Draft genome sequence of a novel Bacillus glycinifermentans strain having antifungal and antibacterial properties

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Draft genome sequence of a novel Bacillus glycinifermentans strain having antifungal and

antibacterial properties

A. Karim, Olivier Poirot, A. Khatoon, M. Aurongzeb

To cite this version:

A. Karim, Olivier Poirot, A. Khatoon, M. Aurongzeb. Draft genome sequence of a novel Bacillus

glycinifermentans strain having antifungal and antibacterial properties. Journal of Global Antimicro-

bial Resistance, Elsevier, 2019, 19, pp.308-310. �10.1016/j.jgar.2019.10.011�. �hal-02409583�

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Draft genome sequence of a novel Bacillus glycinifermentans strain having antifungal and antibacterial properties

A. Karim

^a,

*, O. Poirot

^b

, A. Khatoon

^a

, M. Aurongzeb

^a

aJamil-Ur-RahmanCenterforGenomeResearch,DrPanjwaniCenterforMolecularMedicineandDrugResearch(PCMD),InternationalCenterforChemical andBiologicalSciences(ICCBS),UniversityofKarachi,Karachi75270,Pakistan

bAix-MarseilleUniversité,CNRS,StructuralandGenomicInformationLaboratory,UMR7256(IMMFR3479),163AvenuedeLuminy,Case934,13288, MarseilleCedex9,France

Objectives:Bacillusspp.havebeenusedasbiocontrolagentsagainstsoilbornepathogensbecausethey producesecondarymetabolitesthatexhibitawiderangeofantibacterialorantifungalproperties.Inthis study,anovelstrainofBacillusglycinifermentans sp.(JRCGR-1)wasidentiﬁedand itsgenomewas sequencedandannotated.Thegenomewasexploredforputativegenesinvolvedinantimicrobialactivity.

Methods:Whole-genomesequencingwasperformedonanIlluminaNextSeq500platform.Readquality waschecked byFastQC,paired-end readsweretrimmed using Sickle, and denovo assemblywas performedusingSPAdesv.3.11.11.QUAST5.02wasusedtoassessthequalityofcontigsandscaffolds.

Finally, the assembled scaffoldswere annotated by Prokka v.1.13.Genes involvedin antimicrobial metabolitebiosynthesiswerepredictedusingantiSMASH.Virulenceandantimicrobialresistancegenes werepredicted using BacWGSTdb and the Comprehensive Antibiotic ResistanceDatabase (CARD), respectively.

Results:ThegenomeofB.glycinifermentansJRCGR-1was4700692bpinsizewithaG+Ccontentof 45.52%.Finalassemblyofthegenomeresultedinto84contigsand83scaffolds(>500bplength).Overall, thegenomecomprises5174genes,32tRNAs,4rRNAs,1tmRNAand92misc_RNAs.Elevenputativegene clusters responsiblefor antimicrobial metabolitebiosynthesis were identiﬁed, including genes for biosynthesisofnon-ribosomal lipopeptidesandpolyketides.Virulence andantimicrobial resistance geneswerealsoidentiﬁedinthegenome.

Conclusion:ThepresenceofantimicrobialresistancegenesinthegenomeofB.glycinifermentansJRCGR-1 makesitapotentialbiocontrolagentagainstsoilbornepathogens.

Soilborne pathogens are emerging as a global threat to agriculturalproducts,andeachyearworldwidetheycausehuge economiclosses.Severalchemical-basedfertilisersandpesticides areusedtocontrolsoilbornepathogens.However,withlong-term use thesechemicals arehazardous tohumanhealth and cause environmentpollution.Analternativetoovercomethisproblemis theuseofbiocontrolagents[1].Speciﬁcally,thegenusBacillusisa potentcandidateforbiocontrolagentsagainstsoilbornepathogens as members of this genus producea wide range of secondary metabolites,hydrolyticenzymesandvolatileorganiccompounds withantibacterialorantifungalproperties.

Much workhasbeen donetoexplore thebiochemistry and physiology ofBacillusspp. Advancementsin theﬁeldofwhole-

genome sequencinghavegiven a new dimensiontomicrobial- basedproductsandprocesses.

In2015,twobacterialisolatesfromcheonggukjang,aKorean fermentedsoybeanpastefoodproduct,werefoundtocomprisea novelBacillussp.namedBacillusglycinifermentanssp.nov.[2].To date,onlyﬁveB.glycinifermentansgenomesequencesareavailable inpublicdatabases,includingthreecompletegenomes(GenBank Assembly accession nos. GCA_900093775.1, GCA_002443095.1 andGCA_004103615.1)andtwodraftgenomes(GenBankAssem- blyaccession nos. LECV00000000.1and LECW00000000.2).To thebestofourknowledge,thisistheﬁrstdraftgenomesequenceof

B. glycinifermentansreportedfromPakistan.

TostudythegenomeofB.glycinifermentansstrainJRCGR-1,the strainwas ﬁrst culturedonanutrient agarplate.Colonieswere theninoculatedinto10mLofLuriabrothandwereincubatedat 37Cfor 24h.The opticaldensitywas adjustedtoa McFarland

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standard of 3–4nm. Following incubation, bacterial cells were collectedbycentrifugation(3000gfor15min).Thesupernatant wasremovedwithoutdisturbingthesedimentandtheDNAwas extractedusingacommercialDNAextractionkit(QIAamp¹DNA MiniKit;QIAGEN,Hilden,Germany).AQubit^TMdsDNABRAssaykit (Invitrogen;ThermoFisherScientiﬁc,Eugen,OR,USA)wasusedto calculate the amount of DNA using a Qubit¹ 2.0 ﬂuorometer

(Invitrogen)accordingto themanufacturer’sinstructions.ANextera XTDNALibraryKit(IlluminaInc.,SanDiego,CA,USA)wasusedto constructapaired-endlibrary,andsequencingwasperformedona NextSeq500platform(IlluminaInc.)inpaired-endreadmode.The qualityofthereadswascheckedusingFastQCsoftware.Paired-end readswerethentrimmedusingSickleanddenovoassemblywas performedusingSPAdesv.3.11.11intocontigsandscaffolds(base

Table1

SecondarymetaboliteproﬁleofsixBacillusglycinifermentansstrains.

Strain/metabolitetype Mostsimilarknowncluster Similarity(%)

B.glycinifermentansGO-13

Lanthipeptide GeobacillinII 50

NRPS Lichenysin 100

T3PKS – –

NRPS Fengycin 26

Siderophore – –

β-Lactone Fengycin 53

Lassopeptide – –

NRPS Bacillibactin 53

NRPs,T1PKS,terpene Paenibacterin 60

B. glycinifermentansKJ-17

Thiopeptide,bacteriocin Butirosin 7

NRPS,terpene,T1PKS Paenibacterin 60

T3PKS – –

Lanthipeptide Geobacillin 50

Siderophore – –

NRPS Fengycin 33

NRPS Lichenysin 100

B. glycinifermentansBGLY

Sactipeptide SporulationkillingfactorSkfA 85

NRPS Lichenysin 100

Siderophore – –

Terpene – –

T3PKS – –

NRPS Bacitracin 88

B. glycinifermentansKBNO06PO3352

NRPS Fengycin 26

NRPS,T1PKS,terpene Paenibacterin 60

T3PKS – –

NRPS – –

Lanthipeptide Geobacillin 50

NRPS Lichenysin 100

Siderophore

B.glycinifermentansSRCM103574

Sactipeptide(head-to-tailcyclisedpeptide) SporulationkillingfactorSkfA 85

NRPS Lichenysin 100

Siderophore – –

Terpene – –

T3PKS – –

NRPS Bacitracin 88

B. glycinifermentansJRCGR-1

T3PKS – –

Terpene – –

NRPS Lichenysin 100

Head-to-tail SporulationkillingfactorSkfA 71

Siderophore – –

NRPS Bacitracin 66

Bacteriocin – –

NRPS – –

NRPS Bacitracin 33

NRPS,non-ribosomalpeptidesynthetase.

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quality scores>Q20,k=39).Thequality ofcontigsandscaffoldswas evaluatedbyQUAST5.02.Theassembledcontigswereannotated usingProkka v.1.13[>500bp;e-valuecut-offdefault(10_6)]for rapidannotationofprokaryoticgenomes.tRNAscan-SEwasusedfor thepredictionoftRNA genes.Theplasmidwasassembledusing plasmidSPAdes (base quality scores>Q20, k=55), and gene annotationwasdoneusingProkkav.1.13[>500bp;e-valuecut- offdefault(10_6)].RNAmmerandBarrnapwereusedtoidentify RNAgenes.Putativegenesinvolved inantimicrobialmetabolite biosynthesiswerepredictedusingantiSMASHv.5.0.0rc1.Virulence geneswerepredictedusingtheBacWGSTdbservice(http://bacdb.

org/BacWGSTdb/index.php).Antimicrobialresistancegeneswere predictedusingtheComprehensiveAntibioticResistanceDatabase (CARD)(https://card.mcmaster.ca/home).

A total of 4 851 845 paired-end reads (276-bp) were generated with 32 coverage for strain JRCGR-1. The genome assembly contained 4700692 bp and with an average G+C contentof45.52%.Theﬁnalassemblycontains84contigsand83 scaffolds(>500bplength,N50of135232bp).Themaximumcontig sizewas384553bp.Overall,thegenomecomprises5174genes,32 tRNAs,4 rRNAs,1 tmRNAand 92 misc_RNAs. Theplasmid was assembledinto37scaffoldsusingplasmidSPAdes.Otherfeaturesof theplasmidincludeasizeof1113267bp,27tRNAs,4rRNA,1366 genes,21misc_RNAsand1314CDS(codingsequences).

Genesinvolvedinantimicrobialmetabolitebiosynthesiswere predictedusingantiSMASH.DraftgenomeanalysisofstrainJRCGR- 1revealed11putativegeneclustersresponsibleforantimicrobial metabolitebiosynthesis,amongwhichﬁveencodenon-ribosomal peptidesynthetases(NRPS)(forbiosynthesisofonefengycin,one lichenysin,onebacillibactinandtwobacitracins)andonerelated toterpeneandβ-lactonebiosynthesis.Siderophores(bacillibactin) producedbybacteriaareinvolvedininhibitionofphytopathogen growthbydeprivingthemofessentialiron[3].

SupplementaryFigs.S1andS2showtwoputativegeneclusters ofstrainJRCGR-1(labelasquerysequence)involvedinbiosynthe- sisofsecondarymetabolites. Thesegeneshavesimilaritieswith non-ribosomallipopeptides(lichenysin,surfactin,fengycin,myco- subtilinand plipastatin) and polyketides (basiliskamides). Sup- plementaryFig.S1(A)showstheﬁrstputativegeneclusterofstrain JRCGR-1 involved in secondary metabolite biosynthesis and its similaritytolichenysin(100%),surfactin(47%)andbasiliskamides (9%).ComparisonofthisgeneagainstotherBacillusspp.shows94%

similaritywithB.glycinifermentansstrainGO-13,B.glycinifermen- tansstrainKBNO06PO3352and B.glycinifermentansstrainBGLY [SupplementaryFig. S1(B)]. Similarly, thesecond putative gene cluster presented similarities with fengycin (53% similarity), mycosubtilin (40% similarity) and plipastatin (30% similarity) [Supplementary Fig. S2(A)].Comparison of this predicted gene clusterofstrainJRCGR-1withotherBacillusspp.showedsimilarity between45–50%forsevenBacillusparalicheniformissp.and46%

genesimilarityforBacilluslicheniformisstrainB4123[Fig.S2(B)].

SecondarymetabolitesforfiveotherstrainsofB.glycinifermentans werealso predicted using antiSMASH. The finding reveals that fengycinandlichenysinwerepresentascoregenes,i.e.theywere present in all six strains. A putative gene for bacillibactin was absentinB.glycinifermentansstrainKBNO06PO3352.Sporulation killingfactorSkfAwaspresentinB.glycinifermentansstrainJRCGR- 1 and B. glycinifermentans strain SRCM103574. Details of the secondarymetaboliteprofileofsixstrainsofB.glycinifermentans sp.aregiveninTable1.ThepresenceoftheseNRPSgenescanbe linkedwiththebiocontrolpotentialofbacteria[4].Severalgene clusterwerealsoidentifiedthatcansuppressthegrowthofGram- positivebacteriabyproducingsynthetases(PKS),e.g.oleandomy- cin,β-lactamase, tetracenomycinand tetracycline.Putative bsIA (yuaB)andtasAgenesassociatedwiththeproductionofbiofilm

matrixandfungalcell-wall-degradingenzymes,respectively,were alsoidentified.InthegenomesofsixstrainsofB.glycinifermentans sp.,twoantimicrobialresistancegenes(bcrAandEscherichiacoli ampC1β-lactamase)werepresentasaccessorygenes.Thesegenes were present in three strains of B. licheniformis, including SRCM103574,BGLYandJRCGR-1.ThebcrAgeneisanATP-binding cassette(ABC)transporterfoundmostlyinB.licheniformissp.that confersbacitracinresistance[5].TheE.coliAmpC1β-lactamase resistancemechanismisbasedonantibioticinactivation,whereas forBcrAtheresistancemechanisminvolvesantibioticefflux.Inthe genomesofstrainsKBNO06PO3352andGO-13,onlyE.coliampC1 β-lactamasewaspredicted.Ontheotherhand,strainKJ-17lacksall of thesegenes.Wealsoidentified virulencegenesin thestrain JRCGR-1 genome, including capA, capB, capC and hlyIII, with similaritiesof83.25%,89.30%,88%and82.26%,respectively.

The aforementionedﬁndings revealthat B. glycinifermentans JRCGR-1 might be a potential candidate as a biological control agentagainstplantsoilbornediseases.

GenBankaccessionno

ThedraftgenomesequenceofB.glycinifermentansJRCGR-1was submittedtotheNCBIdatabasewithaccessionno.VHPY0000000.

Funding

This study was supported by funds from the International CenterforChemicalandBiologicalSciences(ICCBS),Universityof Karachi(Karachi,Pakistan),CampusFranceandEmbassyofFrance inPakistan.

Competinginterests Nonedeclared.

Ethicalapproval Notrequired.

Acknowledgments

TheauthorswouldliketothankMatthieuLegendre, Chantal Abergel, Sébastien Santini and Sébastien Nin [Aix-Marseille Université,CNRS,StructuralandGenomicInformationLaboratory, UMR 7256 (IMMFR 3479)] and Georges Massiot[Universityof Reims,Reims,France]fortheirvaluablesupport.

AppendixA.Supplementarydata

Supplementarymaterialrelatedtothisarticlecanbefound,in theonlineversion,atdoi:https://doi.org/10.1016/j.jgar.2019.10.011.

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