Patent Publication Number: US-2006019370-A1

Title: Phage-resistant microorganisms and genetic determinants of phage resistance

Description:
CROSS REFERENCE TO RELATED APPLICATIONS  
      This application is a continuation of application Ser. No. 10/031,953, filed on Jan. 24, 2002, which is a National Stage of PCT/EP00/05503 filed on Jun. 5, 2000, the entire contents of which are hereby incorporated by reference. 
    
    
      The present invention relates to novel bacterial strains, the plasmids derived thereof, a gene sequence included in the plasmids, encoding a protein of the phage resistance system, and a method to confer such a resistance to microorganism cultures.  
     INVENTION BACKGROUND  
      Lactic bacteria have a fundamental role in the manufacturing process of milk derivatives, particularly of the fermented milks and cheeses.  
      Their action takes place in the first phases of cheese transformation, inducing some modifications in the milk and/or in the curd, depending on the production rate and amount of lactic acid obtained from lactose fermentation.  
      The acidifying capacity and total enzyme activity of the lactic bacteria starter culture, used in specific dairy manufacturing steps, are fundamental technological parameters, which determine the organoleptic and structural characteristics of the finished product.  
      These metabolic properties are typical of the different species of lactic bacteria and depend, quantitatively, in different ways, on the number and the degree of vitality of the lactic bacteria in the culture and on their multiplication rate in milk and subsequently in curd.  
      A delay or even worse, a block in the growth starter, can cause serious manufacturing problems, impairing the industrial process as a whole.  
      A more frequent cause of slowed down or completely blocked bacterial replication is due to the presence of bacteriophages, viruses able to replicate inside a bacterial cell. Bacteriophages, or phages, are able to recognize and specifically attack the host cell and, in the lytic cycle, to totally destroy it, releasing dozens or hundreds of other virulent phages able to attack other sensitive bacterial cells.  
      The event, first described in 1935, constitutes to date, one of the most serious problems affecting the cheese industry because when the phage infection starts, the possibility arises that production cannot be completed thus determining huge economic loss.  
      The most promising results for overcoming this problem have been achieved using bacterial cultures comprised of:  
      a) strains with different lysotype (phage sensitivity) used in rotation;  
      b) phage-resistant strains.  
      The first solution, at the moment the most adopted by the starter-producing companies, involves considerable organization efforts from culture providers and users, and, in any case, does not permit a complete standardization of the finished product. This is because it is almost impossible to obtain, to isolate, and to produce bacterial strains with identical technological characteristics but with a different lysotype.  
      The second solution can be obtained by different mechanisms.  
      The phage-resistant strains arise spontaneously in sensitive populations following phage attack.  
      The phage-resistance mechanisms outlined in these strains can be grouped into three categories:  
      1. block of adsorption on the bacterial wall and subsequent block of the phage DNA entry in the cytoplasm;  
      2. phage DNA restriction (enzyme cleavage) upon entry inside the bacterial cell;  
      3. interference with the phage DNA duplication mechanisms upon its entrance inside the bacterial cell (abortive infection).  
      However, spontaneous phage-resistant mutants are generally characterized by a scarce technological aptitude, making them unsuitable for industrial use (King W. R. et al., Appl. Environ. Microbiol., 1983, 45, 1481-1485; Steenson L. R. et al., Dairy Sci., 1986, 69, 2227-2236).  
      Recently inventions have been described which deal with the problem of obtaining phage-resistant strains with high technological properties by means of genetic engineering aimed at introducing genes encoding one or more of the above listed mechanisms in starter strains (U.S. Pat. No. 5,824,523 and U.S. Pat. No. 5,538,864).  
      This approach presents some drawbacks from the industrial point of view because these microorganisms and the foods obtained therefrom are included, at least in the European Community, in the “Novel Foods” category, governed by the EC regulation N o . 258/97 of Jan. 27, 1997.  
      The possibility to obtain phage-resistant culture strains by using natural gene transfer techniques is of particular interest.  
      For this purpose it is necessary to select genetic elements able to recombine and mobilize in vivo and to contemporarily confer elevated phage resistance levels.  
     DESCRIPTION OF THE INVENTION  
      The authors of the present application isolated novel  Streptococcus thermophilus  strains showing phage-resistance. These strains were taxonomically, technologically, and genetically characterized. Furthermore, gene elements responsible for the phage resistance were isolated and characterized.  
      The parental strain, called TO03, was deposited at the BCCM™/lmg Bacteria Collection (Gent-Belgio) at N. P-18384, whilst the corresponding phage-resistant mutant, termed B39, was deposited at the same collection at N. P-18383. Both strains represent the first aspect of the invention. These strains contain the gene information conferring phage resistance, but only in the B39 strain, in which the two plasmids—otherwise contained as two distinct molecules in the wild type—are genetically recombined, phage resistance is observed. The B39 phage-resistant phenotype has, for its use in the dairy-milk field, the same properties as the parental strain; in particular, the acidifying rate in milk is the same as that of the TO03 strain, allowing the use of the B39 strain as starter culture in the same dairy processes in which the parental strain is used.  
      The TO03 strain has two plasmids, termed pCRB33 and pCRB63, in which two ORFs (Open Reading Frames) were found presenting high homology with the “s” subunits, known to be involved in the type I restriction and modification mechanisms. In the TO03 strains these two ORF are incomplete and thus inactive. The above plasmids can recombine, creating the pCRB96 plasmid, in which the incomplete and inactive ORFs give, upon recombination, a complete and active “s” subunit.  
      The pCRB33, pCRB63, and pCRB96 plasmids are further embodiments of the invention.  
      Such plasmids are described in the following example 1. In particular, the complete restriction map is provided for each plasmid.  
      In another aspect, the invention relates to the gene determinant responsible for conferring phage resistance. This determinant corresponds to the ORF of pCRB96 plasmid, encoding the above mentioned “s” subunit, whose sequence is reported in SEQ. ID NO. 1. Such a protein, in the type I restriction and modification systems, confers specificity to the restriction enzyme and methylase. The subunit alone is not able to confer phage resistance; actually transferring it to a heterologous host does not automatically determine phenotype change, the presence of gene encoding the two involved enzymes being necessary. The introduction in a host containing a complete type I R/M of a heterologous s subunit, may result in an enhanced phage resistance, comparable to a complete R/M system. In fact, the s subunit alone can change the system specificity, without inhibiting the pre-existing one. Resistance results from the sum of the effects of the two subunits.  
      The gene determinant can be inserted in any suitable plasmid using conventional techniques (for example as described in Maniatis, T. et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, 1982).  
      A plasmid containing the gene determinant for phage resistance described herein is another object of the invention.  
      The non-conjugative plasmids, such as pCRB96, can be used in conjunction with other plasmids able to mediate their transfer. Therefore, in another aspect, the invention relates to the use of plasmids containing the gene determinant of phage resistance described herein, alone or in combination with a conjugative plasmid, to confer phage resistance to bacteria.  
      Another aspect of the invention relates to a host microorganism in which the plasmid containing the phage resistance gene determinant, in accordance with the invention, is able to replicate. The plasmid can be introduced by conventional techniques, such as conjugative transfer and transformation.  
      Besides  Streptococcus thermophilus,  the host can be  Bacillus subtilis  or  Escherichia coli,  or preferably  Lactococcus lactis.  The introduction by transformation of the s subunit in a heterologous host of genera and species other than  Streptococcus thermophilus,  but endowed with the same industrial interest, can be accomplished by vectors suitable for the host itself. The microorganisms containing the plasmid of the invention are particularly useful in the production of milk derivative such as fermented milks and cheeses. They are used as starter cultures, comprised of single or multiple strains.  
     DETAILED DESCRIPTION OF THE INVENTION  
      The  Streptococcus thermophilus  TO03 strain, used as starter culture in the fresh and pasta filata cheeses, proved to be sensitive to the lytic bacteriophage SST3 attack.  
      The TO03 strain was characterized by:  
      taxonomy, obtained by hybridization with a 23 S rRNA specific probe according to Ehmann et al., 1992 ( FIG. 1 );  
      sugar fermentation profile, obtained by API tunnels, which was positive to glucose, fructose, lactose and sucrose;  
      acidifying capacity in sterile skim milk at 37° C., as determined by continuous pH detection ( FIG. 2 );  
      two extra-chromosomal DNA or plasmids, 3.3 and 6.3 kb in size, named pCRB33 and pCRB63, respectively ( FIG. 3 ).  
      The TO03 strain was attacked by the SST3 lytic phage, with a phage/bacterial cell ratio of 1:10 (m.o.i. 0.1). The surviving cells were plated on M17 agar medium. The plates were then incubated under anaerobiosis at 42° C. overnight. This procedure allowed the identification of 40 colonies of the original bacterial population, constituted of 10 9  CFU/ml. The isolated colonies were further assayed for phage resistance.  
      Also the obtained phage-resistant isolates were characterized by their sugar fermentation profile, their acidifying capacity in milk and extra-chromosomal DNA content.  
      Thus the resistant phage isolates could be divided into two groups:  
      Group R1 comprising isolates containing the two plasmids, pCRB33, pCRB63 and the additional pCRB96 plasmid.  
      Group R2 comprising isolates containing the pCRB33 and the additional pCRB96 plasmid.  
      All isolates were taxonomically identical to the parental strain, showed the same sugar fermentation profile, but grew more slowly in milk, making them of low technological usefulness.  
      An isolate of the R2 group was successively grown in liquid M17 at 30° C., a temperature lower than the optimal one. The bacterial population obtained in these conditions was plated on agar medium and further subjected to phage attack.  
      The CFU, that proved to be stably phage-resistant, about 50% of the isolates, were thus again tested in order to evaluate their acidifying rate in milk. Surprisingly, in one of these colonies, termed B39, an acidifying rate similar to that of the parental strain was observed ( FIG. 2 ).  
      The plasmid profile analysis of this strain demonstrated the presence of the 9.6 kb plasmid, termed pCRB96, alone.  
      In order to study the involvement of the pCRB96 plasmid in phage resistance, we proceeded to the curing of the plasmid itself. The obtained clone, termed C48, was free of plasmids and SST3 phage sensitive. In order to obtain a further confirmation of the role of this plasmid, we proceeded to reintroduce it by conjugation into the C48 clone. However the C48 clone had the same phenotypic characters as the B39 strain, making discrimination between donors and recipients after the conjugation impossible. To overcome this drawback we selected a fusidic acid-resistant clone, starting from C48.  
      The C48 strain was UV irradiated and the surviving cells were seeded in M17 agar medium, containing fusidic acid. The aim was to select a mutant to this type of antibiotic in order to use it as a recipient selector after conjugation.  
      The obtained clone was named TO60.  
      The UV rays did not alter the clone SST3 phage sensitivity. Because the pCRB96 plasmid is conjugative, it was necessary to perform a co-transfer of the plasmid itself mediated by pAMβ 1 . This is a conjugative plasmid and it encodes erithromycin resistance. The subsequent steps, aimed at obtaining the plasmid transfer, are illustrated in  FIG. 4  and can be summarized as follows:  
      the pAMβ 1  plasmid was transferred from the donor strain  Lactobacillus lactis  subsp.  lactis  SH4174, to the B39 clone by a first conjugation. The colonies of this donor strain were counted in M17 plates containing glucose and erithromycin, and incubated at 30° C. under anaerobic conditions. The trans-conjugating colonies were selected on M17 plates containing lactose and erithromycin and incubated at 42° C. in anaerobic conditions;  
      in a second conjugation event we used the B39 clone (containing pAMβ 1 ) as the donor strain and the TO60 clone as the recipient strain. In this case, the expected trans-conjugants were resistant to erithromycin and also to fusidic acid. All the colonies with these characteristics were assayed for phage resistance.  
      Some of them (11 over a total of 350 assayed colonies) proved to be phage resistant. The plasmid content of these clones demonstrated the contemporary presence of the pAMβ 1  and the pCRB96 plasmids.  
      The curing experiments and the pCRB96 plasmid transfer have thus demonstrated that the phage resistance of the clones isolated starting from the TO03 strain was linked to the presence of such plasmid.  
      The phage-resistant clones did not show full resistance against the SST3 phage, but the number of the phage plaques obtained on the plate was reduced compared to the PFU number obtained with the TO03 strain of at least 2 log. In order to identify the type of phage resistance involved, we performed cross-hybridizations between the SST3 phage propagated in the TO03 strain, and the same phage propagated in the B39 strain.  
      For convenience the latter phage was called SST39.  
      As demonstrated in table 1 the phage titrated on the sensitive strain, showed a higher titre than that obtained when the host strain was B39. On the other hand the phage multiplied on the B39 strain produced the same PFU/ml when titrated on the two strains.  
                               TABLE 1                                       SST3 phage   SST39 phage           Host strain   PFU/ml   PFU/ml                          TO03   2 × 10 8     3 × 10 7             B39   3 × 10 6     3 × 10 7             C48   2 × 10 8     3 × 10 7                        
 
      Table 2 outlines the results of the titrations obtained on the sensitive strains and on the strain resistant to the SST39 propagated on TO03 and B39, respectively. It is possible to observe that the SST39 phage lost its ability to attack with high efficiency the phage resistant strain upon propagation on the TO03 strain. This behavior is typical of restriction and modification systems. We thus attributed a role in an R/M system to the pCRB96 plasmid.  
                               TABLE 2                                       SST39/TO03 phage   SST39/B39 phage           Host strain   PFU/ml   PFU/ml                          TO03   2 × 10 7     3 × 10 8             B39   3 × 10 5     3 × 10 8             C48   2 × 10 7     3 × 10 8                        
 
 Plasmid DNA Analysis 
 
      The restriction map analysis of the pCRB33, pCRB63, and pCRB96 plasmids suggested that the latter could be the result of the integration of the two plasmids originally located in the TO03 phage-resistant strain. The first confirmations were obtained in DNA/DNA hybridization experiments. With the latter method, in fact, signals were obtained when the pCRB96 plasmid was hybridized to probes comprised of pCRB33 and pCRB63 fragments.  
      In order to obtain further evidence of the integration event we conducted cloning and sequencing of the pCRB33 plasmid. The plasmid graphic representation is shown in  FIG. 5 . From the sequence analysis we could localize two complete ORFs, indicated in  FIG. 5  ORF 1  and ORF 2 , respectively.  
      The ORF 2  showed a high homology (87%) with respect to the sequence of the RepA protein, located on the pST1 plasmid of the  Streptococcus thermophilus  ST strain (deposit number GENEBANK X65856). A termination region was found downstream from the coding region, constituted of repeated sequences with 86% homology compared to that of the above mentioned RepA. The ORF 1  shows homology in some portions to many s subunits of type I restriction and modification system.  
      The higher homologies were found in a 133 bp region whose sequence contains one of the two conserved motifs from the s subunits. The same homology to the s subunits was found also in a region of 153 bp outside the ORF 1  and 473 bp distant from the end of the first region. These two regions can be considered two repeated direct sequences. We named the first 133 bp sequence DR1, and the second 153 bp sequence DR2.  
      Using a primer set designed on the sequence of the two DR found in pCRB33, the pCRB96 plasmid was amplified by PCR. The amplification product was made up of two fragments of 3.3 and 6.3 kb, respectively. These results induced us to hypothesize that the two DRs would be located in the integration region.  
      In summary pCRB33 contains a gene encoding a protein responsible for replication and probably two DR involved in the integration event.  
      We thus cloned and determined the nucleotide sequence of the pCRB63 plasmid. The sequence analysis did not show any homology to the known genes encoding for phage resistance apart from the ORF 1 , whose sequence showed a region endowed with high homology with respect to the different s subunit of type I restriction and modification systems, exactly as previously demonstrated for the pCRB33 plasmid ORF 1 . The revealed homology also in this case concerned the conserved motifs of the s subunits. Also in the pCRB63 plasmid are present the two DR, exactly as for pCRB33.  
      The pCRB96 plasmid was thus fully sequenced and resulted to be a co-integration product of the pCRB33 and pCRB63 plasmids.  
      The two regions in which the integration takes place are those delimited by the two DRs, whilst the region between them is where the two plasmids are cut and joined together. In fact, in pCRB96 there are 2 regions in which DR1 and DR2 are present. The pCRB33 DR1 is, in this case, associated with the pCRB63 DR2, whilst the smaller plasmid DR2 is associated with the pCRB63 DR1.  
      The ORF 1  ( FIG. 2 ), with high homology to the s subunits of type 1 R/M systems, in particular to the s subunit isolated from  Lactobacillus lactis  IL1403 and the s subunit of LldI of  Lactococcus lactis  subsp.  cremoris  (deposit number GENEBANK AF 034786 and U90222) which are 55% homologous, was located in one of the integration regions. In the case of pCRB96, the sequence, homology, and phenotype demonstrate that the s subunit encoding gene is complete and functional.  
      On the contrary, pCRB33 and pCRB63 contain ORFs with homology to genes encoding the s subunit of type I R/M systems, but they are incomplete and thus not functional. Only pCRB96, by means of integration, has the functional gene, whose sequence is the sum of pCRB33 ORF 1  and pCRB63 ORF 1  portions. The sequence of the entire pCRB96 s subunit is reported in Seq ID N o . 1. 
    
    
     BRIEF DESCRIPTION OF THE FIGURES  
       FIG. 1 : Taxonomic identification of the TO03 and B39 strains  
      Probe used: CATGCCTTCGCTTACGCT  
      Probe and hybridization protocol according to Ehrmann et al. (1992) “Species-specific oligonucleotide probe for the identification of  Streptococcus thermophilus ”, Systematic and Applied Microbiology, 15, 453-455.  
      Hybridization results:  
      A 1 : Model strain of the species  Streptococcus thermophilus  DSM 20617 (positive control);  
      A 2 : DNA extracted from  Lactobacillus helveticus  ATCC 15009 (negative control);  
      B 1 : DNA extracted from  Streptococcus thermophilus  B39;  
      B 2 : DNA extracted from  Streptococcus thermophilus  TO03.  
      Positive signals were obtained from the reference strain DSM 20617 and from the TO03 and B39 strains under investigation, thus confirming to be  Streptococcus thermophilus  species.  
       FIG. 2 : Acidification curve: 1% inoculum, sterile skim milk, 37° C.  
       FIG. 3 : Plasmid profile of the TO03 (well  2 ) and B39 (well  4 ) strains.  
       FIG. 4 : Scheme of the conjugations performed in order to co-transfer pAMβ1 and pCRB96 plasmids.  
       FIG. 5 : Schematic representation of pCRB33.  
      ORF 1 : ORF located from nt 411 to nt 1308, corresponding to 299 amino acids, of the appended pCRB33 nucleotide sequence. This ORF shows homology to different subunits of type I restriction systems.  
      ORF 2 : ORF located from nt 2070 to nt 2960. This ORF shows 87% homology to the pST1 RepA (acc. num. X65856). 
    
    
      The following examples illustrate the invention in further detail.  
     EXAMPLE 1  
     Characterization of the pCRB33, pCRB63, and pCRB96 Plasmids  
      The following tables report the restriction profiles of the pCRB33, pCRB63, and pCRB96 plasmids.  
               TABLE 3                          pCRB33 plasmid, 3375 base pairs:                                         Recognition       Enzyme name   N o . cuts   Site positions   sequence                                     AccI       1233   gt/mkac       AluI       134 138 161 466 622 1137 1670   ag/ct               1714 2259 2421 2745 2932 2982       AseI       2055   at/taat       AsnI       2055   at/taat       AvaII       2199 2213 2227   g/gwcc       DdeI       157 1144 1215 1671 1695 2159   c/tnag               2203 2255       DpnI       985 2253   ga/tc       DraI       2828 3024   ttt/aaa       EcoRI       39 1804   g/aattc       EcoRV       636   gat/atc       FokI       259   ggatg       HaeIII       2157 2173 3092 3275   gg/cc       HindIII       1712 2257 2743   a/agctt       HinfI       1223 1697 2006 2102 2341   g/antc       HpaII       202 2174   c/cgg       KpnI       1565   ggtac/c       MaeI       135 139 669 733 784 1877 2848   c/tag               2854 3084 3269 3277       MaeII       1081 1201 1468 2887 3049 3174   a/cgt               3306       MaeIII   8   712 1082 1177 1752 2499 2872   /gtnac               3045 3307       MboI   2   983 2251   /gatc       MseI   23   8 167 237 243 287 335 358 518   t/taa               616 1095 1329 1904 1996 2055               2195 2378 2447 2636 2741 2827               2965 3023 3180       NotI   1   3090   gc/ggccgc       PstI   1   3101   ctgca/g       PvuII   1   2421   cag/ctg       Sau3AI   2   983 2251   /gatc       Sau96I   3   2199 2213 2227   g/gncc       SpeI   2   1876 3083   a/ctagt       TaqI   12   1 632 638 763 1254 1735 1849   t/cga               2004 2111 2985 3104 3372       XbaI   1   732   t/ctaga                  
 
 wherein: 
 
      r=a or g; k=g or t; h=a or c or t; d=a or g or t; y=c or t; s=c or g; b=c or g or t; n=a or c or g or t; m=a or c; w=a or t; v=a or c or g.  
      The following endonucleases did not cleave the pCRB33 sequence:  
      ApaI, Ava I, BamHI, BclI, BglI, CfoI, ClaI, HaeII, HincII, HindII, HpaI, NcoI, PvuI, SacI, SacII, SalI, SmaI, SohI, XhoI, XmaI.  
               TABLE 4                          pCRB63 plasmid, 6148 base pairs:                             Enzyme           Recognition       name   N o . cuts   Site positions   sequence                                     AccI   4   165 312 2451 4102   gt/mkac       AluI   20   692 850 1358 1487 1518 1965 2264   ag/ct               2270 2471 2625 2913 3155 3667               3717 3744 3793 4038 4270 4648               5530       AseI   2   81 5115   at/taat       AsnI   2   81 5115   at/taat       AvaI   1   2702   c/ycgrg       AvaII   2   200 4368   g/gwcc       CfoI   8   1203 1897 2046 2609 2909 4097   gcg/c               4442 4483       ClaI   2   276 322   at/cgat       DdeI   11   61 120 346 1930 2130 3953 4251   c/tnag               4276 4581 5002 5904       DpnI   7   363 1029 1067 4890 5032 5718   ga/tc               5917       DraI   5   922 2773 4304 5261 5483   ttt/aaa       EcoRI   2   1569 3413   g/aattc       EcoRV   2   274 2510   gat/atc       FokI   6   3875 3905 3911 4961 5500 5565   ggatg       HaeII   2   1898 4098   rgcgc/y       HaeIII   3   2189 3001 5891   gg/cc       HincII   2   2452 3706   gty/rac       HindII   2   2452 3706   gty/rac       HindIII   1   2623   a/agctt       HinfII   12   124 324 344 380 612 1434 2146   g/antc               2453 3955 4255 5355 5448       HpaII   3   194 3396 5812   c/cgg       KpnI   1   580   ggtac/c       MaeI   16   609 1580 1916 2271 2540 2721   c/tag               2910 3597 4079 4122 4184 4622               4985 5231 5272 5945       MaeII   19   88 211 355 877 1529 2092 2318   a/cgt               2545 2827 2877 3283 3349 3367               3624 3686 3887 3987 4192 6016       MaeIII   12   240 341 1094 1260 2011 2273 2878   /gtnac               3350 3590 4056 4773 5379       MboI   7   361 1027 1065 4888 5030 5716   /gatc               5915       MseI   44   81 99 147 207 375 921 1062 1151   t/taa               1481 2219 2410 2711 2772 3020               3092 3204 3221 3443 3491 3544               3644 3694 3750 3825 3937 3993               4003 4141 4229 4303 4407 4530               4550 4750 4847 5015 5115 5260               5455 5482 5548 5626 5630 5666       PvuI   1   364   cgat/cg       SacI   1   3795   gagct/c       SacII   1   2020   ccgc/gg       SaII   1   2450   g/tcgac       Sau3AI   7   361 1027 1065 4888 5030 5716   /gatc               5915       Sau96I   3   200 4368 5889   g/gncc       SpeI   1   4183   a/ctagt       SphI   1   5743   gcatg/c       TaqI   13   276 322 2377 2451 2465 2473 2620   t/cga               2808 2866 3377 3726 4666 5921       XbaI   4   608 2720 4078 4121   t/ctaga                  
 
      r=a or g; k=g or t; h=a or c or t; d=a or g or t; y=c or t; s=c or g; b=c or g or t; n=a or c or g or t; m=a or c; w=a or t; v=a or c or g.  
      The following endonucleases did not cleave the pCRB33 sequence:  
      ApaI, BamHI, BclI, BglI, HpaI, NcoI, PstI, PvuII, SmaI, XhoI, XmaI.  
               TABLE 5                          pCRB96 plasmid, 9515 base pairs:                             Enzyme           Recognition       name   N o . cuts   Site positions   sequence                                     AccI   5   1150 1297 3436 5087 7381   gt/mkac       AluI   33   134 138 161 466 622 1677 1835   ag/ct               2343 2472 2503 2950 3249 3255               3456 3610 3898 4140 4652 4702               4729 4778 5023 5255 5633 6515               7284 7815 7859 8402 8564 8888               9072 9122       AseI   3   1066 6100 8200   at/taat       AsnI   3   1066 6100 8200   at/taat       AvaI   1   3687   c/ycgrg       AvaII   5   1185 5353 8342 8356 8370   g/gwcc       CfoI   8   2188 2882 3031 3594 3894 5082   gcg/c               5427 5468       ClaI   2   1261 1307   at/cgat       DdeI   18   157 1046 1105 1331 2915 3115   c/tnag               4938 5236 5261 5566 5987 6889               7363 7816 7840 8302 8346 8398       DpnI   9   1348 2014 2052 5875 6017 6703   ga/tc               6902 7132 8396       DraI   7   1907 3758 5289 6246 6468 8969   ttt/aaa               9164       EcoRI   4   39 2554 4398 7949   g/aattc       EcoRV   3   636 1259 3495   gat/atc       FokI   7   259 4860 4890 4896 5946 6485   ggatg               6550       HaeII   2   2883 5083   rgcgc/y       HaeIII   7   3174 3986 6876 8300 8316 9232   gg/cc               9415       HincII   2   3437 4691   gty/rac       HindII   2   3437 4691   gty/rac       HindIII   4   3608 7857 8400 8886   a/agctt       HinfII   17   1109 1309 1329 1365 1597 2419   g/antc               3131 3438 4940 5240 6340 6433               7371 7842 8151 8245 8484       HpaII   5   202 1179 4381 6797 8317   c/cgg       KpnI   2   1565 7710   ggtac/c       MaeI   27   135 139 669 733 784 1594 2565   c/tag               2901 3256 3525 3706 3895 4582               5064 5107 5169 5607 5970 6216               6257 6930 8022 8988 8994 9224               9409 9417       MaeII   26   1073 1196 1340 1862 2514 3077   a/cgt               3303 3530 3812 3862 4268 4334               4352 4609 4671 4872 4972 5177               7001 7228 7349 7614 9027 9189               9314 9446       MaeIII   21   712 1225 1326 2079 2245 2996   /gtnac               3258 3863 4335 4575 5041 5758               6364 7229 7325 7897 8415 8642               9012 9185 9447       MboI   9   1346 2012 2050 5873 6015 6701   /gatc               6900 7130 8394       MseI   68   8 167 237 243 287 335 358 518   t/taa               616 1066 1084 1132 1192 1360               1906 2047 2136 2466 3204 3395               3696 3757 4005 4077 4189 4206               4428 4476 4529 4629 4679 4735               4810 4922 4978 4988 5126 5214               5288 5392 5515 5535 5735 5832               6000 6100 6245 6440 6467 6533               6611 6615 6651 7242 7291 7475               8049 8141 8200 8338 8521 8590               8779 8884 8968 9105 9163 9320       NotI   1   9230   gc/ggccgc       PstI   1   9241   ctgca/g       PvuI   1   1349   cgat/cg       PvuII   1   8564   cag/ctg       SacI   1   4780   gagct/c       SacII   1   3005   ccgc/gg       SaII   1   3435   g/tcgac       Sau3AI   9   1346 2012 2050 5873 6015 6701   /gatc               6900 7130 8394       Sau96I   6   1185 5353 6874 8342 8356 8370   g/gncc       SpeI   3   5168 8021 9223   a/ctagt       SphI   1   6728   gcatg/c       TaqI   23   1 638 1261 1307 3362 3436 3450   t/cga               3458 3605 3793 3851 4362 4711               5651 6906 7402 7880 7994 8149               8254 9125 9244 9512       XbaI   5   732 1593 3705 5063 5106   t/ctaga                  
 
      r=a or g; k=g or t; h=a or c or t; d=a or g or t; y=c or t; s=c or g; b=c or g or t; n=a or c or g or t; m=a or c; w=a or t; v=a or c or g.  
      The following endonucleases did not cleave the pCRB96 sequence:  
      ApaI, BamHI, BclI, BglI, HpaI, NcoI, SmaI, XhoI, XmaI.  
     EXAMPLE 2  
     Coniugative Transfers  
      1. In the first conjugation cycle, the cultures of the donor strain  Lactobacillus lactis  SH4174 containing the pAMβ1 plasmid encoding erithromycin resistance, and cultures of the recipient strains  Streptococcus thermophilus  B39, containing the pCRB96 plasmid, are grown.  
      2. In the second conjugation cycle, the donor and recipient strain cultures of  Streptococcus thermophilus  B39 (pAMβ1) containing the pAMβ1 plasmid and the pCRB96 plasmid, and of  Streptococcus thermophilus  TO60, plasmid-free and resistant to fusidic acid, respectively, are grown.  
      Procedure  
      Equal volumes are taken from both cultures and mixed.  
      From this mix 0.2 ml are taken, placed on a Petri dish containing M17 medium without any selection agent, uniformly plated, and incubated from 6 to 30 hours.  
      The bacterial cells grown on this medium are harvested with 1 ml of saline and then appropriate decimal dilutions, in culture media (see table) suitable for selecting donor and recipient strains, and possible trans-conjugants present in the conjugation mix, were seeded on plate.  
               TABLE 6                          First conjugation cycle.                             STRAINS   SELECTION               DONORS   SH4174   30° C., 50 μg/ml erithromycin,               glucosate M17 medium       RECIPIENTS   B39 (pCRB96)   42° C., lactosate M17 medium       TRANS-   B39 (pCRB96-   42° C., lactosate M17       CONJUGATES   pAMβ1)   medium, 10 μg/ml               erithromycin                  
 
     
       
         
           
               
             
               
                 TABLE 7 
               
             
            
               
                   
               
               
                   
               
               
                 Second conjugation cycle. 
               
            
           
           
               
               
               
            
               
                   
                 STRAINS 
                 SELECTION 
               
               
                   
               
               
                 DONORS 
                 B39 (pCRB96-pAMβ1) 
                 42° C., lactosate M17 
               
               
                   
                   
                 medium, 10 μg/ml 
               
               
                   
                   
                 erithromycin 
               
               
                 RECIPIENTS 
                 TO60 
                 42° C., lactosate M17 
               
               
                   
                   
                 medium, 10 μg/ml fusidic 
               
               
                   
                   
                 acid 
               
               
                 TRANS- 
                 TO60 (pCRB96-pAMβ1) 
                 42° C., lactosate M17 
               
               
                 CONJUGATES 
                   
                 medium, 10 μg/ml 
               
               
                   
                   
                 erithromycin, 10 μg/ml 
               
               
                   
                   
                 fusidic acid 
               
               
                   
               
            
           
         
       
     
      Two types of trans-conjugants are expected from the second conjugation cycle:  
      one containing only the conjugative pAMβ1 plasmid and one containing both pCRB96 and pAMβ1 plasmids.  
      Results  
               TABLE 8                          First conjugation cycle.                                         Colony Forming               STRAINS   Units                       DONORS   SH4174        10 7             RECIPIENTS   B39 (pCRB96)        10 9             TRANS-   B39 (pCRB96-   1000           CONJUGATES   pAMβ1)                      
 
     
       
         
           
               
             
               
                 TABLE 9 
               
             
            
               
                   
               
               
                   
               
               
                 Second conjugation cycle. 
               
            
           
           
               
               
               
               
            
               
                   
                   
                 STRAINS 
                 Colony Forming Units 
               
               
                   
                   
               
               
                   
                 DONORS 
                 B39 (pCRB96- 
                 10 8   
               
               
                   
                   
                 pAMβ1) 
               
               
                   
                 RECIPIENTS 
                 TO60 
                 10 8   
               
               
                   
                 TRANS- 
                 TO60 (pAMβ1) 
                 10 4  (pAMβ1) 
               
               
                   
                 CONJUGANTS 
                 TO60 (pCRB96- 
                 50 (pCRB96-pAMβ1) 
               
               
                   
                   
                 pAMβ1) 
               
               
                   
                   
               
            
           
         
       
     
      In the second conjugation cycle only 50 Colony Forming Units were subjected to co-mobilization of the pCRB96 plasmid by the pAMβ1 plasmid.  
      The phage resistance levels of the TO60 trans-conjugants (pCRB96-pAMβ1) were identical to those of B39.  
      The phage sensitivity levels of TO60 and TO60 (pAMβ1) were identical to those of TO03.