Genome Sequences of Serratia Strains Revealed Common Genes in Both Serratomolides Gene Clusters

biology A icle Genome Sequences o Se a ia S ains Re ealed Common Genes in Bo h Se a omolides Gene Clus e s Ca a ina Ma ques-Pe ei a †, Diogo Ne es P oença * and Paula V. Mo ais Depa men o Li e Sciences, Cen e o Mechanical Enginee ing, Ma e ials and P ocesses, Uni e si y o Coimb a, Calçada Ma im de F ei as, 3000-456 Coimb a, Po ugal; [email p o ec ed] (C.M.-P.); [email p o ec ed] (P.V.M.) *Co espondence: [email p o ec ed]; Tel.: +351-239240798 †Cu en add ess: Cen e o Neu oscience and Cell Biology & Cen e o Inno a i e Biomedicine and Bio echnology, Uni e si y o Coimb a, 3004-504 Coimb a, Po ugal. Recei ed: 31 Oc obe 2020; Accep ed: 17 Decembe 2020; Published: 20 Decembe 2020   Simple Summa y: Biosu ac an s a e amphiphilic molecules p oduced by mic oo ganisms wi h a hyd ophilic and a hyd ophobic g oup, able o educe su ace ension. These molecules a e la gely used in he en i onmen al, ood, pha maceu ical, medical, and cleaning indus ies, among o he s. Se a ia s ains a e ubiqui ous mic oo ganisms wi h he abili y o p oduce biosu ac an s, such as se awe ins. These ex acellula lipopep ides a e desc ibed as biocides agains many bac e ia and ungi. This wo k used compa a i e genomics o de e mine he dis ibu ion and o ganiza ion o he se awe ins W1 and W2 biosyn he ic gene clus e s in all he 84 publicly a ailable genomes o he Se a ia genus. He e, he se awe in W1 gene clus e s’ o ganiza ion is epo ed o he i s ime. The se awe in W1 biosyn he ic gene sw W and se awe in W2 biosyn he ic gene sw A we e p esen in 17 and 11 Se a ia genomes, espec i ely. The same genes in he biosyn he ic clus e s ame he sw W and sw A biosyn he ic genes. This wo k iden i ied ou genes common o all se awe in gene clus e s, highligh ing hei key po en ial in he se awe ins biosyn he ic p ocess. Abs ac : Se a ia s ains a e ubiqui ous mic oo ganisms wi h he abili y o p oduce se a omolides, such as se awe ins. These ex acellula lipopep ides a e desc ibed as biocides agains many bac e ia and ungi and may ha e a nema icidal ac i i y agains phy opa hogenic nema odes. Se awe ins W1 and W2 om di e en s ains ha e di e en s uc u es ha migh be co ela ed wi h dis inc genomic o ganiza ions. This wo k used compa a i e genomics o de e mine he dis ibu ion and he o ganiza ion o he se awe ins biosyn he ic gene clus e s in all he 84 publicly a ailable genomes o he Se a ia genus. The se awe in W1 and W2 gene clus e s’ o ganiza ion was es ablished using an iSMASH so wa e and compa ed wi h single and sho da a p e iously desc ibed o YD25 T Se a ia. He e, he se awe in W1 gene clus e s’ o ganiza ion is epo ed o he i s ime. The se awe in W1 biosyn he ic gene sw W was p esen in 17 Se a ia genomes. Eigh y di e en coding sequence (CDS) we e assigned o he W1 gene clus e , 13 being common o all clus e s. The se awe in W2 sw A gene was p esen in 11 Se a ia genomes. The W2 gene clus e s included 68 CDS wi h 24 p esen in all he clus e s. The genomic analysis showed he sw A gene cons i u es i e modules, ou wi h h ee domains and one wi h ou domains, while he sw W gene cons i u es one module wi h ou domains. This wo k iden i ied ou genes common o all se awe in gene clus e s, highligh ing hei essen ial po en ial in he se awe ins biosyn he ic p ocess. Keywo ds: Se a ia; genome; se awe in; biosyn he ic gene clus e ; se a omolides; sw W gene; sw A gene Biology 2020,9, 482; doi:10.3390/biology9120482 www.mdpi.com/jou nal/biology Biology 2020,9, 482 2 o 18 1. In oduc ion Su ac an s a e amphiphilic molecules wi h a hyd ophilic and a hyd ophobic g oup, able o educe su ace ension. These molecules a e la gely used in he en i onmen al, ood, pha maceu ical, medical, and cleaning indus ies, among o he s [ 1 – 3 ]. Biosu ac an s a e seconda y me aboli es p oduced by bac e ia, yeas , o ungi, capable o educing he su ace ension o ex acellula media [ 4 ]. They can ha e a a ie y o s uc u es, mainly di ided in o six majo g oups, namely, glycolipids, lipopolysaccha ides, lipopep ides and phospholipids, and hyd oxyla ed and c oss-linked a y acids [ 5 ]. When compa ed wi h syn he ic su ac an s, biosu ac an s ha e highe biodeg adabili y, lowe oxici y, and highe ac i i y a ex eme condi ions, such as high empe a u es, pH, and salini y [6]. Bac e ial species o he genus Se a ia belonging o he amily En e obac e iaceae a e G am-nega i e, acul a i e anae obic, and od-shaped bac e ia [ 7 , 8 ]. They ha e been isola ed om di e en en i onmen s, such as wa e , soil, plan s, insec s, and e eb a es [ 9 ]. Su aces in hese habi a s ha e a a ie y o cha ac e is ics and can be hyd ophobic, hyd ophilic, ac al, smoo h, and axenic, among o he s. To colonize hem, bac e ia om he genus Se a ia p oduce biosu ac an s (ex acellula lipopep ides) as he se a omolides se awe in W1 [10], se awe in W2 [4], and se awe in W3 [11]. Mu an s o he se a omolides biosyn he ic genes ailed o o m colonies on nu i i e aga pla es and hese lipopep ides we e sugges ed as p omo e s o a new ype o sp eading g ow h [4]. Bo h he se awe in W1 and W2 biosyn he ic genes (sw W and sw A genes) belong o a Non-Ribosomal Pep ide Syn he ase (NRPS) gene clus e [ 12 , 13 ]. Condensa ion, adenyla ion, hiola ion, and hioes e ase domains a e de ec able in bo h se awe in gene clus e s. Along wi h o he p o eins, such as PPTase o acyl ca ie p o eins (ACP) [ 12 , 14 ], se awe in W1 and W2 biosyn he ic p o eins a e able o syn hesize se awe in W1 and W2 lipopep ides. Se awe in W1 has a symme ic s uc u e composed o wo se ine amino acids and wo a y acids (3-hyd oxydecanoic) [ 4 ] while se awe in W2 is a cyclic pep ide con aining i e amino acids. This lipopep ide p omo es lagellum sp eading g ow h, con ibu ing speci ically o su ace bac e ial ansloca ion [ 4 ], and was able o demons a e an imic obial ac i i y agains many bac e ia and ungi and an an i umo al ac i i y agains Hela cells [ 13 ]. The Se a ia s ains p oducing se awe in W1 showed he highes b oad-spec um an imic obial ac i i y agains clinical, ood, and en i onmen al bac e ial pa hogens compa ed wi h he Se a ia s ains p oducing se awe in W2 [ 15 ]. A s ain o S. ma cescens showed he capaci y o inhibi an endophy ic ungus due o i s p oduc ion o compounds, namely, se awe ins [16]. Due o he limi ed in o ma ion on his opic, in his s udy, we pe o med a compa a i e genomic analysis o de e mine he dis ibu ion and he o ganiza ion o he se awe ins biosyn he ic gene clus e s in all he 84 publicly a ailable genomes o he Se a ia genus. He e, we epo a deep analysis o he se awe in W1 and W2 gene clus e s and es ablish hei o ganiza ion using an iSMASH so wa e. In pa icula , o he i s ime, we show he se awe in W1 gene clus e s’ o ganiza ion. These analyses showed he p esence o ou genes common o all he se awe in gene clus e s, highligh ing hei key po en ial in he se awe ins biosyn he ic p ocess. 2. Ma e ials and Me hods 2.1. Bac e ial S ains, Da a Collec ion, and Genomes Eigh y- ou Se a ia genomes a ailable on NCBI, ep esen ing all he publicly a ailable genomes, we e included in his s udy (Table S1), ob ained om 49 di e en hos s and isola ed om 29 coun ies [13,17–49]. Se awe in W1 and W2 biosyn he ic gene clus e p edic ion was pe o med in all he 84 comple e and d a Se a ia genomes publicly a ailable, using he web pla o m an iSMASH 3.0 [ 50 ]. This inpu da ase includes s ains belonging o S. ma cescens,S. lique aciens,S. g imesii,S. nema odiphila, S. plymu hica , and S. u eily ica, as well as s ains no cha ac e ized o he species le el. An iSMASH analysis ou pu was examined o iden i y he se awe ins biosyn he ic genes clus e s. The e o e, o ind he sw W and sw A genes, BLAST analysis was pe o med o all p o ein sequences, codi ied by Biology 2020,9, 482 3 o 18 each gene o he NRPS p edic ing me aboli e clus e s, using he NCBI [ 51 ] da abase. Clus e bounda ies we e p edic ed wi h he CASSIS algo i hm speci ied o he NRPS domains [50]. 2.2. Phylogene ic Analysis The 16S RNA sequences o he 47 Se a ia s ains, which showed he p esence o se awe ins genes in hei genomes (Table 1), we e selec ed o phylogene ic analysis. These sequences we e compa ed wi h he sequences a ailable in he EMBL/GenBank da abase using he BLASTN ne wo k se ices [ 51 ], and wi h sequences a ailable a he Ez axon-e se e (h p://ez axon-e.ezbiocloud.ne /) [ 52 ]. Sequences we e aligned wi hin he SINA alignmen se ice [ 53 ]. Sequences we e included in he 16S RNA-based Li ing T ee P ojec (LTP, elease 115) da abase (h p://www.a b-sil a.de/p ojec s/li ing- ee/) by pa simony implemen ed in he ARB so wa e package e sion 5.5 [ 54 ]. Phylogene ic dend og ams o his s udy s ains and closes e e ence sequences we e cons uc ed using he Neighbo -Joining and Randomized Axele a ed Maximum Likelihood (RAxML) me hod wi h he GTRGAMMA model [ 55 ] included in he ARB so wa e [54]. Table 1. Se a ia s ains wi h he sw W and sw A genes iden i ied h ough an iSMASH so wa e. An iSMASH so wa e was used in 84 Se a ia genomes (see Table S1). X ep esen s he p esence o genes sw W and sw A in se en een and ele en genomes, espec i ely. Bac e ial S ain Accession Numbe sw W sw A Se a ia sp. AS12 CP002774.1 X Se a ia sp. AS13 CP002775.1 X Se a ia sp. FS14 CP005927.1 X Se a ia sp. SCBI CP003424.1 X Se a ia sp. YD25 CP016948.1 X Se a ia sp. SSNIH1 CP026383.1 X Se a ia sp. PWN146 LT575490.1 X Se a ia ma cescens s ain UMH8 CP018927.1 X Se a ia ma cescens s ain IOMTU 115 AB894481.1 X Se a ia ma cescens subsp. ma cescens ATCC 13880 JMPQ01000033.1 X Se a ia ma cescens s ain CDC_813-60 DP21 JOVM01000004.1 X Se a ia nema odiphila DZ0503SBS1 s ain DSM 21420 JPUX00000000.1 X Se a ia ma cescens VGH107 AORJ00000000.1 X Se a ia ma cescens EGD-HP20 AVSR00000000.1 X Se a ia ma cescens WW4 CP003959.1 X Se a ia ma cescens BIDMC 81 JJMZ01000006.1 X Se a ia s ain TEL NODE_13 LDEG01000018.1 X Se a ia plymu hica NBRC 102599TBCTU00000000.1 X Se a ia g imesii isola e BXF1 LT883155.1 X Se a ia g imesii s ain A2 JGVP00000000.1 X Se a ia plymu hica AS9 CP002773.1 X Se a ia ma cescens SM39 AP013063.1 X Se a ia ma cescens SmUNAM836 CP012685.1 X Se a ia ma cescens BIDMC 44 JAPD01000005.1 X Se a ia u eily ica L 5/4 LG59 JSFB01000001.1 X Se a ia ma cescens RSC-14 CP012639.1 X Se a ia ma cescens subsp. ma cescens AH0650_Sm1 AG2 LFJS01000014.1 X Se a ia ma cescens subsp. ma cescens Db11 HG326223.1 X 2.3. Se awe ins Biosyn he ic Gene Clus e s Analysis The NRPS gene clus e , whe e bo h he se awe in W1 and W2 biosyn he ic genes belong, was analyzed he e by using an iSMASH so wa e [ 50 ]. The econs uc ion o he domains, modules, and s uc u es o he se awe ins was pe o med by using Phy e2 idimensional p edic ion [ 56 ], using he se awe in W1 biosyn he ic p o ein om Se a ia sp. AS13, and W2 om Se a ia sp. PWN146. PubChem 2D was used o explo e he chemical in o ma ion o he se awe ins using he bac e ial models men ioned abo e. The conca ena ed amino acid sequences o each se awe in biosyn he ic gene clus e we e o ganized by p o ein iden i ica ion in he same o de o allow a clus e alignmen wi h Clus alW Biology 2020,9, 482 4 o 18 in MEGAX so wa e [ 57 ]. The se awe in W1 biosyn he ic p o eins om 17 Se a ia genomes and se awe in W2 biosyn he ic p o eins om 11 Se a ia genomes we e sepa a ely aligned. The e olu iona y ela ionship be ween he clus e s and be ween he se awe in biosyn he ic p o ein sequences we e es ablished using he Neighbo -Joining me hod, Poisson model [ 58 ], in MEGAX so wa e [ 57 ]. The aligned and o ganized clus e genes we e ep esen ed in an e olu iona y ee acco ding o he size, di ec ion, and accession numbe s iden i ied wi h he NCBI BLAST da abase [ 51 ]. Func ions o he co e p o eins in all clus e s we e sea ched on UniP o [59]. 3. Resul s 3.1. Bac e ial Phylogeny and Compa a i e Genomics o Se a ia spp. All he 84 Se a ia s ains selec ed o his s udy had hei genome publicly a ailable a NCBI. Acco ding o he Neighbo -Joining and Maximum-Likelihood phylogene ic ees based on 16S RNA gene sequences, 14 s ains belong o S. ma cescens, wo s ains belong o S. plymu hica, wo s ains belong o S. g imesii, one s ain belongs o S. lique aciens, one s ain belongs o S. nema odiphila, one s ain belongs o S. u eily ica, and 63 s ains could no be assigned o species le el due o a simila i y lowe han 97% (Figu e 1). Genomes o he Se a ia s ains ha e a size om 5.0 Mbp o 7.7 Mbp and he G +C con en a ies om 45.9 o 60.1 mol%. Biology 2020, 9, x 4 o 19 PubChem 2D was used o explo e he chemical in o ma ion o he se awe ins using he bac e ial models men ioned abo e. The conca ena ed amino acid sequences o each se awe in biosyn he ic gene clus e we e o ganized by p o ein iden i ica ion in he same o de o allow a clus e alignmen wi h Clus alW in MEGAX so wa e [57]. The se awe in W1 biosyn he ic p o eins om 17 Se a ia genomes and se awe in W2 biosyn he ic p o eins om 11 Se a ia genomes we e sepa a ely aligned. The e olu iona y ela ionship be ween he clus e s and be ween he se awe in biosyn he ic p o ein sequences we e es ablished using he Neighbo -Joining me hod, Poisson model [58], in MEGAX so wa e [57]. The aligned and o ganized clus e genes we e ep esen ed in an e olu iona y ee acco ding o he size, di ec ion, and accession numbe s iden i ied wi h he NCBI BLAST da abase [51]. Func ions o he co e p o eins in all clus e s we e sea ched on UniP o [59]. 3. Resul s 3.1. Bac e ial Phylogeny and Compa a i e Genomics o Se a ia spp. All he 84 Se a ia s ains selec ed o his s udy had hei genome publicly a ailable a NCBI. Acco ding o he Neighbo -Joining and Maximum-Likelihood phylogene ic ees based on 16S RNA gene sequences, 14 s ains belong o S. ma cescens, wo s ains belong o S. plymu hica, wo s ains belong o S. g imesii, one s ain belongs o S. lique aciens, one s ain belongs o S. nema odiphila, one s ain belongs o S. u eily ica, and 63 s ains could no be assigned o species le el due o a simila i y lowe han 97% (Figu e 1). Genomes o he Se a ia s ains ha e a size om 5.0 Mbp o 7.7 Mbp and he G + C con en a ies om 45.9 o 60.1 mol%. Figu e 1. A phylogene ic dend og am based on a compa ison o he 16S RNA gene sequence o he Se a ia s ains used in his s udy and he ype o s ains. The ee was c ea ed using he Neighbo - Joining me hod in ARB so wa e. The numbe s on he ee indica e he pe cen ages o boo s ap sampling, de i ed om 1000 eplica ions; alues below 50% a e no shown. In blue a e shown he Se a ia s ains ha showed he p esence o he se awe in W1 biosyn he ic gene clus e and in g een a e shown he Se a ia s ains ha showed he p esence o he se awe in W2 biosyn he ic gene clus e . The ype species Esche ichia coli DSM 30083T was used as he ou g oup. Scale ba , 1 in e ed nucleo ide subs i u ion pe 100 nucleo ides. Figu e 1. A phylogene ic dend og am based on a compa ison o he 16S RNA gene sequence o he Se a ia s ains used in his s udy and he ype o s ains. The ee was c ea ed using he Neighbo -Joining me hod in ARB so wa e. The numbe s on he ee indica e he pe cen ages o boo s ap sampling, de i ed om 1000 eplica ions; alues below 50% a e no shown. In blue a e shown he Se a ia s ains ha showed he p esence o he se awe in W1 biosyn he ic gene clus e and in g een a e shown he Se a ia s ains ha showed he p esence o he se awe in W2 biosyn he ic gene clus e . The ype species Esche ichia coli DSM 30083 T was used as he ou g oup. Scale ba , 1 in e ed nucleo ide subs i u ion pe 100 nucleo ides. Bioin o ma ic analysis h ough an iSMASH so wa e showed ha he se awe in W1 biosyn he ic gene was p esen in 17 Se a ia genomes, s ains ATCC 13880, CDC_813-60 DP21, UMH8, IOMTU 115, DSM 21420, VGH107, EGD-HP20, WW4, FS14, BIDMC81, TEL NODE_13, NBRC 102599 T , BXF1, A2, AS13, AS9, and AS12 (Table 1and Table S2). The se awe in W2 biosyn he ic gene was p esen in 11 Biology 2020,9, 482 5 o 18 Se a ia s ains, PWN146, SSNIH1, SM39, SmUNAM836, BIDMC 44, L 5/4 LG59, RSC-14, AH0650_Sm1 AG2, Db11, SCBI, and YD25T(Table 1and Table S3). 3.2. Se awe in W1 Biosyn he ic Gene Clus e s All se awe in W1 biosyn he ic gene clus e s we e iden i ied as NRPS clus e s. Mo eo e , he bioin o ma ic analysis p edic ed an a chi ec u e including he domains condensa ion (C), adenyla ion (A), hiola ion (T), and hioes e ase (TE) in all he se awe in W1 biosyn he ic genes (Figu e 2). Biology 2020, 9, x 5 o 19 Bioin o ma ic analysis h ough an iSMASH so wa e showed ha he se awe in W1 biosyn he ic gene was p esen in 17 Se a ia genomes, s ains ATCC 13880, CDC_813-60 DP21, UMH8, IOMTU 115, DSM 21420, VGH107, EGD-HP20, WW4, FS14, BIDMC81, TEL NODE_13, NBRC 102599T, BXF1, A2, AS13, AS9, and AS12 (Tables 1 and S2). The se awe in W2 biosyn he ic gene was p esen in 11 Se a ia s ains, PWN146, SSNIH1, SM39, SmUNAM836, BIDMC 44, L 5/4 LG59, RSC- 14, AH0650_Sm1 AG2, Db11, SCBI, and YD25T (Tables 1 and S3). 3.2. Se awe in W1 Biosyn he ic Gene Clus e s All se awe in W1 biosyn he ic gene clus e s we e iden i ied as NRPS clus e s. Mo eo e , he bioin o ma ic analysis p edic ed an a chi ec u e including he domains condensa ion (C), adenyla ion (A), hiola ion (T), and hioes e ase (TE) in all he se awe in W1 biosyn he ic genes (Figu e 2). Figu e 2. Se awe in W1: om he biosyn he ic gene clus e o he s uc u e o se awe in W1. Gene ic o ganiza ion o he genome o Se a ia s ain AS13 by an iSMASH analysis, Phy e2 idimensional p edic ion o se awe in W1 biosyn he ic p o ein, and PubChem 2D s uc u e o se awe in W1. (a) Se awe in W1 biosyn he ic gene clus e wi h iden i ica ion o he s wW co e gene; (b) sw W gene o ganiza ion: C, condensa ion domain; A, adenyla ion domain; T, hiola ion domain; and TE, hioes e ase domain; (c) biosyn he ic p o ein wi h he condensa ion, adenyla ion, hiola ion, and hioes e ase domains; (d) se awe in W1 2D s uc u e. To con i m he iden i ica ion o he sw W gene e ealed by an iSMASH, each sw W was que ied o NCBI BLASTP, in o de o ind he closes ela i e and de e mine he iden i y pe cen age (Tables 2 and S2). The p o ein om he se awe in W1 biosyn he ic gene (sw W) showed an iden i y pe cen age ha anges om 77.79% o 100% as he closes iden i ica ion by using BLASTP (Table 2). Figu e 2. Se awe in W1: om he biosyn he ic gene clus e o he s uc u e o se awe in W1. Gene ic o ganiza ion o he genome o Se a ia s ain AS13 by an iSMASH analysis, Phy e2 idimensional p edic ion o se awe in W1 biosyn he ic p o ein, and PubChem 2D s uc u e o se awe in W1. ( a ) Se awe in W1 biosyn he ic gene clus e wi h iden i ica ion o he s wW co e gene; ( b )sw W gene o ganiza ion: C, condensa ion domain; A, adenyla ion domain; T, hiola ion domain; and TE, hioes e ase domain; ( c ) biosyn he ic p o ein wi h he condensa ion, adenyla ion, hiola ion, and hioes e ase domains; (d) se awe in W1 2D s uc u e. To con i m he iden i ica ion o he sw W gene e ealed by an iSMASH, each sw W was que ied o NCBI BLASTP, in o de o ind he closes ela i e and de e mine he iden i y pe cen age (Table 2 and Table S2). The p o ein om he se awe in W1 biosyn he ic gene (sw W) showed an iden i y pe cen age ha anges om 77.79% o 100% as he closes iden i ica ion by using BLASTP (Table 2). Biology 2020,9, 482 6 o 18 Table 2. The se awe in W1 gene (sw W) o each s ain in his s udy and he accession numbe s and iden i ica ion o he sw W gene’s closes ela i es using BLASTP. Bac e ial S ain sw W Closes Rela i e Genes Accession Numbe Iden i y Pe cen age Se a ia ma cescens EGD-HP20 non- ibosomal pep ide syn he ase ERH70695.1 99.52 Se a ia ma cescens WW4 se awe in W1 syn he ase AGE20181.1 100 Se a ia sp. FS14 pu a i e se awe in W1 syn he ase AIA46701.1 100 Se a ia ma cescens VGH107 amino acid adenyla ion p o ein EMF04443.1 99.12 Se a ia nema odiphila DZ0503SBS1 s ain DSM 21420 non- ibosomal pep ide syn he ase KFF87803.1 99.31 Se a ia ma cescens s ain IOMTU 115 pu a i e se awe in W1 syn he ase BAO21138.1 99.92 Se a ia ma cescens s ain UMH8 non- ibosomal pep ide syn he ase ASM18665.1 99.01 Se a ia ma cescens subsp. ma cescens ATCC 13880 amino acid adenyla ion domain-con aining p o ein KFD14984.1 98.57 Se a ia ma cescens s ain CDC_813-60 DP21 non- ibosomal pep ide syn he ase KFL05097.1 98.63 Se a ia ma cescens BIDMC 81 non- ibosomal pep ide syn he ase EZQ62923.1 95.12 Se a ia s ain TEL NODE_13 non- ibosomal pep ide syn he ase KLE36484.1 95.05 Se a ia plymu hica NBRC 102599T non- ibosomal pep ide syn he ase WP_063202307.1 81.71 Se a ia sp. AS13 non- ibosomal pep ide syn he ase AEG30284.1 81.48 Se a ia plymu hica AS9 non- ibosomal pep ide syn he ase AEF47625.1 81.48 Se a ia sp. AS12 non- ibosomal pep ide syn he ase WP_013814722.1 81.48 Se a ia g imesii isola e BXF1 amino acid adenyla ion domain-con aining p o ein SMZ58711.1 77.79 Se a ia g imesii s ain A2 non- ibosomal pep ide syn he ase KFB89923.1 78.17 Eigh y di e en genes om he se awe in W1 biosyn he ic gene clus e we e iden i ied. Fi een genes a e common o all 17 gene clus e s (Figu e 3and Table S4), such as genes encoding o mu ein hyd olase e ec o p o ein L gB and mu ein hyd olase egula o L gA, bo h wi h hyd olase ac i i y; LysR egula o y p o ein wi h DNA-binding ansc ip ion ac o ac i i y; a sodium-hyd ogen an ipo e and xan hine-u acil- i amin C pe mease, bo h wi h ansmemb ane anspo ac i i y; glyoxalase–bleomycin esis ance p o ein and glu a hione S- ans e ase domain p o ein, bo h wi h a dioxygenase ac i i y; 3-oxoacyl-(acyl-ca ie -p o ein) educ ase; single-s anded DNA-binding p o ein; exonuclease ABC subuni A; mal ose O-ace yl ans e ase; and a oma ic amino acid amino ans e ase. No conside ing s ain NRBC 102599 T , an addi ional se en genes we e ound o be common o all s ains in he se awe in W1 biosyn he ic gene clus e (Figu e 3). Mo eo e , 15 genes a e exclusi e o he S. plymu hica NBRC 102599Tbiosyn he ic gene clus e (Figu e 3). Biology 2020,9, 482 7 o 18 Biology 2020, 9, x 7 o 19 Figu e 3. Se awe in W1 gene clus e analysis. The phylogene ic ela ionship was es ablished wi h Mega X so wa e by he Neighbo -Joining me hod on aligned se awe in W1 gene clus e s o Se a ia s ains ATCC 13880, CDC_813-60 DP21, UMH8, IOMTU 115, DSM 21420, VGH107, EGD-HP20, WW4, FS14, BIDMC81, TEL NODE_13, NBRC 102599, BXF1, A2, AS13, AS9, and AS12. (a) Phylogene ic ee based on p o ein sequences o he se awe in W1 biosyn he ic gene clus e on an es ablished gene ic o ganiza ion. (b) Se awe in W1 biosyn he ic gene clus e s based on na u al gene ic o ganiza ion. (c) Compa ison o he phylogene ic ee based on p o ein sequences o he se awe in W1 biosyn he ic gene clus e s (le ) wi h he phylogene ic ee o he se awe in W1 biosyn hesis p o ein ( igh ). The scale ba o 0.050 in e s he nucleo ide subs i u ions pe 100 nucleo ides. Figu e 3. Se awe in W1 gene clus e analysis. The phylogene ic ela ionship was es ablished wi h Mega X so wa e by he Neighbo -Joining me hod on aligned se awe in W1 gene clus e s o Se a ia s ains ATCC 13880, CDC_813-60 DP21, UMH8, IOMTU 115, DSM 21420, VGH107, EGD-HP20, WW4, FS14, BIDMC81, TEL NODE_13, NBRC 102599, BXF1, A2, AS13, AS9, and AS12. ( a ) Phylogene ic ee based on p o ein sequences o he se awe in W1 biosyn he ic gene clus e on an es ablished gene ic o ganiza ion. ( b ) Se awe in W1 biosyn he ic gene clus e s based on na u al gene ic o ganiza ion. ( c ) Compa ison o he phylogene ic ee based on p o ein sequences o he se awe in W1 biosyn he ic gene clus e s (le ) wi h he phylogene ic ee o he se awe in W1 biosyn hesis p o ein ( igh ). The scale ba o 0.050 in e s he nucleo ide subs i u ions pe 100 nucleo ides. Biology 2020,9, 482 8 o 18 The ela ionship be ween s ains es ablished based on he analysis o he conca ena ed genes o he W1 biosyn he ic gene clus e de ined he same clus e s as he ela ionships de ined based on he sw W gene analysis, excep o S. plymu hica NBRC 102599 T , which is disco dan . The posi ion o S. plymu hica NBRC 102599 T in W1 phylogene ic ee highligh s he di e en gene composi ion o he W1 biosyn he ic clus e o he s ain. On he o he hand, in he sw W phylogene ic ee, S. plymu hica NBRC 102599T o ms a sis e g oup wi h Se a ia s ains AS13, AS9, and AS12 (Figu e 3). In he se awe in W1 biosyn he ic gene clus e , so wa e p edic ion iden i ied ou genes in ol ed in PKS-NRPS (PolyKe ide Syn hases Non-Ribosomal Pep ide Syn he ases), encoding o enoyl educ ase quinone oxido educ ase (only p esen in se en s ains), ke o educ ase 3-oxoacyl-(acyl-ca ie -p o ein) educ ase (p esen in all s ains), enoyl educ ase dehyd ogenase (absen in wo s ains), and a oma ic amino acid amino ans e ase (p esen in all s ains) (Table 3and Table S4, Figu e 3). Table 3. The PKS gene accession numbe s om he sw W biosyn he ic gene clus e s p edic ed by an iSMASH so wa e. Bac e ial S ain Enoyl educ ase Quinone Oxido educ ase Ke o educ ase 3-Oxoacyl-(Acyl-Ca ie -P o ein) Reduc ase Enoyl educ ase Dehyd ogenase A oma ic Amino Acid Amino ans e ase Se a ia ma cescens EGD-HP20 ERH70706.1 ERH70710.1 ERH70714.1 Se a ia ma cescens WW4 AGE20192.1 AGE20197.1 AGE20201.1 Se a ia sp. FS14 AIA46690.1 AIA46685.1 AIA46681.1 Se a ia ma cescens VGH107 EMF04432.1 EMF04427.1 EMF04423.1 Se a ia nema odiphila DZ0503SBS1 s ain DSM 21420 KFF87792.1 KFF87787.1 KFF87783.1 Se a ia ma cescens s ain IOMTU 115 BAO21148.1 BAO21153.1 BAO21155.1 Se a ia ma cescens s ain UMH8 ASM18675.1 ASM18680.1 ASM18684.1 Se a ia ma cescens subsp. Ma cescens ATCC 13880 KFD14974.1 KFD14969.1 KFD14965.1 Se a ia ma cescens s ain CDC_813-60 DP21 KFL04091.1 KFL03204.1 KFL04717.1 Se a ia ma cescens BIDMC 81 EZQ62938.1 EZQ62913.1 EZQ62903.1 Se a ia s ain TEL NODE_13 KLE36470.1 KLE36494.1 KLE36503.1 Se a ia plymu hica NBRC 102599TWP_063202297.1 WP_062868864.1 WP_006328339.1 Se a ia sp. AS13 AEG30270.1 AEG30294.1 AEG30297.1 AEG30301.1 Se a ia plymu hica AS9 AEF47611.1 AEF47635.1 AEF47638.1 AEF47642.1 Se a ia sp. AS12 WP_013814712.1 WP_013814732.1 WP_013814734.1 WP_013814736.1 Se a ia g imesii isola e BXF1 SMZ58698.1 SMZ58721.1 SMZ58727.1 SMZ58731.1 Se a ia g imesii s ain A2 KFB89936.1 KFB89913.1 KFB89904.1 3.3. Se awe in W2 Biosyn he ic Gene Clus e s E e y se awe in W2 biosyn he ic gene (sw A) showed an a chi ec u e composed o i e modules, each wi h a condensa ion (C1, C2, C3, C4, and C5), adenyla ion (A1, A2, A3, A4, and A5), and hiola ion (T1, T2, T3, T4, and T5) domain. Module 5 has an addi ional hioes e ase (TE) domain. This o ganiza ion is sha ed by all he se awe in W2 biosyn he ic genes (Figu e 4). Biology 2020,9, 482 9 o 18 Biology 2020, 9, x 9 o 19 Figu e 4. Se awe in W2: om he biosyn he ic gene clus e o he s uc u e o se awe in W2. Gene ic o ganiza ion o he genome o Se a ia s ain PWN146 by an iSMASH analysis, Phy e2 idimensional p edic ion o he se awe in W2 biosyn he ic p o ein, and PubChem 2D s uc u e o se awe in W2. (a) Se awe in W2 biosyn he ic gene clus e wi h iden i ica ion o he sw A co e gene; (b) sw A gene o ganiza ion: i e modules (M1–M5) wi h C, condensa ion domain; A, adenyla ion domain; T, hiola ion domain; and TE, hioes e ase domain; (c) biosyn he ic p o ein wi h i e modules (M1–M5), each composed o condensa ion, adenyla ion, and hiola ion domains, and in he las module an addi ional hioes e ase domain. To con i m he iden i ica ion o he sw A gene e ealed by an iSMASH, each sw A was, as men ioned abo e, que ied o NCBI BLASTX, in o de o ind he closes ela i e and de e mine he iden i y pe cen age (Tables 4 and S3). The p o ein coded by he biosyn he ic genes o se awe in W2 (sw A) showed an iden i y pe cen age ha anges om 76.38% o 99.4% as he closes iden i ica ion by using BLASTX (Table 4). Table 4. The se awe in W2 gene (sw A) o each s ain in his s udy and he accession numbe s and iden i ica ion o he sw A gene’s closes ela i es using BLASTX. Bac e ial S ain sw A Closes Rela i e Genes Accession Numbe Iden i y Pe cen age Se a ia ma cescens SM39 Se a ia ma cescens SM39 DNA, comple e genome BAO35825.1 76.70 Se a ia ma cescens SmUNAM836 Se a ia ma cescens s ain SmUNAM836, comple e genome ALE98111.1 76.70 Se a ia sp. SSNIH1 Se a ia sp. SSNIH1 ch omosome, comple e genome AUY16873.1 76.70 Se a ia sp. PWN146 Se a ia ma cescens isola e PWN146_assembly genome assembly, ch omosome SAY45247.1 77.02 Se a ia u eily ica L 5/4 LG59 Se a ia ma cescens s ain UMH7, comple e genome KKO55915.1 76.38 Se a ia ma cescens RSC-14 Se a ia ma cescens s ain RSC-14, comple e genome ALD45109.1 76.38 Se a ia ma cescens BIDMC 44 Se a ia ma cescens s ain 1274 genome ETX44761.1 77.35 Se a ia sp. YD25 Se a ia sp. YD25, comple e genome AOF02338.1 93.57 Se a ia sp. SCBI Se a ia sp. SCBI, comple e genome AIM23801.1 93.44 Se a ia ma cescens subsp. ma cescens AH0650_Sm1 AG2 Se a ia ma cescens s ain BWH-23 ch omosome, comple e genome KMU50701.1 99.40 Se a ia ma cescens subsp. ma cescens Db11 Se a ia ma cescens subsp. ma cescens Db11, comple e genome CDG14244.1 99.32 Figu e 4. Se awe in W2: om he biosyn he ic gene clus e o he s uc u e o se awe in W2. Gene ic o ganiza ion o he genome o Se a ia s ain PWN146 by an iSMASH analysis, Phy e2 idimensional p edic ion o he se awe in W2 biosyn he ic p o ein, and PubChem 2D s uc u e o se awe in W2. ( a ) Se awe in W2 biosyn he ic gene clus e wi h iden i ica ion o he sw A co e gene; ( b )sw A gene o ganiza ion: i e modules (M1–M5) wi h C, condensa ion domain; A, adenyla ion domain; T, hiola ion domain; and TE, hioes e ase domain; ( c ) biosyn he ic p o ein wi h i e modules (M1–M5), each composed o condensa ion, adenyla ion, and hiola ion domains, and in he las module an addi ional hioes e ase domain. To con i m he iden i ica ion o he sw A gene e ealed by an iSMASH, each sw A was, as men ioned abo e, que ied o NCBI BLASTX, in o de o ind he closes ela i e and de e mine he iden i y pe cen age (Table 4and Table S3). The p o ein coded by he biosyn he ic genes o se awe in W2 (sw A) showed an iden i y pe cen age ha anges om 76.38% o 99.4% as he closes iden i ica ion by using BLASTX (Table 4). Table 4. The se awe in W2 gene (sw A) o each s ain in his s udy and he accession numbe s and iden i ica ion o he sw A gene’s closes ela i es using BLASTX. Bac e ial S ain sw A Closes Rela i e Genes Accession Numbe Iden i y Pe cen age Se a ia ma cescens SM39 Se a ia ma cescens SM39 DNA, comple e genome BAO35825.1 76.70 Se a ia ma cescens SmUNAM836 Se a ia ma cescens s ain SmUNAM836, comple e genome ALE98111.1 76.70 Se a ia sp. SSNIH1 Se a ia sp. SSNIH1 ch omosome, comple e genome AUY16873.1 76.70 Se a ia sp. PWN146 Se a ia ma cescens isola e PWN146_assembly genome assembly, ch omosome SAY45247.1 77.02 Se a ia u eily ica L 5/4 LG59 Se a ia ma cescens s ain UMH7, comple e genome KKO55915.1 76.38 Se a ia ma cescens RSC-14 Se a ia ma cescens s ain RSC-14, comple e genome ALD45109.1 76.38 Se a ia ma cescens BIDMC 44 Se a ia ma cescens s ain 1274 genome ETX44761.1 77.35 Se a ia sp. YD25 Se a ia sp. YD25, comple e genome AOF02338.1 93.57 Se a ia sp. SCBI Se a ia sp. SCBI, comple e genome AIM23801.1 93.44 Se a ia ma cescens subsp. ma cescens AH0650_Sm1 AG2 Se a ia ma cescens s ain BWH-23 ch omosome, comple e genome KMU50701.1 99.40 Se a ia ma cescens subsp. ma cescens Db11 Se a ia ma cescens subsp. ma cescens Db11, comple e genome CDG14244.1 99.32 Six y-eigh genes we e iden i ied in he se awe in W2 biosyn he ic gene clus e s (Figu e 5and Table S5). Twen y- ou genes we e p esen in all he s ains’ gene clus e s (Figu e 5), namely, he Biology 2020,9, 482 16 o 18 26. 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