PHAGE-RESISTANT MICROORGANISMS AND GENETIC DETERMINANTS OF PHAGE RESISTANCE

Abstract: There are described bacterial strains, plasmids derived thereof, a gene sequence included in the plasmids encoding a phage resistance system protein, and a method to confer phage resistance to microorganism cultures.

Description: PHAGE-RESISTANT MICROORGANISMS AND GENETIC DETERMINANTS OF PHAGE RESISTANCE 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 bactehophages, viruses able to replicate inside a bacterial cell. Bactehophages, 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 (patents US-5,824,523 and US- 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°. 258/97 of January, 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 B39 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 N. 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-i. 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 pAMβi 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-i) 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-i 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 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 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 ORF1 and ORF2, respectively. The ORF2 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 ORF1 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 ORF1 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 ORF1 , 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 ORF1. 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 ORF1 (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 IL-1403 and the s subunit of Lld\ 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 ORF1 and pCRB63 ORF1 portions. The sequence of the entire pCRB96 s subunit is reported in Seq ID N°. 1. BRIEF DESCRIPTION OF THE FIGURES Figure 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: A1 : Model strain of the species Streptococcus thermophilus DSM 20617 (positive control); A2: DNA extracted from Lactobacillus helveticus ATCC 15009 (negative control); B1 : DNA extracted from Streptococcus thermophilus B39; B2: 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. Figure 2: Acidification curve: 1 % inoculum, sterile skim milk, 37°C. Figure 3: Plasmid profile of the TO03 (well 2) and B39 (well 4) strains. Figure 4: Scheme of the conjugations performed in order to co-transfer pAMβl and pCRB96 plasmids. Figure 5: Schematic representation of pCRB33. ORF1 : 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. ORF2: ORF located from nt 2070 to nt 2960. This ORF shows 87% homology to the pST1 RepA (ace. num. X65856). The following examples illustrate the invention in further detail. EXAMPLE 1 Characterization of the DCRB33. PCRB63. and DCRB96 plasmids. The following tables report the restriction profiles of the pCRB33, pCRB63, and pCRB96 plasmids. Table 3: pCRB33 plasmid, 3375 base pairs: (continued) Table 3 (continued) 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: Apal, Ava I, BamHI, Bell, Bgll, Cfol, Clal, Haell, Hindi, Hindll, Hpal, Ncol, Pvul, Sad, Sacll, Sail, Smal, Sohl, Xhol, Xmal. Table 4: pCRB63 plasmid, 6148 base pairs: (continued) Table 4 (continued) (continued) Table 4 (continued) 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: Apal, BamHI, Bell, Bgll, Hpal, Ncol, Pstl, Pvull, Smal, Xhol, Xmal. Table 5: pCRB96 plasmid, 9515 base pairs: (continued) Table 5 (continued) (continued) Table 5 (continued) (continued) Table 5 (continued) 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: Apal, BamHI, Bell, Bgll, Hpal, Ncol, Smal, Xhol, Xmal. EXAMPLE 2 Conjugative transfers 1. In the first conjugation cycle, the cultures of the donor strain Lactobacillus lactis SH4174 containing the pAMβl 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 (pAMβl ) containing the pAMβl 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. Table 7: Second conjugation cycle. Two types of trans-conjugants are expected from the second conjugation cycle: one containing only the conjugative pAMβl plasmid and one containing both pCRB96 and pAMβl plasmids. Results Table 8: First conjugation cycle. Table 9: Second conjugation cycle. In the second conjugation cycle only 50 Colony Forming Units were subjected to co-mobilization of the pCRB96 plasmid by the pAMβl plasmid. The phage resistance levels of the TO60 trans-conjugants (pCRB96- pAMβl ) were identical to those of B39. The phage sensitivity levels of TO60 and TO60 (pAMβl ) were identical to those of TO03.

Claims: CLAIMS 1. Streptococcus thermophilus strains deposited at BCCM/LMG (Gent/Belgium) P-18383 and P-18384. 2. Nucleic acid molecule having the sequence Seq. ID N°. 1. 3. Plasmid containing the nucleic acid molecule of claim 2. 4. Plasmid obtainable from a culture of Streptococcus thermophilus strain N. P-18384 according to claim 1 , having 3375 base pairs and a single restriction site for each Accl, Asel, Asnl, EcoRV, Fokl, Notl, Pstl, Pvull, Xbal nuclease. 5. Plasmid obtainable from a culture of Streptococcus thermophilus strain N. P-18384 according to claim 1 , having 6184 base pairs and a single restriction site for each Aval, Hindlll, Kpnl, Pvul, Sad, Sacll, Sail, Spel, Sphl nuclease. 6. Plasmid obtainable from a culture of Streptococcus thermophilus strain N. P-18383 according to claim 1 , having 9515 base pairs and a single restriction site for each Aval, Notl, Pstl, Pvul, Pvull, Sad, Sacll, Sail, Sphl nuclease. 7. Microorganism containing the plasmid of claims 3-6. 8. Microorganism according to claim 7, selected from Streptococcus thermophilus, Lactobacillus lactis, Bacillus subtilis, Escherichia coli, Lc. lactis subsp. lactis, Lc. lactis subsp. diacetylactis, Lc. Cremoris, Lb. delbruckeii subsp. Lactis, Lb. delbruckeii subsp. bulgahcus, Lb. delbruckeii subsp. delbruckeii, Lb. Helveticus, Lb. casei. 9. Starter culture for milk fermentation, comprising a microorganism of claims 1 , 7 or 8. 10. Use of a plasmid of claims 3-6, alone or in combination with a conjugative plasmid, for conferring phage resistance to a bacterium.

Inventors: BOTTAZZI VITTORIO, CALLEGARI MARIA LUISA, DI LORENZO SIMONA, MOGNA GIOVANNI, MORELLI LORENZO, STROZZI PAOLO, BOTTAZZI, VITTORIO, CALLEGARI, MARIA, LUISA, DI LORENZO, SIMONA, MOGNA, GIOVANNI, MORELLI, LORENZO, STROZZI, PAOLO
Applicants: ANIDRAL S R L, BOTTAZZI VITTORIO, CALLEGARI MARIA LUISA, LORENZO SIMONA DI, MOGNA GIOVANNI, MORELLI LORENZO, STROZZI PAOLO, ANIDRAL S.R.L., BOTTAZZI, VITTORIO, CALLEGARI, MARIA, LUISA, DI LORENZO, SIMONA, MOGNA, GIOVANNI, MORELLI, LORENZO, STROZZI, PAOLO
Application year: 2000
Main classification: C12N1
IPC codes: A23C19, C07K14, C12N15