methicillin-resistant S. aureus , and v ancomycin-r esistant enter ococci (Sc hneider O et al. 2018, Shen et al. 2019 ).Studies demonstrated that berninamycin and its variants produced by Streptomyces species sho w ed antibacterial activity against Gram-positive bacteria (Bagley et al. 2005, Schneider et al. 2018, De BC et al. 2021 ).Strain SKN60 T was cultured on different carbon and nitrogen sources to investigate the possibility of variation in antimicrobial compound production.The cell-fr ee br oth of str ain SKN60 T sho w ed antibacterial activity a gainst indicator str ains of Gr am-positiv e bacteria like S. aureus MTCC 1430, E. faecalis MTCC 439, and E. faecium MTCC 789.Notably, the antibacterial activity was also in alignment with berninamycin.Ho w e v er, thiopeptides wer e also known to hav e antifungal, antiplasmodial, and anticancer activities (Aminake et al. 2011, Ovc hinnik ov et al. 2021 ).In contrast, SKN60 T peptide did not inhibit the growth of yeast and fungi, indicating its potential to differentiate between prokaryotic and eukaryotic cell membranes.
Thiopeptide antibiotics like nosiheptide thiocillin, thiostrepton, and geninthiocin were known to bloc k pr otein synthesis r esulting in the death of cells (Mikolajka et al. 2011 , Cundliffe andThompson 1981 ).Similarly, thiopeptide from strain SKN60 T in this study caused cell death and TEM analysis sho w ed disruption of cell integrity of S. aureus MTCC 1430.Although antimicrobial peptides have gained significant attention over the past two decades, but problems such as haemolysis, cytotoxicity and poor efficacy under in vivo conditions have limited their clinical value.Similarly, SKN60 T peptide sho w ed haemolysis of RBCs at a higher concentr ation that incr eased with pr olonged incubation.The r ecent success in de v eloping analogues of thiopeptide with r emarkable pharmaceutical applications may pave the way for de v eloping drugs targeting antimicrobial-resistant pathogens (Just- Baringo et al. 2014 ).Although berninamycin was reported from S. bernensis , suc h str ain is not av ailable in public domain and it is not v alidated as a species of the genus Streptomyces .This is first study to report the production of berninamycin by an isolate, which is identified as a novel species of the genus Streptomyces .It is important to note that such antimicrobial producing strains are not available in public culture collection, but their genome sequences are deposited in databases .T hus , it hinders the comparison studies, a complete understanding of the antimicrobial production and str ain impr ov ement to incr ease the yield.
Taken together, our results indicate that Streptomyces sp.strain SKN60 T is a novel species isolated from agricultural soil with low sequence identity with all type strains of the genus Streptomyces .In this study, we have reported the production of thiopeptide berninamyin from a type strain as confirmed by mass and sequence analyses .T his bioactive molecule sho w ed inhibitory activity against Gram-positive bacteria.Nevertheless, further research must be carried out to elucidate the molecular mechanisms underl ying the syner gistic effects and the exact mec hanism behind the bactericidal effect.Such information/knowledge will help design more efficient and safer drugs before the clinical trials.

Description of novel species
Streptomyces terrae (terrae.L. Gen.n.Terrae pertaining to soil, referring to isolation source) Strain SKN60 T is an aerobic , Gram-positive , nonmotile filamentous actinobacteria.The substrate mycelium was well-grown and v aried fr om off-white to br ownish on differ ent media.Gr owth is observ ed fr om 20 to 40ºC and pH ranging 4-10.It tolerates up to 8% NaCl and sho w ed a positive reaction for nitrate reduction.Utilization of sugars like arabinose, dulcitol, sucrose, galactose, inos-

Figure 1 .
Figure 1.Phenotypic and genetic c har acterization of strain SKN60 T .(A) Cultural characteristics of SKN60 T strain on ISP-1 to ISP-7 media and Streptom yces a gar showing substr ate m ycelium, aerial m ycelium, sporulation, and secretion of soluble pigment.(B) Phylogenetic r elation of str ain SKN60 T with close r elativ es based on neighbor-joining method using 16S rRNA gene sequences.Bootstr a p v alues > 50% ar e displayed on their r elativ e br anc hes.Kim ur a 2_par ameter method was consider ed to measur e the e v olutionary distances and gamma distribution w as set to 0.5 (C) Intergenomic distances based phylogenomic tree showing relation of strain SKN60 T with other closest relatives.It was performed using GBDP.The analysis sho w ed formation of a distinct clade of strain SKN60 T with Streptomyces roseicoloratus TRM 44457 T .

Figure 2 .
Figure 2. Genome compar ativ e anal yses of str ain SKN60 T with close phylogenetic r elativ es.(A) Circular plot of genome comparison among the phylogenetic r elativ e str ains using CGVie w comparison tool (CCT).Outer most rings showing sense and antisense CDS follo w ed b y three rings sho wing blast hit comparison results outer to inner, SKN60 T vs. TRM 44457 T , SKN60 T vs. ATCC 15227 T , and SKN60 T vs ATCC 31255 T ) (B) Pr oksee based of str ain SKN60 T showing different rings indicating the presence CDS (sense and antisense) in the outer most region, follo w ed b y different contigs .T he contig which is brown in color is the berninamycin encoding region of SKN60 T peptides .T he GC content, GC skew (positive and negative), tRNA, rRNA, r epeated r egion, and tmRNA r egions ar e also shown in the centr al portion of the circular ma p. (C) Flo w er pot dia gr am r epr esentation of the str ain SKN60 T along with its closest strains showing BPGA for core, accessory, and unique gene distributions.

Figure 3 .
Figure 3. Purification and c har acterization of antimicrobial substance produced by SKN60 T .(A) HPLC analysis of crude extract (inset TLC showing multiple bands under UV light 254 nm and antimicrobial activity of HPLC fractions at different retention time).(B) Q-TOF spectrum of the peak showing r epr esentativ e pr ecursor ions (m/z, 1146.3 [M + H], 573.6 [M + 2H] 2 + ).(C) MALDI result of purified AMP sho w ed intact peak of 1169 [M + Na] as sodium adduct (inset TLC showing a single band in UV light and stained with ninhydrin.The bioautogr a phy anal ysis sho w ed inhibition of S. aureus MTCC 1430 using agar o verla y assa y).(D) Organization of genes in BGC and comparison with S. bernensis UC5144 and Streptomyces sp.YIM130001 with their peptide sequence precursors encoded by the structural gene.Arrow in between the peptide sequence indicated the demarcation between leader and core amino acid sequences.

Figure 4 .
Figure 4. Determination of antibacterial and haemolytic activities.(A) Time-kill assay against Gram-positive bacterial strains.(B) TEM visualization of membr ane disintegr ation by antimicr obial substance: (i) untr eated cells of S. aureus MTCC 1430, (ii) S. aureus MTCC 1430 cells treated with 2X MIC of SKN60 T peptide, and (iii) treated with 5X MIC of SKN60 T peptide.(C) Determination of haemolysis by SKN60 T peptide showing lysis of RBCs at higher concentration upon 3 h of incubation.

Table 1 .
MIC v alues of antimicr obial peptide fr om SKN60 T a gainst v arious Gr am-positiv e bacterial test str ains.