The assembly is spread on a medium plate M17 agar containing calcium chloride to 10 mM. In all cases where lysis is observed, 5. Results: Of all 45 Lactococcus strains tested, 12 strains are fully resistant to all tested phages 7 L. Conjugal transfer of genetic elements involved in phage resistance - Fetching 5 clones resistant transconjugants to phage.
The S45 strain was obtained from the S strain itself obtained from the strain L. KAY et al Appl. KAY Journal of Bacteriology, The specific protocol used is described below. La souche obtenue est la souche S The strain obtained is the S45 strain. Then the culture is centrifuged.
The whole is centrifuged. The pellet is resuspended in Electric shock is carried out with 25 uF and 2. The pellet is resuspended in the residual liquid, mixed with soft agar containing the culture medium, glucose 0. These 20 strains carry the resistance trait Rap phages and the ability to ferment lactose Lake. B below for their ability to give transconjugants carrying the Rap and Lake characters. Some characteristics of these strains are presented below in Table I below. The selection of transconjugants is done by the ability to ferment lactose and resistance to erythromycin, streptomycin resistance serves to differentiate donor and receptor.
From overnight culture in M17 medium, with an amount of glucose varies according to the strain concerned, the receiving and donor strains, Dairy Sci. Fractions of These experiments include determining the theoretical conjugation efficiency defined as the ratio between the number of transconjugants obtained and the number of receptor cells added to the beginning of the combination of experience and to estimate the effectiveness of equal conjugation theoretical conjugation efficiency multiplied by the inverse of the multiplication factor receptor during conjugation. Then The same experiment is performed with the recipient strain S45 witness, the only difference being that 0.
A mechanism of resistance to a particular phage in the transconjugant is indicated by the difference as between the phage and the transconjugant strain S45 for this phage, and also by the difference in the size of the plaque for the phage. The diameter of the plaque is also measured.
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This parameter gives quantitative information on yield, number of phages obtained after phage multiplication cycle for a phage that infects bacteria. More lysis range is large plus the yield is high. Phages used to highlight the phage resistance mechanisms are phages and group I homology and the phage Group III homology. Of the 20 crosses made first mating cycle, only 6 crosses were transconjugants, 2 with L.
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Table 2 below specifies the efficacy and transfer the ability to ferment lactose, defined as the ratio between the number of transconjugants obtained and the initial number of receptor for 6 above strains. The control consists of the only strain S The most effective donor strain for conjugative transfer of the capacity to ferment lactose is the S96 strain.
On the other hand, extraction was verified from plasmid content of the transconjugants and electrophoretic analysis continuous field see Example 3 the presence in each of these of the plasmid pVA One of the 6 transconjugants, the resulting cross x S88 S45, proved sensitive to the action of streptomycin: it seems that there was transfer of plasmid pVA in principle not conjugative, the S45 strain to the S88 strain.
This transconjugant was eliminated. The main results obtained with the selected transconjugants, by implementing the technique described in A 2 , are collated in Table 3 below, which specifies for these and the S45 strain control the phage title and diameter lysis range. Note also that for the transconjugant S, 13 clones were tested, of which only two the clone 6 had a phage resistance mechanism. This is not necessarily transferred along with the ability to ferment lactose. The latter, probably present on a plasmid is not necessarily on the same plasmid as the phage resistance mechanism, which are conjugative mobilized during the conjugation the plasmid carrying the ability to ferment lactose with formation of a cointegrate.
Highlighting plasmids carrying phage resistance mechanisms. A range of concentrations of novobiocin in M17 medium containing 0.
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Then the contents of the two tubes whose concentration is the closest to the minimum inhibitory concentration MIC , is spread with inoculating loop on M17 medium containing 0. In microtiter plates 96 tanks Incubation is performed in an oven such that it has an exponential growth phase. Les plasmides sont extraits par une technique de lyse alcaline. The plasmids are extracted by an alkaline lysis technique.
The same volume of 2 x M17 medium is added and cultures are left in the oven for 1 hour. They are then centrifuged. The pellet is resuspended in the residual liquid. The whole is centrifuged in centrifuge Eppendorf tubes. The pellet is taken up in NaOH 0. Then are added The aqueous phase is precipitated with 2.
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This DNA is pure enough to be treated with restriction enzymes. KAY et al J. La souche L. The L. Kay et ses collaborateurs Kay and colleagues The new strain obtained was named S The conjugation protocol is that used in Example 1. Les souches S77 et S toutes deux L.
The S77 and S strains both L. Plasmid extraction and analysis by gel electrophoresis performed using the technique described in A 5 did not allow to visualize the plasmids responsible characters Lake and Rap these results are explained by the large size of these plasmids, which can only be analyzed by pulsed field technique described in example 4.
On constate : We aknowledge :. Isolation and characterization of plasmids bearing phage-resistance mechanisms. The tank contained 2. The migration is carried out at V constant, with a pulse time and a migration time determined in advance according to the desired separation of the DNA fragments. A stock of phage lambda purified on cesium chloride is dosed at nm. The "inserts" thus formed are suspended in a solution composed of: 0. Incubation is carried out in an oven until there is an optical density of 0. The pellet is washed one time with 5 ml of SCM buffer 0.
The cells are centrifuged and the pellet is taken up in Immediately, this solution is distributed into plastic matrices provided for this purpose, by aliquot of The "inserts" thus obtained are placed in 2. Two other dialysis are then carried out under the same conditions but without PMSF. The method of McClelland et al is, for its part, to include the cells in the exponential growth phase in agarose low melting point and then to treatment with lysozyme and proteinase K and SDS. Le traitement des cellules au lysozyme avant leur inclusion dans l'agar permet d'augmenter la concentration d'ADN dans l'agarose.
Treatment of cells with lysozyme prior to their inclusion in agar increases the concentration of DNA in agarose. A third or a quarter of an "insert" is placed in an Eppendorf tube in the presence of 1 ml of hydrolysis buffer recommended by the manufacturer. The "inserts" are incubated 24 hours at the temperature optimum of the enzyme activity. It is necessary to use "inserts" prepared according to the technique which allows to have 10 times more DNA see 3 above.
These agarose pieces are then treated with restriction enzymes as an "insert" classic. DNA hydrolyzed with ApaI restriction enzyme from a bacterial strain migrates pulsed field. This technique, derived from a conventional method Hintermann et al. The main interest of the agarose gel electrophoresis pulsed-field technique is to allow separation of large DNA fragments, in particular of more than 20 kb in size.
The total DNA of the transconjugants S clone 1, clone 1 S, S clone 6, clone 1 and S S clone 1 and the recipient strain control , curates and not cures were hydrolyzed using the ApaI restriction enzyme and the large Fragements obtained were analyzed by pulsed field gel electrophoresis on agarose gel. There was thus revealed supernumerary fragments present in the transconjugants and not in the recipient strain.
Supernumerary 72 kb fragment therefore carries a phage resistance mechanism and supernumerary 57 kb fragment Lake character.
Supernumerary fragments of kb, kb, kb, 66 kb and 72 kb are therefore carriers of a phage resistance mechanism.