Identification of Four Phage Resistance Plasmids from Lactococcus lactis subsp. cremoris HO2

The bacteriophage-host sensitivity patterns of 16 strains of Lactococcus lactis originally isolated from a mixed strain Cheddar cheese starter culture were determined. Using phages obtained from cheese factory whey, four of the strains were found to be highly phage resistant. One of these isolates, Lactococcus lactis subsp. cremoris HO2, was studied in detail to determine the mechanisms responsible for the phage insensitivity phenotypes. Conjugal transfer of plasmid DNA from strain HO2 allowed a function to be assigned to four of its six plasmids. A 46-kb molecule, designated pCI646, was found to harbor the lactose utilization genes, while this and plasmids of 58 kb (pCI658), 42 kb (pCI642), and 4.5 kb (pCI605) were shown to be responsible for the phage resistance phenotypes observed against the small isometric-headed phage 712 (936 phage species) and the prolate-headed phage c2 (c2 species). pCI658 was found to mediate an adsorption-blocking mechanism and was also responsible for the fluffy pellet phenotype of cells containing the molecule. pCI642 and pCI605 were both shown to be required for the operation of a restriction-modification system.     

Physical analysis of in vivo pCI301 fusion plasmids in Lactococcus lactis subsp. lactis

Abstract In vivo fusion plasmids identified following conjugative mobilization of pCI301, the 75-kilobase (kb) lactose-proteinase plasmid of Lactococcus lactis subsp. lactis UC317, were characterized. These plasmids (95 kb) were generated from fusion-deletion events involving pCI301 and the 38-kb UC317-derived cryptic plasmid, pCI303. Recombinant plasmids were separable into distinct classes based on their associated phenotypes and restriction maps. The formation of pCI301: : pCI303 composite plasmids within strain UC317 was also demonstrated.     

Cloning and partial sequencing of the proteinase gene complex from Lactococcus lactis subsp. lactis UC317.

The proteinase genes from Lactococcus lactis subsp. lactis UC317 were identified on a plasmid, pCI310, which is a deletion derivative of a cointegrate between pCI301, the 75 kb Lac Prt plasmid from UC317 and the 38.5 kb cryptic plasmid from that strain. The prt genes were cloned using a replacement cloning strategy whereby fragments from pCI310 were exchanged with the equivalent fragments in pNZ521, which contains the cloned proteinase genes from L. lactis subsp. lactis SK112. This generated two plasmids which encoded a cell-envelope-associated and a secreted proteinase, respectively. Specific regions of the UC317 structural prtP gene known to encode seven of the amino acids essential for substrate cleavage specificity were sequenced and compared with the known sequences of prt genes from L. lactis strains SK112, Wg2 and NCDO763. In spite of various differences that were detected in the nucleotide sequence of this region, it appears that these seven amino acids in strains UC317 and NCDO763 are identical, and represent a combination of three of the amino acids from SK112 and four from Wg2. These results indicate that the UC317 proteinase is a natural hybrid of the SK112 and Wg2 proteinases.     

Relationship between Lactococcus lactis subsp. lactis 952, an industrial lactococcal starter culture and strains 712, ML3 and C2

Lactococcus lactis subsp. lactis 952, used as a component of a cheese starter culture, was found to be closely related to the 712/ML3/C2 group of strains. Plasmid profiles of the four isolates were very similar and factory phages isolated against strain 952 could produce plaques on all the other strains at the same titres. Treatment with mitomycin C failed to induce phage from strain 952 although homology was observed between φT712 and the chromosome of strain 952. Pulse field gel electrophoresis of the four strains revealed only minor differences in their genomic DNA. The clumping phenomenon observed during lactose transfer in strains 712 and ML3 was also evident in strain 952. The ability of strain 952 to mobilize non-conjugative vectors such as pGB301 was also examined and analysis of pGB301 transconjugants revealed that some contained novel enlarged plasmids not present in either the donor or the recipient. Preliminary characterization of these enlarged plasmids suggested the involvement of a sex factor type element in strain 952.     

The use of cadmium resistance on the phage-resistance plasmid pNP40 facilitates selection for its horizontal transfer to industrial dairy starter lactococci

AIMS: To facilitate the horizontal transfer and selection of phage-resistance plasmids in industrial lactococci. METHODS AND RESULTS: Cadmium-resistance properties similar to those previously identified in Lactococcus were linked to the well-known phage-resistance plasmid pNP40. This finding was exploited to facilitate delivery of the plasmid to an industrial cheese starter Lactococcus lactis DPC4268. Additionally, 25 different cadmium-sensitive cheese starter lactococci were also identified as potential recipients for the phage-resistance plasmid pNP40, and also the plasmids pAH90/pAH82 which also encode cadmium resistance. All three plasmids were successfully conjugated to strain DPC4268. Cheddar cheese was manufactured in industry with the pNP40 phage-resistant transconjugant. SIGNIFICANCE AND IMPACT OF THE STUDY: Food-grade enhancement of phage resistance in industrial starter strains has been made simpler by the use of this selection, especially since the majority of potential recipient starter strains analysed were cadmium sensitive.     

Lactococcus lactis DPC5598, a plasmid-free derivative of a commercial starter,provides a valuable alternative host for culture improvement studies

AIMS: To generate a plasmid-free derivative of an extensively used industrial starter strain Lactococcus lactis DPC4268, which could be used as a backbone strain for starter improvement programmes. METHODS AND RESULTS: DPC4268 containing four large plasmids was subjected to high temperature plasmid curing resulting in derivatives, each with a different plasmid complement of one, two or three different plasmids in addition to a plasmid-free derivative. Industrially relevant phenotypes were assigned to each plasmid on the basis of detailed phenotypic and genetic analyses and these were (a) proteinase activity (Prt, 60 kb) (b) lactose fermentation (Lac, 55 kb) (c) bacteriophage adsorption inhibition (Ads, 44 kb) and (d) type I restriction/modification (R/M, 40 kb). The plasmid-free variant of DPC4268 was shown to be transformable at frequencies comparable to the common laboratory strain L. lactis MG1614. Furthermore its genome was demonstrated to be significantly different from the laboratory strains L. lactis MG1614 and the recently sequenced L. lactis IL1403 genomes by pulsed-field gel electrophoresis. CONCLUSIONS: This study produced an easily transformable plasmid-free derivative which was genomically different from both MG1614 and IL1403. In addition, important plasmid-borne industrial traits, including two phage-resistance mechanisms, were identified in DPC4268. SIGNIFICANCE AND IMPACT OF THE STUDY: L. DPC4268 is a vitally important commercial strain used in the manufacture of Cheddar cheese. The generation of a plasmid-free derivative may provide an important backbone strain as a basis for future strain improvement purposes.     

pAMbeta1-Associated Mobilization of Proteinase Plasmids from Lactococcus lactis subsp. lactis UC317 and L. lactis subsp. cremoris UC205

A combination of plasmid curing and DNA-DNA hybridization data facilitated the identification of proteinase plasmids of 75 (pCI301) and 35 kilobases (pCI203) in the multi-plasmid-containing strains Lactococcus lactis subsp. lactis UC317 and L. lactis subsp. cremoris UC205, respectively. Both plasmids were transferred by conjugation to a plasmid-free background only after introduction of the conjugative streptococcal plasmid, pAMbeta1. All Prt transconjugants from matings involving either donor contained enlarged recombinant Prt plasmids. UC317-derived transconjugants were separable into different classes based on the presence of differently sized cointegrate plasmids and on segregation of the pCI301-derived Lac and Prt markers. All UC205-derived transconjugants harbored a single enlarged plasmid that was a cointegrate between pCI203 and pAMbeta1. The identification of prt genes on pCI301 and pCI203 derivatives was achieved by a combination of restriction enzyme and hybridization analyses.     

Isolation and characterization of large lactococcal phage resistance plasmids by pulsed-field gel electrophoresis

Abstract Five phage-resistant Lactococcus lactis strains were able to transfer by conjugation the lactose-fermenting ability (Lac⁺) to a plasmid-free Lac⁻ L. lactis strain. In each case, some Lac⁺ transconjugants were phage-resistant and contained one or two additional plasmids of high molecular mass, as demonstrated by pulsed-field gel electrophoresis. Plasmids pPF144 (144 kb), pPF107 (107 kb), pPF118 (118 kb), pPF72 (72 kb) and pPF66 (66 kb) were characterized: they are conjugative (Tra⁺), they confer a phage-resistant phenotype and they bear lactose-fermenting ability (Lactose plasmid) except for the last two. Plasmids pPF144, pPF107 and pPF118 resulted probably from a cointegrate formation between the Lactose plasmid and another plasmid of the donor strain, whereas pPF72, pPF66 and the Lactose plasmid were distinct in the corresponding transconjugants. Plasmids pPF72 and pPF66 produced a bacteriocin. At 30°C, the phage resistance conferred by the plasmids was complete against small isometric-headed phage and partial against prolate-headed phage, except for pPF107 whose phage resistance mechanism was totally effective against both types of phages, but was completely inactivated at 40°C. Restriction maps of four of the plasmids were constructed using pulsed-field gel electrophoresis.     

Integration and excision of plasmid DNA in Lactococcus lactis subsp. lactis

The capacity of the 75-kb lactose-proteinase plasmid pCI301 from Lactococcus lactis subsp. lactis UC317 to recombine with the lactococcal chromosome was examined. Low-frequency integration of pCI301 sequences was detected following protoplast transformation of strain MG136Sm with total plasmid DNA from strain UC317. Excision of integrated sequences was subsequently observed at a low level. Excised sequences were rescued through recombination with and mobilization by the conjugative enterococcal plasmid pAMB1. Transconjugants harboring novel recombinant pCI301::pAMB1 plasmids, both pAMB1 and a pCI301 derivative, and pAMB1 only were isolated. The latter represents a class of transconjugant in which an elevated level of reintegration of pCI301 DNA in the recipient chromosome has occurred.     

Increasing phage resistance of cheese starters : a case study using Lactococcus lactis DPC4268

This study serves as an example of strategies used to increase the phage resistance of an important Irish Cheddar cheese starter, Lactococcus lactis DPC4268. It describes the emergence and persistence of a lytic bacteriophage, 4268, that has a relatively large burst size and exhibits no homology to the most common phage types encountered in Irish cheese plants. Inherent difficulties were encountered that prevented the effective introduction of conjugative phage-resistance plasmids pNP40 and pMRC01 to strain DPC4268. In fact, pNP40-associated Abi systems were naturally present in six of 19 starters. Control of phage 4268 was eventually achieved by generating a mutant of DPC4268, which was subsequently used for cheese manufacture.     

Characterization and classification of virulent lactococcal bacteriophages isolated from a Cheddar cheese plant

C.N. CASEY, E. MORGAN, C. DALY AND G.F. FITZGERALD, 1993. Twenty-two bacteriophages, isolated from cheese-vat whey samples over a period of 4 years, were found to be active against one or more of four different strains of Lactococcus lactis subsp. cremoris used in a defined strain starter system in an Irish Cheddar cheese factory. All the phages were small isometric-headed with non-contractile tails, a baseplate and a collar; they had genome sizes of approximately 30 kb, and belonged to a single DNA hybridization group. All 22 phages could be classified into four distinct groups based on restriction analysis which overlapped perfectly with those based on host range. Each group of phage examined showed cross-reactive host ranges. None of the phage DNAs hybridized to the chromosomes of any of the seven cultures used in the factory during the four cheesemaking seasons, and neither could phages be induced from any of the strains by mitomycin-C or ultraviolet light treatment.     

Sequence and analysis of the 60 kb conjugative, bacteriocin-producing plasmid pMRC01 from Lactococcus lactis DPC3147

The complete sequence of pMRC01, a large conjugative plasmid from Lactococcus lactis ssp. lactis DPC3147, has been determined. Using a shotgun sequencing approach, the 60 232 bp plasmid sequence was obtained by the assembly of 1056 underlying sequences (sevenfold average redundancy). Sixty-four open reading frames (ORFs) were identified. Analysis of the gene organization of pMRC01 suggests that the plasmid can be divided into three functional domains, with each approximately 20 kb region separated by insertion sequence (IS) elements. The three regions are (i) the conjugative transfer region, including a 16-gene Tra (transfer) operon; (ii) the bacteriocin production region, including an operon responsible for the synthesis of the novel bacteriocin lacticin 3147; and (iii) the phage resistance and plasmid replication region of the plasmid. The complete sequence of pMRC01 provides important information about these industrially relevant phenotypes and gives insight into the structure, function and evolution of large Gram-positive conjugative plasmids in general. The completely sequenced pMRC01 plasmid should also provide a useful framework for the design of novel plasmids to be incorporated into starter strain improvement programmes for the dairy industry.     

Comparison of the acidifying activity of Lactococcus lactis subsp. lactis strains isolated from goat's milk and Valdeteja cheese

AIMS: This work was carried out to study the acid production by Lactococcus lactis subsp. lactis strains isolated from goat's milk and goat cheese (Valdeteja variety) in order to select a suitable starter culture for industrial goat cheese manufacturing. METHODS AND RESULTS: The titrable acidity of 45 Lactococcus lactis subsp. lactis strains isolated from a home-made batch of Valdeteja cheese with excellent sensory characteristics was measured over a period of 18 h. The strains were divided into two groups depending on the acid production rate: 20 fast acid producer (F) strains and 25 slow acid producer (S) strains. The kinetic parameters (lag phase, maximum acid production rate and value of upper asymptote curve) of the acid production curves for F and S strains were significantly (P < 0.001) different. CONCLUSIONS: Significant (P < 0.001) differences between titrable acidity of F and S strains were observed after the second hour of incubation. SIGNIFICANCE AND IMPACT OF THE STUDY: An F strain acetoin producer (Lactococcus lactis subsp. lactis 470Ch2) was selected as autochthonous starter culture for industrial Valdeteja goat cheese manufacturing.     

Analysis of a region from the bacteriophage resistance plasmid pCI528 involved in its conjugative mobilization between Lactococcus strains.

A 10-kb HindIII fragment of pCI528 cloned into the nonconjugative shuttle vector pCI3340 could be transferred by conjugative mobilization from Lactococcus lactis subsp. lactis MG1363, whereas other HindIII fragments of pCI528 or the vector alone were nonmobilizable. Subcloning of this 10-kb region identified a 4.4-kb BglII-EcoRI fragment which contained all the DNA essential for transfer. Sequence analysis of a 2-kb region within this 4.4 kb-segment revealed a region rich in inverted repeats and two potential overlapping open reading frames, one of which demonstrated homology to mobilization proteins of two nonconjugative staphylococcal plasmids.     

Plasmid content and bacteriocin production by five strains of Lactococcus lactis isolated from semi-hard homemade cheese

In this study, the plasmid content and bacteriocin production of natural isolates of lactococci were investigated. Five bacteriocin producing lactococcal strains (Lactococcus lactis subsp. lactis BGMN1-2, BGMN1-3, BGMN1-5, BGMN1-6, and BGMN2-7) were isolated as nonstarter microflora of semi-hard homemade cheese and characterized. All isolates contained a number of plasmids. It was shown that lcnB structural genes for bacteriocin lactococcin B were located on large plasmids in all isolates. In the strains BGMN1-3 and BGMN1-5 proteinase prtP genes collocated with lcnB. Furthermore, these strains produced two additional bacteriocins (LsbA and LsbB) with genes responsible for their production and immunity located on the small rolling circle-replicating plasmid pMN5. Using deletion experiments of pMN5, minimal replicon of the plasmid and involvement of a bacteriocin locus in plasmid maintenance were identified. In addition, plasmid curing experiments showed that genes for catabolism or transport of 10 carbohydrates in the strain BGMN1-5 were plasmid located.     

一个含有乳链菌肽抗性基因的乳酸乳球菌质粒pTS50的鉴定

在添加乳链菌肽、乳糖及溴甲酚紫的M1 7选择培养基上 ,从 1 97个新鲜牛奶样品中筛选到 3株乳链菌肽抗性菌株 ,PCR扩增证实它们都含有乳链菌肽抗性基因。菌种生理生化特性鉴定及特异性 1 6SrDNAPCR扩增产物的序列测定结果表明这 3株菌都属于乳酸乳球菌乳酸亚种。质粒转化实验发现乳酸乳球菌乳酸亚种TS 1 640中的乳链菌肽抗性基因位于一个约47kb的大质粒pTS50上。BamHI、EcoRI、HindⅢ、NcoI、PstⅠ酶切分析和Southern杂交 ,进一步将乳链菌肽抗性基因定位于pTS50的一个约 1 9kbEcoRI酶切片段中     

DNA-DNA homology among lactose- and sucrose-fermenting transconjugants from Lactococcus lactis strains exhibiting reduced bacteriophage sensitivity.

DNA-DNA homology between a reduced bacteriophage sensitivity (Rbs+) probe and DNA from both Rbs+ and Rbs- Lactococcus lactis strains was examined. Homology was detected between the probe and five plasmids (pCI750, pCC34, pEB56, pNP2, and pJS88) isolated from lactose-positive Rbs+ transconjugants and between the probe and genomic DNA of a sucrose-positive Rbs+ transconjugant. Additionally, hybridizations conducted between the probe and plasmids reported to encode abortive bacteriophage infection indicated homology with pTR2030 but not with pBF61 and pGBK17. The results suggest that a common genetic determinant(s) may be present in a variety of lactococcal plasmids coding for Rbs+.     

DNA-DNA homology among lactose- and sucrose-fermenting transconjugants from Lactococcus lactis strains exhibiting reduced bacteriophage sensitivity

DNA-DNA homology between a reduced bacteriophage sensitivity (Rbs+) probe and DNA from both Rbs+ and Rbs- Lactococcus lactis strains was examined. Homology was detected between the probe and five plasmids (pCI750, pCC34, pEB56, pNP2, and pJS88) isolated from lactose-positive Rbs+ transconjugants and between the probe and genomic DNA of a sucrose-positive Rbs+ transconjugant. Additionally, hybridizations conducted between the probe and plasmids reported to encode abortive bacteriophage infection indicated homology with pTR2030 but not with pBF61 and pGBK17. The results suggest that a common genetic determinant(s) may be present in a variety of lactococcal plasmids coding for Rbs+.