Inducible cellulase production from an organic solvent tolerant Bacillus sp. SV1 and evolutionary divergence of endoglucanase in different species of the genus Bacillus

h tt p : / / w w w . b j m i c r o b i o l . c o m . b r /

Fungal

and

Bacterial

Physiology

Inducible

cellulase

production

from

an

organic

solvent

tolerant

Bacillus

sp.

SV1

and

evolutionary

divergence

of

endoglucanase

in

different

species

of

the

genus

Bacillus

Lebin

Thomas,

Hari

Ram,

Ved

Pal

Singh

∗

UniversityofDelhi,DepartmentofBotany,Delhi,India

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received16October2016 Accepted6May2017

Availableonline6November2017 AssociateEditor:LucySeldin

Keywords: Bacillus Cellulases

Conservedsignatureindels

a

b

s

t

r

a

c

t

Bacteriaareimportantsourcesofcellulaseswithvariousindustrialandbiotechnological applications.Inviewofthis,anon-hemolyticbacterialstrain,toleranttovarious environ-mentalpollutants(heavymetalsandorganicsolvents),showinghighcellulolyticindex(7.89) wasisolatedfromcattleshedsoilandidentifiedasBacillussp.SV1(99.27%pairwise

similar-itywithBacilluskorlensis).Extracellularcellulasesshowedthepresenceofendoglucanase,

totalcellulaseand␤-glucosidaseactivities.Cellulaseproductionwasinducedinpresence ofcellulose(3.3timesCMCase,2.9timesFPaseand2.1times␤-glucosidase),andenhanced (115.1%CMCase)bylow-costcornsteepsolids.Aninsilicoinvestigationofendoglucanase (EC3.2.1.4)proteinsequencesofthreeBacillusspp.asquery,revealedtheirsimilaritieswith membersofninebacterialphylaandtoEukaryota(representedbyArthropodaand Nema-toda),andalsohighlightedofaconvergentanddivergentevolutionfromotherenzymes ofdifferentsubstrate[(1,3)-linkedbeta-d-glucans,xylanandchitosan]specificities. Char-acteristicconservedsignatureindelswereobservedamongmembersofActinobacteria(7 aainsert)andFirmicutes(9aainsert)thatservedasapotentialtoolinsupportoftheir relatednessinphylogenetictrees.

©2017SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/

licenses/by-nc-nd/4.0/).

Introduction

Glycoside hydrolases or glycosidases are enzymes that hydrolyzeO-,N-andS-linkedglycosides,whichhavediverse industrial applications. Cellulases are glycoside hydrolases that hydrolyze O-linked glycosides,i.e. the ␤-1,4-glycosidic

∗ _{Correspondingauthor.}

E-mail:[email protected](V.P.Singh).

bonds betweentwo glucose residues ofcellobiose units in cellulose. They form a synergistically functioning enzyme system, that chiefly consist of endoglucanase (EC 3.2.1.4), exoglucanase/cellobiohydrolase(EC3.2.1.176/EC3.2.1.91)and beta-glucosidase (EC 3.2.1.21). Cellulases have important industrialapplicationsintextileprocessing,asdetergent com-ponents,aspartofmaceratingenzymesinfoodprocessing,

https://doi.org/10.1016/j.bjm.2017.05.010

1517-8382/©2017SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

inproductionofanimalfeed,forremovingbacterialbiofilms, andmorerecentlyinbiorefineryforproductionofvalue-added chemicalsandbiofuelsfromrenewablebiomassfeedstocks.1,2

Majority of the cellulases are commonly obtained from fungi,butbacteriaareconsideredimportantbecauseoftheir highgrowth rate,productionofeffectivecomplexenzymes and ease of genetic engineering for enhancing enzyme production.3_{Amongvarioussources,anexcellentcellulolytic}

environmentisthecattlerumenwhichcontainsaverydense and complex mixture of cellulolytic bacteria (in majority), fungi and protozoa.4 _{The bacterial species able to utilize}

crystalline cellulose are known frequently from the phyla Firmicutes,ActinobacteriaandProteobacteria.5_Within

Firmi-cutes,mostspeciesofBacillusareknowntobeendoglucanase producers, in that, they are able to hydrolyze amorphous forms of cellulose like carboxymethyl cellulose. However, relatively onlyfew strains of Bacillus are known to utilize crystallineformsofcelluloseandproducemultiplecellulase enzymes.3,6,7

In addition to the use in industrial fermentations, the prefatory potential of suchmicroorganisms in other areas likebioremediationcouldalsobestudiedfortheirmultiple applications. Organic solvents are often used in the man-ufactureofpharmaceuticalproducts, paints,varnishesand adhesives,andtheycausesignificantairandwaterpollution, andland contamination.8 _{Microorganismsthatare tolerant}

tothedestructiveeffectsofsolventshavebeenexploredfor theirpotential inindustrial andenvironmental biotechnol-ogytocarryoutbioremediationandbiocatalyticprocesses.9

Moreover, pollution due to the heavy metals from mining andindustrialwastes,vehicleemissionsandfertilizershave negativeconsequencesonthe hydrosphere,and oneofthe bestproceduresconsideredinremovingthetoxicmetalsfrom theenvironmentisusingmetaltolerantbacteria.10,11 _Thus,

itisrequiredtofindmicroorganismsthatapartfrombeing representativesforproductionofanumberofeconomic prod-uctsi.e.enzymes,arealsocapableoftoleratingenvironmental pollutants.12

Conserved signature indels (CSIs)in protein sequences, characterized by their defined size and being flanked on both sides by conserved regions, are rare and specific geneticchangesthatprovideusefulphylogeneticmarkersfor understanding evolutionary relationships among different organisms.13 _{Presently, relationship of cellulolytic}

bacte-ria among different phyla is not well known. One way of establishing this is by studying protein sequences among various organisms, which can indicate specific patterns occurring in a particular or several group of species. CSIs specificfor differentlineages can providedefinitive means ofdifferentiationoforganismsofthesegroups,whilethose shared among different taxa enable the determination of phylogenetic relationships based on multiple protein sequences.14

Thus,in the present study a cellulase producing bacte-rialstrainofBacillussp.SV1wasisolatedfromacattleshed soil.Itstolerancetodifferentenvironmentalpollutants includ-ing toxic heavy metals and organic solvents was checked. Factorseffectingcellulaseproduction,differentcellulose sub-stratehydrolyzingcapacityoftheextracellularcellulaseand cellulase genedetection were studied. Cellulase sequences

from three species of Bacillus were used as query to ana-lyzephylogeneticrelationshipsandoccurrenceofCSIsamong differenttaxa.

Materials

and

methods

Chemicals

Chemical reagents of analytical gradewere obtained from Sigma–Aldrich (St. Louis, MO, USA), Hi-Media laboratories (Mumbai,India)andFisherScientific(Mumbai,India).

Isolationofcellulolyticbacterium

Soilsamplewascollectedinsterilevialsfromacattleshed (locatedinSoniaVihar,NewDelhi–28.70743◦Nand77.25993◦ E)andstoredat4◦C.Onegramsoilwassuspendedin50mLof sterilizedliquidmediumIV(pH7.0)containing(g/L):NaNO3 – 1; K2HPO4 – 1; KCl – 1; MgSO4 – 0.5; yeast extract – 0.5; sodiumcarboxymethylcellulose (Na-CMC,1500–3000cP) – 10.15 _{Actidione (100ppm) was included to prevent}

fun-gal growth. Enrichment of cellulolytic bacteria was done at 37◦C and 180rpm in an incubator shaker (Kuhner LT-X) for 48h. This suspension after serial dilution in 0.87% (w/v)NaClsolutionwasspread(100␮L)onnutrientagar(NA) (g/L: peptone,5;sodium chloride,5;beefextract, 1.5;yeast extract, 1.5;agar, 15)platesandincubated at37◦Cfor48h, fromwhichtendiscreetcolonieswereselectedandpurified on NA.Eachofthe tenisolateswerethen pointinoculated on solid medium IV (pH 7.0) containing (g/L): NaNO3, 1; K2HPO4,1;KCl,1;MgSO4,0.5;yeastextract,0.5;sodium car-boxymethylcellulose(Na-CMC,1500–3000cP),10;agar,16,and incubatedat37◦Cfor48h,followedbyCongored(1mg/mL) staining–NaCl (1M) de-stainingand measurement of cellu-lolyticindex(CI)(ratioofthediameteroftheclearingzone around the colony tothe diameter of colony).16 _Hemolytic

behaviorsoftheisolateswereexamined bystreakingthem on sterile sheep blood agar plates and incubating at37◦C for 24h.17 _{Out often, oneisolate, designated as SV1, was}

selected forfurther study based on CI and non-hemolytic nature.

Characterizationandphylogenyofthecellulolyticisolate

Morphological,physiologicalandbiochemicalcharactersfor the isolate SV1 were studied.Genomic DNA was extracted usingaFermentasGeneJETgenomicDNApurificationkit.The universal primer pair, 27f (5 -AGAGTTTGATCCTGGCTCAG-3) and 1492r (5-GGTTACCTTGTTACGACTT-3) was used to amplify16SrRNAgene.18_{PCRparametersadoptedwere:initial}

denaturationat94◦C(3min),followedby30cyclesof denatur-ationat94◦C(30s),annealingat55◦C(30s)andextensionat 72◦C(2.5min),withthefinalextensionat72◦C(6min). Ampli-fiedregionwassequencedwithanAppliedBiosystems(ABI) Prism310GeneticAnalyzer.Theobtainedsequencewasused toblastintheEzTaxon-eserver,followedbymultiplesequence alignment of the retrieved closest neighbors reference sequences.19 _{Basedonlowestcalculated Bayesian}

informa-tioncriterionscores,Kimura2-parametermodelwasusedfor constructingamaximumlikelihoodphylogenetic tree(with

thebootstraptestof1000replicates).20_{Thenon-uniformityof}

evolutionaryratesamongsitesweremodeledusingadiscrete gammadistribution(+G)with5ratecategoriesandby assum-ingthatacertainfractionofsitesareevolutionarilyinvariable (+I).AllevolutionaryanalyseswereconductedinMEGA6.21

The16SrRNAgenesequencewassubmittedtothe Gen-Bank database using the BankIt submission tool, and the obtainedaccessionnumberisKJ024372.

Antibioticsusceptibilitypattern

ThesusceptibilityoftheisolateSV1tovariousantibioticswas testedusingthestandarddiscdiffusionmethod.12_Bacterial

culture (0.1mL of 1.5 OD600) in nutrient broth (NB) (g/L: peptone,5;sodiumchloride,5;beefextract,1.5;yeastextract, 1.5)wasspreadonNAplates,andthensingleantibioticdiscs wereplacedonthe surface,followedbyincubationat37◦C for 24h. The antibiotics used were (␮g/disc or units/disc) were:Streptomycin(10),Ciprofloxacin(5),Erythromycin(10), Ofloxacin(5), Norfloxacin (10),Ampicillin (10), Carbenicillin (100), Penicillin-G (2units/disc), Tetracycline (10), Cefazolin (30), Cefaclor (30), Cefotaxime/Cephotaxime (30), Amikacin (30),Cefuroxime(30),Chloramphenicol(30),Rifampicin(30), Ceftriaxone (30), Gentamicin (10), Kanamycin (5) and Cef-tazidime(30).Thediametersofgrowthinhibitionzones inclu-siveofthediameterofdiscsweremeasured,andcategorized assusceptible(≥21mm),intermediate(16–20mm)orresistant (≤15mm).12

Toleranceagainstenvironmentalpollutants

Heavy metaltolerance ofthe isolate SV1 bymeasurement ofminimuminhibitoryconcentration(MIC),wasdetermined bygrowingbacteria on NAplates incorporated with differ-entheavymetals(␮g/mL),followedbyincubationat37◦Cfor 24h.12 _{Theheavymetalions(salts,␮g/mL) usedwere:Pb}2+ (leadnitrate,0–500),Cu2+_{(cupricsulphate,0–200),Hg}2+ (mer-curicchloride,0–100),Cr3+_{(chromiumchloridehexahydrate,} 0–600),Zn2+ _{(zinc chloride, 0–200),Co}2+ _{(cobaltous chloride} hexahydrate,0–200)andNi2+_{(nickel(II)chloride,hexahydrate,} 0–300).

Thetolerance of the isolate SV1 to organic solvents of different logPow was also examined. Bacterial growth was

monitoredinNBcontainingdifferentsolvents(10%, v/v)at 37◦C, 180rpm for 48h.22 _{The organic solvents used were}

methanol, ethanol, acetone, n-propanol, isopropanol, ethyl acetate,butanol,benzene,chloroform,toluene,p-xylene, n-hexane,1-decanolandisooctane.NBwithoutanysolventwas usedasthecontrol.

Preparationofinoculumandextracellularcrudecellulase

IsolateSV1wassub-culturedinNBat30◦C,200rpmfor48h, and theobtained brothculture(OD600 about 1.5) wasused as inoculum at 1% (v/v) in all fermentation runs, carried outin250-mLErlenmeyerflasks.Afterfermentation,culture aliquots(1–2mL)were centrifuged at5000×g for15minat 4◦C(ThermoScientific,HeraeusFRESCO21),andtheobtained supernatantwasusedascrudecellulase.

Differentmediaandphysiologicalparameterseffecting growthandcellulaseproduction

Theeffectsofsevendifferentmediaoncellgrowthand cellu-laseproductionat37◦Cand180rpmfor48hwereestimated. Themedia(g/L)withinitialpH7.0usedwere:

I (NH4)2SO4–1;K2HPO4–1;MgSO4–0.5;NaCl–0.1;CMC– 10.23

II KH2PO4–4;Na2HPO4–4;tryptone–2;MgSO4–0.2;CaCl2 –0.001;FeSO4·7H2O–0.001;CMC–10.24

III Yeast extract – 2.5; K2HPO4, NaCl – 1; MgSO4 – 0.2; (NH4)2SO4–0.6;CMC–10.25

IV NaNO3–1;K2HPO4–1;KCl–1;MgSO4–0.5;yeastextract –0.5;CMC–10.15

V MgSO4 – 0.2; K2HPO4 – 1; KH2PO4 – 1; NH4NO3 – 1; FeCl3·7H2O–0.05;CaCl2–0.02;CMC–10.26

VI (NH4)2SO4 – 1; KH2PO4 – 3; MgSO4 – 0.1; K2HPO4 – 7; trisodiumcitrate–0.5;yeastextract–0.2;CMC–10.27

VII Yeastextract–7;KH2PO4–4;Na2HPO4–4;MgSO4–0.2; CaCl2–0.001;FeSO4·7H2O–0.004;CMC–10.28

The optimization of physiological conditions, includ-ing pH (6.0–9.5), temperature (25–40◦C) and agitation rate (0–250rpm)onbacterialgrowthandproductionofcellulases wastheninvestigatedthroughtheone-factor-at-a-time(OFAT) approachinmediumVII.

Estimationofinduciblenatureofcellulasesynthesis

Inducible natureofcellulase synthesiswas investigatedin mediumVII(initialpH7.5,at30◦C,200rpm,48h):(a)inthe absence/presence ofCMC,(b)by replacingCMC withother carbonsources(monosaccharides,disaccharides, polysaccha-rides and sugar alcohols), and (c) by supplementing CMC withothercarbonsources(monosaccharides,disaccharides, polysaccharidesandsugaralcohols)asco-substrates.15

Variousnutritionalfactorseffectingbacterialgrowthand cellulaseproduction

Effectsofdifferentorganicnitrogen(caseinhydrolysatebroth, corn steep solid, beef extract, tryptone, peptone, glycine andbactocasaminoacids)sourcesat0.7%(w/v)inplaceof yeastextract,andofdifferentconcentrationsofCaCl2(fused, 0–5g/L)andFeSO4·7H2O(0–2.9g/L),onbacterialgrowth and cellulaseproductioninmediumVIIwerestudiedatinitialpH 7.5,30◦Cand200rpmfor48h.

Analyticalmethodsandstatisticalanalysis

Cellulases (CELs) were assayed using the following sub-strates, (a) for CMCase/endoglucanase activity: 1.8mL of 1% (w/v) Na-CMC prepared in phosphate buffer (0.05M, pH7), (b)forFPase/totalcellulase activity:Whatmangrade 1 filter paper strip (1×3cm) in 1.8mL phosphate buffer (0.05M, pH 7), and (c) for ␤-glucosidase activity: 2mL of 0.15g/LpNPG (p-nitrophenyl-␤-d-glucopyranoside) in phos-phate buffer (0.05M, pH 7). For reaction, 0.2mL of crude cellulase(replacedbyphosphatebufferincontrol)wasmixed

withsubstratesandincubatedat40◦Cfor1h.Thereducing sugars(glucoseequivalent)forCMCaseand FPaseactivities were estimated at 575nm, using 3mL of freshly prepared dinitrosalicylicacid reagent (g/100mL: sodium hydroxide– 1;dinitrosalicylicacid – 1;crystalline phenol– 0.2;sodium sulfite – 0.05), followed bykeeping ina boiling waterbath for15min, and then adding 1mLof 40% (w/v) solution of potassiumsodiumtartarate(Rochellesalt)priortocooling.29

For ␤-glucosidase activity, the reaction was terminated by 1M Na2CO3 solution(2mL) andp-nitrophenolreleasedwas estimatedat400nminaUV–visiblespectrophotometer (Shi-madzu250 1PC, version 2.33).30 Oneunit ofCELs activity wasexpressedas1␮Mofglucose(forCMCaseandFPase)and

p-nitrophenol(for␤-glucosidase)liberatedpermLcrude

cellu-laseperminute.Theformulausedforcalculatingtheactivities ofCELsis

(␮Mofglucose/p-nitrophenolreleased)×(totalassayvolume)×(dilutionfactor) (volumeofcrudecellulaseused)×(volumeincuvette)×(incubationtime) where␮Mofglucose/p-nitrophenolreleasedisobtainedfrom

thestandardcurve,totalassayvolumeis2mL(CMCaseand FPase activities)/2.2mL (␤-glucosidase activity), volume of crudecellulaseusedis0.2mL,volumeincuvetteis1mLand incubationtimeis60min.

Allexperimentswereperformedintriplicate,andvalues arepresentedasthemean±standarderrorofthemean(SEM). Thestatisticalanalysisofdatabyone-wayANOVA,followedby Duncan’smultiple-comparisontest(p<0.05)wasdoneusing theSPSS16.0software(SPSSInc.,Chicago,IL,USA).

Detectionofcellulasegene

Twopairofprimerswereused,pair1:BsFw(forwardprimer: 5-GGAATTCCATATGAAACGGTCAATCTC-3)andBsRv(reverse primer: 5-TGCGGCCGCCTAATTTGGTTCTGTTCCC-3),31 and pair 2: CelBF (forward primer: 5 -CCATGGATCATGAGGAT-GTGAAAACTC-3)andCelBR(reverseprimer:5 -CTCGAGTGA-ATTGGTTGTCTGAGCTG-3).32_{ThePCRreactionswerecarried}

outinafinalvolumeof25␮LcontaininggenomicDNA(2␮L), 10× NH4 buffer (2.5␮L),50mM MgCl2 (2␮L),10mM dNTPs (1␮L),10mMofeachforward/reverseprimer(1␮Leach),10U taqDNApolymerase(0.3␮L)andsteriledoubledistilledwater (15.2␮L).PCRamplificationswereperformedwiththe follow-ingparameters:initialdenaturationat94◦C(3min),followed by30cyclesofdenaturationat94◦C(30s),annealingat52.7◦C (30s)andextensionat72◦C(2min),withthefinalextensionat 72◦C(7min).ThePCRproductswereloadedandrunon1.0% agarosegels.

Insilicoinvestigationoftheevolutionarydivergencein endoglucanaseofBacillusspp.

The protein sequence of endoglucanase component (EC 3.2.1.4) of cellulases from three species of Bacillus: Bacillus

agaradhaerens (UniProt accession– O85465, 400aa), Bacillus

cellulolyticus(UniProtaccession–E6TWN7,488aa)and

Bacil-lus subtilis (UniProt accession – P07983, 499 aa) was taken

asquery.EachsequencewasusedtoblastintheUniProtKB database,withdefaultparametersofE-Thresholdvalue(0.01), matrix(auto),filtering(none),gapped(yes)and hits(500).33

Theobtained500proteinreferencesequencesfromeachblast were retrieved in FASTA format, from which fragmented, hypothetical,redundantanduncharacterizedsequenceswere deleted,whiletheremainingreferencesequenceswerethen aligned by MUSCLE using MEGA6. Within the alignment, presence of conserved signature indels (CSIs) that were surroundedbyconservedregionswerestudied.The neighbor-joining phylogenetic trees with 1000bootstrap replications were constructedbycomputing the evolutionarydistances usingthe Poissoncorrectionmethod,34 _{corroboratedbythe}

Jones–Taylor–Thornton (JTT) matrix-basedmodel, and were intheunitsofnumberofaminoacidsubstitutionspersite.35 Theratevariationamongsiteswasmodeledwithagamma distribution (shape parameter=1) for the JTT model. Fur-ther,completedeletionofgaps,homogeneouspatternamong

lineages and uniform rates among sites were used. The propensity (totalfrequenciesofoccurrence)ofaminoacids wascalculatedamongthereferencesinMEGA6.

Results

Cellulolyticbacterialcharacterizationandphylogenetic analysis

TheisolatedstrainSV1showedaveryhighcellulolyticindex (CI)of7.89ontheplateassay(SupplementaryFig.1).Italso showednon-hemolyticpatternonthesheepbloodagarplate (SupplementaryFig.2).Themorphological,physiologicaland biochemicalcharacterswerestudiedforthestrainSV1,which havebeengiveninSupplementaryTable1.

Theobtained16SrRNAgenesequence(1375bp)ofstrain SV1 in the EzTaxon-e blast gavemaximum pairwise simi-laritieswithBacillusspp.,includingBacilluskorlensis(99.27%),

Bacillusberingensis(98.61%),Bacilluskyonggiensis(97.58%),

Bacil-luscirculans(96.85%),Bacillusnealsonii(96.78%),Bacillusfirmus

(96.72%)andBacilluseiseniae(96.63%).Inthemaximum like-lihoodphylogenetictree,strainSV1formedamonophyletic groupwithB.korlensisandB.beringensis(SupplementaryFig. 3).TheestimatedmajorfattyacidcomponentsofstrainSV1 were iso-C15:0 (34.02%),C16:1 ␻11c(18.11%) andanteiso-C15:0 (11.80%),whichwasclosetoitsnearestneighborB.korlensis (SupplementaryTable2).Hence,basedontheseresults,strain SV1wasdesignatedasBacillussp.SV1.

Fromthe twoprimerpairofcellulasegene,pair1(BsFw andBsRv)wasabletodetectthepresenceofcellulasegeneof about1000bpinBacillussp.SV1(SupplementaryFig.4).

Antibioticsusceptibilitypatternandtolerancestoheavy metalsandorganicsolvents

Strain SV1was susceptibletoall theantibioticstested and showedanintermediateresistanceagainstceftazidime (Sup-plementaryTable1).Itshowed tolerancetomultipleheavy

Table1–ThetoleranceofthecellulolyticstrainSV1to organicsolventsofdifferentlogPowvalues.

Organicsolvent LogPowa Growth(O.D.at600nm)b

Control – 0.31±0.11 Methanol −0.77 0.01±0.00 Ethanol −0.58 0.01±0.00 Acetone −0.23 0.01±0.00 n-Propanol 0.25 0.01±0.00 Isopropanol 0.26 0.01±0.00 Ethylacetate 0.73 0.01±0.00 Butanol 0.88 0.02±0.00 Benzene 2.13 0.39±0.00 Chloroform 2.24 0.02±0.00 Toluene 2.73 0.01±0.00 p-Xylene 3.1 0.18±0.01 n-Hexane 3.5 0.76±0.11 1-Decanol 4.1 0.05±0.01 Isooctane 4.7 0.61±0.03

SEM,Standarderrorofthemean. a _LogP

owisthelogarithmofoctanol–waterpartitioncoefficient,P, ofthesolvent.

b _{Opticaldensity(OD)at600nmwasmeasuredafter48hofgrowth} at37◦_{C,180rpminnutrientbrothcontaining10%(v/v)solvents.} Thevaluesarerepresentedasmean±SEM.

metalions,asinferredfrombacterialgrowth.Theminimum inhibitoryconcentration(MIC)ofheavymetals(␮g/mL)was: Pb2+ _{(100), Cu}2+ _{(100), Hg}2+ _{(50), Cr}3+ _{(400), Zn}2+ _{(50), Co}2+ (150)andNi2+_{(200).Italsoshowedhightoleranceagainstthe} organicsolventswhichhadlogPow>2.0includingisooctane,

n-hexane,benzeneandp-xylene(Table1).

Estimationofdifferentmediaandphysiological parametersongrowthandcellulaseproduction

Thevariedeffectsofdifferentmediaonbacterialgrowthand cellulase productionare shownin SupplementaryFig. 5. It wasobservedthatmediumVIIsupportedmaximumgrowth (of3.6times)andcellulaseproduction(of1.5timesCMCase, 1.1timesFPaseand1.6times␤-glucosidase)thanthe enrich-mentmediumIV,respectively.However,inothermedia,both growth and cellulase productionwere very low. Maximum growthoccurredatpH7.0,30◦Cand100rpm,whilemaximum cellulaseproductionoccurredatpH7.5,30◦Cand200rpmin mediumVII(SupplementaryFig.5b–d).

Induciblenatureofcellulasesynthesis

CellulaseproductionwashigherinpresenceofCMCthanin itsabsence(3.3timesCMCase,2.9timesFPaseand2.1times ␤-glucosidase)orreplacement(Fig.1).Theobservedpattern ofcellulaseproductionwasdifferentinconditionswhenCMC wasreplaced(Fig.1)orusedasco-substrate(Fig.2)with dif-ferent carbon sources. When CMC was replaced, raffinose, aviceland xylanpromoted higher␤-glucosidaseproduction thanCMCase;galactosecompletelysuppressedCMCaseand FPaseproductions(Fig.1).Amongco-substrates,xylose,xylan and pectinenhancedCMCase (1.7times, 1.6times and 1.2 times) and FPase (3.1 times, 1.9 times and 3.8 times) pro-ductionsrespectively,overthecontrolcontainingonlyCMC

(Fig.2).However,nicotinicacid,citricacid,guaiacol,glucose, fructoseandcellobioseasco-substrateswereinhibitory.The presenceoftheco-substratessodiumacetate,chitin, starch (potato),glycerolandsorbitoldidnotsignificantlyaffectthe CMCaseproduction,whilesodiumacetate,galactose,lactose andchitindidnotsignificantlyaffectthe␤-glucosidase pro-duction.

Effectsofdifferentnutritionalfactorsongrowthand cellulaseproduction

Presence ofan appropriateorganic nitrogen sourceand of metals(CaCl2andFeSO4·7H2O)washighlypreferredfor cel-lulaseproduction,aswasobservedamongdifferentmedia. Hence, absence or presence of different organic nitrogen sourceswas investigatedfor theireffects on cellulase pro-duction.Absenceoforganicnitrogensource(growth–9.8%, CMCase–10.8%,FPase–18.8%,␤-glucosidase–9.8%)and pres-enceofglycine(growth–6.0%,CMCase–11.0%,FPase–0.0%, ␤-glucosidase–9.7%)andbactocasaminoacids(growth–9.4%, CMCase–5.9%,FPase–38.1%,␤-glucosidase–10.5%)caused drasticdecreaseinbothgrowthandcellulaseproduction,than inthecontrolcontainingyeastextract(Fig.3A).Inthepresence ofcornsteepsolids,growth(145.6%)andCMCaseproduction (115.1%) wasmaximum, comparedtothe control.Presence ofCaCl2(upto5mg/L)causedenhancementin␤-glucosidase production(1.5times)comparedtoitsabsence,whilehigher concentrations(>100mg/L)wereinhibitory(Fig.3B).The pres-enceofFeSO4·7H2O(upto36mg/L)wasfoundtobeessential forincreasing productionsofCMCase(of 1.5times)and ␤-glucosidase (of1.1times)than itsabsence, whereas higher concentrations (>108mg/L) were inhibitoryforbothgrowth andcellulaseproduction(Fig.3C).

Insilicoinvestigationofevolutionarydivergencein endoglucanasecomponentofcellulasesofBacillusspp.

TheproteinsequenceblastofeachBacillusspp.cellulasequery intheUniProtKBdatabaseincludedreferencesthatbelonged tovarious taxa withinthedomain Bacteriaand Eukaryota. Bacterial references belonged tophyla Proteobacteria, Bac-teroidetes,Firmicutes,Actinobacteria,Spirochaete, Fibrobac-teres,Ignavibacteriae,PlanctomycetesandVerrucomicrobia. Eukaryota references included members of Arthropoda (Psacothea, Apriona, Anoplophora)and Nematoda(Ditylenchus,

Heterodera, Radopholus, Aphelenchoides, Pratylenchus). Among

the references, different enzymes including endo-1,3-beta-glucanase,endo-1,4-beta-xylanaseandchitosanasewerealso present.Thephylogeneticrelationshipofthesereferencesis indicatedintheneighbor-joiningtreesforeachquery(Fig.4; SupplementaryFigs. 6and 7).Inall thethreetrees,similar taxawere foundtobegroupedtogether,andeukaryota ref-erences had acommon ancestorwiththe members ofthe phylumBacteroidetes.

Duringalignmentofreferencesobtainedfromblastofthe threeBacillusspp.cellulasequeries(B.agaradhaerens,B.

cellu-lolyticusandB.subtilis),characteristictaxon-specificconserved

signatureindels(CSIs)wereobserved.AmongB.cellulolyticus references,a7aalonginsert,specifictoActinobacteriaand to one Bacteroidetes member Niastella koreensis was found

140.0 2.5 2.0 1.5 1.0 .5 .0 CMCase FPase β-Glucosidase Growth

120.0

100.0

Cels activity (U/mL)

Growth (OD at 600 nm)

Without C.M.C. _{C.M.C. (control)}

D (+) Xylose

D (+) Galactose

D-Fructose

Sucrose Lactose Maltose

D (+) Cellobiose D (+) T rehalose D (+) Melibiose D (+)-Raf finose A vicel Cellulose powder

Oat spelt xylan

Polycaccharides Disaccharides

Monosaccharides

Different carbon sources (%)

Sugar alcohols

Chitin Pectin

Starch (potato) Starch (corn) _{Starch (soluble)}

Glycerol D-Sorbitol (D-Glucitol) Mannitol D-(+)-Glucose (Dextrose) 80.0 60.0 40.0 20.0 .0 T risaccharide

Fig.1–EffectsofdifferentcarbonsourcesoncellulaseproductionandbacterialgrowthofthecellulolyticBacillussp.SV1.All valuesarerepresentedasmeanoftriplicateexperiments±SEM,takenafter48hofbacterialculturingat30◦C,200rpmin mediumVII(initialpH7.5).

(Fig.5A).N.koreensisalsobranchedwithinthemonophyletic

groupofActinobacteriamembers(Fig.4).Further,amongB.

subtilisreferences,an8aalonginsert specificonlyto

Acti-nobacteriamembers (Fig.5B)and an8aa(Fig. 6A)and a9

aa (Fig. 6B)long inserts specifictocertain members of Fir-micutes (species of Caldicellulosiruptor, Caldanaerobacter and

Clostridium)wereobserved.Asimilar9aalonginsertshared

amongthesethreemembersofFirmicuteswasalsoobserved

180 160 140 120 100 80 60 40 20 0

Cels activity (U/mL)

Without C.M.C. _{C.M.C. (control)} Nicotinic acid

Citric acid Sodium accetate Guaicol D (+) Xylose D (+) Galactose D-Fructose

Sucrose Lactose Maltose

D (+) Cellobiose D (+) T rehalose D (+) Melibiose D (+)-Raf finose A vicel Cellulose powder

Oat spelt xylan

Chitin Pectin

Starch (potato) Starch (corn) _{Starch (soluble)}

Glycerol D-Sorbitol (D-Glucitol) Mannitol D-(+)-Glucose (Dextrose) 2.5 3 2 1.5 1 .5 0 Growth (OD at 600 nm)

CMCase FPase β-Glucosidase Growth

Polycaccharides Disaccharides

T

risaccharide

Monosaccharides

Additional carbon sources (1%) with CMC (1%)

Sugar alcohols

Fig.2–Effectsofdifferentadditionalcarbonsourcesasco-substratestoCMConcellulaseproductionandbacterialgrowth ofthecellulolyticBacillussp.SV1.Allvaluesarerepresentedasmeanoftriplicateexperiments±SEM,takenafter48hof bacterialculturingat30◦C,200rpminmediumVII(initialpH7.5).

140.0 3.0

A

C

B

2.0 1.8 1.6 1.4 1.2 1.0 .8 .6 .4 .2 .0 2.0 120.0 100.0 80.0 60.0 40.0 20.0 .0 0 0.0010.005 0.01 0.05 0.1 0.5 1 5 0 0.004 0.012 0.036 0.108 0.324 0.972 2.916 120.0 100.0 80.0 60.0 40.0 20.0 .0 1.8 1.6 1.4 1.2 1.0 .8 .6 .4 .2 .0 2.5 2.0 1.5 1.0 .5 .0 120.0 100.0 80.0 60.0 40.0 20.0 0.0

Cels activity (U/mL)

Relative cels activities (%)

Cels activity (U/ml)

Growth (O.D. at 600 nm) Growth (O.D. at 600 nm)

Growth (O.D. at 600 nm)

Without organic nitrogen Casein hydrolysate broth

Corn steep solids

Beef extract

Y

east extract, type I

(control)

T

ryptone

Organic nitrogen source (0.7% w/v)

[CaCL2] (g/L) [Feso4.7H2o] (g/L)

Peptone Glycine

Bacto casamino acids

CMCase FPase β-Glucosidase Growth

Fig.3–Effectsofdifferentorganicnitrogensource(A),andconcentrationsofCaCl2(B)andFeSO4·7H2O(C)oncellulase

productionandgrowthofBacillussp.SV1.Allvaluesarerepresentedasmeanoftriplicateexperiments±SEM,takenafter 48hofbacterialculturingat30◦C,200rpminmediumVII(initialpH7.5).In(A),theproductionofcellulasesisexpressedas relativeCELsactivity,definedasthepercentageoftheactivitiesobservedwithrespecttothecontrol(containingyeast extract).

inB.agaradhaerensreferences(SupplementaryTable3).Even

inthephylogenetic trees,speciesofCaldicellulosiruptor,

Cal-danaerobacterandClostridiumformedmonophyleticgrouping

(SupplementaryFigs.6and7).CSIsbothspecifictoandshared amongthemembersofActinobacteriaandFirmicuteswere foundwithinthereferencesofB.agaradhaerensandB.subtilis cellulasequeries(SupplementaryTable3).

Amongthedifferentaminoacidresidues,propensityof ala-nine,glycineandserinewashigh,whereasthatofcysteine, methionine and histidine was very low, though variations werealsoobservedindistributionofresiduesindifferent ref-erences(SupplementaryTable4).Forinstance,thedistribution ofthecatalyticresidueaspartatewaslowintheCystobacter (Proteobacteria), Hymenobacter (Bacteroidetes) and the Acti-nobacteria Verrucosispora and Actinoplanes,but very high in FirmicutesmembersCoprococcus,Butyrivibrioandinthe Acti-nobacteriaBifidobacterium.

Discussion

Inthepresentstudy,acellulaseproducingbacteriumwas iso-latedfromsoilcollectedfromacattleshedarea.Thesoilhere isusuallymixedwithcattledung,duetowhichdifferent

cel-lulolyticorganismsfromthecattlerumenmightbeavailable. Itwasconsideredtoisolatecellulaseproducingbacterianot onlybasedonhighcellulolyticindex(CI),butalsoonits non-hemolyticbehaviorwhichprovidessaferworkingconditions andlesserriskoftherapeutichurdles.15,17_{Mostofthebacteria}

thatarereportedfortheproductionofcellulases,donothave informationonthenon-pathogenicbehavior(intermsof non-hemolyticbehavior)ofthebacteria.Therefore,ifthisbehavior ofthebacteriaisalreadystudied,thatmakesthebacteriamore safertouse,causinglesserobstacles fortherapyand treat-mentifanyinfectioniscaused.Amongtheisolates,somehad lowgrowthwithlowCI,somehadhighCIbutshowedeither ␣-or␤-hemolyticbehaviorsonsheepbloodagarplates,and somehadlowCI with␥(non-hemolytic)behavioronblood agar plates.OnlythestrainSV1wasconsideredasithada veryhighCI(7.89)andnon-hemolyticbehavior.TheCIknown from cellulolyticbacteria (members ofProteobacteria, Acti-nobacteria,Firmicutes andBacteroidetes)isolated from the gut of the larvae ofHolotrichia parallela rangedfrom 1.1to 9.0.36,37_{TheCIknownforsomeofthecellulolyticbacteriaare}

BacillusmegateriumRU4(6.5),38_{ParacoccusyeeiRA2(3.0),}38

Bacil-lussp.KC2(6.86),39 _{Bacillus sp.KC4 (4.37),}39 _{Bacillussp.KC8}

(3.95),39_{actinomycetes(1.1–2.3).}40_{StrainSV1wassusceptible}

Fig.4–Neighbor-joiningphylogenetictreeofcellulase(endoglucanase)referencesofBacilluscellulosilyticusbasedonthe aminoacidsequences.HighlightedregionsincludecladebetweenendoglucanaseofBacteroidesovatusandxylanaseof

Bacteroidesxylanisolvens,cladebetweenendoglucanaseand1,3-betaglucanaseofPaenibacilluspolymyxa,anda7amino acid(aa)insertsharedamongmembersofActinobacteriaandNiastellakoreensis(ofBacteroidetes).Symbolsand abbreviations:Firmicutes(opencircle),Proteobacteria(closedsquare),Spirochaete(opentriangle),Ignavibacteriae(Ign.), Planctomycete(Pla.),Bacteroidetes(closedcircle),Verrucomicrobiae(Ver.),Eukaryota(closedtriangle),Actinobacteria(open square),Fibrobacteres(Fib.).Bar,0.1substitutionspernucleotideposition.

utilizationforapplicationincellulase production.However, itshowedanintermediateresistanceagainstceftazidime.It is considered that ceftazidime resistance in bacteria arise byeitherthehorizontalacquisitionof␤-lactamasesorfrom an altered expression of the chromosomal wide-spectrum classC␤-lactamase.41,42_{Duetoitstoleranceagainstthetoxic}

heavymetalsCr,Ni,Coandothermetals,somephysiological mechanismmightbeinvolved,whichmayincludetheir degra-dationintonon-toxicforms.Thedifferentmechanisms,often plasmid-associated,known are activeefflux, sequestration, complexation,biosorption,ionicinteraction,changingtoxic

tonon-toxicforms,enzymaticmetalreduction, immobiliza-tion andprecipitation.43,44 _{ThereductionofCrtonon-toxic}

formshavebeen known from variousbacteria likeBacillus,

Pseudomonas,Escherichiacoli,Exiguobacterium,Desulfovibrioand

Microbacterium,whileaCr(VI)reducingBacillussp.FM1wasalso

foundtobetoleranttomultipleheavymetalsincludingCu,Co,

Cd,NiandZn.45_{B.circulansEB1hasbeenusedforthe}

biosorp-tionofMn,Zn,Cu,NiandCo,thusmakingitsuitableforthe bioremediationofmetalcontaminatedaqueoussystems.46_A

nickeltolerantB.megateriumSR28C,thatshowedtoleranceto other metals (Cu,Zn, Cd,Pb, Cr), wasfound tohaveplant

A

B

Bacteroidetes

Actinobacteria

Fig.5–Conservedsignatureindels(CSIs)shared(A)amongmembersofActinobacteriaandoneBacteroidetesmember,and (B)specificallyamongmembersofActinobacteria.

growthpromotingpropertybydecreasingNitoxicityinsoils, whichisusefulforphytostabilization.47_{Furthermore,allthe}

organicsolventsthat thestrainSV1 wastolerantto (isooc-tane, n-hexane,benzene and p-xylene)had alogPow value

greaterthan2.0,whichindicatesoftheirmorehydrophobicity. Besidesshowingtolerance,thestrainSV1alsohadenhanced growthinthepresenceofthesolventsn-hexane,isooctane and benzene,which suggests ofits ability toeither utilize ordegradethesesolvents.Similarly,thetolerancetoorganic solventswithhigherlogPowvalueshasalsobeenfoundin

sev-eralspecies ofBacillus usedforenzymeproductions, some ofwhichareBacillusaquimaris(benzene,heptane),22_Bacillus

sphaericus(n-benzene,toluene,ethylbenzeneandp-xylene),48

BacilluslicheniformisPAL05(benzeneandtoluene),49_B.

licheni-formisYP1A (tetradecane and decane),50 and Bacillus cereus

BG1(hexane).51 _{Thus, the strain SV1 could bevaluable for}

thebioremediationofmulti-metalororganicsolventpolluted areas.

Thevariedeffectsofdifferentmediaonbacterialgrowth andcellulaseproductionclearlyreflectedonthedrasticeffects thatdifferentcomponentscouldhave.MediumIVusedfor iso-latingthebacteriumsupportedlesscellulaseproductionthan mediumVII.Bothofthesemediahadseveralcompositional differences, the major being the increased concentration

of yeastextract and presenceofCaCl2 and FeSO4·7H2O in mediumVII.MediumIIalsocontainedCaCl2andFeSO4·7H2O, buthad adifferentorganicnitrogensourcetryptone,which supportedlesscellulaseproductionthatwasalsoconfirmed inthestudyofdifferentorganicnitrogensourcesthateffected cellulaseproduction.MediumIandVhadnoorganicnitrogen source,duetowhichtherewasverylessgrowthandcellulase productioninthem.MediumVIhadverylowconcentrationof yeastextractthanmediumIII,andthusgrowthandFPase pro-ductionobservedwasverylowinmediumVIthaninmedium III.TheratioofC/N,withboththeirtypeandrelative concen-tration,canbeimportantintheproductionofenzymes.Low levelofnitrogencanbeinadequate,whilehigherlevelscanbe detrimentalforenzymeproduction,whichcouldalsovaryfor differentstrainsproducingvariousenzymes.52ForstrainSV1, themediaI,II,IV,VandVIIhaveaC/Nratioof1.0,whilemedia IIIandVIhavetheC/Nratioof0.5.Moreover,whenonlytheC source(Na-CMC)wasprovidedandtheorganicnitrogenwas removed,theproductionofcellulaseswerereduced.Similarly, theC/Nratioof1.0wasfoundtogivemaximumproduction ofanotherenzyme,amylasefromB.licheniformisSPT27.52_In

contrast,forTrichodermareeseiZU-02,thecellulaseproduction wasfoundtoincreasewithincreasingC/Nratioofupto8.0,53

Caldicellulosiruptor spp. Caldicellulosiruptor spp. Caldanaerobacter spp. Caldanaerobacter spp. Clostridium spp. Clostridium spp.

A

B

Fig.6–Conservedsignatureindels(CSIs)shared(AandB)amongmembersofFirmicutesincludingthespeciesof

Caldicellulosiruptor,CaldanaerobacterandClostridium.

Table2–ThecomparisonoftheproductionofcellulasesofstrainSV1withdifferentcellulolyticstrainsofBacillus,using variouscellulosicsubstrates.

Cellulolyticstrains Cellulosicsubstrateused Productionofcellulases(U/mL) Reference

Bacillussp.SV1 Na-CMC CMCase–107.45,FPase–26.79and␤-glucosidase–80.90 Thisstudy

BacilluspumilusEB3 CMC FPase–0.05,CMCase–0.31and␤-glucosidase–0.18 3

Bacillussp.SMIA-2 Sugarcanebagasse CMCase–0.29 6

BacillussubtilisAS3 CMC CMCase–0.57 65

Bacillusthuringiensisvar.israelensis CMC CMCase–0.07,FPase–0.01 66

Bacillusthuringiensisvar.thompsoni CMC CMCase–0.06,FPase–0.01 66

BacillusamyloliquefaciensSS35 CMC CMCase–0.13–0.53 67

BacillusvallismortisRG-07 Sugarcanebagasse CMCase–4105.00 68

BacilluspumilusEB3 Oilpalmemptyfruitbunch CMCase–0.06 69

Na-CMC,Sodium-carboxymethylcellulose;CMC,Carboxymethylcellulose;CMCase,Carboxymethylcellulase;FPase,Filterpapercellulase.

highataC/Nratiobelow20.2.54_{Thus,presenceoftheorganic}

nitrogenyeastextractandthemetalsCaCl2andFeSO4·7H2O wereessentialforbothgrowthandcellulaseproductionbythe strainSV1.Yeastextractisacomplexhydrolysateofyeasts, whichprovidesnitrogenouscompounds,carbon,sulfur,trace nutrients, vitaminB complex and other important growth factors.55,56_{Theorganicnitrogensourcesthatpromotedvery}

lowgrowth and cellulaseproductionare amongthosethat havesimplercomponentscontainingeithersingleaminoacid (glycine)ormixturesofaminoacidandpeptides(casamino acids, tryptone, peptone and casein hydrolysate broth). However, beef extract (mixture of amino acids, peptides,

organic acids,mineralsandsomevitamins)causedslightly moregrowthandcellulaseproductionthanglycine,casamino acids,tryptone,peptoneandcaseinhydrolysatebroth.Among theorganicnitrogenotherthanyeastextract,onlycornsteep solidscouldenhancemaximumgrowthandCMCase produc-tion.Cornsteepsolidsarelow-costby-productsthatcontain amino acids, vitamins and minerals, which has also been found to cause high CMCaseproduction from Streptomyces

malaysiensis,57_{Streptomycesdrozdowiczii,}58_andT.reesei.59_The

enhanced productionofcellulases inthe presenceof CMC thanitsabsenceorreplacementbyothercarbonsources,and the suppressedCMCase productionin presenceof glucose

andcellobiose(whichareamongtheend-productsof cellu-losehydrolysis)indicatedcellulasesynthesistobeinducible andundercataboliterepression,respectively.Thus,cellulase productionwas increasedonly for the requirement of the utilizationofcellulose,butwhensimplesugarslikeglucose, fructoseandcellobiosewerepresent,bacteriautilizedthese easily metabolizable sugars and did not produce cellulase eitherduetoits non-requirement(in condition whenCMC wasreplaced)orforutilizinglesscomplexsugars(in condi-tion when CMC was a co-substrate withmonosaccharides and disaccharides).60 _{Moreover, glucose and cellobiose are}

amongtheend-productsofcellulosehydrolysiswhich also causeacataboliterepressionofcellulasesynthesis.Certain co-substratesdid not significantlyaffect the production of cellulases,whichindicatedthatcellulasesmighthavebeen co-producedwithotherenzymesneededtoutilizedifferent substrates for supporting bacterialgrowth. The strain also utilizedmicrocrystallinecellulose(avicel)fortheproduction ofdifferent cellulases especially higher ␤-glucosidase than with CMC. Raffinose as a single carbon source enhanced ␤-glucosidase production. This trisaccharide comprising of galactose,glucoseandfructose,hasbeenusedasaninducer for maximum production of another cellulase component CMCasefrom onlyB. subtilisAU-1, whereit was suggested that raffinose was initially hydrolyzed into its component sugars,whichtheninducedhigherCMCaseproduction.61 _A

combinationofcellulolyticbacteria(Streptomyces,Cohnellaand

Cellulosimicrobium)havebeenusedtoreducethesoybeanmeal

non-starch polysaccharide containing raffinose.62 _However,

raffinosehasalsobeenfoundtosuppresstheproductionof CMCasefromPenicilliumexpansum.63_{Theenhancedcellulase}

productioncausedbytheco-substratesxylose,xylan,pectin and cellulose powder might be attributedto the fact that plantcelluloseismostlyinassociationwithdifferent polysac-charidesofhemicelluloseandpectin,duetowhichinorderto getaccesstocellulose,theproductionofcellulolyticenzymes might be controlled notonly in the presence of cellulose, butalsohemicellulose(likexylan)andpectin.64_Theoptimal

cellulase produced by strain SV 1 was 107.45±2.12U/mL (CMCase), 26.79±0.27U/mL (FPase) and 80.90±4.05U/mL (␤-glucosidase), which is comparable to that observed in differentstrains of Bacillus sp.,utilizing differentcellulosic substrates(Table2).

Therearefewcellulaseproducingbacterialspecies(though majorityarefungi)thatareusedinthemarketsforthe pro-ductionandusageofcellulases.ThespeciesofBacillusisone ofthem. Therefore, an evolutionarystudy of the cellulase aminoacidsequenceofdifferentcellulolyticspeciesof Bacil-luswasstudiedtoknowitsrelationshipwithothercellulolytic organisms.Thiswas donebytheuse ofphylogenetictrees andconservedsignatureindels.Theproteinsequenceblastof

eachBacillusspp.cellulasequery(endoglucanase,EC3.2.1.4)

includedreferencesofalmostninebacterialphyla.Reference cellulasesofEukaryarepresentedbyArthropodaand Nema-toda indicated oftheir identity withthe bacterial queries, whichwassupportedbythemhavingcommonancestorwith thebacterialphylumBacteroidetesinthephylogenetictrees. Suchsimilarityissuggestedtobeeitherduetothe involve-mentofhorizontaltransferofgenesfromsymbioticorganisms orfromaconvergentevolutioneventwhichwaspassedon

from anow extinct ancestor.70 _{Thequery cellulase, which}

causes endohydrolysis of(1,4)-beta-d-glucosidic linkagesin cellulose, showed similarity in blast and formed clade in phylogenetictree(Fig.4)withanothertypeofcellulase, endo-1,3-beta-glucanase(EC3.2.1.6),whichcausesendohydrolysis of(1,3)- or(1,4)-linkagesinbeta-d-glucans. Their similarity mightbeattributedwithaconvergentevolutionthatoccurred fromtheselectivepressureoftheabundantavailabilityof cel-lulose. Cellulase query alsoshowed similarity in blast and formedcladeinphylogenetictree(Fig.4,SupplementaryFigs. 6and7)withfunctionallydifferentenzymes, endo-1,4-beta-xylanase(EC3.2.1.8)andchitosanase(EC3.2.1.132).Bothare glycosidehydrolases,whicheitherhydrolyzethebeta-1,4 link-ages between xylan (xylanase) or between d-glucosamine residues ina partlyacetylatedchitosan (chitosanase).This is highly indicative of a divergent evolution among these enzymes fromabasicproteinfoldsoastoincorporate dif-ferentsubstrates.64

Thesharingoftheconservedsignatureinsertionsand dele-tions (CSIs) observed between N. koreensis (a Bacteroidetes member) anddifferentmembers ofActinobacteria(Fig. 5A) wasalsosupportedbyitsbranchingwithinthemonophyletic group of Actinobacteria (Fig. 4). This CSI of 7 aa insert musthavebeenintroducedinthecellulaseoftheircommon ancestor.Themonophyleticgroupingofthespeciesof

Caldicel-lulosiruptor,CaldanaerobacterandClostridium (Supplementary

Figs.6and7)wassupportedbytheirsharingofan8aa(Fig.6A) and9aa(Fig.6B)(SupplementaryTable3)inserts,whichmust havebeenacquiredandthenlaterdivergedfromtheir ances-tralspeciesduringevolution.CSIsobservedtobespecificto differentbacteriaphylaincludingActinobacteriaand Firmi-cutes(SupplementaryTable3)wouldbeimportanttoolsfor characterizationanddistinguishingofthemembersoftheir commonevolutionarydecent.Similarly,evolutionary relation-shipsamongcellulases,usingCSIsarealsoknownforvarious speciesofArchaea,BacteriaandEukarya.71_Fromthe

differ-entaminoacidresiduesincellulase,frequencyofoccurrence ofalanine,glycineandserinewerehigh,howeverthereason for veryhigh and low variationsobserved inseveral refer-encesforcertainresiduesisnotknown.Suchvariationswere distributedamongthemembersofProteobacteria, Actinobac-teria, Firmicutes,Bacteroidetes, Fibrobacteres,and insome NematodaandoneArthropoda(Psacothea).

Conclusion

A cellulolytic and non-hemolytic soil bacterium,

Bacillus sp. SV1 showed susceptibility to various

antibiotics, and tolerances to toxic heavy metals (Cr3+_>Ni2+_>Co2+_>Pb2+_=Cu2+_>Hg2+_=Zn2+_{) and organic} solvents (n-hexane>isooctane>benzene>p-xylene). Extra-cellularly synthesized cellulases of the strain SV1 had endoglucanase, filter paper and ␤-glucosidase activities, which were increased in the presence of cellulose. CMC replaced by raffinose, avicel and xylan promoted higher ␤-glucosidase production, while xylose, xylan and pectin as co-substratestoCMC enhancedCMCaseand FPase pro-ductions. Thelow-costcorn steep solidscaused maximum growth andproductionofCMCase,whichalsorequiredthe

presenceofCaCl2andFeSO4·7H2O.Therefore,thestrainSV1 haspotentialinthe productionofcomplete cellulases ina cheapermedium,and alsoforprefatorybioremediation(of multiple heavy metals and organic solvents) applications. An evolutionary relationship of the cellulase of different species of Bacillus was studied using their protein amino acidsequences.TheproteinsequenceblastofthreeBacillus spp.cellulase(EC3.2.1.4)showedthatithadsimilaritieswith members of bacterial and eukaryotic phyla, with another cellulase enzyme (endo-1,3-beta-glucanase), and with two other hydrolase (endo-1,4-beta-xylanase and chitosanase) enzymes.Within thealignment ofthe reference cellulases forthequeries,conservedsignatureindelswereobservedfor themembersofActinobacteriaand Firmicutes,whichwere insupportoftheirevolutionarydecentinphylogenetictrees.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgements

FellowshipsfromC.S.I.R.,NewDelhitoLebinThomas,from U.G.C.,NewDelhitoHariRam,andtheDST-purseandR& DgrantsfromtheUniversityofDelhiaregratefully acknowl-edged.

Appendix

A.

Supplementary

data

Supplementarydataassociatedwiththisarticlecanbefound, intheonlineversion,atdoi:10.1016/j.bjm.2017.05.010.

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