Patent Application: US-53090300-A

Abstract:
the invention concerns a plasmid having a replication mode which is not of the rcr type , and capable of being transferred in stable form into host lactic acid bacteria belonging at least to three different kinds .

Description:
the strains employed in these studies are listed in table i . they were propagated by conventional microbiological methods in culture media and at temperatures which were recommended for each species . when necessary , erythromycin or ampicillin were added to the culture media at the rate of 5 μg of erythromycin / ml , in the case of the cultures of lactic acid bacteria , and of 150 μg of erythromycin / ml and 75 μg of ampicillin / ml in the case of the escherichia coli cultures . the plasmids which were characterized , used and constructed are described in table ii . the general molecular biological techniques employed have been described by sambrook et al . ( 1989 ). the restriction and modification enzymes were used in accordance with the recommendations of the supplier ( gibco - brl ). the electroporation protocols which were implemented for transforming lactobacillus and pediococcus are described by berthier et al . ( 1996 ) and raya et al . ( 1992 ), respectively . the technique of raya et al . ( 1992 ) was used for transforming leuconostoc ; in this case , the bacteria are firstly cultured in the presence of 0 . 6 g of dl - threonine / l . for preparing the plasmid ptxl1 , a plasmid extract was obtained from the leuconostoc mesenteroides strain y110 using the method described by muriana and klaenhammer ( 1991 ) and then purified from an agarose gel of the ® seaplaque gtg ( fcm bioproducts ) type using the ® prep - a - gene ( bio - rad ) kit . the computer analysis of the nucleic acid and protein sequences which were determined was carried out using the ® gcg ( usa ) software . activity antagonistic to l . ivanovii was detected using the “ drop test ” method . a soft bhi medium ( difco ) agar ( 0 . 6 % agar ), which has been seeded to the extent of 0 . 2 % with a culture of l . ivanovii clip257 which is in the exponential phase of growth , is used to cover a bhi medium which contains 1 . 5 % agar . five μl of a supernatant from a culture are then adsorbed onto the surface of the “ soft ” agar after the latter has solidified . the results are read after 18 hours of incubation at 30 ° c . in order to test the protein nature of the antagonists which are detected , 5 μl of a 10 mg / ml solution of ® proteinase k ( sigma ) are adsorbed at about 2 cm from the site at which the culture supernatant was deposited . the presence of an antagonist is manifested by the appearance of a halo ( circular shape ) of growth inhibition . this inhibition halo will be partially destroyed ( crescent shape ) in the presence of proteinase k if the antagonist is protein in nature . the active molecule produced by the clone dsm20484 ( pfbyc070 ) was purified using the method described by biet et al ., ( 1997 ). its molecular mass was then determined by mass spectrometric analysis ( perkin elmer sciex api 165 ). the strain y110 was isolated in the laboratory ( ibmig , poitier university ) from unpasteurized goat milk samples and selected on account of its antagonistic property with regard to listeria ssp . its taxonomy was determined by the “ deutsche sammlung von mikroorganismen ” ( dsm , germany ); strain y110 is strain of leuconostoc mesenteroides subspecies mesenteroides . its biochemical characteristics are given in table iii . the presence of extra chromosomal ( or plasmid ) dna in strain y110 was determined . this strain harbours five plasmids which are characteristic for the strain . the plasmid which has a size of approximately 2 . 6 kbp and which is designated ptxl1 is the subject of the remainder of the study . the available molecular biological tools are not suitable for carrying out a direct analysis in ln . mesenteroides . molecular analysis of plasmid ptxl1 therefore requires it to be initially cloned into e . coli , which is a species which is routinely employed for this type of analysis . the cloning involves separating plasmid ptxl1 from the other plasmids harboured by strain y110 and fusing plasmid ptxl1 to the cloning vector pbluescript ii sk +( pbsskii +, stratagene ). the plasmids belonging to strain y110 were separated by electrophoresis on an 0 . 8 % ® seaplaque gtg agarose gel . the portion of the gel containing plasmid ptxl1 was isolated and the plasmid was purified . fusing plasmid ptxl1 and the cloning vector pbsskii + requires a preliminary study in order to define the unique restriction sites which are common to these two plasmids . the restriction sites contained in pbsskii + are described in the literature ( stratagene ). in order to search for the unique restriction sites in ptxl1 , a portion of plasmid ptxl1 , obtained as described above , is acted on by a variety of restriction enzymes . it was possible to define unique restriction sites for the restriction enzymes sali , spei and sspi in ptxl1 ( fig1 ). plasmid ptxl1 and the cloning vector pbsskii + were linearized with the restriction enzyme sali and then purified using the ® prep - a - gene dna purification kit . the two plasmids which had thus been linearized were then ligated using t4 dna polymerase and subsequently introduced into the strain e . coli dh5α . the resulting fusion plasmid ptxl1 / pbsskii + was designated pfbyc050 and has a size of 5 . 6 kb . the complete nucleic acid sequence of plasmid ptxl1 was determined by the method of sanger et al . ( 1977 ) using the pfbyc050 construct and employing the ® autoread ( pharmacia ) sequencing kit and an automated dna sequencer ( alf , pharmacia ). the construct pfbyc051 was prepared by the method described for pfbyc050 but using the restriction enzyme spei . this construct was prepared in order to verify the sequence of plasmid ptxl1 at the sali restriction site ( in particular in order to ensure that the complete plasmid had been cloned ). in addition , the complete nucleic acid sequence of plasmid ptxl1 was determined once again using the pfbyc051 construct , thereby enabling the initially obtained sequence to be confirmed . the nucleic acid sequence of ptxl1 was compared with the nucleic acid sequences which are listed in the genbank ( usa ) and embl ( germany ) databases . a region of ptxl1 of approximately 200 bp in size displays strong sequence identity with the pwv01 - type rcr plasmid family ( leenhouts et al ., 1991 ). the remainder of the sequence does not display any identity with the sequences contained in the databases . this homology with the pwv01 - type plasmids would suggest a replication in accordance with the rolling circle model ( gruss and ehrlich , 1989 ). however , components which are essential to this mode of replication are absent from ptxl1 , in particular the gene which encodes the replicase , which is a component which is obligatory for rcr replication . furthermore , it has never been possible to detect a single - stranded form of plasmid ptxl1 , whereas this intermediate type is generally detected in cells which harbour an rcr plasmid . finally , the inventors subsequently demonstrated that the sequence displaying homologies with rcr plasmids was not required for replicating the plasmid ( see fig2 plasmid fby ). the fine analysis of the sequence , in particular the search for open reading frames and inverted sequences and direct repeats , was carried out on the minimum replicon , as described below . the lactic acid bacteria are genetically transformed using the electroporation method ( bio - rad ). the efficiency of this transformation is very dependent on the strains employed . in l . lactis , it usually varies from 10 to 10 6 transformants per μg of dna depending on the receptor strain . after optimizing the transformation technique described by raya et al . ( 1992 ), the inventors were able to select a strain ( ln . mesenteroides dsm20484 ) which displayed an elevated degree of transformability ( approximately 10 5 transformants per μg of dna ). nevertheless , the transformation rate of the strains is still very low . for example , the transformation rate for dsm20484 is less than 1 bacterium transformed per 10 5 bacteria treated . it is therefore absolutely necessary to introduce a marker for selecting the bacteria which are transformed during the electroporation . genes for resistance to antibiotics are normally used as positive selection markers . the gene for resistance to erythromycin , derived from plasmid pamβ1 , is known to function in lactic acid bacteria , in particular in leuconostoc bacteria . consequently , the gene encoding resistance to erythromycin ( eryr ), as obtained from the cloning vector pghost91ss1 ( maguin et al ., 1996 ), was introduced into the unique xbai restriction site in pfbyc050 as shown in fig1 . this construct , termed pfbyc050e , was initially obtained in e . coli and then transferred successfully into dsm20484 . elimination of the pbsskii + replicon plasmid pfbyc050e is therefore operational in the dsm20484 strain . however , in order to be able to confirm that the functions supplied by plasmid ptxl1 are responsible for the replication of this plasmid , the replication functions carried by pbsskii + have to be eliminated . this deletion was effected by cleaving twice with the restriction enzyme pvuii , as shown in fig1 . this double cleavage excises a fragment which constitutes more than 85 % of the pbsskii +, including all its replication functions . the plasmid resulting from this manipulation , termed pfbyc50e , was successfully introduced into the dsm20484 strain , thereby unequivocally demonstrating the ability of the replicon to function in this configuration , which can be likened to a cloning vector . in order to delimit more closely the elements encoded by ptxl1 and required for its replication , various fragments of the plasmid were fused with the vector pbsskii +. a marker gene ( gene for resistance to erythromycin ) was then introduced into these constructs . these plasmids were obtained in e . coli as a consequence of the replication functions supplied by pbsskii + and are shown in table ii and in fig2 . the replication functions supplied by pbsskii + were therefore not eliminated since the inventors have demonstrated above that these functions do not play any role in this configuration . subsequently , these constructs were used for transforming dsm20484 . given the high transformation efficiency obtained for this strain ( from 104 to 105 transformants per μg of dna ), the view was taken that the totality of the replication functions was not present in the cloned ptxl1 fragment when no transformant was obtained . the results which were obtained , and which are shown in fig2 made it possible to define a minimum fragment of 1346 bp in size which was delimited by the sali and sspi restriction sites and which contained all the elements which were required for ptxl1 to replicate autonomously . this fragment , which was termed the minimum replicon , comprises the sequence seq id no . 1 . the literature is full of examples of plasmids which have been isolated from lactic acid bacteria , and which encode proteins , termed replicases , which play an essential role in the replication of these plasmids . orfs were therefore sought in the ptxl1 minimum replicon . two short orfs were found ( fig3 ): orf1 ( nucleotides 1000 to 1341 ) and orf2 ( nucleotides 883 to 640 ). sequences resembling ribosome binding sites ( rbss ), which are required for translating orfs into proteins , were detected in their immediate vicinity . however , no translation initiation codon is located at an appropriate distance from these potential rbss , suggesting that these orfs might not be translated . the protein sequences were nevertheless deduced from these orfs and compared with the protein sequences contained in the genbank ( usa ) and swissprot ( embl , germany ) databases . they are in no way similar to proteins which have already been described . orf1 and orf2 are affected by the cleavage with afliii which is carried out in order to obtain pfbyc068e ( fig2 ). this construct is unable to replicate autonomously , and it is therefore possible that these sequences play a role in the replication of ptxl1 . it is known from the literature that dr and / or ir sequences are involved in the replication of a large number of plasmids . they possess the distinctive characteristic of being able to induce the formation of secondary structures . while some of them have been described , they are not present in ptxl1 . on the other hand , two ir sequences ( referenced ir1 and ir2 , fig3 ) were detected within the ptxl1 minimum replicon . they are capable of generating secondary structures whose calculated δg 0 values are − 10 . 6 and − 22 . 4 kcal / mol . the presence of 3 dr sequences , referenced dr1 , dr2 and dr3 in fig3 is also to be noted the region containing the ir1 and dr1 elements is absent in plasmid pfbyc065 , which does not replicate in dsm20484 . it is therefore possible that these sequences have a role in ptxl1 replication . the majority of cloning vectors of alimentary quality which have been developed for the lactic acid bacteria are derived from plasmids of the rcr type and prove to be unstable in the absence of selection pressure . in order to assess the stability of plasmid pfbyc50e , a clone of dsm20484 harbouring pfbyc50e was isolated on selective mrs culture medium ( containing 10 μg of erythromycin / ml ). this clone was then used to inoculate non - selective mrs media ( not containing any erythromycin ) successively , at 1 : 1000 dilution , so as to multiply the clone over at least 100 generations . the stability of the plasmid is then assessed by the proportion of subclones which have retained the plasmid . this measurement is performed by comparing a dilution of the final culture ( after 100 generations ) which is spread on selective medium and on non - selective medium . the stability of the plasmid is expressed as a percentage and corresponds to : number   of   subclones   obtained   on   selective   medium number   of   subclones   obtained   on   non  -  selective   medium × 100 the results presented in table iv clearly demonstrate the stability of plasmid pfbyc50e as compared with that of the rcr plasmid pfr18 . the host spectrum of the vectors which have been developed from plasmids which are of the theta type and which have been isolated from lactic acid bacteria is generally narrow . attempts were made to transform bacteria belonging to the main lactic acid groups , which are also those most frequently employed industrially , i . e . lactobacillus , pediococcus and leuconostoc , with plasmid pfbyc50e . the strains selected were chosen in accordance with their level of transformability ( lactobacillus sake 23k ; pediococcus acidilactici pac1 . 0 and leuconostoc mesenteroides dsm20484 ) when using the most suitable transformation protocols . as table v shows , it was possible to obtain transformants with all the strains . plasmid pfbyc50e and plasmid pfbyc018e , which is a vector which was constructed from a plasmid of the rcr type derived from ln mesenteroides , were used to assess the efficiency of the transformation of the strain lb . sake 23k by electroporation . these two plasmids possess the same selection marker and were prepared from the same host strain . equivalent quantities of plasmids and the same preparation of electrocompetent bacteria were employed . based on three independent experiments , the transformation yields obtained with plasmid pfbyc50e are approximately 10 4 transformants per μg of dna , that is an efficiency which is approximately 1000 times greater than that obtained with the vector which was constructed from pfr18 . 7 . use of the replicon for producing a bacteriocin in lactic acid bacteria the bacteriocins are antibacterial peptides . in particular , mesentericin y105 is a bacteriocin which is produced by ln mesenteroides y105 and which displays activity against listeria . its genetic determinants have been described ( fremaux et al ., 1995 ), with this study demonstrating that its production in the extracellular medium depends on a specific transport system . in order to avoid the potential problems linked to this transport system when heterologously expressing the bacteriocin , a gene assembly was constructed which is capable of secreting mesentericin y105 ( biet et al ., 1997 ). this is because secretion is a universal transport system in bacteria . this gene assembly was then introduced into a plasmid derived from ptxl1 . in order to enable mesentericin y105 to be secreted , several genetic elements , which function in lactic acid bacteria , were combined as described in fig4 and by biet et al . ( 1997 ). these elements are : 1 . the constitutive promoter p59 , derived from l . lactis ( van der vossen et al ., 1987 ); 2 . the ribosome binding site ( rbs ) which is located upstream of the structural gene for divergicin a ( dvna ), which is produced by carnobacterium divergens lv13 ( worobo et al ., 1995 ); 3 . the part of the structural gene for divergicin a ( dvna ) which encodes the signal peptide ( worobo et al ., 1995 ); 4 . the part of the structural gene for mesentericin y105 ( mesy ) which encodes the mature part of mesentericin y105 ( fremaux et al ., 1995 ); 5 . the mesi gene , which encodes the protein for immunity to mesentericin y105 . construction of the plasmid which enables mesentericin y105 to be secreted construction of pfbyc051e : the construction was performed by introducing a bamhi fragment , encoding resistance to erythromycin , into plasmid pfbyco51 , as described for pfbyc050e in fig1 . construction of pfbyc070 : the dna fragment enabling mesentericin y105 to be secreted ( described in fig4 ) was introduced between the smai and hindiii restriction sites in plasmid pfbyc051e . this construction was performed directly in the ln . mesenteroides dsm20484 strain , given the toxicity of mesentericin y105 when cloned in e . coli . expression of mesentericin y105 by the dsm20484 clone harbouring plasmid pfbyc070 it was possible to use the drop test method to visualize the presence of an active antagonist of l . ivanovii clip257 in culture supernatants derived from the dsm20484 strain harbouring pfbyc070 . the protein nature of the active molecule was demonstrated by its sensitivity to proteinase k . the antagonist was then purified to 99 % purity using the method developed and described by biet et al ., ( 1997 ) and its molecular mass was determined by spectrometry . the value obtained , i . e . 3868 ± 0 . 1 da , corresponds exactly to the theoretical mass calculated from the reduced form of mesentericin y105 . * percentage of clones which were isolated after 100 generations and which harboured the plasmid berthier , f ., m . zagorec , m . champomier - vergès , s . d . ehrlich , and f . morel - deville . 1996 . efficient transformation of lactobacillus sake by electroporation . microbiology . 142 : 1273 - 1279 . biet , f ., j . m . berjeaud , r . w . worobo , y . cenatiempo , and c . fremaux . 1997 . heterologous expression of mesentericin y105 using the general secretion pathway ( gst ) or the dedicated transport system ( dts ) in gram - positive and gram - negative bacteria . soon to be submitted for publication in microbiology . biet f ., cenatiempo , and c . fremaux 1997 , nucleotide sequence analysis and characterization of pfr18 a small cryptic plasmid for leuconostoc mesneteroides ssp . mesenteroides fr52 and its use as a vector . submitted for publication . fremaux , c ., y . héchard , and y . cenatiempo . 1995 . mesentericin y105 gene clusters in leuconostos mesenteroides y105 . microbiology . 141 : 1637 - 1645 . frère , j ., m . novel , and g . novel . 1993 . molecular analysis of the lactococcus lactis subspecies lactis cnrz270 bidirectional theta replicating lactose plasmid pucl22 . molecular microbiology . 10 ( 5 ): 1113 - 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