Horizontal transfer of iturin A operon, itu, to Bacillus subtilis 168 and conversion into an iturin

10.1128/AAC.49.11.4641-4648.2005.2005, 49(11):4641. DOI:Antimicrob. Agents Chemother. and Makoto ShodaKenji Tsuge, Satoka Inoue, Takashi Ano, Mitsuhiro Itayainto an Iturin A Producer168 and Conversion Bacillus subtilis to ,itu Horizontal Transfer of Iturin A Operon, /content/49/11/4641Updated information and services can be found at: These include:REFERENCES/content/49/11/4641#ref-list-1at: This article cites 33 articles, 14 of which can be accessed free CONTENT ALERTSmore»articles cite this article), Receive: RSS Feeds, eTOCs, free email alerts (when new /site/misc/reprints.xhtml Information about commercial reprint orders: /site/subscriptions/To subscribe to to another ASM Journal go to: on July 14, 2014 by NANKAI UNIVERSITY/Downloaded fromA NTIMICROBIAL A GENTS AND C HEMOTHERAPY,Nov.2005,p.4641–4648Vol.49,No.11 0066-4804/05/$08.00ϩ0doi:10.1128/AAC.49.11.4641–4648.2005Copyright©2005,American Society for Microbiology.All Rights Reserved.Horizontal Transfer of Iturin A Operon,itu,to Bacillus subtilis168 and Conversion into an Iturin A ProducerKenji Tsuge,1,2Satoka Inoue,1Takashi Ano,1Mitsuhiro Itaya,2and Makoto Shoda1* Chemical Resources Laboratory,Tokyo Institute of Technology,4259Nagatsuta,Midori-ku,Yokohama226-8503,Japan,1and Mitsubishi Kagaku Institute of Life Sciences,11Minamiooya,Machida,Tokyo194-8511,Japan2 Received17December2004/Returned for modification30January2005/Accepted23August2005 Iturin A and its derivatives are lipopeptide antibiotics produced by Bacillus subtilis and several closely relatedbacteria.Three iturin group operons(i.e.,iturin A,mycosubtilin,and bacillomycin D)of those antibiotic-producing strains have been cloned and sequenced thus far,strongly implying the horizontal transfer of theseoperons.To examine the nature of such horizontal transfer in terms of antibiotic production,a42-kb regionof the B.subtilis RB14genome,which contains a complete38-kb iturin A operon,was transferred via competentcell transformation to the genome of a non-iturin A producer,B.subtilis168,using a method based ondouble-crossover homologous recombination with two short landing pad sequences(LPSs)in the genome.Therecombinant was positively selected by confirming the elimination of the c I repressor gene,which was localizedbetween the two LPSs and substituted by the transferred segment.The iturin A operon-transferred strain168was then converted into an iturin A producer by the introduction of an sfp gene,which encodes4؅-phospho-pantetheinyl transferase and is mutated in strain168.By inserting the pleiotropic regulator degQ,theproductivity of iturin A increased sevenfold and was restored to about half that of the donor strain RB14,without the transfer of additional genes,such as regulatory or self-resistance genes.Horizontal gene transfer is thought to be a ubiquitous event (18).In terms of antibiotic production genes,the␤-lactam-class antibiotic genes are found in some organelles of fungi and in gram-positive and gram-negative bacteria and are supported to horizontally transfer from bacteria to fungi(1).In contrast, the syringomycin operon of Pseudomonas syringae,which en-codes nonribosomal peptide synthetases,is postulated to be horizontally transferred from a eukaryote to a prokaryote(6). The number of possible examples of the transferred antibiotic production gene is increasing,and studies of successful artifi-cial horizontal transfer of antibiotic production genes to a heterologous host have been reported(4,22,29).However, due to the limited numbers of artificial horizontal transfer, there is little knowledge of the genes required for the conver-sion from a heterologous host into an antibiotic producer. Many Bacillus subtilis strains simultaneously produce some lipopeptide antibiotics,whose peptide moiety is synthesized nonribosomally by large template enzyme complexes(e.g., B.subtilis RB14[33]).Such lipopeptide antibiotics identified thus far have been divided into three groups according to their structure,as follows:the surfactin group(28),the plipastatin and fengycin group(34),and the iturin group(10,24,27). Iturin group lipopeptides are composed of seven␣-amino acids and one␤-amino acid with a long lipid moiety and are potent antifungal agents(24).Three distinct operons that belong to the iturin group have been cloned and sequenced thus far:the mycosubtilin operon of B.subtilis ATCC6633(3),the iturin A operon of B.subtilis RB14(33),and the bacillomycin D operon of B.amyloliquefaciens FZB42(19).All of these operons are composed of a putative transcriptional unit with four genes:one small gene that encodes malonyl-coenzyme A transferase and is probably responsible for␤-amino acid synthesis and three large genes that encode large template enzymes for the synthesis of peptides with defined sequences and chiralities. Although the percent amino acid sequence identities between the operons range from approximately75%to85%,theflank-ing regions of the operons are quite different from each other, as shown in Fig.1.Theflanking region of the mycosubtilin operon is identical to that of the plipastatin operon of B.subtilis168(3).On the other hand,the iturin A and bacil-lomycin D operons areflanked by sequences homologous to xynD of strain168(19,33)(Fig.1).Thesefindings strongly indicate the dynamic features of the iturin group operons. Although168does not produce lipopeptides due to its mu-tation of the4Ј-phosphopantetheine transferase gene sfp, which is responsible for the conversion of nascent antibiotic synthetase to its active form(21),it is a potential producer of two lipopeptide antibiotics,surfactin and plipastatin.We pre-viously converted168into a coproducer of surfactin and plipastatin by introducing sfp and the pleiotropic regulator gene degQ,which is also mutated in168(32).However,on the basis of whole-genome sequence data,168does not have iturin group operons(20).To investigate the nature of the horizontal transfer of an antibiotic producer gene in terms of antibiotic production,we transferred the iturin A operon of RB14to the non-iturin-producing strain168,using a positive-selection system that employs the c I repressor gene of lambda phage as a reverse marker(14,15,16).In this study,although sfp is necessary,we show that the iturin A operon is essentially the only operon required for conversion of strain168into an iturin A producer. We also demonstrate that,in the presence of degQ,an iturin A production almost comparable to that of the donor strain was achieved.*Corresponding author.Mailing address:4259Nagatsuta,Midori-ku, Yokohama226-8503,Japan.Phone:81-45-924-5274.Fax:81-45-924-5276.E-mail:mshoda@res.titech.ac.jp.4641 on July 14, 2014 by NANKAI UNIVERSITY / Downloaded fromFIG.1.(A)Structure and flanking regions of the iturin A,mycosubtilin,and bacillomycin D operons.Shaded bridges between different strains connect regions homologous with the iturin A operon of RB14,while dotted bridges connect regions homologous with the plipastatin operon of 168.Solid black regions in the iturin A and bacillomycin D operons indicate regions homologous with the xynD gene of 168.Vertical lines,horizontal lines,and areas not lined represent B.subtilis RB14DNA,B.amyloliquefaciens FZB42DNA (19),and B.subtilis ATCC 6633DNA (3),respectively.Fengycin is another name for plipastatin (19).(B)Whole-genome map of strain 168(diagonal lines).The counterparts of the relevant genes in panel A are mapped.(C)Strain development.The positive-selection system is also indicated.The CI repressor in 6234/cI represses P r-neo4642TSUGE ET AL.A NTIMICROB .A GENTS C HEMOTHER .on July 14, 2014 by NANKAI UNIVERSITY/Downloaded fromMATERIALS AND METHODSBacterial strains,phage,plasmids,and media.The bacterial strains,phage,and plasmids used in this study are listed in Table 1.Luria-Bertani (LB)medium was used for the cultivation of Escherichia coli and B.subtilis (30).When necessary,antibiotics were added at the following concentrations:ampicillin,50␮g/ml;chloramphenicol,5␮g/ml;erythromycin,10␮g/ml;tetracycline,20␮g/ml;and neomycin,3␮g/ml.Number 3S (no.3S)medium,containing (per liter)10g of Polypeptone S (Nihon Pharmaceutical Co.),10g of glucose,1g of KH 2PO 4,and 0.5g of MgSO 4·7H 2O (pH 6.8),was used for lipopeptide production in liquid culture.Transformation and DNA manipulation.The B.subtilis strain was trans-formed by the method of Anagnostopoulos and Spizizen (2),as described previously (31).Routine DNA manipulation,E.coli transformation,contour-clamped homogeneous electric-field (CHEF)pulsed-field gel electrophoresis,and Southern hybridization were performed as described previously (32).The CHEF conditions were as follows:field strength,3V/cm;pulse time,45s;and running time,44h.Construction of LPA.A landing pad sequence array (LPA),in which the c I repressor gene and spectinomycin resistance gene are flanked by two landing pad sequences (LPSs),was constructed in plasmid pBR322in E.coli as follows.A 9-kb EcoRI fragment with a downstream edge of the iturin A operon was obtained from recombinant phage ␭69,derived from a lambda DASH II phage library of the RB14chromosome (33),and cloned into the EcoRI site of pBR322to obtain plasmid pBRE9k.An 8-kb HindIII fragment,with an upstream edge of the iturin A operon and a transposon,was obtained from pBRHd8k (33)and ligated to the largest fragment of the products of the EcoRV digestion of pBRE9k,generating the plasmid pBRE4H8.pBRE4H8was digested with both AgeI and CpoI,and then the largest fragment (10kb)obtained was purified by electrophoresis.The obtained fragment was dephosphorylated by alkaline phos-phatase from E.coli (Toyobo,Inc.,Japan),blunted with a DNA blunting kitexpression,resulting in neomycin susceptibility.However,once substitution of the c I gene by transformed DNA takes place,the transformant becomes neomycin resistant.Solid black regions show the tetracycline (labeled pBR)and ampicillin (labeled 322)resistance gene sides of the split pBR322sequence.The genes cat and spc represent the chloramphenicol and spectinomycin resistance genes,respectively.The genes c I and P r-neo indicate the CI repressor gene and the P r promoter-driven neomycin resistance gene,respectively.BEST6234,6234/cI,and 6234/itu also have a P r-neo cassette in their genome;however,this cassette is absent in the RM/iS2and RM/iSd series.The NeoR/S labels at the far right indicate neomycin resistance (R)or sensitivity (S).LPS,landing pad sequence;LPA,LPSs array.TABLE 1.Bacteria,phage,and plasmids used in this studyStrain,phage,or plasmidCharacteristic(s)aReference or sourceE.coli strain JA221F ϪhsdR hsdM ϩtrp leu lacY recA111B.subtilis strains RB14IT ϩSF ϩPL ϩ33168trpC2sfp 0degQ 0,IT ϪSF ϪPL Ϫ20RM125leuB8arg-15⌬SP ␤R(hsdR-cotA ϩ-purB ϩ)202-5sfp 0degQ 0,IT ϪSF ϪPL Ϫ13BEST6225RM125⌬(yvfC-yveP )::P r-neo ,IT ϪSF ϪPL ϪThis study BEST6234RM125⌬(yvfC-yveP )::P r -neo leuB ::pBR322::cat ,Cm r Nm r IT ϪSF ϪPL ϪThis study 6234/cI BEST6234;leuB ::pBR322::[(yxjCDEF RB14-ituD )-(c I-spc )-(ituC-xynD RB14)],Sp r Cm s Nm s IT ϪSF ϪPL ϪThis study 6234/itu BEST6234(host)ϫRB14(DNA);leuB ::pBR322::(yxjCDEF RB14-ituD ϩA ϩB ϩC ϩ-xynD RB14),Nm r Sp sIT ϪSF ϪPL ϪThisstudyRM/iS26234/itu::pMMN6(sfp ϩcat )R[⌬(yvfC-yveP )::P r -neo ]RM125,Cm r Nm s IT ϩSF ϩPL ϩThis study RM/iSd4,-12,-14,and -16RM/iS2::pUC19HP1NmrF(degQ YB8ϩneo ),Cm r Nm r IT ϩSF ϩPL ϩ,clone numbers 4,12,14,and 16This study RM/Sp6RM125::pMMN6(sfp ϩ,cat ),Cm r IT ϪSF ϩPL ϩThis study Phage ␭69Lambda DASH II cloned downstream edge of iturin A operon33Plasmids pBR322Cloning vector,Ap r Tc r30pNEXT4PN-2P r-neo cassette introduction plasmid,Ap r Nm rThis study RSF2124.B.leuB EcoRI fragment of B.subtilis leuB gene cloned into ColiE125pBR322Cmcat cassette cloned into EcoRI site of pBR322,Ap r Tc r Cm r 12pBMAP103-322CA RSF2124.B.leuB-inserted pBRCm in the leuB gene This study pCISP303B#6c I-spc cassette cloned into pBR322,Ap r Tc r Sp r15pBRE9k 9-kb EcoRI fragment from ␭69containing downstream edge of iturin A operon (i.e.,ituC-xynD RB14)cloned in EcoRI site of pBR322,Ap r Tc rThis study pBRHd8k 8-kb HindIII fragment from strain 1006containing promoter region of iturin A operon (i.e.,yxjCDEF RB14-ituDA )inserted into HindIII site of pBR322,Ap r Cm r33pBRE4H8pBRE9k-inserted 8-kb HindIII fragment from pBRHd8k into EcoRV site by replacing small EcoRV fragments,Ap r Cm rThis study pBRE2H4cI (LPA)pBRE4H8-inserted 2.5-kb SmaI fragment containing c I-spc cassette from pCISP303B#6into AgeI-CpoI site by replacing a small fragment,Ap r Sp r This study pMMN6E.coli plasmid containing functional sfp ,Ap r Cm r26pUC19HPINmrFdegQ of strain YB8cloned into E.coli plasmid,Ap r Nm r32aIT,iturin A production;SF,surfactin production;PL,plipastatin production;Ap r ,ampicillin resistant;Tc r ,tetracycline resistant;Cm r or Cm s ,chloramphenicol resistant or sensitive;Nm r or Nm s ,neomycin resistant or sensitive;Sp r or Sp s ,spectinomycin resistant or sensitive;cat ,chloramphenicol resistance gene;neo ,neomycin resistance gene;spc ,spectinomycin resistance gene.V OL .49,2005HORIZONTAL TRANSFER OF ITURIN A OPERON4643on July 14, 2014 by NANKAI UNIVERSITY/Downloaded from(Takara Shuzo Co.,Japan),and then ligated to the SmaI fragment of the c I-spc cassette from pCISP303B#6(15),resulting in plasmid pBRE2H4cI,containing the LPA.Preparation of a recipient strain containing the LPA.The P r-neo cassette introduction plasmid was constructed with a substitutional insertion of an EcoRI fragment of P r-neo from pBEST515C(14)between two NotI sites in pNEXT4 (11).The resulting plasmid,pNEXT4PN-2,was used to transform B.subtilis RM125,a strain168derivative that lacks a restriction/modification system,gen-erating BEST6225.The LPA carried by pBRE2H4cI was inserted into the leuB region in the BEST6225genome as follows.Plasmid pBR322Cm(12),which harbors a chloramphenicol resistance gene(cat)cassette in the EcoRI site of pBR322,was linearized at a unique PvuII site and then inserted into a unique BamHI site in the leuB gene of plasmid RSF2124.B.leuB(25)by using a T4DNA polymerase-based blunting kit.The resulting plasmid,pBMAP103-322CA,was used to transform BEST6225,generating BEST6234,which has pBR322Cm inserted in the genomic leuB region(Fig.1C).BEST6234was transformed with pBRE2H4cI and selected using spectinomycin.Transformants were assayed for chloramphenicol sensitivity by plate replication,and then a chloramphenicol-sensitive strain,which resulted from the double-crossover recombination be-tween pBR322Cm and pBRE2H4cI(Fig.1C),was selected.The obtained strain was designated6234/cI and used as the recipient of the iturin A operon.Iturin A operon transfer.High-molecular-weight whole DNA of strain RB14 was prepared according to a method reported previously(11).One hundred microliters of competent cell culture of6234/cI was mixed with10␮l of1␮g/␮l RB14chromosome.Following incubation at37°C for30min,300␮l of LB medium was added to the culture,which was incubated with gentle agitation at 37°C for3h to allow the expression of neomycin resistance.The culture was then plated on LB plates containing neomycin and incubated at30°C overnight. Colonies that appeared on the plates were streaked on two LB plates,one containing spectinomycin and the other containing neomycin,for the screening of spectinomycin-sensitive colonies.Selected colonies were then picked up with a toothpick and inoculated in25␮l of PCR solution(5U of TaKaRa Ex Taq DNA polymerase[Takara Shuzo,Kyoto,Japan],10␮l of Ex Taq buffer,and8␮l of deoxynucleoside triphosphate solution[2.5mM each])with the primers ITUP1-F(5Ј-AGCTTAGGGAACAATTGTCATCGGGGCTTC-3Ј,positioned from nucleotide15353to15383of the iturin A operon sequence[DDBJ/EMBL/ GenBank accession no.AB050629])and ITUP2-R(5Ј-TCAGATAGGCCGCC ATATCGGAATGATTCG-3Ј,complementary sequence positioned from nucle-otide17326to17355of AB050629),which are able to detect a2-kb region that includes the intergenic sequence between ituA and ituB.The colony PCR con-ditions were as follows:96°C for5min;30cycles of96°C for30s,60°C for30s, and72°C for150s.Introduction of sfp and degQ.The sfp-harboring E.coli plasmid pMMN6(26) was inserted into the genome of6234/itu by Campbell-type insertion.In this transformation,genomic DNA of RM125was simultaneously transferred to remove P r-neo from the yvfC-yveP region for the following experiment.Thus,a chloramphenicol-resistant,neomycin-sensitive colony was selected and desig-nated RM/iS2.This strain harbors pMMN6in the sfp0region.We did not determine in which site the actual insertion in RM/iS2occurred.The degQ YB8-containing E.coli plasmid pUC19HP1NmrF(32)was transformed into the RM/ iS2strain and selected for neomycin.Since pUC19HP1NmrF has three potential sites for Campbell-type insertion in the RM/iS2genome(one is degQ0and the others are ampicillin resistance genes in genomic pBR322and pMMN6),several transformants were selected and designated the RM/iSd series.Quantitative analysis of iturin A,plipastatin,and surfactin.The culture (40ml of no.3S medium,30°C)of the B.subtilis strain was acidified to pH2.0 with12N HCl.Then,the precipitate formed was collected by centrifugation and extracted with methanol.Iturin A,plipastatin,and surfactin in the extracted solution were quantified by reversed-phase high-performance liquid chromatog-raphy(HPLC)using a two-eluent gradient as described previously(32,33).For the detailed composition analysis of the fatty moiety of the␤-amino acid of iturin A,the methanol extract was subjected to another reversed-phase HPLC using one eluent as described previously(8).RESULTSHorizontal transfer of iturin A synthetase operon to strain 168.To horizontally transfer the complete iturin A operon (42kb)in one step,B.subtilis BEST6234,which was con-structed from RM125(13),a derivative of168,was used. Briefly,strain BEST6234has two functional units in its ge-nome.One is a P r-neo cassette,which is a neomycin resistancegene(neo)whose original promoter was replaced with the P r promoter from lambda phage to control neo expression by a c I gene of lambda phage.This cassette was substitutionally in-serted between the NotI sites of yvfC and yveP.The other is pBR322Cm,containing a chloramphenicol resistance gene,in-serted into the BamHI site in the leuB coding sequence(Fig.1). Prior to the horizontal transfer of the iturin A operon,two landing pad sequences(2kb and4kb),corresponding to the two edges of the region to be transferred and bracketing a cassette of the c I repressor gene with the spectinomycin resis-tance gene(spc)between them,were assembled in pBR322in E.coli and then localized in the genomic pBR322sequence in the leuB gene of BEST6234,as shown in Fig.1.The resulting strain,6234/cI,was cultivated to develop competent cells,sup-plemented with whole chromosomal DNA of RB14,and then plated onto neomycin-containing LB plates.About100neo-mycin-resistant transformants were then screened for sensitiv-ity to spectinomycin,and20transformants showing a neomy-cin-resistant and spectinomycin-susceptible phenotype were selected.These transformants were subjected to a colony PCR assay using specific primers for the internal region of the iturin A operon.Two of them yielded a2-kb PCR product,and one of these was chosen for further experiments and named6234/ itu.To confirm the transfer of the iturin A operon to the leuB region of the recipient strain,the whole chromosome of6234/ itu was digested with NotI or SfiI,fractionated by pulsed-field gel electrophoresis,and then subjected to Southern hybridiza-tion analysis using pBRE4H8as the probe.As shown in Fig.2, strong signals from6234/itu were observed because pBRE4H8 has12-kb-long iturin A operon sequences plus the pBR322 sequence.On the other hand,BEST6234showed weak bands, which are due to hybridization between the genomic copy of pBR322and the pBR322sequences of pBRE4H8.The sizes of the observed bands are consistent with the expected values from the whole-genome sequence(Fig.2).6234/itu was thus confirmed to have integrated the42-kb region containing the iturin A operon into the leuB region.Conversion of iturin A operon-transferred strain into an iturin A producer.In a previous study,we demonstrated that the4Ј-phosphopantetheinyl transferase sfp gene is essential for iturin A production as well as for surfactin production in the RB14strain(7,9).Since strain168lacks a functional4Ј-phosphopantetheinyl transferase gene(sfp),a functional sfp gene is introduced by a Campbell-like insertion of the sfp-containing plasmid pMMN6 (26).When sfp was transferred to6234/itu,the resulting strain, RM/iS2,produced iturin A at a concentration of8␮g/ml in no. 3S medium at30°C for120h,while the control strain,6234/itu, and RM/Sp6did not produce iturin A(Fig.3).However,the production of iturin A by RM/iS2was13-fold less than that by RB14(105␮g/ml).In our previous study,we also demonstrated that the intro-duction of the degQYB8gene into the sfpϩstrain168derivative causes plipastatin hyperproduction(32).Analogous with ourprevious study,we examined the effect of degQYB8on iturin A production by the sfpϩstrain RM/iS2.By transformation withplasmid pUC19HP1NmrF,which carries degQYB8,28transfor-4644TSUGE ET AL.A NTIMICROB.A GENTS C HEMOTHER.on July 14, 2014 by NANKAI UNIVERSITY /Downloaded fromFIG.2.Confirmation of iturin A operon transfer.(A)Southern hybridization analysis.SfiI and NotI digestion of BEST6234and 6234/itu genomic DNA prepared in an agarose gel block were fractionated by CHEF pulsed-field gel electrophoresis and subjected to Southern hybridization analysis using pBRE4H8as the probe.Letters correspond to labeled portions of the map in panel B.EtBr,ethidium bromide.(B)Physical map around leuB of iturin A operon-transferred strain.Lettered fragments on the map were actually observed as bands on the photographs in panel A.This figure is drawn on the basis of the whole-genome sequence of strain 168(DDBJ/EMBL/GenBank accession no.AL009126).Diagonal and vertical lines represent B.subtilis 168DNA and B.subtilis RB14DNA,respectively.V OL .49,2005HORIZONTAL TRANSFER OF ITURIN A OPERON 4645on July 14, 2014 by NANKAI UNIVERSITY/Downloaded frommants (RM/iSd series)were obtained,and 4of them,randomly selected (RM/iSd4,-12,-14,and -16),were subjected to further investigation.Southern hybridization analysis was performed to analyze the integration sites of pUC19HP1NmrF in these transformants.In each of them,the integration site of the plasmid was confirmed to be into one of the two ampicillin resistance genes that are in the genomic copies of pBR322(RM/iSd4,-12,and -14)or pMMN6(RM/iSd16)and not into the degQ 0genomic allele (data not shown).This occurred probably because the length of degQ YB8in pUC19HP1NmrF is 0.5kb,shorter than the 1-kb-long ampicillin resistance gene present in the vector portion of pUC19HP1NmrF.In consid-eration of the two integration sites of pUC19HP1NmrF in RM/iS2,all of the four transformants of the RM/iSd series were assayed for iturin A production.The production levels by the four strains ranged from 51to 64␮g/ml,which was six-to eightfold higher than that of RM/iS2(not containing degQ YB8)and one-half that of RB14.The time course of iturin A pro-duction,as well as those of surfactin and plipastatin production in the RM/iSd series strains,was similar to that of iturin A production in RB14(data not shown).A slight difference was observed in the detailed peak com-positions of the iturin A produced,which was caused by a structural difference of the ␤-amino acids in iturin A (Fig.3).Generally,the highest peak of iturin A produced by RB14was peak 1,which corresponds to the n -C 14-␤-amino acid,while that of the RM/iSd series strains was peak 2,which corre-sponds to the anteiso -C 15-␤-amino acid.As well as the differ-ences in iturin A production,the fatty acid compositions of surfactin and plipastatin produced by RB14were different from those seen for the 168derivatives.DISCUSSIONUsing a positive-selection system,we transferred a 42-kb region that contains a 38-kb complete iturin A operon that had no selectable markers.In the previous artificial transfer of the subtilin operon or the bacitracin operon into strain 168,trans-formants were obtained from a donor DNA which had a se-lection marker inserted into the relevant operon at the original host level for the direct selection of the transferred recombi-nant (4,22).It is implied that the donor of this type ofhori-FIG.3.HPLC peak patterns of three lipopeptides (iturin A [IT],surfactin [SF],and plipastatin [PL])(top)and those focused on iturin A (bottom)produced by RM125(A),6234/itu (B),RM/Sp6(C),RM/iS2(D),RM/iSd12as represented by four RM/iSd strains (E),and RB14(F)in no.3S medium at 30°C for 120h.Two distinct HPLC conditions were used to analyze one sample.The top chromatographs were obtained with a two-eluent gradient,while bottom chromatographs were obtained with one eluent.Peaks 1,2,3,and 4correspond to iturin A whose ␤-amino acids are n -C 14-␤-amino acid,anteiso -C 15-␤-amino acid,iso -C 15-␤-amino acid,and n -C 16-␤-amino acid,respectively.4646TSUGE ET AL.A NTIMICROB .A GENTS C HEMOTHER .on July 14, 2014 by NANKAI UNIVERSITY/Downloaded fromzontal transfer is limited to the organisms that have established genetic transformation to introduce the selectable marker in the donor genome prior to horizontal transfer.In our study,we performed the positive-selection system,which employs the CI repressor and a P r promoter-driven neomycin resistance gene (P r-neo)to detect transfer.In this system,the CI repressor in the host strain usually represses P r-neo expression,causing a neomycin-susceptible phenotype.However,once the trans-ferred DNA is substituted for the c I gene,the host strain is no longer able to repress the P r-neo expression and becomes neo-mycin resistant.Since this system does not require any selec-tion marker in the donor DNA,it is suitable for the study of general horizontal transfer(14,15,16).Of the utmost relevance in this study was to identify which genes are required for the conversion of a heterologous host into an antibiotic producer.Several genes are thought to be involved in antibiotic production:for example,regulator genes that control the conditional expression of antibiotic synthetase genes;antibiotic synthetase or structural genes;modification genes;and self-resistance,or efflux pump,genes.In general, these antibiotic production-related genes would form a cluster in the genome.In particular,synthetase genes are usually in one transcriptional unit that is expressed from one promoter in a polycistronic manner.This centered regulation appears to reflect the need for a strict expression control of synthetases. However,Guenzi et al.demonstrated that by inserting a sec-ondary promoter into the surfactin operon,coordinate tran-scription of surfactin synthesis is not necessary for surfactin production(5).From the viewpoint of horizontal transfer,the cluster feature is reasonable,due to the very low frequency of transformation of multiple-donor regions.If these genes are separated into several parts,the simultaneous transfer of sev-eral genes necessary for conversion into a producer may be quite rare.When the cluster feature is a consequence of hor-izontal transfer,it is probable that the transfer of one cluster may be sufficient for conversion into a producer.Indeed,in a previous horizontal transfer of a bacitracin operon to strain 168,the transferred genes contained self-resistance genes and their regulator genes as well as synthetase genes,all of which are members of the cluster(4).In the transfer of the antibiotic subtilin,the transferred segment has not only the structural gene of the antibiotic but also the regulator genes of the struc-tural gene,modification gene,immunity gene,and efflux gene, which are also of the same cluster(17,22,23).Thus,we postulated that the iturin A operon,composed of one tran-scriptional unit encoding four synthetase genes,has the ability to completely convert a heterologous host into an iturin A producer upon transfer of this operon.The6234/itu transformant carrying the iturin A operon was not able to produce iturin A due to the presence of a null sfp allele in this strain.We previously demonstrated that the sfp homologue gene lpa-14is essential for iturin A production in RB14(7,9).When sfp was introduced into the6234/itu strain, the strain produced iturin A,although at a very low level(Fig.3). 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