Variable bacteriocin production in the commercial starter Lactococcus lactis DPC4275 is linked to the formation of the cointegrate plasmid pMRC02

Lactococcus lactis DPC4275 is a bacteriocin-producing transconjugant of the industrial starter strain DPC4268. Strain DPC4275 was generated through conjugal transfer by mating DPC4268 with L. lactis MG1363 containing the 60-kb plasmid pMRC01, which encodes the genetic determinants for the lantibiotic lacticin 3147 and for a phage resistance mechanism of the abortive infection type. The many significant applications of this strain prompted a genetic analysis of its apparently unstable bacteriocin-producing phenotype. Increased levels of lacticin 3147 produced by DPC4275 were associated with the appearance of an 80-kb plasmid, designated pMRC02, which was derived from DNA originating from pMRC01 (60 kb) and a resident DPC4268 proteinase plasmid, pMT60 (60 kb). Indeed, pMRC02 was shown to be derived from the insertion of a 17-kb fragment of pMRC01, encompassing the lacticin 3147 operon, into pMT60. The presence of pMRC02 at a high copy number was found to correlate with increased levels of lacticin 3147 in DPC4275 compared to the wild-type containing pMRC01. Subsequent transfer of pMRC02 into the plasmid-free strain MG1363 by electroporation allowed a direct phenotypic comparison with pMRC01, also studied in the MG1363 background. Plasmid pMRC02 displayed phage resistance similar to that by pMRC01, although it was less potent, as demonstrated by a larger plaque size for phage c2 infection of MG1363(pMRC02). While this locus is flanked by IS946 elements, the sequencing of pMT60-pMRC01 junction sites established that this event was unlikely to be insertion sequence mediated and most probably occurred by homologous recombination followed by deletion of most of pMRC01. This was not a random occurrence, as nine other transconjugants investigated were found to have the same junction sites. Such derivatives of commercial strains producing increased levels of bacteriocin could be exploited as protection cultures for food applications.     

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.     

Improvement of phage defence in Lactococcus lactis by introduction of the plasmid encoded restriction and modification system LlaAI

AIMS: To study the ability of the plasmid-encoded restriction and modification (R/M) system LlaAI to function as a bacteriophage resistance mechanism in Lactococcus lactis during milk fermentations. METHODS AND RESULTS: Plasmid pAIcat4, carrying the R/M system LlaAI and a chloramphenicol resistance cassette, was introduced into the plasmid-free strain L. lactis MG1614 and the industrial strain L. lactis 964. By measuring changes in conductivity the influence of different phage on the growth was determined. CONCLUSIONS: The plasmid-encoded R/M system LlaAI significantly improves the bacteriophage resistance of L. lactis during milk fermentations. SIGNIFICANCE AND IMPACT OF THE STUDY: It is essential to determine the potential of a phage defence mechanism in L. lactis starter culture strains during growth in milk before steps are taken to improve starter cultures. This study shows that LlaAI is useful for improvement of starter cultures.     

Thiol-stimulated secretion of riboflavin and porphyrins by Escherichia coli

Abstract Streptococcus lactis strain IL594 contains 9 plasmids, designated pIL1 to pIL9. On the basis of protoplast-induced curing experiments we showed that derivatives containing pIL7 (31 kb) were resistant to UV-irradiation while derivatives lacking pIL7 were sensitive. The pIL7-determined UV-protection was confirmed by cotransfer of the plasmid and of the character into a plasmid-free derivative of S. lactis IL594. Moreover, prophage induction required higher UV-fluence in this derivative carrying pIL7 than in the plasmid-free strain. This is the first report of a plasmid-mediated UV-protection in group N streptococci.     

Citrate permease gene expression in Lactococcus lactis subsp. lactis strains IL1403 and MG1363

Abstract Citrate permease gene expression in the plasmid-free Lactococcus lactis strains IL1403 and MG1363 was studied. The ability to transport citrate results in diacetyl and acetoin production in IL1403 but not in MG1363. Citrate lyase, α-acetolactate decarboxylase, diacetyl and acetoin reductase were detected in IL1403. These data show that L. lactis ssp. lactis strain IL1403 is a citrate permease mutant of the biovar. diacetylactis . Immunological analysis revealed the α-and β-subunits of citrate lyase not only in IL1403 but also in MG1363 where no citrate lyase activity was found.     

Variable Bacteriocin Production in the Commercial Starter Lactococcus lactis DPC4275 Is Linked to the Formation of the Cointegrate Plasmid pMRC02

Lactococcus lactis DPC4275 is a bacteriocin-producing transconjugant of the industrial starter strain DPC4268. Strain DPC4275 was generated through conjugal transfer by mating DPC4268 with L. lactis MG1363 containing the 60-kb plasmid pMRC01, which encodes the genetic determinants for the lantibiotic lacticin 3147 and for a phage resistance mechanism of the abortive infection type. The many significant applications of this strain prompted a genetic analysis of its apparently unstable bacteriocin-producing phenotype. Increased levels of lacticin 3147 produced by DPC4275 were associated with the appearance of an 80-kb plasmid, designated pMRC02, which was derived from DNA originating from pMRC01 (60 kb) and a resident DPC4268 proteinase plasmid, pMT60 (60 kb). Indeed, pMRC02 was shown to be derived from the insertion of a 17-kb fragment of pMRC01, encompassing the lacticin 3147 operon, into pMT60. The presence of pMRC02 at a high copy number was found to correlate with increased levels of lacticin 3147 in DPC4275 compared to the wild-type containing pMRC01. Subsequent transfer of pMRC02 into the plasmid-free strain MG1363 by electroporation allowed a direct phenotypic comparison with pMRC01, also studied in the MG1363 background. Plasmid pMRC02 displayed phage resistance similar to that by pMRC01, although it was less potent, as demonstrated by a larger plaque size for phage c2 infection of MG1363(pMRC02). While this locus is flanked by IS946 elements, the sequencing of pMT60-pMRC01 junction sites established that this event was unlikely to be insertion sequence mediated and most probably occurred by homologous recombination followed by deletion of most of pMRC01. This was not a random occurrence, as nine other transconjugants investigated were found to have the same junction sites. Such derivatives of commercial strains producing increased levels of bacteriocin could be exploited as protection cultures for food applications.     

Physical and genetic map of the chromosome of Lactococcus lactis subsp. lactis IL1403.

A combined physical and genetic map of the chromosome of Lactococcus lactis subsp. lactis IL1403 was determined. We constructed a restriction map for the NotI, ApaI, and SmaI enzymes. The order of the restriction fragments was determined by using the randomly integrative plasmid pRL1 and by performing indirect end-labeling experiments. The strain IL1403 chromosome was found to be circular and 2,420 kb in size. A total of 24 chromosomal markers were mapped on the chromosome by performing hybridization experiments with gene probes for L. lactis and various other bacteria. Integration of pRC1-derived plasmids via homologous recombination allowed more precise location of some lactococcal genes and allowed us to determine the orientation of these genes on the chromosome. Recurrent sequences, such as insertion elements and rRNA gene (rrn) clusters, were also mapped. At least seven copies of IS1076 were present and were located on 50% of the chromosome. In contrast, no copy of ISS1RS was detected. Six ribosomal operons were found on the strain IL1403 chromosome; five were located on 16% of the chromosome and were transcribed in the same direction. A comparison of the physical maps of L. lactis subsp. lactis IL1403 and DL11 showed that these two strains are closely related and that the variable regions are located mainly near the rrn gene clusters. In contrast, despite major restriction pattern dissimilarities between L. lactis IL1403 and MG1363, the overall genetic organization of the genome seems to be conserved between these two strains.     

Protoplast transformation of group N streptococci with cryptic plasmids

Abstract By using an extension to group N streptococci of a contransformation procedure we have introduced 4 different-sized cryptic plasmids for Streptococcus lactis into the plasmid-free S. lactis IL1403. A mixture of 4 cryptic plasmids with an indicator plasmid (pHV1301) conferring erythromycin resistance was used for IL1403 protoplast transformation. Under such conditions, 41.5% of the erythromycin-resistant transformants were contransformed with one of the cryptic plasmids in addition to pHV1301. Indicator plasmid pHV1301 was later spontaneously segregated from doubly transformed cells. This protocol should be very useful for constructing lactic streptococcal strains bearing any phenotypically cryptic plasmid.     

限制和修饰系统LlaBIII在构建抗噬菌体菌株中的作用

限制和修饰 (ｒｅｓｔｒｉｃｔｉｏｎａｎｄｍｏｄｉｆｉｃａｔｉｏｎ ,Ｒ Ｍ)系统是指由限制性内切酶和甲基化酶组成的单亚基或多亚基复合酶系统 ,两者通常成对出现 ,具有相同的ＤＮＡ识别位点 ,其作用相反。Ｒ Ｍ系统在原核生物中普遍存在 ,在保护细胞免遭外源病毒侵害方面具有重要作用[1] 。作为发酵剂的乳酸乳球菌在乳制品发酵中具有重要作用 ,但这类菌株极易遭受噬菌体感染 ,导致菌株产酸力降低 ,甚至发酵失败 ,造成严重的经济损失。所以在乳制品发酵过程中防止噬菌体感染就成为十分重要的问题。通过自然筛选或诱变处理等手段筛选噬菌…     

Adaptative potential of the Lactococcus lactis IL594 strain encoded in its 7 plasmids

The extrachromosomal gene pool plays a significant role both in evolution and in the environmental adaptation of bacteria. The L. lactis subsp. lactis IL594 strain contains seven plasmids, named pIL1 to pIL7, and is the parental strain of the plasmid-free L. lactis IL1403, which is one of the best characterized lactococcal strains of LAB. Complete nucleotide sequences of pIL1 (6,382 bp), pIL2 (8,277 bp), pIL3 (19,244 bp), pIL4 (48,979), pIL5 (23,395), pIL6 (28,435 bp) and pIL7 (28,546) were established and deposited in the generally accessible database (GeneBank). Nine highly homologous repB-containing replicons, belonging to the lactococcal theta-type replicons, have been identified on the seven plasmids. Moreover, a putative region involved in conjugative plasmid mobilization was found on four plasmids, through identification of the presence of mob genes and/or oriT sequences. Detailed bioinformatic analysis of the plasmid nucleotide sequences provided new insight into the repertoire of plasmid-encoded functions in L. lactis, and indicated that plasmid genes from IL594 strain can be important for L. lactis adaptation to specific environmental conditions (e.g. genes coding for proteins involved in DNA repair or cold shock response) as well as for technological processes (e.g. genes encoding citrate and lactose utilization, oligopeptide transport, restriction-modification system). Moreover, global gene analysis indicated cooperation between plasmid- and chromosome-encoded metabolic pathways.     

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.     

限制和修饰系统LlaBⅢ在构建抗噬菌体菌株中的作用

限制和修饰(restriction and modification,R/M)系统是指由限制性内切酶和甲基化酶组成的单亚基或多亚基复合酶系统,两者通常成对出现,具有相同的DNA识别位点,其作用相反.R/M系统在原核生物中普遍存在,在保护细胞免遭外源病毒侵害方面具有重要作用[1].     

Influence of lactococcal plasmid on the specific growth rate of host cells

AIMS: To investigate a lactococcal plasmid responsible for a reduction in growth rate of its host cell. METHODS AND RESULTS:Lactococcus lactis subsp. lactis biovar. diacetylactis DRC1 carries a high number of plasmids. The DRC1 wild-type strain was found to grow more slowly than a plasmid-free derivative of DRC1. The plasmids extracted from DRC1 together with an indicator plasmid were cotransformed into the plasmid-free strain DRC1021. A 7.4-kb cryptic plasmid, designated pDR1-1, was found to significantly affect the maximum specific growth rate ( micro max) of the host cell. Polymerase chain reaction (PCR) analysis was carried out in order to detect the presence of pDR1-1 in the other L. lactis strains. The micro max of the single pDR1-1-positive strain was determined to be the same as that of DRC1. SIGNIFICANCE AND IMPACT OF THE STUDY: These results suggest that pDR1-1 (or a pDR1-1-like plasmid) is a critical factor in the reduction of the micro max of DRC1, and that its effect on the micro max is significantly greater than that of any other coexisting plasmid.     

A food-grade cloning vector for lactic acid bacteria based on the nisin immunity gene nisI

A new food-grade cloning vector for lactic acid bacteria was constructed using the nisin immunity gene nisI as a selection marker. The food-grade plasmid, pLEB590, was constructed entirely of lactococcal DNA: the pSH71 replicon, the nisI gene, and the constitutive promoter P45 for nisI expression. Electroporation into Lactococcus lactis MG1614 with 60 international units (IU) nisin/ml selection yielded approximately 10⁵ transformants/μg DNA. MG1614 carrying pLEB590 was shown to be able to grow in medium containing a maximum of 250 IU nisin/ml. Plasmid pLEB590 was succesfully transformed into an industrial L. lactis cheese starter carrying multiple cryptic plasmids. Suitability for molecular cloning was confirmed by cloning and expressing the proline iminopeptidase gene pepI from Lactobacillus helveticus in L. lactis and Lb. plantarum. These results show that the food-grade expression system reported in this paper has potential for expression of foreign genes in lactic acid bacteria in order to construct improved starter bacteria for food applications. Electronic Publication     

Inhibition of tyrosine-sensitive 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase by glyphosate in Candida maltosa

Abstract A gene block controlling sucrose-fermenting ability, nisin resistance and nisin production was found to be transmissible by a conjugation-like process. The 'pSN' (sucrose nisin) plasmid was transferred from 8 different nisin-producing donor strains into MG1614, a plasmid-free derivative of Streptococcus lactis 712. In the new host low yields of a plasmid of approx. 30 MDa were isolated but its authenticity as a pSN plasmid has not yet been established. Possibilities for increased nisin yield by genetic manipulation in S. lactis 712 must exist.     

Conjugal mobilization of streptococcal plasmid pMV158 between strains of Lactococcus lactis subsp. lactis.

pMV158, a non-self-transmissible plasmid encoding tetracycline resistance, was conjugally transferred from Enterococcus faecalis JH203 to Lactococcus lactis subsp. lactis IL1403. This transfer appeared to be dependent on the cotransfer of the conjugative plasmids pAM beta 1 or pIP501. Intraspecies conjugal transfer of pMV158 also occurred in strain IL1403. In contrast to the transfer from E. faecalis, transfer in IL1403 did not require the presence of a conjugative plasmid in the donor strain but, rather, appeared to be dependent on putative chromosomal functions in strain IL1403. The transfer of pMV158 from strain IL1403 required the presence of an active pMV158-encoded protein, which showed homology to the Pre (plasmid recombination enzyme) proteins encoded by several small plasmids extracted from Staphylococcus aureus, such as pT181.     

Physical and genetic map of the Lactococcus lactis subsp. cremoris MG1363 chromosome: comparison with that of Lactococcus lactis subsp. lactis IL 1403 reveals a large genome inversion.

A physical and genetic map of the chromosome of the Lactococcus lactis subsp. cremoris reference strain MG1363 was established. The physical map was constructed for NotI, ApaI, and SmaI enzymes by using a strategy that combines creation of new rare restriction sites by the random-integration vector pRL1 and ordering of restriction fragments by indirect end-labeling experiments. The MG1363 chromosome appeared to be circular and 2,560 kb long. Seventy-seven chromosomal markers were located on the physical map by hybridization experiments. Integration via homologous recombination of pRC1-derived plasmids allowed a more precise location of some lactococcal genes and determination of their orientation on the chromosome. The MG1363 chromosome contains six rRNA operons; five are clustered within 15% of the chromosome and transcribed in the same direction. Comparison of the L. lactis subsp. cremoris MG1363 physical map with those of the two L. lactis subsp. lactis strains IL1403 and DL11 revealed a high degree of restriction polymorphism. At the genetic organization level, despite an overall conservation of gene organization, strain MG1363 presents a large inversion of half of the genome in the region containing the rRNA operons.     

Proteome analysis of a Lactococcus lactis strain overexpressing gapA suggests that the gene product is an auxiliary glyceraldehyde 3-phosphate dehydrogenase

The sequence of the genome from the Lactococcus lactis subspecies lactis strain IL1403 shows the presence of two reading frames, gapA and gapB, putatively encoding glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Previous proteomic analysis of the L. lactis subspecies cremoris strain MG1363 has revealed two neighbouring protein spots, GapBI and GapBII, with amino terminal sequences identical to the product of gapA from the L. lactis subspecies cremoris strain LM0230 and that of the two IL1403 sequences. In order to assign the two protein spots to their respective genes we constructed an L. lactis strain that overexpessed the gapA gene derived from MG1363 upon nisin induction. Compared to the wild-type, the overexpressing strain had a 3.4-fold elevated level of specific GAPDH activity when grown in the presence of nisin. In both MG1363 and the gapA overexpressing strain the GAPDH activity was specific for NAD. No NADP dependent activity was detected. Proteome analysis of the gapA overexpressing strain revealed two new protein spots, GapAI and GapAII, not previously detected in proteome analysis of MG1363. Results from mass spectrometry analysis of GapA and GapB and comparison with the deduced protein sequences for the GAPDH isozymes from the genome sequence of strain IL1403 allowed us to assign GapA and GapB to their apparent IL1403 homologues encoded by gapA and gapB, respectively. Furthermore, we suggest that a homologue of a gapB product, represented by GapB, is the main source of GAPDH activity in L. lactis during normal growth.     

Linear plasmids pWR1A and pWR1B of the yeast Wingea robertsiae are associated with a killer phenotype

Wingea robertsiae CBS6693 (synonym Debaryomyces robertsiae) was previously reported to harbor two cryptic linear plasmids, designated pWR1A (8.3 kb) and pWR1B (14.6 kb). Reexamination of a putative plasmid encoded killer phenotype involved UV-curing as well as a highly sensitive toxin assay. Killer activities of concentrated culture supernatants prepared from both, a plasmid carrying and a cured plasmid-free strain, were examined in liquid media. Supernatants collected from plasmid carrying strains subjected to cultures of the plasmid-free derivative had clear concentration-dependent inhibitory effects, whereas plasmid harboring cells were not affected. Incubation at 65 degrees C for 10 min totally destroyed the toxin. Since supernatants prepared from the plasmid-free strain did not possess such killer activity and the presence of the plasmids confered resistance, toxin as well as immunity functions appear plasmid encoded. Beyond this, chitin affinity chromatography and Western blot analysis proved plasmid specific expression and secretion of a protein displaying similarities to the alpha-subunit of the Kluyveromyces lactis killer toxin. The assay applied in this study will most probably allow disclosure of other hidden killer phenomena, which may have escaped detection by conventionally applied plate assays.