A novel actinobacterium, designated strain SMC 277T, was isolated from the clay soil in paddy field of Chonburi Province, Thailand, and characterized using polyphasic taxonomy. Strain SMC 277T formed straight chains of nonmotile cylindrical spores with smooth surface developed on aerial mycelia. The typical chemotaxonomic properties of members of the genus Streptomyces were observed in strain SMC 277T, e.g., cell wall peptidoglycan, whole cell sugars, major menaquinones, cellular fatty acids, and polar lipids. Chemotaxonomic data combined with mycelium and spore morphologies supported the assignment of strain SMC 277T to the genus Streptomyces. The results of comparative analysis of the 16S rRNA gene sequences confirmed that strain SMC 277T represented a member of the genus Streptomyces. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain SMC 277T shared the highest sequence similarity with Streptomyces bambusae NBRC 110903T (98.8%). Genome sequencing revealed a genome size of 6.55 Mbp and a digital G+C content of 73.4 mol%. In addition to the differences in phenotypic characteristics (morphology and physiology), values of ANI (ANIb and ANIm), AAI and dDDH between strain SMC 277T and its closest relative S. bambusae NBRC 110903T were 81.84, 86.77, 76.91 and 26.1%, respectively. Genome annotation and secondary metabolite gene cluster analysis predicted that SMC 277T contained 35 biosynthetic gene clusters encoding diverse bioactive secondary metabolites. It is in agreement with observed antimicrobial activity against drug-resistant bacteria associated with nosocomial infections (methicillin-resistant Staphylococcus aureus, extended-spectrum β-lactamase producing Klebsiella pneumoniae, and multidrug-resistant Acinetobacter baumannii). On the basis of these genotypic and phenotypic characteristics, strain SMC 277T can be characterized to represent a novel species of the genus Streptomyces, for which the name Streptomyces antimicrobicus is proposed. The type strain is SMC 277T (= TBRC 15568T = NBRC 115422T).
Citation: Chanama M, Suriyachadkun C, Chanama S (2023) Streptomyces antimicrobicus sp. nov., a novel clay soil-derived actinobacterium producing antimicrobials against drug-resistant bacteria. PLoS ONE 18(5): e0286365. https://doi.org/10.1371/journal.pone.0286365
Editor: Paula V. Morais, Universidade de Coimbra, PORTUGAL
Received: October 26, 2022; Accepted: May 10, 2023; Published: May 31, 2023
Copyright: © 2023 Chanama et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper and its Supporting Information files.
Funding: This research project is supported by Mahidol University. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
The genus Streptomyces classified in the family Streptomycetaceae of the suborder Streptomycineae [1,2] was proposed by Waksman and Henrici , and emended subsequently by Rainey et al.  and Kim et al. . It is the largest genus of the Actinobacteria with 699 validly published and correct species, based on the LPSN (List of Prokaryotic names with Standing in Nomenclature, https://lpsn.dsmz.de/genus/streptomyces) website at the time of writing. Members of the genus Streptomyces are identified as aerobic, gram-stain-positive, non-acid-fast bacteria that form extensively branched substrate and aerial mycelia. They have long chains of spores, contain LL-diaminopimelic acid in their cell walls, major amounts of saturated, iso-and anteiso-fatty acids, and typically possess either MK-9(H6) or MK-9(H8) menaquinones. The predominant polar lipids contain diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol and phosphatidylinositol mannoside. The high genomic DNA G+C contents (66–78 mol%) are observed .
Obviously, Streptomyces strains are promising resources to produce bioactive compounds as antibiotics [7–10]. Several novel bioactive compounds with diverse antimicrobial activities have been reported from various species of the genus Streptomyces isolated from soils in different areas, for example, albocycline-type macrolides , benditerpenoic acid , and pyrimidomycin . Until now, bacteria causing nosocomial infections still remain public health crisis and health security threats due to increasing antibiotic resistance . Therefore, discovering novel Streptomyces species with potential bioactivities is crucial.
In this study, a polyphasic taxonomy, including morphological, physiological, chemotaxonomic and genotypic characterization was conducted to classify a novel actinomycete strain, designated SMC 277T, isolated from the clay soil in paddy field of Chonburi Province, Thailand. The inhibitory activities against drug-resistant bacteria of nosocomial infections, and biosynthetic potential of the strain were also investigated. Strain SMC 277T represents a novel species of the genus Streptomyces, for which the name Streptomyces antimicrobicus sp. nov. is proposed.
Materials and methods
Isolation and culture conditions
During an investigation of actinomycete diversity from soils in Thailand, strain SMC 277T was isolated from a sample of clay soil collected from a paddy field of Chonburi Province. The sample was kept at -20°C before being air-dried at 37°C for 7 days. The strain was isolated using the standard dilution plate method and grown on humic acid-salts vitamin agar  supplemented with cycloheximide (50 mg/L) and nystatin (50 mg/L). After incubating at 28°C for 7 days, the colony of strain SMC 277T was selected and then subcultivated on International Streptomyces Project (ISP) 3 medium (oatmeal agar) . A pure culture was maintained in glycerol (20%, v/v) at -80°C. The type strain of the closest species, Streptomyces bambusae NBRC 110903T was obtained from Thailand Bioresource Research Center (TBRC). The strain was cultured under the same conditions, and used to compare polyphasic characteristics.
Morphological characterization of strain SMC 277T and the type strain, Streptomyces bambusae NBRC 110903T, was performed after growth on various International Streptomyces Project (ISP) media (ISP 2–7), as described by Shirling and Gottlieb , after incubating at 28°C for 14 days. The Inter-Society Color Council National Bureau of Standards (ISCC-NBS) color charts  were used to determine the color of aerial and substrate mycelium, and soluble pigments. Morphology of mycelia and spores was observed after cultivating on modified soil extract agar  at 28°C for 10 days under both a light microscope (model CX31, Olympus, Japan) with a x50 working distance objective lens (model SLMPLN50X, Olympus, Japan) and a scanning electron microscope (SEM) (model JSM-IT500HR, JEOL, Japan). The sample for SEM was prepared by fixing an agar block on which the culture grew with 2% osmium tetroxide vapor, followed by dehydration through a graded ethanol series, and finally dried using a critical point dryer (Leica model EM CPD300, Austria). The dried sample was placed on the stub and coated with gold using sputter coater (Balzers model SCD040, Germany) for visualization under SEM.
Physiology and biochemical properties
Growth at different temperatures (20, 25, 30, 37, 40 and 45°C) was assessed on ISP 2 medium after incubating at 28°C for 14 days. The pH range at pH 4.0 to 11.0 (intervals of 1.0 pH unit) and NaCl tolerance with 0.5, 1, 2, 3, 4 and 5% (w/v) for growth were determined using ISP 2 medium as a basal medium after 14 days of incubating at 28°C. Using carbohydrates as a sole carbon source was examined on ISP 9 medium as a basal medium supplemented with a final concentration of 1% (w/v) of the carbon sources . The hydrolysis of various substrates was evaluated using a basal medium and method recommended by Gordon et al. . Gelatin liquefaction, milk peptonization and coagulation, nitrate reduction and hydrolysis of starch, xanthine and hypoxanthine were determined by cultivating on various media as described by Arai , and Williams and Cross . Enzyme activities were examined using the API-ZYM system (bioMérieux) according to the manufacturer instructions.
Biomass of strain SMC 277T and its closest relative, S. bambusae NBRC 110903T used for chemotaxonomic analysis was obtained from culture grown in glucose-yeast extract broth  on a rotary shaker at 28°C, 250 rpm for 7 days. Cells were harvested using centrifugation, washed three times with sterile distilled water before freeze drying. The isomer of diaminopimelic acid in the cell wall was determined using the method of Staneck and Roberts . The compositions of reducing sugar in whole-cell hydrolysates were analyzed using cellulose TLC as described by Komagata and Suzuki . Total polar lipids in whole cells were extracted and analyzed according to the method of Minnikin et al. . Cellular fatty acids were prepared and analyzed following the instructions of the RTSBA6 method of the Microbial Identification System (MIDI; Sherlock, Version 6.4) . Menaquinones were extracted and purified using the method of Collins et al. , and analyzed using reverse-phase HPLC [Cosmosil 5C18 column (4.6x150 mm); Nacalai Tesque] with a mixture of methanol and 2-propanol (2:1, v/v) as elution solvent .
Genomic and phylogenetic characterization
Genomic DNAs of strain SMC 277T and Streptomyces bambusae NBRC 110903T were extracted according to a modified method of Saito and Miura  from cells grown in glucose-yeast extract broth at 28°C, 250 rpm for 5 days. Freeze-dried cells were lysed by grinding with mortar and pestle, instead of lysozyme. The 16S rRNA gene of strain SMC 277T was amplified using PCR with 27F and 1492R primers as described by Monciardini et al. . The amplified 16S rRNA gene was purified and directly sequenced by SolGent (Seoul, South Korea) using the ABI373OXL platform and 4 sequencing primers (2F: 5′ ACGGGAGGCAGCAGTG 3′, 3F: 5′ AACACCGGTGGCGAAG 3′, 4F: 5′ CGTCAAGTCATCATGCCC 3′, 4R: 5′ CCTACGWGCYCTTTACGCC 3′). The nucleotide sequences obtained from all primers were assembled using the Cap contig assembly program, an accessory application in the Bioedit Sequence Alignment Editor Software (Version 7.2.5) . The Basic Local Alignment Search Tool (BLAST) analysis retrieved from the nucleotide databases of NCBI (https://blast.ncbi.nlm.nih.gov/Blast.cgi;  was used to compare the almost-complete 16S rRNA gene sequence of strain SMC 277T with sequences of all validly published species of the genus Streptomyces. Based on pairwise alignment using the NCBI BLAST database , the 16S rRNA gene sequence similarities between species were calculated. Multiple alignments were carried out using the CLUSTAL W Program  in Bioedit Sequence Alignment Editor Software (Version 7.2.5) . Nocardioides albus KCTC 9186T was taken as an outgroup. The MEGA Software Package, Version 10.1.7  was used to construct a phylogenetic tree using neighbor-joining , maximum-parsimony , and maximum-likelihood  methods. The neighbor-joining result was calculated according to Kimura’s two-parameter model with complete deletion. The search method for maximum-parsimony was subtree-pruning-regrafting. The Tamura 3-parameter plus gamma distributed with invariant sites was used for maximum-likelihood. The statistical reliability of the tree topology was evaluated using bootstrap analysis with 1000 replications .
Whole genomes of strain SMC 277T and S. bambusae NBRC 110903T were sequenced using Illumina NovaSeq platform at Novogene (Beijing, China). Libraries of genomic DNA were prepared using a TruSeq Nano DNA kit (Illumina) and pooled libraries were subjected to multiplexed paired-end sequencing. Raw paired-end sequences were used for quality control, and their adapter and primer sequences were trimmed using the FASTP Program . The cleaned sequences were then used to assemble the genome with SPAdes (Version 3.10.1) . The assembled genome was annotated using the NCBI Prokaryotic Genome Annotation Pipeline [43–45]. The average nucleotide identity (ANI) values, i.e. ANI-BLAST (ANIb) and ANI-MUMmer (ANIm) of the whole genome of strain SMC 277T with closely related type strains were calculated using the JSpeciesWS web server . The average AAI of strain SMC 277T compared with closely related type strains was calculated using the online server AAI calculator (http://enve-omics.ce.gatech.edu/aai/) . The Type (Strain) Genome Server (TYGS), a free bioinformatics platform available at https://tygs.dsmz.de, was used to analyze the whole genome-based taxonomic . The tree was inferred using FastME 184.108.40.206  from the Genome BLAST Distance Phylogeny Approach (GBDP) distances calculated from genome sequences, and branch support was inferred from 100 pseudo-bootstrap replicates. Digital DNA-DNA hybridization (dDDH) values between strain SMC 277T and each of the closely related type strains was calculated using the recommended settings of the Genome-to-Genome Distance Calculator (GGDC), Version 2.1 of TYGS [48,50]. The genomic DNA G+C content (mol%) was determined from the whole genome data sequencing.
Genome annotation and secondary metabolite gene cluster analysis
Genome assemblies of strain SMC 277T, and closely related type strains, including S. bambusae NBRC 110903T, S. toxytricini NBRC 12823T, S. cirratus NBRC 13398T, S. vinaceus ATCC 27476T, S. nojiriensis JCM 3382T, S. yangpuensis DSM 100336T, S. virginiae NBRC 12827T, and S. amritsarensis MTCC 11845T were downloaded from NCBI. Protein-coding sequences (CDS) were predicted using Prodigal 2.6.3 . Subsequently, all the annotated protein sequences were grouped to clusters of orthologous groups (COGs) using WebMGA . The predicted secondary metabolite biosynthesis gene clusters were obtained using antiSMASH , and BLAST known cluster function was enabled to find known substances matched from repository of known biosynthetic gene clusters. ClustVis , a tool for hierarchical clustering, was used to create heatmaps and visualize differences of the COGs and biosynthesis gene clusters among Streptomyces species.
Target microorganisms for antimicrobial bioassays were bacteria associated with nosocomial infections, called ESKAPE pathogens (Enterococcus faecalis, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter aerogenes). Drug-sensitive bacteria, i.e., E. faecalis DMST 2860, S. aureus DMST 8840, K. pneumoniae DMST 7592, A. baumannii DMST 10437, P. aeruginosa DMST 4739 and E. aerogenes DMST 8841, were purchased from the National Institute of Health of Thailand, whereas drug-resistant bacteria, i.e., methicillin-resistant Staphylococcus aureus (MRSA) AMH 10, extended-spectrum β-lactamase (ESBL) producing Klebsiella pneumoniae AMH 20 and multidrug-resistant (MDR) Acinetobacter baumannii AMH 30, were kindly provided by Ananda Mahidol Hospital (AMH), Lopburi Province, Thailand.
Strain SMC 277T was precultured in a 125 ml flask containing 10 ml of SCM medium  for 3 days at 28°C with 250 rpm shaking. Each 1% (v/v) of the seed culture was then transferred to each 250 ml flask containing 50 ml of SCM medium, and incubated at 28°C, 250 rpm for 7, 14 and 21 days separately. Each supernatant was added with 5 ml of Diaion® HP-20 resin (Sigma). After leaving the suspension for 2 h under shaking, the resin was washed with 25 ml of water, filtered, and eluted with 25 ml of 80% methanol. The methanolic extract was evaporated to dryness, and dissolved with 100 μl of 67% methanol. Each 5 μl of the extract was transferred onto a paper disk (diameter of 6 mm, Whatman) to examine bioactivity. For bacterial targets, single colonies grown on tryptic soy agar (Becton Dickinson) at 37°C overnight were picked, adjusted to 108 CFU/ml with tryptic soy broth (Becton Dickinson) and swabbed on the entire surface of Muller Hinton agar (MHA) (Becton Dickinson). The paper disk with extract was placed on the surface of MHA plates containing the target microorganisms. A disk with 5 μl of 67% methanol was used as a negative control. The plate was incubated at 37°C for 24 h and then measured for the diameter of zone of inhibition. Each test was performed in triplicate.
Results and discussion
Phylogenetic analysis based on 16S rRNA genes
Strain SMC 277T isolated from the clay soil in paddy field collected from Chonburi Province, Thailand was used to characterize the taxonomic status using polyphasic approaches. The 16S rRNA gene is generally considered to be highly conserved, and can be used to identify prokaryotes at the genus and species level which are evaluated at 97.0 and 98.7%, respectively . Firstly, the almost-complete 16S rRNA gene sequence of strain SMC 277T (1476 bp, Genbank accession number OK380049) was obtained from the amplification of its gene using PCR, and analyzed to compare nucleotide sequence similarity with sequences of currently and validly published type strains using NCBI BLAST. The pairwise alignments of strain SMC 277T showed the sequence similarity to those of members of genus Streptomyces with the top 41 Streptomyces spp. ranking from 98.8–97.6%, e.g., Streptomyces bambusae NBRC 110903T (98.8%), S. griseocarneus DSM 40004T (98.4%), S. coerulescens NBRC 12758T (98.3%), S. abikoensis NBRC 13860T (98.2%), S. yangpuensis DSM 100336T (98.2%). The sequence similarity of the strain SMC 277T was the most matched to that of Streptomyces bambusae NBRC 110903T (98.8%), which this value was slightly higher than defined value of species delineation (98.7%) for determining the bacterial strains to the same genomic species . Hence, this result provides us a clue to predict that SMC 277T has genetic differences from any other type strains of species of the genus Streptomyces, and may represent a novel species of the genus.
Moreover, the phylogenetic trees reconstructed by all algorithms of neighbor-joining (Fig 1), maximum-parsimony (S1A Fig) and maximum-likelihood (S1B Fig) yielded similar topology indicating that SMC 277T formed a monophyletic clade with only S. bambusae NBRC 110903T, with high bootstrap support at 96, 92 and 97%, respectively. For this reason, S. bambusae NBRC 110903T was selected as the closest phylogenetic relative for comparative analysis. Even though the results of phylogenetic relationship exhibited that strain SMC 277T belonged to the genus Streptomyces with S. bambusae NBRC 110903T as the closest relative, overall genomic relatedness indices (ORGIs) should be further calculated between genome sequences of strain SMC 277T and closely related type strains in order to delineate SMC 277T as a new species.
Nocardioides albus KCTC 9186T was used as an outgroup. Asterisks (*) indicate the branches of the tree that were found using maximum-parsimony and maximum-likelihood methods. The numbers on the branches indicate the percentage bootstrap values of 1,000 replicates, and only values >50% are shown. Bar 0.010 substitutions per nucleotide position.
Genome features and genotypic analysis
It has been proven that the information of genome sequences is objective and reliable for the prokaryotic taxonomy . The overall genomic relatedness indices (OGRIs) including average nucleotide identity (ANI), average amino acid identity (AAI) and digital DNA-DNA hybridization (dDDH) can be used to examine if a strain belongs to a known species. Therefore, OGRI values should be combined with 16S rRNA gene sequence similarity for a systematic process to identify and recognize a new species .
On the basis of genome sequencing, the draft genomes of strain SMC 277T (284 contigs), and S. bambusae NBRC 110903T (169 contigs) have been submitted to GenBank with accession numbers of JAJAUY000000000 and JAJAUZ000000000, respectively, and are publicly available. The draft genome sequence of strain SMC 277T yielded the size of 6.55 Mbp with an average in silico DNA G+C content of 73.4 mol%, and total of 5,809 protein-CDSs and 78 RNAs, whereas the genome size of S. bambusae NBRC 110903T was 8.31 Mbp with DNA G+C content of 73.0 mol%, and total of 7,269 CDSs and 75 RNAs. To further clarify the relationship between strain SMC 277T and closely related type strains, we obtained genomic data for 30 of the 41 strains with the highest 16S rRNA gene sequence similarity to strain SMC 277T (S1 Table). The whole genome phylogeny of strain SMC 277T indicated that it constituted a member of the genus Streptomyces, and was clearly separated from S. bambusae NBRC 110903T (Fig 2). Both average nucleotide identity (ANI) values, namely, ANIb and ANIm, of strain SMC 277T and S. bambusae NBRC 110903T were 81.84% and 86.77%, respectively. The average amino acid identity (AAI) value of strain SMC 277T with S. bambusae NBRC 110903T was 76.91%. The digital DNA-DNA hybridization (dDDH) value between the genomes of strain SMC 277T and S. bambusae NBRC 110903T was 26.1% (C.I. model 23.7 to 28.6%). The ANI (ANIb and ANIm), AAI and dDDH values of strain SMC 277T and other closely related species exhibited in the phylogenomic tree were shown in S1 Table. Clearly, these ANI, AAI and dDDH values were below the thresholds of 95, 95 and 70%, respectively, for prokaryotic species delineation [57,58]. The results of OGRIs (ANI, AAI and dDDH), and phylogenomic relationship were sufficient to categorize strain SMC 277T as representing a distinct species from previously described Streptomyces species.
Myxococcus fulvus DSM 16525T was used as an outgroup. Tree inferred with FastME 220.127.116.11  from GBDP distances calculated from genome sequences. The branch lengths are scaled in terms of GBDP distance formula d5. The numbers above branches are GBDP pseudo-bootstrap support values >60% from 100 replications, with an average branch support of 85.3%. The tree was rooted at the midpoint .
Morphological and physiological characteristics
To support the novelty at the species level of SMC 277T, phenotypic characteristics including morphology and physiology were performed. It developed straight spore chains on only aerial mycelium. Each spore was smooth-surfaced, and measured 0.5 to 0.7 by 1.1 to 1.3 μm in size (Fig 3). The mycelium and spore morphologies of SMC 277T were consistent with members of the genus Streptomyces. Strain SMC 277T exhibited good growth on ISP 2 and ISP 3 media, moderate growth on ISP 4 media, and poor growth on ISP 5, ISP 6 and ISP 7 media. The color of the substrate mycelium on these media was pale yellow to grayish greenish yellow. No aerial mycelium was produced on ISP 6, while the aerial mycelium with yellowish white to pinkish gray was produced on other growth media. The diffusible pigment was not observed on all tested media (S2 Table). The phenotypic comparison between SMC 277T and the closest relative, S. bambusae NBRC 110903T revealed differential characteristics that enabled SMC 277T to be readily distinguished from the closest relative (Table 1). Unlike S. bambusae NBRC 110903T, strain SMC 277T was able to grow at the maximum temperature of 40°C. Even though SMC 277T and S. bambusae NBRC 110903T were capable of growing at the maximum tolerance to NaCl of 4% (w/v), the growth capability of SMC 277T was higher than that of S. bambusae NBRC 110903T at 0.5, 1 and 2%. Moreover, hydrolysis of xanthine, gelatin liquefaction, nitrate reduction, milk coagulation and peptonization, the use of D-arabinose, cellobiose, D-galactose, D-mannitol, D-trehalose and D-xylose as sole carbon sources, and the capabilities to produce various enzymes, such as alkaline phosphatase, cystine arylamidase, α-glucosidase and α-mannosidase were the apparent characteristics for separating between SMC 277T and S. bambusae NBRC 110903T. Additionally, morphological and physiological characteristics of strain SMC 277T and other closely related Streptomyces species showing 16S rRNA similarity values below cut-off point of 98.7%, including S. griseocarneus DSM 40004T, S. coerulescens NBRC 12758T, S. abikoensis NBRC 13860T, S. yangpuensis DSM 100336T, S. virginiae NBRC 12827T, S. amritsarensis MTCC 11845T, S. toxytricini NBRC 12823T and S. cirratus NBRC 13398T were also presented in S2 Table. These informative data have been supporting evidence for phenotypic differences between SMC 277T and each of these closely related type strains. Therefore, strain SMC 277T could be distinguished from other Streptomyces taxa, and represented as a novel species.
The strain exhibited straight spore chains with smooth spore surfaces on its aerial mycelia. It was grown on modified soil extract agar at 28°C for 10 days. Bar, 5 μm.
Chemotaxonomy is also one of key information for describing a new taxon. Hence, these characteristics were investigated to confirm the taxonomic affiliation of strain SMC 277T at the genus level. Strain SMC 277T exhibited chemotaxonomic characteristics that were typical profiles of the genus Streptomyces. Strain SMC 277T contained LL-diaminopimelic acid in the cell wall peptidoglycan (S2 Fig), which was a common feature of members of the genus Streptomyces . Whole cell sugars consisted of glucose, galactose, mannose and ribose (S3 Fig). The major menaquinones were detected as MK-9(H8) (79.5%) and MK-9(H6) (20.5%). The major menaquinones of SMC 277T were similar to those found in the closest relative, S. bambusae NBRC 110903T, with different proportions . Polar lipids consisting of phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylinositol mannoside and phospholipids were detected in the cells (S4 Fig). The predominant cellular fatty acids (>10%) were iso-C16:0 (26.4%), anteiso-C15:0 (20.9%) and anteiso-C17:0 (10.8%) (Table 2). The pattern of major fatty acids in the cells of SMC 277T was similar to those predominant fatty acids in S. bambusae NBRC 110903T, the closest relative with different proportions (Table 2). Therefore, the chemotaxonomic analysis confirmed that strain SMC 277T represented a member of the genus Streptomyces.
Genome annotation and secondary metabolite gene cluster analysis
A total of 23 functional categories of COG proteins were observed in genomes of strain SMC 277T, the closest relative, S. bambusae NBRC 110903T, and other closely related type strains in the same phylogenomic clade as SMC 277T, including, S. toxytricini NBRC 12823T, S. cirratus NBRC 13398T, S. vinaceus ATCC 27476T, S. nojiriensis JCM 3382T, S. yangpuensis DSM 100336T, S. virginiae NBRC 12827T and S. amritsarensis MTCC 11845T. The three most abundant proteins were associated with general cellular function, transcription and amino acid transport and metabolism, respectively (S3 Table). In addition, hierarchical clustering of heatmap (Fig 4) showed different COG proteins clustered in different strains. In fact, strain SMC 277T, S. bambusae NBRC 110903T, S. vinaceus ATCC 27476T, S. cirratus NBRC 13398T and S. toxytricini NBRC 12823T were clustered in a group having a widespread abundance of COG proteins, whereas S. virginiae NBRC 12827T, showed enrichment in inorganic ion transport and metabolism; coenzyme transport and metabolism; translation, ribosomal structure and biogenesis; intracellular trafficking, secretion and vesicular transport; energy production and conversion; amino acid transport and metabolism; carbohydrate transport and metabolism; lipid transport and metabolism; and secondary metabolite biosynthesis and transport and catabolism. On the other hand, S. yangpuensis DSM 100336T, S. amritsarensis MTCC 11845T and S. nojiriensis JCM 3382T showed enrichment in general function prediction; transcription; defense mechanisms; cell cycle control, cell division, chromosome partitioning; signal transduction mechanisms; posttranslational modification, protein turnover, chaperones; RNA processing and modification; and unknown function.
Heatmap shows a data matrix where coloring gives an overview of the numeric differences from a reference.
Genomes of strain SMC 277T and closely related type strains mentioned as above were also scanned with antiSMASH to explore the known and putative secondary metabolite biosynthetic potential in their genome sequences. Altogether, 27 types of secondary metabolites and a total of 285 secondary metabolite biosynthesis gene clusters were identified (Fig 5 and S4 Table). The average number of BGCs of all Streptomyces strains was 32. The most abundant BGCs were hybrid clusters followed by terpene. However, S. antimicrobicus SMC 277T exhibited 35 gene clusters with NRPSs as the dominant classes of predicted BGCs. Interestingly, by comparing secondary metabolite clusters and their products from the antiSMASH database among all Streptomyces strains, S. antimicrobicus SMC 277T revealed 7 BCGs showing less than 50% similarity to known BCGs, including guadinomine, totopotensamide, SCO-2138, JBIR-78, griseoviridin, salinamide A and indigoidine. These BCGs were not present in any of the other analyzed strains. This suggested that S. antimicrobicus SMC 277T has the potential to produce novel metabolites.
Antibacterial activity of strain SMC 277T
To further explain the biosynthetic potential of strain SMC 277T, antimicrobial activity was assayed against target microoganisms including ESKAPE bacterial pathogens associated with nosocomial infections. The antimicrobial activity profile of strain SMC 277T is shown in Table 3. The results indicated that the extract from strain SMC 277T was able to inhibit the growth of several drug-sensitive bacteria, i.e., S. aureus DMST 8840, K. pneumoniae DMST 7592, A. baumannii DMST 10437 and E. aerogenes DMST 8841, and drug-resistant bacteria, i.e., MRSA strain AMH 10, ESBL producing K. pneumoniae AMH 20 and MDR A. baumannii AMH 30, when cultured for 7, 14 and 21 days. The inhibitory activity was not observed against E. faecalis DMST 2860 and P. aeruginosa DMST 4739. The largest zone of inhibition was observed against A. baumannii DMST 10437. These results indicated that strain SMC 277T may prove a promising candidate to produce secondary metabolites with a wide capability of antimicrobial activities against bacterial pathogens.
A novel Streptomyces strain with antibacterial activity, SMC 277T, was isolated from the clay soil in paddy field of Chonburi Province, Thailand. The 16S rRNA gene sequence similarity, mycelium and spore morphologies, and chemotaxonomic properties were comparable to those of validly published Streptomyces taxa. Differences in phenotypic characteristics (morphology on various cultivation media, and physiology) and low ORGI values (ANI values <95%, AAI values <95% and dDDH <70%) clearly distinguished strain SMC 277T from its closest phylogenetic neighbor. Genome annotation and secondary metabolite gene cluster analysis revealed that strain SMC 277T harbored NRPSs as the dominant classes of predicted BGCs and displayed 7 putative products as novel metabolites. This finding together with the observed antibacterial activity profile of the strain suggested that SMC 277T is capable of producing bioactive metabolites against drug-resistant bacteria associated with nosocomial infections. Based on the analysis of polyphasic taxonomy in this study, they clearly distinguished strain SMC 277T from the currently and validly published species of Streptomyces. Therefore, strain SMC 277T should be recognized as representing a novel species of the genus Streptomyces, for which the name Streptomyces antimicrobicus sp. nov. is proposed. The species description of S. antimicrobicus sp. nov. is provided in Table 4.
a. Maximum parsimony tree based on 16S rRNA gene sequences showing the phylogenetic position of Streptomyces antimicrobicus SMC 277T relative to the top 41 closely related species of the genus Streptomyces. Nocardioides albus KCTC 9186T was used as an outgroup. Numerals at nodes indicate bootstrap percentages derived from 1000 replications, and only values greater than 50% are indicated. b. Maximum-likelihood tree based on 16S rRNA gene sequences showing the phylogenetic position of Streptomyces antimicrobicus SMC 277T relative to the top 41 closely related species of the genus Streptomyces. Nocardioides albus KCTC 9186T was used as an outgroup. Numerals at nodes indicate bootstrap percentages derived from 1000 replications, and only values greater than 50% are indicated.
S2 Fig. LL-diaminopimelic acid in the cell-wall peptidoglycan of Streptomyces antimicrobicus SMC 277T.
S3 Fig. The compositions of reducing sugar in whole-cell hydrolysates of Streptomyces antimicrobicus SMC 277T analyzed using cellulose TLC.
Its whole cell sugars consisted of glucose, galactose, mannose and ribose.
S4 Fig. Polar lipid profiles of Streptomyces antimicrobicus SMC 277T separated by 2-dimensional thin layer chromatography, and stained with phosphomolybdic acid (for detection of total lipids), Dittmer & Lester reagent (phospholipids), ninhydrin (amines), and anisaldehyde (sugars).
Abbreviations: DPG, diphosphatidylglycerol; PG, phosphatidylglycerol; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PIM, phosphatidylinositol mannoside; PL, phospholipid.
S1 Table. The pairwise 16S rRNA gene sequence and overall genomic relatedness indices (ANIb, ANIm, AAI and dDDH values) of Streptomyces antimicrobicus SMC 277T and the closest relative, Streptomyces bambusae NBRC 110903T as well as other closely related type strains.
S2 Table. Morphological and physiological characteristics of Streptomyces antimicrobicus SMC 277T and the closest relative, Streptomyces bambusae NBRC 110903T, as well as other closely related type strains.
Strains: 1, SMC 277T; 2, S. bambusae NBRC 110903T; 3, S. griseocarneus DSM 40004T (data from Reimer et al. , but some from Wen et al.  and Benedict et al.  as indicated by a and b, respectively); 4, S. coerulescens NBRC 12758T (data from Reimer et al. ); 5, S. abikoensis NBRC 13860T (data from Reimer et al. , but some from Sujarit et al.  and Mingma et al.  as indicated by c and d, respectively; 6, S. yangpuensis DSM 100336T (data from Tang et al. ); 7, S. virginiae NBRC 12827T (data from Reimer et al. , but some from Komaki et al.  as indicated by e; 8, S. amritsarensis MTCC 11845T (data from Sharma et al. , but some from Tang et al.  as indicated by f; 9, S. toxytricini NBRC 12823T (data from Reimer et al. , but some from Tamura et al.  as indicated by g; 10, S. cirratus NBRC 13398T (data from Reimer et al. ), but some from Koshiyama et al.  as indicated by h. All data were generated in the present study unless indicated otherwise. +, positive; -, negative; w, weakly positive; N, No; Y, Yes; ND, not determined.
S3 Table. Functional classification of protein-coding genes presented in genomes of Streptomyces antimicrobicus SMC 277T and closely related type strains by the abundance of clusters of orthologous groups (COGs).
Strains: 1, SMC 277T; 2, S. bambusae NBRC 110903T; 3, S. toxytricini NBRC 12823T; 4, S. cirratus NBRC 13398T; 5, S. vinaceus ATCC 27476T; 6, S. nojiriensis JCM 3382T; 7, S. yangpuensis DSM 100336T; 8, S. virginiae NBRC 12827T; 9, S. amritsarensis MTCC 11845T.
S4 Table. Number and type of putative secondary metabolite biosynthesis gene clusters presented in various genomes of Streptomyces antimicrobicus SMC 277T and closely related type strains.
A hybrid cluster contains more than one type of secondary metabolite biosynthesis genes in the clusters. Strains: 1, SMC 277T; 2, S. bambusae NBRC 110903T; 3, S. toxytricini NBRC 12823T; 4, S. cirratus NBRC 13398T; 5, S. vinaceus ATCC 27476T; 6, S. nojiriensis JCM 3382T; 7, S. yangpuensis DSM 100336T; 8, S. virginiae NBRC 12827T; 9, S. amritsarensis MTCC 11845T.
We would like to thank Dr. Stefano Donadio of NAICONS, Milan, Italy for critically reading this manuscript and his valuable suggestions.
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