Lactococcus raffinolactis BGTRK10-1, an...

Research ArticleLraI from Lactococcus raffinolactis BGTRK10-1an Isoschizomer of EcoRI Exhibits IonConcentration-Dependent Specific Star Activity

Marija Miljkovic1 Milka Malesevic1 Brankica Filipic12

Goran Vukotic13 andMilan Kojic 1

1 Institute of Molecular Genetics and Genetic Engineering University of Belgrade Belgrade Serbia2Faculty of Pharmacy University of Belgrade Belgrade Serbia3Faculty of Biology University of Belgrade Belgrade Serbia

Correspondence should be addressed to Milan Kojic mkojicimggebgacrs

Received 22 November 2017 Accepted 2 January 2018 Published 29 January 2018

Academic Editor Gamal Enan

Copyright copy 2018 MarijaMiljkovic et alThis is an open access article distributed under theCreative CommonsAttribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Restriction enzymes are the main defence system against foreign DNA in charge of preserving genome integrity Lactococcusraffinolactis BGTRK10-1 expresses LraI Type II restriction-modification enzyme whose activity is similar to that shown for EcoRILraI methyltransferase protects DNA from EcoRI cleavage The gene encoding LraI endonuclease was cloned and overexpressedin E coli Purified enzyme showed the highest specific activity at lower temperatures (between 13∘C and 37∘C) and was stable afterstorage at minus20∘C in 50 glycerol The concentration of monovalent ions in the reaction buffer required for optimal activity of LraIrestriction enzyme was 100mM or higher The recognition and cleavage sequence for LraI restriction enzyme was determinedas 51015840-GAATTC-31015840 indicating that LraI restriction enzyme is an isoschizomer of EcoRI In the reaction buffer with a lower saltconcentration LraI exhibits star activity and specifically recognizes and cuts another alternative sequence 51015840-AAATTC-31015840 leavingthe same sticky ends on fragments as EcoRI which makes them clonable into a linearized vector Phylogenetic analysis based onsequence alignment pointed out the common origin of LraI restriction-modification system with previously described EcoRI-likerestriction-modification systems

1 Introduction

Restrictionendonucleases are generally accompanied by acog-natemethyltransferase [1]Bothenzymesworking together forma restriction-modification system (RM system) RM systemsare important for the maintenance of the genome integrityof prokaryotic organisms The range of biological processesthat utilize RM system also includes involvement in DNAtransposition [2] and recombination [3] In addition there isevidence that the genes for restriction and modification en-zymes may act together as selfish elements [4 5]

Restriction endonucleases exhibit high sequence speci-ficity in substrate binding and use versatile DNA cleavagemechanisms and thus are excellent model systems for under-standing DNA recognition and phosphodiester bond hydrol-ysis Restriction endonucleases are classified according to

their subunit composition cofactor requirement recognitionsite cleavage site and mode of action to define the differenttypes (I II III and IV) Restriction endonucleases Type IIare essential tools for recombinant DNA technology It seemsunlikely that todayrsquos modern molecular biology and thebiotechnology industry would have developed without TypeII restriction enzymes Because of their great importancein gene analysis and cloning there is a constant need todiscover new ones According to data from the REBASE[6 httprebasenebcom] which summarizes all informationknown about every restriction enzyme and any associatedprotein there are more than 3945 biochemically or geneti-cally characterized restriction enzymes and out of 3834 TypeII restriction enzymes 299 distinct specificities are known By2010 six hundred and forty-one restriction enzymes werecommercially available including 235 distinct specificities [7]

HindawiBioMed Research InternationalVolume 2018 Article ID 5657085 10 pageshttpsdoiorg10115520185657085

2 BioMed Research International

Because of the large number of sequenced genomes rate ofdiscovery of new putative restriction and modification genesis rising rapidly In contrast the number of restriction en-zymes that are biochemically characterized has actuallydropped down to the level that was three decades ago

Restriction endonucleases Type II are homodimeric ortetrameric enzymes that cleaveDNAat defined sites of 4ndash8 bpin length and require Mg2+ ions for catalysis [8] For many ofrestriction endonucleases Type II it was found that modifiedconditions (lower ionic strength higher pH presence ofdifferent metallic cofactors and organic solvents) coulddecrease their substrate specificity [9ndash13] Under nonoptimalrestriction conditions these endonucleases can usually cleavedegenerate sequences which differ from standard recogni-tion sites at only one nucleotide This alteration in digestionspecificity causing cleavage of DNA at novel similar but notidentical sequences is defined as enzyme star activity Mod-ified specificity of restriction enzyme (star) activity couldbe exploited to facilitate recombinant DNA techniques sincethe same enzyme in controlled conditions could recognizedifferent DNA sequences and cleave at additional positions[9]

Restriction endonucleases with identical recognition sitesisolated from different organisms are termed isoschizomers[7 14] The RsrI endonuclease found in Rhodobacter sphae-roides is an isoschizomer of the EcoRI Both enzymes recog-nize the sequence GAATTC and cleave it at the same position(GAATTC) and are sharing 50 amino acid sequenceidentity [15] Interesting MunI recognizes the sequenceCAATTG which differs from the recognition sequence ofEcoRI (and RsrI) only in the external base pairs Comparisonof the MunI amino acid sequence with that of EcoRI andRsrI revealed only a low level of overall similarity whereforesequence homology between EcoRI and RsrI has a strongersignificance [16]

This work describes for the first time the occurrence ofEcoRI-like restriction-modification genes in lactococci Theobjective was to clone purify and biochemically and genet-ically characterize novel lactococcal LraI restriction enzymeLraI restriction enzyme was overexpressed and purified tothe homogeneity from E coli using pMAL expression andpurification system Results demonstrate that LraI restrictionenzyme although an isoschizomer of EcoRI shows differentcharacteristics One of characteristics that could be furtherexploited is star activity of LraI that is limited to one variantof the recognition site which after cleavage leaves identicalcohesive ends as EcoRI and LraI restriction enzymes so thatthe fragments obtained after digestion could be cloned with-out additional processing

2 Material and Methods

21 Bacterial Strains and Culture Conditions Lactococcusraffinolactis BGTRK10-1 was isolated from autochthonoussweet kajmak produced from sheep milk without the use ofstarter cultures in a household of the Vlasic mountain regioncentral Bosnia and Herzegovina [17] (Table 1) Preliminarystrain classification was done according to its fermentationability using API 50CHL (Api System SA Bio-Merieux

Montelieu-Vercieu France) temperature of growth (30∘C37∘C and 45∘C) growth in the presence of salt (4 and65) and pH tolerance Final taxonomic classification ofBGTRK10-1 was performed by sequencing of amplified 16SrDNAusing primers previously described [18]The strainwasgrown inM17medium (MerckGmbHDarmstadt Germany)supplemented with D-glucose (05 wv) (GM17) at 30∘CEscherichia coli DH5120572 HB101 and ER2523 strains weregrown aerobically in Luria-Bertani (LB) broth at 37∘C unlessotherwise specified Solid medium was made by adding175 (wv) agar (Torlak Belgrade Serbia) to the liquidmedia Antibiotics were used at the following concentra-tions erythromycin 300 120583gml and ampicillin 100 120583gml forselection and maintaining of transformants The 5-bromo-4-chloro-3-indolyl-120573-D-galacto-pyranoside (X-Gal) (Fermen-tas Vilnius Lithuania) was added to LB medium platesfor bluewhite colour screening of colonies with clonedfragments at final concentration of 40 120583gml

22 Construction of Cosmid Library of L raffinolactisBGTRK10-1 Total DNA isolated from the L raffinolactisBGTRK10-1 was partially digested with XbaI restrictionenzyme Incubation was carried out during 1 h and wasstopped in different time intervals by adding EDTA Opti-mally digestedDNA giving fragments 30ndash40 kb was purifiedand ligated overnight at 16∘C with the pAZILcos vector [19]predigested with XbaI restriction enzyme and dephospho-rylated Ligation for formed concatemers of high molecularweight was checked on agarose gel and encapsulated intophage particles using packaging kit (Agilent Technologies)Encapsulated cosmids were transfected into E coli HB101magnesium cells and selection of clones was done on LAplates containing erythromycin 300 120583gml Constructed cos-mid library in E coli was stored in LB containing 15 (vv)glycerol at minus80∘C

23 DNA Manipulations Total DNA from L raffinolactisBGTRK10-1 was isolated by modified method described byHopwood et al [20] the logarithmic phase cells werepretreated with lysozyme (4mgml for 15min at 37∘C) priorto treatment with SDS For plasmid isolation from E coli theQIAprep Spin Miniprep kit was used according to the manu-facturerrsquos recommendations (Qiagen Hilden Germany)Standard heat-shock transformation was used for plasmidtransfer into E coli [21] Digestion with restriction enzymeswas conducted according to the supplierrsquos instructions (Ther-mo Fisher Scientific) The DNA fragments from agarose gelswere purified using QIAqick Gel extraction kit as describedby the manufacturer (Qiagen Hilden Germany) DNA wasligated with T4 DNA ligase (Agilent technologies USA) ac-cording to the manufacturerrsquos recommendations PlatinumTaq DNA Polymerase High Fidelity (Thermo Fisher Scien-tific Waltham MA USA) was used to amplify DNAfragments by PCR in GeneAmp PCR system 2700 thermalcycler (Applied Biosystems Foster City CA USA) PCRproducts were purified with QiAquick PCR purification kit(Qiagen Hilden Germany) according to the manufacturerrsquosrecommendations DNA sequencing was done by theMacro-genSequencing Service (MacrogenEuropeTheNetherlands)

BioMed Research International 3

Table 1 Bacterial strains and plasmids used in this study

Strains or plasmids Relevant characteristics Source or referenceLactococcus raffinolactis

BGTRK10-1 Natural isolate from autochthonous sweet kajmak [17]Escherichia coli

DH5120572 supE44 ΔlacU169 (oslash80 lacZΔM15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1 [21]

HB101 Fminus hsdS20 (rBminus mBminus) supE44 recA13 ara-14 proA2 lacY1 rpsL20(SmR) xyl-5

mtl-1 galK2 lacY1 120582minus [24]

ER2523 fhuA2 [lon] ompT gal sulA11 R(mcr- 73miniTn10- -TetS) 2 [dcm]R(zgb-210Tn10- -TetS) endA1 Δ(mcrC-mrr)114IS10

New England BiolabsLtd UK

ER2523pAZIL-LraRM Competent cells obtained by transformation of ER2523 cells withpAZIL-LraRM This study

PlasmidspAZIL 7109 bp Emr shuttle cloning vector [19]

pAZIL-LraRM PCR fragments of LraI operon from BGTRK10-1 cloned into pAZIL vectorpredigested with SmaI This study

pAZIL-LraIlowast672pBS DNA fragment of 672 bp obtained after digestion of pBluescipt SK+ withLraIlowast activity cloned into pAZIL vector predigested with LraI This study

pAZILcos 8194 bp Emr shuttle cosmid vector [19]pAZILcosLra Cosmid selected from total XbaI cosmid library of BGTRK10-1 This studypBluescript SK+ 2958 bp Ampr cloning vector Stratagene

pBSLraCla ClaI fragment of 4239 bp obtained from pAZILcosLra cloned intopBluescript SK+ vector This study

pMAL-c5X 5677 bp pMB1 origin lacImalE bla Factor Xa cleavage site New England BiolabsLtd UK

pMAL-cX5LraI-29PCR fragment of LraI restriction endonuclease from pAZIL-LraRM clonedinto pMAL-c5X vector predigested with XmnI and HindIII restrictionenzymes

This study


This study


This study

The primers used in PCR for amplification of LraI operon(lraIR and lraIM genes) were as follows LraRM-Fw(51015840-GTATAGAAAGAAGAATCG-31015840) and LraRM-Rev(51015840-GCAGGGTAATGTTCCTCAC-31015840) while followingprimers were used for overexpression of LraI restrictionenzyme (lraIR gene) in pMALc5X vector LraI-Fw (51015840-ATG-TCGAGAAAAAATCAGTCG-31015840) and LraI-Rev (51015840-CTC-AAGCTTTCTAATTAATCCTTTTTTGC-31015840HindIII restric-tion site is underlined) Total DNA (1 ng) was mixed with179 120583l of bidistilled water 25 120583l of 10x PCR buffer (ThermoFisher Scientific) 1120583l dNTP mix (10mM) 15 120583l of MgCl

2

(25mM) 1 120583l (10 pmol) of each primer and 01120583l ofPlatinum Taq DNA Polymerase High Fidelity Performedusing the GeneAmp 2700 PCR Cycler (Applied Biosystems)the PCR programs consisted of initial denaturation (5minat 96∘C) 30 cycles of denaturation (30 s at 96∘C) annealing(30 s at 40∘C) and polymerization (2 or 1min at 68∘C) andan additional extension step of 5min at 68∘C PCR fragmentsof LraI operon amplified using Platinum Taq DNAPolymerase High Fidelity were cloned into pAZIL vectorpredigested with SmaI

24 Recombinant LraI Restriction Endonuclease Overexpres-sion in E coli and Purification PCR fragment consisted oflraIR gene (from ATG to stop codon) obtained by LraI-FwLraI-Rev primers and Platinum Taq DNA PolymeraseHigh Fidelity was purified digested with HindIII and clonedinto pMAL-c5X vector digested with XmnI and HindIIIrestriction enzymes and transformed into ER2523 competentcells (New England Biolabs Ltd UK) previously transformedwith pAZIL-LraRM construct (Table 1) Transformants wereselected on LA Petri dishes containing 2 glucose ampicillin100 120583gml and erythromycin 300 120583gml at 23∘C Confirma-tion of fragment presence in adequate orientation was ob-tained by restriction enzyme analysis (with SacI and HindIIIdigestion) and sequencing Expression of recombinant pro-tein was carried out at 23∘C by induction with 01mMisopropyl 120573-D-1-thiogalactopyranoside (IPTG) Purification(cell lysis affinity chromatography and cleavage of fusionprotein with Xa protease) was performed according to man-ufacturer instruction (pMAL Protein Fusion amp PurificationSystem New England Biolabs Ltd UK) Purified recombi-nant LraI restriction endonucleasewas stored atminus20∘C inCM


buffer (20mM Tris-HCl pH74 200mM NaCl 1mM EDTAand 1mM DTT) containing 50 glycerol

25 Endonuclease Assays Endonuclease activity was assayedby incubating various amounts of purified LraI enzymein buffer recommended for use with EcoRI (50mM Tris-HCl pH 75 10mM magnesium chloride 100mM sodiumchloride 002 Triton X-100 supplemented with 100 120583gmlBSA Thermo Fisher Scientific) containing 1120583g of pBluesciptSK+ plasmid DNA per 50 120583l reaction mixture for 1 h at 37∘COne unit of enzyme activitywas defined as amount of purifiedLraI enzyme whichwas able to completely cut 1 120583g of plasmidDNA for 1 h

Influence of reaction buffer composition on LraI enzymeactivity was assayed in different commercial buffers (Buffer B(blue 10mM Tris-HCl pH 75 10mM magnesium chlo-ride 100 120583gml BSA) Buffer G (green 10mM Tris-HCl pH75 10mM magnesium chloride 50mM sodium chloride100 120583gml BSA) Buffer O (orange 50mM Tris-HCl pH75 10mM magnesium chloride 100mM sodium chloride100 120583gml BSA) Buffer R (red 10mM Tris-HCl pH 8510mM magnesium chloride 100mM potassium chloride100 120583gml BSA) Buffer Tango (yellow 33mM TrisndashacetatepH 79 10mMmagnesium acetate 66mMpotassium acetate100 120583gml BSA) and buffer recommended for usewithEcoRIThermo Fisher Scientific) used for reaction with 1U ofpurified LraI enzyme and 1 120583g of plasmidDNA for 1 h at 37∘CTo measure the activity of purified LraI enzyme at differenttemperatures 1 U of purified LraI enzyme was incubatedwith 1 120583g of plasmid DNA for 1 h at different temperatures(13∘C 23∘C 30∘C 37∘C 45∘C 60∘C and 80∘C) In all endo-nuclease activity assays commercial EcoRI restriction enzyme(Thermo Fisher Scientific) was used as control Reactionswere stopped by addition 110 volume of stop solution(50mM EDTA pH 8 50 glycerol 002 orange G) andproducts were analyzed by electrophoresis in 1 agarose gels

26 Determination of LraI Cleavage Site To determine theprecise positions and nucleotide sequence of cleavage siteswithin double stranded DNA for the LraI restriction enzymepBluescript SK+ was used as template Plasmid was digestedwith recombinant enzyme LraI complete digestion wasconfirmed by agarose gel electrophoresis and digest wassequenced with M13F and M13R primers (Macrogen EuropeThe Netherlands) Simultaneously as control the wholeexperiment was conducted with commercial EcoRI restric-tion enzyme (Thermo Fisher Scientific)

27 Bioinformatic Analysis of LraI Homologs Sequencesearches on the NCBI nucleotide and protein databases wereconducted with BLAST [22] using lraIRLraIR and lraIMLraIM sequences The phylogenetic inferences between re-striction andmethylase enzymeswereobtained byMEGAver-sion 60 (httpwwwmegasoftwarenet)The first 30 proteinreference sequences of EcoRI-like endonuclease or methyl-transferase enzymes chosen according to results of BLASTPsearch and LraI restrictase and LraImethylase sequences sep-arately were trimmed and aligned using Clustal W [23] withdefault parameters The phylogenetic trees were constructed

by the maximum-likelihood (ML) method using a Tamura-Nei model Bootstrapping of 1000 replicates was used to inferconfidence levels of ML trees

The nucleotide sequences of DNA fragments carryinggenes encoding LraI restriction-modification system and 16SrRNA from L raffinolactis BGTRK10-1 were submitted toENA GenBank under accession numbers LT222052 andLT854837 respectively

3 Results and Discussion

31 Identification of LraI (EcoRI-Like) Methylase Activity inL raffinolactis BGTRK10-1 The mesophilic lactic acid bac-terium L raffinolactis is prevalent in dairy foods such as rawmilks natural dairy starter cultures and a great variety ofcheeses L raffinolactis BGTRK10-1 is a natural isolate fromautochthonous young sweet kajmak produced in the Vlasicmountain region of central Bosnia and Herzegovina [17]Strain BGTRK10-1 was selected because of its strong autoag-gregation phenotype In order to construct cosmid library ofstrainBGTRK10-1 to clone aggregation ability coding gene(s)total DNA of the strain was digested with several restrictionenzymes (including EcoRI) It has been observed that theEcoRI did not cut isolated DNA in several attempts unlikethe other used restriction enzymes It was suspected that thestrain possesses RM system (named Lra L raffinolactis) thatrecognizes the same DNA sequence as EcoRI RM systemHence the methylase activity of the strain L raffinolactisBGTRK10-1 which protects its DNA from the digestion byEcoRI restriction enzyme was quite accidentally discoveredduring routine laboratory work

Restriction endonucleases commonly known as restric-tion enzymes are ubiquitously present in prokaryotes Themain function of restriction enzymes is the protection againstforeign genetic material especially against bacteriophageDNA Several restriction-modification systems have beenidentified in lactococci Most of them are plasmid encodedand function as phage-resistance mechanism which is veryimportant for the strains used in the dairy industry in termsof preventing phage infection and cell lysis [25ndash29]

32 Selection of Clone Carrying LraI RMOperon from CosmidLibrary Cosmid DNA was isolated from total XbaI cosmidlibrary in E coli HB101 and 1 120583g of DNA mix from totalcosmid clones was subjected to digestion with EcoRI restric-tion enzyme and after that directly transformed into DH5120572competent cells Cosmid DNA isolated from obtained trans-formants was rechecked for resistance to EcoRI restrictionenzyme digestion One cosmid named pAZILcosLra pro-viding resistance to EcoRI restriction enzyme digestion wasselected for further analyses subcloning and DNA sequenc-ing (Table 1)

33 LraI Operon for RM System Provides Resistance to EcoRIRestriction Enzyme Digestion To localize the minimumgenetic unit on the cosmid pAZILcosLra that is responsiblefor the resistance to digestion with EcoRI restriction enzymethe cosmid pAZILcosLra was digested with several restric-tion enzymes (XbaI-generated four fragmentsHindIII-three


fragmentsClaI-four fragments and EcoRV-three fragments)and then subcloned into pBluescript SK+ vector digestedwith corresponding restriction enzymes Only one constructpBSLraCla (obtained with ClaI) was able to reestablish theresistance to EcoRI restriction enzyme digestion (Table 1)The ClaI DNA fragment of 4239 bp carrying complete infor-mation for resistance to EcoRI digestion was completelysequenced by primer walking Four complete (EcoRI-likeendonuclease EcoRI-like methylase hypothetical proteinand site specific integrase) one truncated (pentapeptide re-peat containing protein) and one partial (N(5)-(carboxyeth-yl) ornithine synthase) open reading frame (ORFs) wererevealed on ClaI DNA fragment (Figure 1) Position of LraIRM operon in genome of strain BGTRK10-1 indicates thepossibility that the operon was acquired by horizontal genetransfer the conserved lactococcal gene for pentapeptiderepeat containing protein is interrupted in themiddle by LraIRM operon and immediately after methylase gene is locatedgene for site specific integraseThis event that occurred in thedistant past is indicated by the fact that additional mutationswere accumulated within the first part of the gene forpentapeptide repeat containing protein most probably dueto its nonfunctionality The distance between the restrictaseand the methylase genes is 10 nucleotides without promoterand ribosomal binding site and in other EcoRI-like oper-ons strongly indicates polycistronic RNA transcription fromupstream promoter and translation from consensus RBS(AGGAGA) 4 nucleotides distant fromATG codon of restric-tase gene

To confirm the functionality of LraI restriction-modi-fication operon a region that includes both (lraIR and lraIM)genes was amplified using LraRM-Fwand LraRM-Rev prim-ers (for details see Section 22) and cloned into pAZIL vectorsgiving construct pAZIL-LraRM Construct carrying onlythese two genes was completely sequencedwhile resistance toEcoRI restriction enzyme digestion was confirmed in vitro

34 Cloning Overexpression and Purification of LraI Restric-tion Endonuclease Plasmid clone pAZIL-LraRMwas used asmatrix for amplification of the open reading frame encodingLraI restriction endonuclease with primers LraI-Fw andLraI-Rev Since HindIII restriction site has been integratedinto LraI-Rev primer obtained amplified fragment was firsttreated withHindIII to provide directed cloning of PCR frag-ment into expression vector pMAL-c5X which was digestedwith XmnI and HindIII restriction enzymes Ligation mixwas transformed into ER2523 cells which were previouslytransformed with a pAZIL-LraRM vector expressing LraImethylase in order to protect transformed cells from thenuclease activity of LraI towards their own Transformantsof ER2523pAZIL-LraRMwith pMAL-cX5LraI were success-fully obtained when selection was carried out at 23∘C onLA selective plates (erythromycin 300 120583gml and ampicillin100 120583gml) containing 2 glucose in order to minimiseexpression of enzymesThree clones (namedpMAL-cX5LraI-29 pMAL-cX5LraI-31 and pMAL-cX5LraI-42 Table 1) wereselected for restriction enzyme analysis complete sequenc-ing and overexpression of enzyme LraI restriction nuclease

was successfully overexpressed in all three clones by over-night induction with 01mM IPTG at 23∘C and purified usingamylose resins and cleaved by Xa protease (which cleavesfusion protein between maltose binding protein and cloneproviding release of exactly the same protein as natural) Theoverexpression of LraI restriction enzyme under aforemen-tioned conditions (overnight induction with 01mM IPTG at23∘C) represents the result that is similar to results observedby other researchers [30] The possible explanation for thiscould be the expression of restriction enzymes is toxic athigher temperatures

Purified LraI restriction enzymes from all three cloneswere stored at minus20∘C in CM buffer with 50 glycerol

35 Functional Analysis Determination of Ionic Strengthand Temperature Optimum of the Purified LraI EndonucleaseActivity Considering that LraI RM system provided com-plete protection against digestion of EcoRI endonucleaseit was assumed that it recognizes and cleaves the identicalnucleotide sequence Since plasmid pBluescipt SK+ containsone EcoRI restriction site in polycloning region it was usedfor functional analysis of purified LraI restriction enzymeSpecific activity (1 U) of purified LraI restriction enzymewas determined in EcoRI reaction buffer (Thermo FisherScientific) at 37∘C Different levels of LraI restriction enzymeexpression were observed in selected clones but specificactivities (U120583g of purified proteins) were almost the same(1U50 plusmn 5 ng) among the clones pMAL-cX5LraI-29 pMAL-cX5LraI-31 and pMAL-cX5LraI-42 (Figure 2)

To determine optimal temperature for LraI activitypurified enzyme was incubated with pBluescipt SK+ vectorat temperatures ranging from 13∘C to 80∘C LraI enzymeshowed the highest activity at lower temperatures (between13∘C and 37∘C) while at 45∘C and higher temperatures it par-tially cut DNA (Figure 3) However this is in agreement withthe optimal growth temperature of strain BGTRK1-10 (30∘C)Briefly since the first description of EcoRI restriction enzymein 1970 [31] more than 500 isoschizomers have been reportedor predicted with very high levels of identity (50ndash70) point-ing to the widespread distribution among species of differentPhyla and indicating possible common origin It seems thatsome specific characteristics such as optimum working tem-perature diverged depending on the optimum growth tem-perature of the enzyme producing bacteria which is why wethink that LraI exhibits better activity at lower temperaturesIt was found that commercial EcoRI (used as control) showedhigh activity at 45∘C in contrast to LraI enzyme pointing tothe difference between these two enzymes

In addition stability of LraI enzyme was tested afterdifferent period of storage at minus20∘C LraI enzyme did not loseactivity after storage for more than six months at minus20∘C inCM buffer with 50 glycerol

To establish the optimal salt concentration in the reactionbuffer for LraI enzyme activity different commercial buffersBuffer B BufferG BufferO BufferR Buffer Tango and bufferrecommended for use with EcoRI (Thermo Fisher Scientific)were used LraI enzyme exhibited high and specific activity inbuffers with 100mMand higher salt concentrations (Figure 4buffer recommended for use with EcoRI Buffer 2x Tango


ClaI 1

Δorf1 lraIR lraIM hyp1 intSS par ceo

ClaI4239XhoI 4168

EcoRI 3908 EcoRV 3275HincII 3284

HincII 2001

SpeI 1474

HincII 2110 XmnI 3069XmnI 2282

XmnI 2400

HindIII 2814 ScaI 3845

Figure 1 Schematic presentation ofClaIDNA fragment of 4239 bp carrying complete information for providing resistance toEcoRI digestionClaI DNA fragment containing following ORFs truncated gene for pentapeptide repeat containing protein (Δorf1) EcoRI-like endonuclease(lraIR) EcoRI-like methylase (lraIM) hypothetical protein (hyp1) site specific integrase (intSS) and partial gene for N(5)-(carboxyethyl)ornithine synthase (par ceo)

LraI

29

LraI

31

LraI

42

EcoR

I

Ladd

er

(bp)

30002000

1000

1500

500

100

1200

Figure 2 LraI activity assay Digestion of pBluescipt SK+ by puri-fied LraI restriction enzyme LraI 29 LraI 31 and LraI 42 fromclones pMAL-cX5LraI-29 pMAL-cX5LraI-31 and pMAL-cX5LraI-42 respectively

100

1000

300010000

500

EcoR

ILr

aI

EcoR

ILr

aIEc

oRI

LraI

EcoR

ILr

aI

EcoR

ILr

aIEc

oRI

LraI

EcoR

ILr

aI

(bp)

13∘C 23

∘C 30∘C 45

∘C 60∘C 80

∘C37∘C

Ladd

er

Figure 3 Determination of temperature optimum of the purifiedLraI restriction enzyme activity Commercial EcoRI restrictionenzyme was used in control reactions

and Buffer O) except in Buffer R (red) (10mM Tris-HCl pH85 10mMmagnesium chloride 100mMpotassium chlorideand 100 120583gml BSA) (Figure 4 Buffer R) It was noticedthat in buffers with lower ionic strengths the LraI enzymeexhibited a specific star activity cutting the vector at anotherposition (Figure 4 Buffer 1x Tango Buffer B Buffer G)

100

1000

30002500

10000

500800

(bp)

EcoR

ILr

aIEc

oRI

LraI

EcoR

ILr

aIEc

oRI

LraI

EcoR

ILr

aIEc

oRI

LraI

EcoR

I

LraI

Ladd

er

Ladd

er

Buffer OBuffer GBuffer BBuffer

Buffer R Undigested1x Tango 2x TangoEcoRI

Figure 4 Determination of influence of ionic strength in differentreaction buffers on the LraI restriction enzyme activity CommercialEcoRI restriction enzyme was used in control reactions L ladder(GeneRuler DNA Ladder Mix Thermo Fisher Scientific) Blackarrows present fragment obtained by LraIlowast activity white arrowspresent undigested plasmid DNA in EcoRI (Thermo Fisher Scien-tific) digestion

It is interesting that in Buffer B and Buffer G commercialEcoRI restriction enzyme exhibited weaker activity partialdigestion

36 LraI Is an Isoschizomer of EcoRI Sequencing of doublestranded cleaved DNA by the LraI enzyme has shown thatthe LraI enzyme recognizes the identical nucleotide sequence(51015840-GAATTC-31015840) as expected and cuts it at the sameposition (between G and A) like EcoRI enzyme (Figure 5)leaving identical sticky ends The same cleavage results wereobtained with LraI enzymes purified from all three cloneswhich supported our conclusion that LraI enzyme is anisoschizomer of EcoRI

37 Determination of the Cleavage Site of LraIlowast Activity Oneof the important characteristics of restriction enzymes istheir high sequence specificity in order to adequately providethe function of protecting the genome integrity In additionit was established that restriction enzymes in nonoptimalconditions could exhibit a modified specificity so that thesame restriction enzyme could recognize and cleave DNA atadditional positions to canonical one [32] For EcoRI restric-tion enzyme it was detected that in at low ionic strength


TGGATCCCCGGGCTGCAGGAATTA

BamHI SmaI PstI Dig EcoRI

110100

(a)

TGGATCCCCGGGCTGCAGGAATTA110100


(b)

Figure 5 Determination of the LraI cleavage site 51015840-GAATTC-31015840of pBluescipt SK+ by DNA sequencing (a) Sequence of pBluesciptSK+ predigested with LraI restriction enzyme and (b) with EcoRIobtained using M13R primer The colour-coded sequence tracesare A (green) T (red) C (blue) and G (black) The extra Abase was added at the end of the cleaved template by the TaqDNA polymerase used for sequencing due to template-independentterminal nucleotide transferase activity of Taq DNA polymerase

and high pH enzyme is transformed so that it recognizes anddigests the shortened tetranucleotide sequence 51015840-AATT-31015840[32]This activity is termed star activity and is usually labeledas ldquolowastrdquo adjacent to the enzyme name and was also detected forother restriction enzymes and could be used to cleave DNAat additional sites for cloning purposes In order to be usedfor cloning star restriction enzyme activity should (i) berestricted to limited number of sequence variants (ii) providethe same sticky ends for ligation into the vector and (iii) becontrolled by changeable conditions

To determine the cleavage site of LraIlowast activity thefragment of 672 bp obtained after digestion of pBluesciptSK+ in buffer with low ionic strength (1x Tango ThermoFisher Scientific) (Figure 6(a)) was cloned into pAZIL vectorpredigested with LraI Isolated plasmids (named pAZIL-LraIlowast672pBS Table 1) from selected white colonies were firstchecked for the presence of DNA fragment of 672 bp byrestriction analysis with LraI enzyme and then sequencedThe sequence of the entire fragment as well as the adjacentregions of the vector showed that the cloned 672 bp fragmentoriginating frompBluescipt SK+was cut out byLraIlowast enzymeactivity at positions (701 EcoRI) and at additional position(29) in which the 51015840-AAATTC-31015840 sequence is present Thesame sequencing results were obtained for three independentclones confirming that additional recognition and cleavagesite for LraIlowast activity is 51015840-AAATTC-31015840 sequence To besure that only AAATTC sequence is recognized and cleavedby LraIlowast activity but not other sequences with alternativechanges within the GAATTC site a search analysis for thepresence of other alternatives in plasmid sequences wasperformed Since all alternatives with one nucleotide changeof recognition sequence exist in pBluescipt SK+ (in addi-tion to AAATTC at position 29 the following recognition

sequences GAATTG at position 647 CAATTC at position850 TAATTC at position 2824 were found) but were notcleaved we conclude that LraIlowast activity specifically recog-nizes and cleaves only one variant of degenerate recognitionsequence (51015840-AAATTC-31015840) To further test the conclusionobtained on pBluescipt SK+ the plasmid pAZIL sequencewas analyzed and subjected to digestion by LraI enzymeunder conditions that induce star activityTheobtained diges-tion results were completely in correlation with the predictedexpectations (nine positionsfragments) (Figure 6(b)) so wecould conclude that LraIlowast activity is limited to only onevariant of the recognition sequence giving identical cohesiveends as in optimal conditionsmaking the resulting fragmentsafter LraIlowast activity clonable without further processing intoLraI or EcoRI treated vectors

LraI restriction enzyme star activity meets all the givenrequirements it recognizes only one variant of the sequencewhich can enable more precise restriction mapping andcloning provides the same cohesive ends compatible withEcoRI (contained by most cloning vectors) and is completelycontrolled by low ion concentration andor high pH Bothfactors which induce star activity of LraI restriction enzymelow ionic strength (Buffer 1x Tango) and buffer with higherpH (Buffer R pH 85) also influence on EcoRI but incontrast LraIlowast recognizes only one additional sequence ex-pressing more specific star activity

38 Phylogenetic Similarity of LraI Restriction-ModificationEnzymes with Others Belonging to EcoRI-Like Group Asearch for number of restriction enzymes recognizing 51015840-GAATTC-31015840 sequence present in REBASE revealed 526putative EcoRI-like proteins Protein BLAST analysis showedthat 196 restriction enzymes in NCBI database (from variousmicroorganisms) share more than 50 identity on at least50 protein coverage with LraI enzyme Highest identity wasobserved with restriction endonucleases from streptococci(Streptococcus suis 68 Streptococcus dysgalactiae 64 andStreptococcus mutans 64) It is interesting that similar buthigher identity was observed for LraI methylase again withstreptococci (Streptococcus pseudopneumoniae 74 Strepto-coccus suis 73 Streptococcus mutans 73 and Streptococ-cus dysgalactiae 68) Similar percentage of identity wasobserved also at nucleotide level (about 70) for both LraIrestrictase and methylase genes The fact that most EcoRIisoschizomers unlike other restriction enzymes share a highlevel of identity indicates their common origin [7]

Phylogenetic trees were constructed for both LraI restric-tase (Figure 7(a)) and methylase (Figure 7(b)) enzymesPhylogenetic analysis showed that LraI homologs (restrictionendonucleases andmethylases) can be divided into twomainbranches one (that could be additionally subdivided com-prising close homologs (Gram-negative bacteria and Cyano-phyta) and the other comprising homologs from speciesbelonging to Firmicutes phylumThe position of genus Fibro-bacter (phylum Fibrobacteres) which consists of only twospecies is interesting its restrictase enzyme belongs to onebranch while methylase protein belongs to the other (Fig-ure 7)


L 1 2 3 4 5 6 7 L

(a)

L 1 2 3 4 5 6 7 L

(b)

Figure 6 Determination of the cleavage site of LraIlowast activity (a) Digestion of pBluescipt SK+ vector by LraI 29 (1 and 4) LraI 31 (2 and 5)and LraI 42 (3 and 6) in buffer recommended for use with EcoRI (1 2 and 3) and in 1x Tango Buffer (4 5 and 6) 7 undigested pBluesciptSK+ digestion of pAZIL vector by LraI 29 (1 and 4) LraI 31 (2 and 5) and LraI 42 (3 and 6) in buffer recommended for use with EcoRI (1 2and 3) and in 1x Tango Buffer (4 5 and 6) 7 undigested pAZIL vector L ladder (GeneRuler DNA Ladder Mix Thermo Fisher Scientific)

92

9289

28

78

21

20

30

37

38

38

25

24

52

5188

83

22

56

4379

19

99

99

99

9949

98

005

Lactococcus raffinolactisStreptococcus mutans

Streptococcus dysgalactiaeStreptococcus suis

Streptococcus pseudopneumoniaePseudobutyrivibrio sp ACV-2Clostridioides difficile

Fibrobacter sp UWB4Helicobacter japonicus

Parabacteroides distasonisPrevotella pleuritidisPrevotella amnii

Porphyromonas gingivalisCapnocytophaga sp oral taxon 863

Microcystis aeruginosa

Anabaena

Polaribacter sp SA4-10Thermoflexibacter ruberRiemerella anatipestife

Bacteroidetes bacterium 37-13Pseudomonas sp Irchel s3a18

Vibrio choleraePseudomonas mendocina

Salmonella entericaAcinetobacter baumannii

Enterobacter timonensisEscherichia coli

Dolichospermum circinale

Flavobacterium psychrophilum DSM 3660

Candidatus Levybacteria bacterium RIFCSPLOWO2 12 F Candidatus Staskawiczbacteria bacterium RIFOXYC1 F

(a)

99

99

48

86

9997

95

9575

79

60

70

7667

16

14

35

39

99946

2528

68

21

35

3639

005

Lactococcus raffinolactis

Streptococcus suis

Streptococcus mutans

Streptococcus dysgalactiaeListeria innocuaFibrobacter sp UWOS

Prevotella amniiPorphyromonas gingivalis

Prevotella sp oral taxon 317Capnocytophaga sp oral taxon 863


Enterobacter timonensisPseudomonas

Vibrio choleraeThermoflexibacter ruber

Riemerella anatipestiferPolaribacter sp SA4-10

Flavobacterium columnareBacteroidetes bacterium 37-13Candidatus Woesebacteria bacterium GW2011 GWA1 39

Microcystis panniformis FACHB-1757Microcystis aeruginosa

Desulfobacteraceae bacterium IS3Candidatus Achromatium palustre

Arcobacter sp LAnabaena sp 90

Dolichospermum circinaleDolichospermum compactum

Streptococcus pseudopneumoniae

Cycloclasticus sp symbiont of Bathymodiolus hecke

(b)

Figure 7 Phylogenetic similarity of LraI RM system with other belonging to EcoRI-like group constructed using a Tamura-Nei model (a)Phylogenetic tree for LraI restrictase (b) phylogenetic tree for LraI methylase enzymes


4 Conclusions

We identified a potent Type II restriction endonuclease in Lraffinolactis BGTRK10-1 named LraI The recognition andcleavage sequence for LraI restriction enzyme was deter-mined as 51015840-GAATTC-31015840 indicating that LraI restrictionenzyme is an isoschizomer of EcoRI but with different char-acteristics One of characteristics that has been thoroughlystudied is star activity of LraI restriction enzyme that islimited to one variant of the recognition site and cuts anotheralternative sequence 51015840-AAATTC-31015840 leaving the same stickyends on fragments as EcoRI making the fragments obtainedafter digestion easy to clone without additional processing

Conflicts of Interest

The authors declare no conflicts of interest

Acknowledgments

TheMinistry of Education Science and Technological Devel-opment of the Republic of Serbia Serbia supported this work(Grant No 173019)

References

[1] T A Bickle andD H Kruger ldquoBiology of DNA restrictionrdquoMi-crobiology andMolecular Biology Reviews vol 57 no 2 pp 434ndash450 1993

[2] A B Hickman Y Li S VMathew EWMay N L Craig and FDyda ldquoUnexpected structural diversity in DNA recombinationThe restriction endonuclease connectionrdquoMolecular Cell vol 5no 6 pp 1025ndash1034 2000

[3] M Mucke G Grelle J Behlke R Kraft D H Kruger and MReuter ldquoEcoRII A restriction enzyme evolving recombinationfunctionsrdquo EMBO Journal vol 21 no 19 pp 5262ndash5268 2002

[4] T Naito K Kusano and I Kobayashi ldquoSelfish behavior of re-striction-modification systemsrdquo Science vol 267 no 5199 pp897ndash899 1995

[5] I Kobayashi ldquoBehavior of restriction-modification systems asselfish mobile elements and their impact on genome evolutionrdquoNucleic Acids Research vol 29 no 18 pp 3742ndash3756 2001

[6] R J Roberts T Vincze J Posfai and D Macelis ldquoREBASEmdashadatabase for DNA restriction andmodification enzymes genesand genomesrdquo Nucleic Acids Research vol 38 pp D234ndashD2362010

[7] A Pingoud G G Wilson and W Wende ldquoType II restrictionendonucleases - A historical perspective and morerdquo NucleicAcids Research vol 42 no 12 pp 7489ndash7527 2014

[8] A Pingoud and A Jeltsch ldquoStructure and function of type II re-striction endonucleasesrdquo Nucleic Acids Research vol 29 no 18pp 3705ndash3727 2001

[9] T I Tikchonenko E V Karamov B A Zavizion and B S Nar-oditsky ldquoEcoRI activity Enzyme modification or activation ofaccompanying endonucleaserdquo Gene vol 4 no 3 pp 195ndash2121978

[10] M Nasri and D Thomas ldquoRelaxation of recognition sequenceof specific endonuclease HlndIIIrdquo Nucleic Acids Research vol14 no 2 pp 811ndash821 1986

[11] M Nasri and D Thomas ldquoAlteration of the specificity of pvuIIrestriction endonucleaserdquoNucleic Acids Research vol 15 no 19pp 7677ndash7687 1987

[12] F Barany ldquoThe TaqI rsquostarrsquo reaction strand preferences revealhydrogen-bond donor and acceptor sites in canonical sequencerecognitionrdquo Gene vol 65 no 2 pp 149ndash165 1988

[13] J Bitinaite and I Schildkraut ldquoSelf-generated DNA terminirelax the specificity of SgrAl restriction endonucleaserdquo Proceed-ings of the National Acadamy of Sciences of the United States ofAmerica vol 99 no 3 pp 1164ndash1169 2002

[14] R J Roberts andKMurray ldquoRestriction endonucleaserdquoCriticalReviews in Biochemistry and Molecular Biology vol 4 no 2 pp123ndash164 1976

[15] F H Stephenson B T Ballard H W Boyer J M Rosenbergand P J Greene ldquoComparison of the nucleotide and amino acidsequences of the RsrI and EcoRI restriction endonucleasesrdquoGene vol 85 no 1 pp 1ndash13 1989

[16] T Chuluunbaatar T Ivanenko-JohnstonM Fuxreiter et al ldquoAnEcoRI-RsrI chimeric restriction endonuclease retains parentalsequence specificityrdquo Biochimica et Biophysica Acta (BBA) -Proteins and Proteomics vol 1774 no 5 pp 583ndash594 2007

[17] A Terzic-Vidojevic S Mihajlovic G Uzelac et al ldquoCharacter-ization of lactic acid bacteria isolated from artisanal Travnikyoung cheeses sweet creams and sweet kajmaks over fourseasonsrdquo Food Microbiology vol 39 pp 27ndash38 2014

[18] B Jovcic J Begovic J Lozo L Topisirovic and M Kojic ldquoDy-namics of sodium dodecyl sulfate utilization and antibioticsusceptibility of strain Pseudomonas sp ATCC19151rdquo Archivesof Biological Sciences vol 61 no 2 pp 159ndash164 2009

[19] M Kojic B Jovcic I Strahinic et al ldquoCloning and expression ofa novel lactococcal aggregation factor from Lactococcus lactissubsp lactis BGKP1rdquo BMC Microbiology vol 11 article 2652011

[20] D A Hopwood J M Bib J F Chater et al Genetic Manipula-tion of Streptomyces A Laboratory Manual 1985

[21] D Hanahan ldquoStudies on transformation of Escherichia coliwithplasmidsrdquo Journal of Molecular Biology vol 166 no 4 pp 557ndash580 1983

[22] S F Altschul T L Madden A A Schaffer et al ldquoGappedBLAST and PSI-BLAST a new generation of protein databasesearch programsrdquo Nucleic Acids Research vol 25 no 17 pp3389ndash3402 1997

[23] M A Larkin G Blackshields N P Brown et al ldquoClustalW andclustal X version 20rdquo Bioinformatics vol 23 no 21 pp 2947-2948 2007

[24] H W Boyer and D Roulland-dussoix ldquoA complementationanalysis of the restriction and modification of DNA in Escheri-chia colirdquo Journal of Molecular Biology vol 41 no 3 pp 459ndash472 1969

[25] C Hill K Pierce and T R Klaenhammer ldquoThe conjugativeplasmid pTR2030 encodes two bacteriophage defense mech-anisms in lactococci restriction modification (R+M+) andabortive infection (Hsp+)rdquo Applied and Environmental Micro-biology vol 55 no 9 pp 2416ndash2419 1989

[26] S Moineau S Pandian and T R Klaenhammer ldquoRestric-tionmodification systems and restriction endonucleases aremore effective on lactococcal bacteriophages that have emergedrecently in the dairy industryrdquo Applied and EnvironmentalMicrobiology vol 59 no 1 pp 197ndash202 1993

[27] D J OrsquoSullivan K Zagula and T R Klaenhammer ldquoIn vivorestriction by LlaI is encoded by three genes arranged in anoperon with llaIM on the conjugative Lactococcus plasmid


pTR2030rdquo Journal of Bacteriology vol 177 no 1 pp 134ndash1431995

[28] N Nyengaard F K Vogensen and J Josephsen ldquoRestriction-modification systems in lactococcus lactisrdquo Gene vol 157 no1-2 pp 13ndash18 1995

[29] A Madsen and J Josephsen ldquoCloning and characterization ofthe lactococcal plasmid-encoded type II restrictionmodifica-tion system LlaDIIrdquo Applied and Environmental Microbiologyvol 64 no 7 pp 2424ndash2431 1998

[30] D-H Chung J R Huddleston J Farkas and J WestphelingldquoIdentification and characterization of CbeI a novel ther-mostable restriction enzyme from Caldicellulosiruptor besciiDSM 6725 and a member of a new subfamily of HaeIII-likeenzymesrdquo Journal of Industrial Microbiology and Biotechnologyvol 38 no 11 pp 1867ndash1877 2011

[31] R J Roberts ldquoHow restriction enzymes became the workhorsesof molecular biologyrdquo Proceedings of the National Acadamy ofSciences of the United States of America vol 102 no 17 pp 5905ndash5908 2005

[32] B Polisky P Greene D E Garfin B J McCarthy H M Good-man and H W Boyer ldquoSpecificity of substrate recognition bythe EcoRI restriction endonucleaserdquo Proceedings of the NationalAcadamy of Sciences of the United States of America vol 72 no9 pp 3310ndash3314 1975

Hindawiwwwhindawicom

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Anatomy Research International

PeptidesInternational Journal of



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The Scientific World Journal

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BioinformaticsAdvances in

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Cell BiologyInternational Journal of



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ArchaeaHindawiwwwhindawicom Volume 2018


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Advances in

Virolog y Stem Cells International



Enzyme Research



MicrobiologyHindawiwwwhindawicom

Nucleic AcidsJournal of

Volume 2018

Submit your manuscripts atwwwhindawicom


Because of the large number of sequenced genomes rate ofdiscovery of new putative restriction and modification genesis rising rapidly In contrast the number of restriction en-zymes that are biochemically characterized has actuallydropped down to the level that was three decades ago

Restriction endonucleases Type II are homodimeric ortetrameric enzymes that cleaveDNAat defined sites of 4ndash8 bpin length and require Mg2+ ions for catalysis [8] For many ofrestriction endonucleases Type II it was found that modifiedconditions (lower ionic strength higher pH presence ofdifferent metallic cofactors and organic solvents) coulddecrease their substrate specificity [9ndash13] Under nonoptimalrestriction conditions these endonucleases can usually cleavedegenerate sequences which differ from standard recogni-tion sites at only one nucleotide This alteration in digestionspecificity causing cleavage of DNA at novel similar but notidentical sequences is defined as enzyme star activity Mod-ified specificity of restriction enzyme (star) activity couldbe exploited to facilitate recombinant DNA techniques sincethe same enzyme in controlled conditions could recognizedifferent DNA sequences and cleave at additional positions[9]

Restriction endonucleases with identical recognition sitesisolated from different organisms are termed isoschizomers[7 14] The RsrI endonuclease found in Rhodobacter sphae-roides is an isoschizomer of the EcoRI Both enzymes recog-nize the sequence GAATTC and cleave it at the same position(GAATTC) and are sharing 50 amino acid sequenceidentity [15] Interesting MunI recognizes the sequenceCAATTG which differs from the recognition sequence ofEcoRI (and RsrI) only in the external base pairs Comparisonof the MunI amino acid sequence with that of EcoRI andRsrI revealed only a low level of overall similarity whereforesequence homology between EcoRI and RsrI has a strongersignificance [16]

This work describes for the first time the occurrence ofEcoRI-like restriction-modification genes in lactococci Theobjective was to clone purify and biochemically and genet-ically characterize novel lactococcal LraI restriction enzymeLraI restriction enzyme was overexpressed and purified tothe homogeneity from E coli using pMAL expression andpurification system Results demonstrate that LraI restrictionenzyme although an isoschizomer of EcoRI shows differentcharacteristics One of characteristics that could be furtherexploited is star activity of LraI that is limited to one variantof the recognition site which after cleavage leaves identicalcohesive ends as EcoRI and LraI restriction enzymes so thatthe fragments obtained after digestion could be cloned with-out additional processing

2 Material and Methods

21 Bacterial Strains and Culture Conditions Lactococcusraffinolactis BGTRK10-1 was isolated from autochthonoussweet kajmak produced from sheep milk without the use ofstarter cultures in a household of the Vlasic mountain regioncentral Bosnia and Herzegovina [17] (Table 1) Preliminarystrain classification was done according to its fermentationability using API 50CHL (Api System SA Bio-Merieux

Montelieu-Vercieu France) temperature of growth (30∘C37∘C and 45∘C) growth in the presence of salt (4 and65) and pH tolerance Final taxonomic classification ofBGTRK10-1 was performed by sequencing of amplified 16SrDNAusing primers previously described [18]The strainwasgrown inM17medium (MerckGmbHDarmstadt Germany)supplemented with D-glucose (05 wv) (GM17) at 30∘CEscherichia coli DH5120572 HB101 and ER2523 strains weregrown aerobically in Luria-Bertani (LB) broth at 37∘C unlessotherwise specified Solid medium was made by adding175 (wv) agar (Torlak Belgrade Serbia) to the liquidmedia Antibiotics were used at the following concentra-tions erythromycin 300 120583gml and ampicillin 100 120583gml forselection and maintaining of transformants The 5-bromo-4-chloro-3-indolyl-120573-D-galacto-pyranoside (X-Gal) (Fermen-tas Vilnius Lithuania) was added to LB medium platesfor bluewhite colour screening of colonies with clonedfragments at final concentration of 40 120583gml

22 Construction of Cosmid Library of L raffinolactisBGTRK10-1 Total DNA isolated from the L raffinolactisBGTRK10-1 was partially digested with XbaI restrictionenzyme Incubation was carried out during 1 h and wasstopped in different time intervals by adding EDTA Opti-mally digestedDNA giving fragments 30ndash40 kb was purifiedand ligated overnight at 16∘C with the pAZILcos vector [19]predigested with XbaI restriction enzyme and dephospho-rylated Ligation for formed concatemers of high molecularweight was checked on agarose gel and encapsulated intophage particles using packaging kit (Agilent Technologies)Encapsulated cosmids were transfected into E coli HB101magnesium cells and selection of clones was done on LAplates containing erythromycin 300 120583gml Constructed cos-mid library in E coli was stored in LB containing 15 (vv)glycerol at minus80∘C

23 DNA Manipulations Total DNA from L raffinolactisBGTRK10-1 was isolated by modified method described byHopwood et al [20] the logarithmic phase cells werepretreated with lysozyme (4mgml for 15min at 37∘C) priorto treatment with SDS For plasmid isolation from E coli theQIAprep Spin Miniprep kit was used according to the manu-facturerrsquos recommendations (Qiagen Hilden Germany)Standard heat-shock transformation was used for plasmidtransfer into E coli [21] Digestion with restriction enzymeswas conducted according to the supplierrsquos instructions (Ther-mo Fisher Scientific) The DNA fragments from agarose gelswere purified using QIAqick Gel extraction kit as describedby the manufacturer (Qiagen Hilden Germany) DNA wasligated with T4 DNA ligase (Agilent technologies USA) ac-cording to the manufacturerrsquos recommendations PlatinumTaq DNA Polymerase High Fidelity (Thermo Fisher Scien-tific Waltham MA USA) was used to amplify DNAfragments by PCR in GeneAmp PCR system 2700 thermalcycler (Applied Biosystems Foster City CA USA) PCRproducts were purified with QiAquick PCR purification kit(Qiagen Hilden Germany) according to the manufacturerrsquosrecommendations DNA sequencing was done by theMacro-genSequencing Service (MacrogenEuropeTheNetherlands)


















This study


This study


This study


2



























ClaI 1


ClaI4239XhoI 4168


HincII 2001

SpeI 1474


XmnI 2400



LraI

29

LraI

31

LraI

42

EcoR

I

Ladd

er

(bp)

30002000

1000

1500

500

100

1200


100

1000

300010000

500

EcoR

ILr

aI

EcoR

ILr

aIEc

oRI

LraI

EcoR

ILr

aI

EcoR

ILr

aIEc

oRI

LraI

EcoR

ILr

aI

(bp)

13∘C 23

∘C 30∘C 45

∘C 60∘C 80

∘C37∘C

Ladd

er



100

1000

30002500

10000

500800

(bp)

EcoR

ILr

aIEc

oRI

LraI

EcoR

ILr

aIEc

oRI

LraI

EcoR

ILr

aIEc

oRI

LraI

EcoR

I

LraI

Ladd

er

Ladd

er










110100

(a)



(b)









L 1 2 3 4 5 6 7 L

(a)

L 1 2 3 4 5 6 7 L

(b)


92

9289

28

78

21

20

30

37

38

38

25

24

52

5188

83

22

56

4379

19

99

99

99

9949

98

005








Anabaena









(a)

99

99

48

86

9997

95

9575

79

60

70

7667

16

14

35

39

99946

2528

68

21

35

3639

005


Streptococcus suis
















(b)



4 Conclusions




Acknowledgments


References





































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018



















This study


This study


This study


2



























ClaI 1


ClaI4239XhoI 4168


HincII 2001

SpeI 1474


XmnI 2400



LraI

29

LraI

31

LraI

42

EcoR

I

Ladd

er

(bp)

30002000

1000

1500

500

100

1200


100

1000

300010000

500

EcoR

ILr

aI

EcoR

ILr

aIEc

oRI

LraI

EcoR

ILr

aI

EcoR

ILr

aIEc

oRI

LraI

EcoR

ILr

aI

(bp)

13∘C 23

∘C 30∘C 45

∘C 60∘C 80

∘C37∘C

Ladd

er



100

1000

30002500

10000

500800

(bp)

EcoR

ILr

aIEc

oRI

LraI

EcoR

ILr

aIEc

oRI

LraI

EcoR

ILr

aIEc

oRI

LraI

EcoR

I

LraI

Ladd

er

Ladd

er










110100

(a)



(b)









L 1 2 3 4 5 6 7 L

(a)

L 1 2 3 4 5 6 7 L

(b)


92

9289

28

78

21

20

30

37

38

38

25

24

52

5188

83

22

56

4379

19

99

99

99

9949

98

005








Anabaena









(a)

99

99

48

86

9997

95

9575

79

60

70

7667

16

14

35

39

99946

2528

68

21

35

3639

005


Streptococcus suis
















(b)



4 Conclusions




Acknowledgments


References





































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018


























ClaI 1


ClaI4239XhoI 4168


HincII 2001

SpeI 1474


XmnI 2400



LraI

29

LraI

31

LraI

42

EcoR

I

Ladd

er

(bp)

30002000

1000

1500

500

100

1200


100

1000

300010000

500

EcoR

ILr

aI

EcoR

ILr

aIEc

oRI

LraI

EcoR

ILr

aI

EcoR

ILr

aIEc

oRI

LraI

EcoR

ILr

aI

(bp)

13∘C 23

∘C 30∘C 45

∘C 60∘C 80

∘C37∘C

Ladd

er



100

1000

30002500

10000

500800

(bp)

EcoR

ILr

aIEc

oRI

LraI

EcoR

ILr

aIEc

oRI

LraI

EcoR

ILr

aIEc

oRI

LraI

EcoR

I

LraI

Ladd

er

Ladd

er










110100

(a)



(b)









L 1 2 3 4 5 6 7 L

(a)

L 1 2 3 4 5 6 7 L

(b)


92

9289

28

78

21

20

30

37

38

38

25

24

52

5188

83

22

56

4379

19

99

99

99

9949

98

005








Anabaena









(a)

99

99

48

86

9997

95

9575

79

60

70

7667

16

14

35

39

99946

2528

68

21

35

3639

005


Streptococcus suis
















(b)



4 Conclusions




Acknowledgments


References





































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018





110100

(a)



(b)









L 1 2 3 4 5 6 7 L

(a)

L 1 2 3 4 5 6 7 L

(b)


92

9289

28

78

21

20

30

37

38

38

25

24

52

5188

83

22

56

4379

19

99

99

99

9949

98

005








Anabaena









(a)

99

99

48

86

9997

95

9575

79

60

70

7667

16

14

35

39

99946

2528

68

21

35

3639

005


Streptococcus suis
















(b)



4 Conclusions




Acknowledgments


References





































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018



L 1 2 3 4 5 6 7 L

(a)

L 1 2 3 4 5 6 7 L

(b)


92

9289

28

78

21

20

30

37

38

38

25

24

52

5188

83

22

56

4379

19

99

99

99

9949

98

005








Anabaena









(a)

99

99

48

86

9997

95

9575

79

60

70

7667

16

14

35

39

99946

2528

68

21

35

3639

005


Streptococcus suis
















(b)



4 Conclusions




Acknowledgments


References





































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018



4 Conclusions




Acknowledgments


References





































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018











Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018


Lactococcus raffinolactis BGTRK10-1, an...

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