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Article history:Received 29 December 2015Received in revised form 29 August2016Accepted 2 September 2016Available online xxx

Keywords:MicromonosporaAnti-microbial producingBacterial genomicsSecondary metabolite

A B S T R A C T

Micromonospora genus produces > 700 bioactive compounds of medical relevance. In spite of its ability to produce highnumber of bioactive compounds, no genome sequence is available with comprehensive secondary metabolite gene clus-ters analysis for anti-microbial producing Micromonospora strains. Thus, here we contribute the full genome sequenceof Micromonospora sp. HK10 strain, which has high antibacterial activity against several important human pathogenslike, Mycobacterium abscessus, Mycobacterium smegmatis, Bacillus subtillis, Staphylococcus aureus, Proteus vulgaris,Pseudomonas aeruginosa, Salmonella and Escherichia coli. We have generated whole genome sequence data of Mi-cromonospora sp. HK10 strain using Illumina NexSeq 500 sequencing platform (2 × 150 bp paired end library) and as-sembled it de novo. The sequencing of HK10 genome enables identification of various genetic clusters associated withknown- and probably unknown- antimicrobial compounds, which can pave the way for new antimicrobial scaffolds.

© 2016 Published by Elsevier Ltd.

Gene xxx (2016) xxx-xxx

Contents lists available at ScienceDirect

Genejournal homepage: www.elsevier.com

Research paper

Complete genome sequencing and comparative analyses of broad-spectrumantimicrobial-producing Micromonospora sp. HK10Madhumita Talukdar a, 1, Dhrubajyoti Das a, 1, Chiranjeeta Borah a, Tarun Chandra Bora a,Hari Prasanna Deka Boruah a, Anil Kumar Singh a, b, ⁎

a Department of Biotechnology, CSIR-North East Institute of Science and Technology, Jorhat 785006, Indiab Academy of Scientific and Innovative Research, Rafi Marg, New Delhi 110001, India

1. Introduction

Micromonospora is a genus of Gram-positive bacteria that tax-onomically belong to the family of Micromonosporaceae with 63species (Genilloud and Genus, 2012; Hirsch and Valdés, 2009;Genilloud, 2015). The members of Micromonospora genus are widelydistributed in diverse geographical habitats e.g., soil, fresh water, ma-rine sediment, rocks, nitrogen fixing nodules from leguminous andactinorhizal plants (Das et al., 2008; Charan et al., 2004; Trujilloet al., 2010; Valdés et al., 2005; Carro et al., 2013; Trujillo et al.,2006; Trujillo et al., 2007) etc. Micromonospora belongs to actino-mycetes group, which are prolific producers of > 700 bioactive com-pounds including gentamicin and calicheamicin (Charan et al., 2004;Bérdy, 2005; Igarashi et al., 2011a; Igarashi et al., 2007; Igarashiet al., 2011b; Furumai et al., 2000; Furumai et al., 2002). Bioac-tive compounds produced by Micromonospora have been studied con-siderably for their critical roles in human health and medi

Abbreviation: COG, clusters of orthologous groups; KEGG, Kyoto encyclopediaof genes and genomes; HGT, horizontal gene transfer; GI, genetic island; IS, in-sertion sequence; CRISPRs, clustered regularly interspaced short palindromic re-peats; SRP, signal recognition particle; Sec, sec secretion system; PKS, polyketidesynthase; NRPS, non-ribosomal peptide synthetase⁎⁎ Corresponding author at: CSIR-North East Institute of Science & Technology, Jorhat785006, Assam, India.Email address: 1010anil@gmail.com (A.K. Singh)1 Co-first authors.

cines (Bérdy, 2005; Igarashi et al., 2011a; Igarashi et al., 2007;Igarashi et al., 2011b; Furumai et al., 2000; Furumai et al., 2002;Shimotohno et al., 1993; Tomita and Tamaoki, 1980; Lam et al.,1996). Advancement of new generation sequencing and improved ac-curacy of gene annotation, have opened up a new avenue for thediscovery of novel secondary metabolite encoding gene clusters andtheir activities. Although Micromonospora genus belong to the or-der Actinomycetales along with Streptomyces genus, which are wellknown for production of secondary metabolites, a limited numbersof studies have been focused on the secondary metabolites and thebiosynthetic gene clusters of this genus. Despite the production of var-ious useful antibiotics by Micromonospora species, the full genomesof only limited number of strains have been available to date. Thegenomes of Micromonospora aurantiaca ATCC 27029T (accessionno. NC_014391.1) and Micromonospora sp. ATCC 39149 (accessionno. GG657738) isolated from soil and Micromonospora sp. L5 andMicromonospora lupini Lupac 08, isolated from nitrogen fixing rootnodules of Casuarina equisetifolia (Valdés et al., 2005; Trujillo etal., 2014) and Lupinus angustifolius (Trujillo et al., 2010; Trujilloet al., 2014) respectively, are available in public database. Intriguedby its potential bioactivity, we contribute here a genome sequence ofMicromonospora sp. strain HK10, a soil isolate from Kaziranga Na-tional Park, Assam, India (Talukdar et al., 2016). HK10 strain showshigh antibacterial activity against both Gram-negative and Gram-posi-tive bacteria (Talukdar et al., 2012). Since secondary metabolites playcrucial roles in several ecological functions for example, chemicalcommunication, nutrient attainment, defense mechanisms etcdeletefull stop here.. (Wietz et al., 2013), we analyzed ge

http://dx.doi.org/10.1016/j.gene.2016.09.0050378-1119/© 2016 Published by Elsevier Ltd.

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netic information related to its unknown active secondary metabolitesand respective genetic determinants. HK10 genome was comparedwithin other Micromonospora (Micromonospora sp. L5 and M. auran-tiaca ATCC20029T) and also with other actinobacteria such as, Salin-ispora arenicola CNS-205, Streptomyces coelicolor A3 (Hirsch andValdés, 2009), Nocardia brasiliensis ATCC 700358 and Rhodococcuspyridinivorans SB3094, which are known to produce bioactive com-pounds. The comparative genome analysis with other strains revealedsome significant genetic differences. Furthermore, we compared var-ious critical systems for their function and distributions of genes liketwo component regulatory systems, secretion systems and transportersmechanisms, which help the microbes to survive and adapt with theirsurrounding environments.

2. Material and methods

2.1. Strains, media and antagonism test

The antibacterial assay was performed against various testpathogens: Salmonella sp. (IDH377), enterotoxigenic Escherichia coliE9 (serotype O146) (Pazhani et al., 2011), Mycobacterium smegma-tis ATCC700084 and Mycobacterium abscessus ATCC19977T. Theantibacterial activity of Micromonospora sp. HK10 was tested usingagar diffusion assay method (Gramer et al., 1976; Forbes et al., 1990)by observing presence of inhibition zone (in mm) around the well.For extraction of crude compound, the pure isolate was cultured into500 ml Erlenmeyer flasks containing 100 ml of CSPY liquid medium(K2HPO4 0.5 g, casein 3 g, maize starch 10 g, peptone 1 g, malt ex-tract 10 g, yeast extract 1 g, distilled water 1 l. pH 7.5) for differ-ent time intervals such as (7, 10, 12 and 14 days) at 28 ± 2 °C at200 rpm. The culture broth was solvent extracted with equal volumeof chloroform (1:1) (v/v) in separating funnels by shaking vigorouslyfor 15 min and the solvent part was dried to yield the crude extract.The dried crude extract was dissolved in 20% dimethyl sulphoxide(DMSO) and antimicrobial bioassay was carried out against the testpathogens. 70 μL of fresh inoculum from each culture was spread onthe plates. Wells were punctured on freshly spread bacterial culture onMuller Hinton Agar (HiMedia, India) using sterile cork borer and thebored wells were filled with the 100 μg/ml extract of Micromonosporasp. HK10. All the plates were incubated at 37 °C for 48 h. Growth in-hibition of test pathogens was measured with standard scale, the meanvalues of inhibition zones were taken and compared with 50 μg/mlneomycin sulfate (HiMedia, India).

2.2. HK10 whole genome sequencing, assembly and annotation

The Micromonospora sp. HK10 genome was sequenced with theIllumina NextSeq 500 system using a paired-end 2 × 150-bp library.The raw data was trimmed using Trimmomatic version 0.30 (Bolger etal., 2014) for high-quality read length (cutoff quality score 20). A totalof 8,896,194 high-quality, vector-filtered reads were used for assem-bly with CLC Genomics Workbench version 6 (CLC bio, Denmark)and the draft genome thus obtained was annotated for protein cod-ing genes with the help of Glimmer (Delcher et al., 2007) and Prodi-gal v2.60 (Hyatt et al., 2010). This whole-genome shotgun project hasbeen deposited at GenBank.

2.3. Complete genomic analysis of Micromonospora sp. HK10

The genome sequence was submitted to the Integrated MicrobialGenomes (IMG) server (http://img.jgi.doe.gov) of the Joint Genome

Institute (JGI) for more detail analysis and genome comparison(Markowitz et al., 2014a; Markowitz et al., 2014b). A one-samplet-test was carried out by using OriginPro 8 software to evaluate thestatistically significant differences of gene abundance in each COGcategory between Micromonospora sp. HK10 and other five genomesof IMG database: M. aurantiaca ATCC 27029T (accession no.NC_014391.1), Micromonospora sp. L5 (accession no.NC_014815.1), Micromonospora sp. ATCC 39149 (accession no.GG657738.1), M. lupini Lupac 08 (accession no. HF570108), and Mi-cromonospora sp. CNB394 (Biosample no. SAMN02441007). Ge-nomic alignment of the Micromonospora sp. HK10 was performedwith Micromonospora sp. L5 and M. aurantiaca ATCC20029T andwith four secondary metabolite producing actinobacteria viz., Salin-ispora arenicola CNS-205, Streptomyces coelicolor A3 (Hirsch andValdés, 2009), Nocardia brasiliensis ATCC 700358 and Rhodococcuspyridinivorans (SB3094) strains using MAUVE software (Darling etal., 2010) and average nucleotide sequence identity (ANI) score wasvalidated by NCBI-Blastn. Complete genome sequence was analyzedusing IMG server (http://img.jgi.doe.gov) and antiSMASH (Medemaet al., 2011; Blin et al., 2013; Weber et al., 2015) was used to de-tect the secondary metabolite gene clusters. A Venn diagram of uniqueand shared genes present in the respective genomes was calculatedusing DrawVenn (http://bioinformatics.psb.ugent.be/webtools/Venn/). HK10 genome was examined for horizontal gene transfer (HGT)which plays critical roles in shaping the genome assisted by trans-posons and other origins. We performed two analyses: (i) GIs predic-tion by IslandViewer (using Dinucleotide-mobility analysis (DIMOB)and IslandPath tools) (Dhillon et al., 2015) and (ii) detection of inser-tion sequences from HK10 genome using IS Finder database (https://www-is.biotoul.fr/). The DIMOB analyses is based on dinucleotidebias and mobility genes to more accurately predicted regions, termedDIMOB islands (DIMOB-Is) that may have been acquired by HGT.IslandPath combine these parameters using additional information oftRNAs and G + C percentage. All the predicted GI contents were com-pared by BLAST to identify the kind of genes acquired by HGT inHK10 strain from other bacteria. The draft genome of Micromono-spora sp. HK10 was examined by CRT, PILERCR (Bland et al., 2007;Edgar, 2007), and CRISPR Finder tool (Grissa et al., 2007) to detectthe presence of CRISPR elements, which confer immunity against in-coming DNA, including bacteriophages. Transport system analyses ofHK10 genome was performed by comparing each predicted proteinagainst transporter classification database (TCDB) (http://www.tcdb.org/).

2.4. Phylogenetic analysis of Micromonospora sp. HK10 strain

Total of 67 almost complete 16S rRNA sequences were acquiredfrom public databases NCBI Genbank and aligned by ClustalW us-ing 1.6 DNA matrix (Thompson et al., 1994). The evolutionary his-tory was inferred by using the Maximum Likelihood method (Kimura,1980; Felsenstein, 1985) based on 1000 bootstrap replicates. Strep-tomyces coelicolor A3 (Hirsch and Valdés, 2009)T was used as anoutgroup. Evolutionary analyses were conducted in MEGA7 software(Kumar et al., 2016).

2.5. Nucleotide sequence accession number

HK10 Whole Genome Shotgun project has been deposited atDDBJ/EMBL/GenBank under the accession number NZ_JT-GL00000000.1.

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3. Results and discussion

3.1. Genome characteristics and comparative analysis of HK10strain with other Micromonospora strains

The final de novo genome assembly of Micromonospora sp. HK10contains 294 contigs with a total size of 6,911,179 bp (50% of theentire assembly of contigs i.e., “N50” equal or larger than 61,860 bpand the longest contig assembled measures 352,445 bp) with aver-age G + C content of 73.39% (Table 1). The genome contains a totalof 6282 predicted protein coding genes/CDSs, having 3 rRNAs and58 tRNAs (Table 1). From total predicted protein coding genes, 4644genes (73.93%) were allocated with a biological function while 1552genes (24.71%) were categorized as of unknown function. Further, outof these 6282 CDS, 3598 genes (57.27%) were observed under theclusters of orthologous groups (COGs) and assigned to 25 differentcategories (Fig. 1, Table S1 & S2). A one-sample t-test was used toevaluate if there were statistically significant differences in the geneabundances of each COG categories between the genomes of HK10and the other five Micromonospora strains present in IMG database:M. aurantiaca ATCC 27029T, Micromonospora sp. L5, Micromono-spora sp. ATCC 39149, M. lupini Lupac 08, and Micromonospora sp.CNB394 (Fig. 1, Table S1).

The results showed that the abundances of the proteins responsi-ble for carbohydrate transport and metabolism (B, 9.11%), cell motil-ity (D, 0.37%), posttranslational modification, protein turnover, chap-erones (S, 4%), signal transduction mechanisms (W, 5.6%) are signif-icantly (P < 0.005) higher than the average levels, which are 8.71%,0.265%, 3.79% and 5.38%, respectively.

The abundances of the proteins responsible for chromatin struc-ture and dynamics (F, 0.02%), coenzyme transport and metabolism(G, 5.99%), defense mechanisms (H, 0.02%), cytoskeleton (I, 2.92%),inorganic ion transport and metabolism (N, 4.49%), lipid transportand metabolism (P, 5.03%), mobilome: prophages, transposons (Q,0.37%), nucleotide transport and metabolism (R, 2.36%), RNA pro-cessing and modification (T. 0.02%) and secondary metabolite biosyn-thesis, transport and catabolism (V, 3.88%) are significantly(P < 0.005) lower than the average levels, 0.03%, 6.26%, 0.033%,2.95%, 4.88%, 5.43%, 0.51%, 2.44%, 0.023%, and 4.26%, respec-tively (Fig. 1, Table S1).

The genomic characteristics of HK10 strain and five other Mi-cromonospora genomes available in the public databases includingM. aurantiaca ATCC 27029T, Micromonospora sp. L5, Micromono-spora sp. ATCC 39149, M. lupini Lupac 08, and Micromonospora

Table 1General statistics of Micromonospora sp. HK10 genome.

Feature Genome

DNA, total number of bases 6911179DNA coding number of bases 6125905DNA G + C content (%) 73.39Genes total number 6282Protein coding genes 6196Protein coding genes with function prediction 4644Protein coding genes without function prediction 1552RNA genes 86rRNA genes 35S rRNA 116S rRNA 123S rRNA 1tRNA genes 58Other RNA genes 25

sp. CNB394 were compared (Table S3). The important differencesobserved between the six strains were GC percentage and numberof tRNA genes. HK10 genome has particularly highest GC content(73.39%) and second highest number of tRNAs (Nikaido andTakatsuka, 1794) compared to other five strains (Table S3). Severalreports in bacteria indicate that increase in GC content correlates witha higher temperature optimum and a broader tolerance range for aspecies (Nishio et al., 2003; Musto et al., 2006). Thus high GC con-tent in HK10 might give more thermo stability for adaptation to theimmediate environment and high numbers of tRNA genes can be seenas a potential for high protein synthesis and good growth rate (Rocha,2004) in Micromonospora strains but it is matter of future investiga-tion.

A complete genome sequence makes the fundamental hereditarycontent of an organism finite and is the most suitable state for anygenome sequence with less error bars. Therefore, we have furthercompared HK10 genome sequence with two fully sequenced genomesavailable in the database i.e., M. aurantiaca ATCC 27029T and Mi-cromonospora sp. L5. Our analysis disclosed that the highest number(6360) of annotated genes present in M. aurantiaca ATCC 27029T fol-lowed by Micromonospora sp. L5 and HK10 strain (6326 and 6282respectively) (Table S3). There were highest 60 predicted gene clus-ters having 861 genes (13.6% of total genome) in Micromonosporasp. L5, followed by M. aurantiaca ATCC 27029T with 893 genes(14.04% of total genome) and HK10 strain detected with lowest 49gene clusters with 721 genes (11.48% of the total genome). Func-tional annotation using the Kyoto encyclopedia of genes and genomes(KEGG) exposed the total protein coding genes connected to KEGGpathways: lowest 1213 (19.31%) in Micromonospora sp. HK10, fol-lowed by 1247 (19.61%) of M. aurantiaca ATCC 27029T and high-est 1277 (20.19%) of Micromonospora sp. L5. Similarly analysis withKEGG Orthology suggested, lowest number (2118) of genes (33.72%)found in Micromonospora sp. HK10, followed by 2237 (35.36%) and2216 (34.84%) number of genes in Micromonospora sp. L5 and M.aurantiaca ATCC 27029T, respectively.

Pair-wise genome synteny studies of these strains also revealeda high degree of positional conservation between the three genomes(Fig. S1). Although the three genomes have a considerable amountof genetic similarity, they have encountered with many inversion andtranslocations. HK10 genome possesses large number of non-con-served regions and unique genomic regions as compared to M. au-rantiaca ATCC 27029T and Micromonospora sp. L5. The genomiccomparison by average nucleotide sequence identity confirmed thatHK10 strain has the highest similarity to Micromonospora sp. L5(with 95% identity and 48% coverage), followed by M. aurantiacaATCC 27029T (with 95% identity and 47.9% coverage). To evaluategenetic similarity of strains, the comparative Venn diagrams of thetotal protein coding genes with predicted function was analyzed. In-terestingly, HK10 strain contains the highest number (4644, 73.93%)of protein coding genes with predicted function in comparison toMicromonospora sp. L5 (4358, 68.1%) and M. aurantiaca ATCC27029T (4259, 66.97%) (Fig. S2). Our analysis also found that HK10genome shared highest common genes (992 genes, 11.1%) with M.aurantiaca ATCC 27029T and lowest common genes (925, 10.2%)with Micromonospora sp. L5 (Fig. S2). Unique genes comparison ofHK10 strain has shown highest 3643 genes (78.4%) with Micromono-spora sp. L5 followed by 3252 genes (76.3%) with M. aurantiacaATCC 27029T. Similarly, HK10 strain contains highest 3710 numberof genes (79.8%), compared to Micromonospora sp. L5 3337 num-bers of genes (76.5%) (Fig. S2). These genetic similarities revealedthe closeness of HK10 strain with both strains.

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Fig. 1. (A) Comparison of COG functional categories between Micromonospora sp. HK10 and other five Micromonospora genomes- Micromonospora sp. HK10, Micromonosporasp. L5, M. aurantiaca ATCC 27029T, Micromonospora sp. ATCC 39149, M. lupini Lupac 08 and Micromonospora sp. CNB394 (from the center to the outer side). (B) Comparisonof COG functional categories between Micromonospora sp. HK10 and four actinobacteria genomes- Micromonospora sp. HK10, S. coelicolor A3 (Hirsch and Valdés, 2009), S.arenicola CNS-205, R. pyridinivorans SB3094, N. brasiliensis ATCC 700358) from IMG database, (from the center to outer side).

3.2. Genomic comparison of Micromonospora sp. HK10 with otheractinobacteria

Since Micromonosporaceae family is well known for productionof a number of medically relevant bioactive compounds, it is interest-ing to understand the genetic composition of other secondary metabo-lite producing actinobacteria, viz. Salinispora arenicola CNS-205(Accession no.: NC_009953), Streptomyces coelicolor A3 (Hirsch andValdés, 2009) (Accession no.: AL645882), Nocardia brasiliensisATCC700358 (Accession no.: NC_018681) and Rhodococcus pyri-dinivorans SB3094 (Accession no.: NC_023150). One-sample t-testwas carried out between Micromonospora sp. HK10 and above fouractinobacteria (Fig. 1, Table S2) suggests that the HK10 strain hassignificantly higher abundance of genes of carbohydrate transport and

metabolism (B, 9.11%), cell motility (D, 0.37%), chaperons (S, 4%),and signal transduction mechanisms (W, 5.6%) compared to otheractinobacteria strains (Fig. 1, Table S2). On contrary, abundance ofgenes categorized under cell cycle control, cell division, chromo-some partitioning (C, 0.81%), coenzyme transport and metabolism(G, 5.99%), defense mechanisms (H, 0.02%), cytoskeleton (I, 2.92%),energy production and conversion (J, 5.89%), inorganic ion trans-port and metabolism (N, 4.49%), lipid transport and metabolism (P,5.03%), mobiolome: prophages, transposons (Q, 0.37%), nucleotidetransport and metabolism (R, 2.36%), RNA processing and modifi-cation (T, 0.02%), secondary metabolite biosynthesis, transport andcatabolism (V, 3.88%), transcription (X, 10.31%) were significantlylower (Table S2). These results revealed a common feature in the sec-ondary metabolite producing bacteria, which is the low abundance of

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genes in secondary metabolites biosynthesis and high abundance ofgenes in carbohydrate metabolism.

Further we carried out a pair wise comparative genome analysis ofHK10 strain between S. arenicola, S. coelicolor, N. brasiliensis and R.pyridinivorans strains using progressive Mauve aligner (Darling et al.,2010) (Fig. S3). Although the five genomes share a notable amountof genetic characteristics, they have encountered various inversionsand translocations and Micromonospora sp. HK10 contains the high-est number of non-conserved regions. The genomic comparison by av-erage nucleotide sequence identity confirmed that HK10 strain has thehighest similarity to S. arenicola. The order of similarity was S. areni-cola (identity score of 92%, coverage of 33%) > S. coelicolor (identityscore of 85%, coverage of 10%) > N. brasiliensis (similarity score of85%, coverage of 5%) > R. pyridinivorans (similarity score of 86%,coverage of 4%). These results are consistent with their phylogenic re-lation of HK10 strain and S. arenicola, as both genera belong to Mi-cromonosporaceae family.

Total protein coding genes with- and without- predicted func-tion is shown as: (4644/1552), (6485/2009), (5364/2846), (3703/1204)and (3316/1790) in Micromonospora sp. HK10, N. brasiliensis, S.coelicolor, R. pyridinivorans, S. arenicola, respectively. Here, wehave compared ‘total protein coding genes with predicted function’to validate the similarity between HK10 and the other actinobacte-ria genomes. Our analysis revealed the similarity in following order,R. pyridinivorans (1953 genes, 42.13% similarity) > N. brasiliensis(1405 genes, 30.31% similarity) > S. arenicola (856 genes, 18.46%similarity) > S. coelicolor (819 genes, 17.66% similarity) (Fig. S4).Surprisingly, this analysis brought the highest sharing (1953 proteincoding genes) between HK10 and R. pyridinivorans strains, but aboveresults (Fig. S3) was found just opposite. These variations might bearising because former results appeared after total genome analysis(Fig. S3) however, latter one utilizing only part of the genome foranalysis. It is also noted that actinobacteria genomes have high num-ber of genes categorized under ‘without predicted function’ category,shown as S. coelicolor, 2846 > N. brasiliensis, 2009 > S. arenicola,1790 > R. pyridinivorans, 1204, which is also accountable for thesedifferences.

3.3. Genomic Island and horizontal gene transfer

A total of 18 genomic islands (GIs) were predicted by IslandViewer method (Dhillon et al., 2015) and their precise location isshown in Fig. S5 and Table S4. These 18 GIs encode a total of113 (1.7%) genes in 1,34,243 bp from various sources viz. eukary-ote, virus and other bacteria. All the identified genes from these GIsare summarized in Table S4, including genes encoding for numer-ous modifying genes i.e., hydrolases, acetyl transferases, dehydroge-nase, methyl transferase, phosphotransferase, glycosyl transferase, he-licase, glycosyl hydrolase. Thus, it shows that HK10 strain evolvedby receiving GIs, which make them genetically richer and equippedwith secondary metabolite modifying enzymes and eventually makingthem potential producer of several bioactive compounds. As per Is-landPath-DIMOB GI-1 (4,549,755 bp to 4,571,443 bp) was found tobe the prominent island representing 16 different genes while GI-3(7,115,968 bp to 7,130,234 bp) was having 8 genes, which also in-cludes transposes IS 116/IS 110/IS902 family genes (Table S4). Pres-ence of three putative transposase (from GI-3, Ga0063173_03998,Ga0063173_6177 and Ga0063173_03269) and one phase integrasefamily gene (from GI-4, Ga0063173_04552) or their fragments werescattered in the genome that could support potential active HGT inthe strain (Table S4). Interestingly only three GIs (GI-1, GI-11 andGI-16) had the similar DNA composition with the Micromonospora

genus in general while the rest of the GIs showed similarity with var-ious phyla of bacteria such as, Actinobacteria: Actinoplanes (GI-2,GI-13, GI-15 and GI-17), Salinispora (GI-3 and GI-18), Streptomyces(GI-4), Verrucosispora (GI-5, GI-7, GI-12), Frankia (GI-8), Strep-tosporangium (GI-14); Chlorobi: Chlorobium tepidum (GI-6); Planc-tomyces: Planctomyces limnophilus (GI-9); and Proteobacteria: Thio-cystis violascens (GI-10) (Table S4). IMG server analysis revealedthat HK10 strain has acquired lesser genes (48 genes) by recent hori-zontal transfer than Micromonospora sp. L5 (61 genes) and M. auran-tiaca ATCC 27029T (67 genes) strains (Fig. 2 and Table S5). Over-all horizontal gene transfer from phylum Proteobacteria is predomi-nantly observed in all Micromonospora genome sequences. Interest-ingly HK10 genome has some unique gene transfer from Armatimon-adetes phylum, which was absent in other available genome sequences(Table S5).

Moreover, HK10 genome contains 23 mobile genetic elementsfrom 11 different IS families i.e., IS 110, IS 1380, IS 21, IS 256, IS3, IS 4, IS 5, IS 630, IS As1, IS L3 and IS NCY (Fig. S6 and TableS6). IS 110 family transposases are predominantly present in HK10genome.

Clustered regularly interspaced short palindromic repeats(CRISPRs) are structures, which conceal complex biological mech-anisms to account for their transfer, evolution and behavior. Theyhave probably played an important role in the evolution of archaeaand bacteria by providing a defense mechanism against foreign DNA(Horvath and Barrangou, 1994). Using CRT and PILERCR tools(Bland et al., 2007; Edgar, 2007) a total of 10 and with CRISPRdatabase (Grissa et al., 2007) a total of 14 CRISPR sequences inHK10 genome were identified (Table S7). Notably no CRISPR se-quences were found in Micromonospora sp. L5, M. aurantiaca ATCC27029T and M. lupini Lupac 08 strains. The presence of numberof CRISPR sequences might be responsible for resistance of for-eign DNA sequences in HK10 strain compared to other availablegenomes of Micromonospora where no CRISPR sequences have beenobserved. Number of possible roles for CRISPR sequences have beensuggested such as, chromosomal rearrangement, modulation of ex-pression of neighboring genes, target for DNA binding proteins, repli-con partitioning, and DNA repair (Makarova et al., 2002). Numer-ous in silico studies have been reported homology between spacer se-quences and extra chromosomal elements (like viruses and plasmids),which led to the hypothesis that CRISPR may provide adaptive immu-nity against foreign genetic elements (Horvath and Barrangou, 1994;Makarova et al., 2006). These studies lead us to speculate that pres-ence of CRISPR in HK10 strain might help in adaptation under vari-able environmental conditions.

3.4. Antimicrobial activity of Micromonospora sp. HK10 andcomplete secondary metabolism encoding genes

HK10 strain has already been reported to possess a broad range ofantibacterial activity against various Gram-positive and Gram-nega-tive bacteria (Talukdar et al., 2012). To reaffirm this broad spectrumphenotype, we have evaluated antibacterial activity of HK10 strainagainst different clinical pathogens such as, Mycobacterium smegma-tis ATCC 700084, Mycobacterium abscessus ATCC 19977T, Salmo-nella sp. IDH377, and enterotoxigenic Escherichia coli E9. The zoneof inhibition was determined as 27 mm, 19 mm, 19 mm and 18 mmfor M. smegmatis, M. abscessus, Salmonella sp. and E. coli respec-tively, compared to 19 mm, 17 mm, 13 mm and 15 mm against M.smegmatis, M. abscessus, Salmonella sp. IDH377 and E. coli E9,respectively using as positive control neomycin sulfate (Fig. 3). Al-though the concentration of HK10 extract used was double (100 μg/

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Fig. 2. Comparative analysis of horizontally transferred genes of Micromonospora HK10 with other five Micromonospora genomes from IMG database.

Fig. 3. Antimicrobial activity of Micromonospora sp. HK10-chloroform cell extracts (HK10) tested against (i) M. smegmatis, (ii) M. abscessus, (iii) Salmonella sp. IDH 377 (iv)Enterotoxigenic E. coli E9 strains, 50 μg/ml neomycin sulfate (NS) used as positive control and only DMSO used as a control (C).

ml) than the neomycin sulfate (50 μg/ml), reasonably higher inhibi-tion zones of HK10 extracts against all test pathogens clearly indicatesthe effectiveness of secondary metabolite produced by HK10. Sec-ondary metabolite production of HK10 strain was opti

mized using different media at various time interval (data not shown)and 10 days old culture of HK10 strain grown on CSPY mediaemerged as the best for antibacterial activity. The antimicrobial ac-tivity of HK10 was absent when other media such as Mueller Hinton

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broth (Himedia), Luria Bertani broth (Himedia), Nutrient broth (Hi-media) and Starch Casein broth (Himedia) was used.

To investigate the secondary metabolite synthesis potential ofHK10 strain, secondary metabolite encoding gene clusters was pre-dicted using IMG server. HK10 genome possesses 49 secondarymetabolite gene clusters, which are encoded by 721 genes with11.48% of genome coverage (Fig. 4, Table S8). Total 49 gene clustersinclude 13 polyketide synthase (PKS) genes, 2 non-ribosomal pep-tide synthetase (NRPS)-PKS genes and 34 encoding other secondarymetabolites including siderophores (Fig. 4, Table S8). Interestingly,the length of ten gene clusters exceeds > 25 Kb. The presence of largegene clusters in HK10 genome indicates that these clustering conferan ecological advantage and each gene of the cluster contributes toan enzymatic step in their respective cellular metabolic process. Theseclusters also imply transcriptional co-regulation of genes, which canbe controlled at different levels of expression. HK10 genome is excep-tionally rich in pks gene cluster (total 13) among sequenced genomes,providing evidence that HK10 strain harbors an extensive secondarymetabolite metabolism (Fig. 4, Table S8). To understand the overallsecondary metabolite synthesis potential of HK10 strain, these clus-ters were compared with Micromonospora sp. L5 and M. aurantiacaATCC 27029T (Table S9). Compared to M. aurantiaca ATCC 27029T,HK10 genome had 8 unique gene clusters, 30 conserved gene clus-ters (similarity > 60%) and 11 clusters were partially conserved (sim-ilarity > 30%) (Table S9). Similarly 9 gene clusters (Cluster 7: Puta-tive, Cluster: 19: Putative, cluster 22: Lantipeptide, Cluster 29: Puta-tive, Cluster 44: Lantipeptide, Cluster 45: Putative, Cluster 47: Lan-tipeptide; NRPS, T1 PKS, Cluster 48: Putative, Cluster 49: Putative)were found to be unique when HK10 genome was compared with Mi-cromonospora sp. L5, along with 29 conserved gene clusters and 11partially conserved clusters (Table S9, Fig. 5, Table S10).

Overall seven gene clusters have been found unique in HK10genome (Fig. 5). Our HGT analysis of HK10 genome has confirmedthat cluster 7 (present in GI-17), cluster 22 (present in GI-8) andcluster 44 (present in GI-3) have been horizontally transferred from

Actinoplanes sp., Frankia sp. and Salinispora arenicola respectively(Table S4 & S10).

To explore the biosynthetic cluster closeness of HK10 strain withother secondary metabolite producing strains, predicted gene clustershave been compared with other actinobacteria. Our results revealedthe similarity between HK10 strain and other actinobacteria in the fol-lowing order Salinispora arenicola (16 conserved, 13 partially con-served, 20 absent) > Streptomyces coelicolor (3 conserved, 15 par-tially conserved, 31 absent) > Nocardia brasiliensis (4 partially con-served and 45 absent) > Rhodococcus pyridinivorans (4 partially con-served and 45 absent) (Table S9). Interestingly seven gene clusters(cluster 7, cluster 19, cluster 22, cluster 44, cluster 45, cluster 47 andcluster 48) of HK10 strain, did not show any similarity with otheractinobacteria used in this study. These clusters might be interestingto explore in future for their secondary metabolite products. Thesesecondary metabolite encoding gene clusters were again revalidatedby antiSMASH cluster predicting tool (Medema et al., 2011; Blinet al., 2013; Weber et al., 2015). After analysis of HK10 genome,total twenty-two secondary metabolite encoding gene clusters havebeen discovered. Interestingly, antiSMASH analysis of HK10 genomeemerged with one more lantipeptide gene cluster (Cluster 1) that wasabsent in our earlier IMG prediction (Table S8). There could be twopossible reasons that cause the differences: (Genilloud and Genus,2012) in IMG the results of biosynthetic cluster prediction is affiliatedwith antiSMASH v2 but in our prediction with online versions of anti-SMASH, we have used the online version of antiSMASH, i.e., version3, which now annotates more classes of clusters including lantipep-tides, (Hirsch and Valdés, 2009) the number of results also depends onthe parameters of running the antiSMASH, for example, IMG allowantiSMASH to predict putative clusters based on an implementationof Clusterfinder and this is not available by default on antiSMASH.

Our analysis revealed five annotated biosynthetic gene clusters(Fig. S7): lantipeptide (cluster 1), PKS-hybrid (cluster 4), terpene(cluster 8), PKS III (cluster 13) and lantipeptide-siderophore (clus-ter 18), showing high level of similarity with SapB biosynthetic genecluster of S. coelicolor (100%), Chlorothricin biosynthetic gene clus

Fig. 4. (A) Comparative analysis of total genes distributed under categories of: -present in secondary metabolite clusters/-not present in secondary metabolite clusters in selectedMicromonospora spp. (B) Comparative analysis of total predicted secondary metabolite gene clusters types (PKS, NRPS, PKS-NRPS, OTHER) present in selected Micromonosporasps.

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Fig. 5. Seven unique biosynthetic gene clusters present in Micromonospora sp. HK10: (A) Cluster-7 (B) Cluster-19 (C) Cluster-22 (D) Cluster-44 (E) Cluster-45 (F) Cluster-47 (G)Cluster-48.

ter of Streptomyces antibioticus (44%), Sioxanthin biosynthetic genecluster of Salinispora tropica (80%), Alkyl-O-Dihydroger-anyl-Methoxyhydroquinones biosynthetic gene cluster of M. auranti-aca ATCC 27029T (71%), and Desferrioxamine B biosynthetic genecluster of Streptomyces griseus (80%) (Table S11). These five clustersfurther aligned with other secondary metabolite producing strains ofthe database and all the results have been shown in Fig. S7. Lantipep-tide encoding cluster 1, PKS-hybrid encoding cluster 4 and lantipep-tide-siderophore encoding cluster 18 have been showing highest gene

similarity with Streptomyces davawensis (19%), Streptomyces antibi-oticus (58%) and Streptomyces fradiae (30%) strains respectively,whereas terpene encoded cluster 8 and PKS III encoding cluster 13are more similar to Micromonospora sp. ATCC 39149 (29%) and M.aurantiaca ATCC 27029T (82%) strains (Fig. S7). Overall our studyprovides critical genetic insights into the HK10 strain, which has an-tibacterial activity against a wide range of bacteria, including My-cobacteria and can produce several bioactive secondary metabolites.

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The antibacterial activity of HK10 strain and its link with predictedsecondary metabolite gene clusters is our undergoing research work.

3.5. Secretion systems of Micromonospora sp. HK10

Secretion systems are essential for translocation of various proteinsacross the cell membrane which plays crucial roles like host-pathogeninteractions and colonization (Driessen and Nouwen, 2008). Proteinsin Gram-positive bacteria predominantly translocated by conservedSec translocase system or twin-arginine translocation (Tat) system.Sometimes the type VII or ESX secretion system has been also re-ported in some Gram positive bacteria. Similar to them HK10 genomeencodes all the Sec components (Table S12), viz. the essential pro-teins SecY (Ga0063173_02940) and SecE (Ga0063173_02979), alongwith SecD (Ga0063173_02596) and SecF (Ga0063173_02597). Bothco- and post-translational routes of targeting secretory proteins to theSec translocon are present in HK10 strain. HK10 genome also encodesSecA protein (Ga0063173_02270, Ga0063173_06183) like Strepto-myces lividans, which has been shown in vitro to be required for thesecretion of the Sec-dependent α-amylase (Blanco et al., 1998). HK10genome also contains ffh gene (Ga0063173_03995), encodes for sig-nal recognition particle subunit SRP54 and ftsY (Ga0063173_03984),encoding the SRP receptor. Mutations in ftsY gene affect sporulationand antibiotic production in S. coelicolor (Shen et al., 2008).

HK10 genome showed presence of various Tat pathways ho-mologs. Similar to other actinobacteria, TatA (Ga0063173_05927),and TatC (Ga0063173_05926) are found adjacent to each other whileTatB (Ga0063173_05707) was located distantly. Unlike M. lupini lu-pac 08 genome, which has no TatB homologue (Trujillo et al., 2014),HK10 Tat secretion pathway is very similar with Micromonospora sp.L5 and M. aurantiaca ATCC 27029T. The Tat pathway is responsi-ble for export of number of pre-folded proteins across bacterial mem-branes (Lee et al., 2006). Tat proteins are also involved in differentcellular activities including cell envelope biogenesis, metal acquisi-tion, anaerobic metabolism and detoxification and virulence (Lee etal., 2006). Hence, presence of Tat pathway in Micromonospora strainsmight useful in dealing with the changing environment around them.

HK10 genome has total 6 genes (Ga0063173_00846,Ga0063173_00894, Ga0063173_00895, Ga0063173_03797,Ga0063173_03798, Ga0063173_03804), which were categorized un-der RD1 or ESX-1/Snm protein secretion system family (Table S13 &S14). Like different Streptomyces, the function of these genes in Mi-cromonospora genus is currently unknown, but a significant physio-logical role is suggested by analogy to similar systems in other organ-isms.

T4SS have been mainly studied in Gram-negative bacteria but inGram-positive bacteria the information is very limited so far. How-ever, Type IV (Conjugal DNA-Protein transfer or VirB) secretorypathway (IVSP) family gene (Ga0063173_02432) was observed inHK10 genome (Table S14) and similar type of gene (virB4) wasalso reported in M. lupini Lupac 08, an endophytic actinobacterium(Trujillo et al., 2014), but was interestingly absent in most of thesequenced Micromonospora species (data not shown), including Mi-cromonospora sp. L5 and M. aurantiaca ATCC 27029T strains. Theprecise role of IVSP family genes in Micromonospora genus is matterof future research.

3.6. Transporters of HK10 strain

Various active transporter systems are involved in transport ofnutrient elements, detoxification and defense mechanisms. HK10genome contains total 74 families of transporters covered by total 587genes (9.3% of total CDS), which are involved in the transport sys-tems for carbohydrates, lipid, amino acids, nucleotides, aromatic com-pounds, metals and inorganic ions (Tables S13 &S14). HK10 genomehas emerged with the lowest 587 (9.3%) number of transporter geneswith 49 predicted metabolite clusters whereas M. aurantiaca ATCC27029T and Micromonospora sp. L5 contain 630 (9.9%) genes with 59predicted secondary metabolite gene clusters and 658 (10.4%) with 60clusters, respectively. The soil actinomycetes, S. coelicolor has alsototal 55 predicted secondary metabolite gene clusters with total 834transporter genes (10.1% genome coverage) (Bentley et al., 2002).These transporters of HK10 strain are potentially involved in metabo-lite export and are consistent with such extensive secondary metab-olism. HK10 genome contains proton-dependent oligopeptide trans-porter (POT) (Ga0063173_06054), responsible for peptide transport(Table S14), which regulates the expression of secondary metabolitegenes (Lorenzana et al., 2004). Major facilitator superfamily (MFS)transporters show specificity for many vital components like nutri-ents, peptides, nucleotides, drugs, Krebs cycle metabolites, osmo-lites, siderophores (efflux & uptake) organic and inorganic anions,etc. (Law et al., 2008). MFS transporter PenT regulates penicillinproduction by enhancing the translocation of penicillin precursorsacross fungal cellular membrane (Yang et al., 2012). RND (Resis-tance-Nodulation-Division) family transporters are widespread espe-cially among Gram-negative bacteria, and catalyze the active effluxof many antibiotics and chemotherapeutic agents and might help tosurvive against different toxin compounds (Nikaido and Takatsuka,1794). Interestingly HK10 genome contains highest number of MFS(Zhao et al., 2004) and RND (Trujillo et al., 2007) transporters whencompared with Micromonospora sp. L5 (61 MFS & 9 RND) and M.aurantiaca ATCC 27029T (58 MFS and 8 RND). Moreover, mem-brane fusion protein (MFP) transporter (Li and Nikaido, 2009)(Ga0063173_03875) presents only in HK10, which was not found inboth Micromonospora sp. L5 and M. aurantiaca ATCC 27029T strains(Table S14).

HK10 genome contains some other transporters, viz. Major intrin-sic protein (MIP) family (TC:1.A.8), Lactate permease (LctP) fam-ily (TC:2.A.14) and Zinc (Zn2 +)-Iron (Fe2 +) permease (ZIP) family(TC:2.A.5), which was absent in both Micromonospora sp. L5 andM. aurantiaca ATCC 27029T strains (Table S12). In contrast, severaltransporters were absent in HK10 strain e.g., Urea transporter (UT)family (TC:1.A.28), Betaine/Carnitine/Choline transporter (BCCT)family (TC:2.A.15), Ca2 +:Cation Antiporter (CaCA) family(TC:2.A.19), Arsenical resistance-3 (ACR3) family (TC:2.A.59),Monovalent cation (K+ or Na+):Proton antiporter-3 (CPA3) family(TC:2.A.63), Auxin efflux carrier (AEC) family (TC:2.A.69), L-Ly-sine exporter (LysE) family (TC:2.A.75), Gluconate:H+ Symporter(GntP) family (TC:2.A.8), the Tricarboxylate Transporter (TTT) Fam-ily (TC:2.A.80), putative cobalt transporter (CbtAB) family(TC:9.B.69) (Table S13).

3.7. Salt tolerant characteristic of HK10 strain

Genomic background of Salinispora suggests its evolution froma terrestrial environment (Penn and Jensen, 2012) which raises thequestion whether Micromonospora has some common genes relatedto marine environment. There are Micromonospora species now

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which have been reported from marine environment (Das et al., 2008;Phongsopitanun et al., 2015; Supong et al., 2013; Maldonado et al.,2009; Zhao et al., 2004; Mincer et al., 2002) and produce various an-timicrobials of medical importance (Charan et al., 2004; Thi et al.,2016; Carlson et al., 2013; Kyeremeh et al., 2014). We have con-firmed the salt tolerance ability of HK10 strain by growing themon higher concentration (0.5–3.0%) of sodium chloride and best an-tibacterial activity was observed at 2.5% sodium chloride (data notshown). This characteristic suggests that certain genes may be adaptedfrom marine microbes. Putative potassium uptake protein (Trk)(Ga0063173_05704) which might be useful in marine adaptation(Schlosser et al., 1993; Nakamura et al., 1994) and NDH protein(Ga0063173_00883) constitutes a part of proton pumping NADH de-hydrogenase and creates a proton-motive force for generation of ATP(Schlosser et al., 1993). HK10 genome also contains gene encod-ing mechanosensitive channels MscL (Ga0063173_00971) and MscS(Ga0063173_02549, Ga0063173_04307, Ga0063173_06285) (TableS15 & S16), which enable growth at low osmotic pressure (Bucarey etal., 2012).These genes are also present in Micromonospora sp. L5 andM. aurantiaca ATCC 27029T strains. Presence of these genes in HK10strain could suggest an adaptation from marine bacteria and might behelpful in salt tolerance in adverse environmental condition.

3.8. Signal transduction and two-component system

The full list of genes involved in signal transduction and two-com-ponent systems (TCS) in HK10 strain are given in Table S15 &S16. Various studies have shown that biosynthesis of antibiotic andmany other secondary metabolites are strongly down regulated byinorganic phosphate (Sola-Landa et al., 2003; Ishige et al., 2003;Lefevre et al., 1997; Braibant and Content, 2001; Glover et al., 2007).In HK10 strain, the inorganic phosphate depriving condition mightbe controlled by senX3-regX3 two-component system. Although thetwo-component system senX3-regX3 (Ga0063173_04526 andGa0063173_04527) was found in HK10 strain, the two-componentsystem phoR-phoP was absent. The alkaline phosphatase, phoA gene(Ga0063173_03810) and ABC phosphate transporter operon pstSCAB(Ga0063173_04482, Ga0063173_04481, Ga0063173_04480,Ga0063173_04479) was also present in HK10 genome. We have alsoobserved a few more genes of phosphate transporter for example,phosphate starvation-inducible protein, PhoH (Ga0063173_04785)and phosphate transport system protein, phoU (Ga0063173_04525).Instead of alkaline phosphatase, phoA gene, alkaline phosphatase,phoD gene (Micau_4701 and Micau_4898) was present in M. auran-tica.

K+ intracellular cation is required for various physiologicalprocesses like turgor homeostasis (Epstein, 1986), pH regulation(Booth, 1985), gene expression (Prince and Villarejo, 1990) etc. Forregulation of K+ ion and adaptation under various stress conditions,HK10 genome has KdpD-KdpE (Ga0063173_03480,Ga0063173_03479) two-component system and K+-transporting AT-Pase genes kdpABC (Ga0063173_03483, Ga0063173_03482,Ga0063173_03481), whereas KdpF gene was absent in HK10 genomelike E. coli (Prince and Villarejo, 1990).

HK10 genome has temperature regulator mechanism DesK-DesRtwo-component system, which works when the ambient temperaturedrops below 30 °C (Aguilar et al., 2001). Similar DesK-DesR sys-tem is also found in both Micromonospora sp. L5 and M. aurantiacaATCC 27029T. Beside this, tricarboxylic transporter TctA-TctB-TctCoperon (ML5_4359, ML5_4358, ML5_4357) is only present in Mi-cromonospora sp. L5 and not found in both HK10 strain and M. au-rantiaca ATCC 27029T. We have also observed gene related to fu

marate metabolism fumarate reductase, frdA (Ga0063173_02826,Ga0063173_03575) present in HK10 genome, which was absent inboth Micromonospora sp. L5 and M. aurantiaca ATCC 27029T

strains.

3.9. Phylogenetic analysis of Micromonospora

The phylogenetic position based on 16S rRNA gene sequenceanalysis of strain HK10 indicated that it was placed in a monophyleticclade with closest relatives, M. auratinigra TT1-11T (99% of iden-tity with 97% coverage), M. peucetia DSM 43363T (98% of identitywith 97% coverage) and M. chaiyaphumensis MC5-1T (99% of iden-tity with 94% coverage).

On habitat basis HK10 strain was clearly positioned within thegenus Micromonospora and form a sub group together with other soilisolates (M. auratinigra TT1-11T, M. peucetia DSM 43363T and M.chaiyaphumensis MC5-1T) (Fig. S8).

4. Conclusions

Despite the ability to produce several useful antibiotics, very fewgenome sequences of Micromonospora are available in the database.We contribute with a 6,911,179 bp genome of a soil isolate Mi-cromonospora sp. HK10, with broad spectrum antibacterial activityagainst Gram-negative and Gram-positive bacteria including entero-toxigenic E. coli, Salmonella sp., M. abscessus and M. smegmatis.HK10 genome analysis emerged with total 49 secondary metabo-lite gene clusters (encoding mainly PKS, NRPS, siderophore, terpeneetc) including comparatively higher (Igarashi et al., 2007) PKS and 7unique gene clusters, which can be exploited for conceiving the newbioactive molecules. Further, this study also provides a new geneticinsight into horizontal gene transfer (18-genetic islands with 3 newsecondary metabolite genetic cluster), secretion system, CRISPRs,two-component system, salt tolerant behavior and transport mecha-nism in Micromonospora. This study will certainly enhance our mole-cular understanding about secondary metabolite engineering and envi-ronmental adaptations of Micromonospora and other related microbes.

Acknowledgement

This work was funded by the Council of Scientific and IndustrialResearch (CSIR), India (CSC131, BSC109, and HCP0001). M.T.,D.D., and C.B. are supported by a research fellowship from the CSIR,India. We thank the Director, CSIR-North East Institute of Scienceand Technology (CSIR-NEIST), Jorhat, India, for his consistent en-couragement and support. We are grateful to Dr. T. Ramamurthy, Na-tional Institute of Cholera and Enteric Diseases, Kolkata, India, for hiskind gift of Salmonella sp. (ID377) and Escherichia coli E9 (serotype0146) strains used in this study. Authors are also very thankful to Prof.Paul Jensen for his kind permission to use the Micromonospora sp.CNB394 genome sequence in this study.

Appendix A. Supplementary data

Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.gene.2016.09.005.

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