Molecular analysis of the Lactococcus lactis subspecies lactis CNRZ270 bidirectional theta replicating lactose plasmid pUCL22

pUCL22 is the lactose protease plasmid of Lactococcus lactis ssp. lactis CNRZ270. The nucleotide sequence of its replication region Rep22 contains a non-transcribed region, the replication origin, followed by a gene encoding a putative 388-amino-acid protein named Rep22A. The promoter regions of the rep22A and pC194 cat genes share strong similarities and the pUCL22 replicon exerted trans or cis negative control on the pC194 cat gene expression in L. lactis. We suggest that Rep22A binds to its own promoter as well as to the pC194 cat promoter and thus is autoregulated. We show that pUCL22 replicates mainly by a bidirectional theta mechanism in L. lactis, and is representative of a widely distributed replicon family, members of which could be co-resident. We propose that compatibility between these closely related replicons results from minor replication protein modifications coupled with base changes in their respective binding sites, supporting the co-existence of numerous related replicons in lactococcal strains.     

Genetic analysis of the minimal replicon of the Lactococcus lactis subsp. lactis biovar diacetylactis citrate plasmid

pBLI is a conjugative linear extrachromosomal element of 43 kb previously isolated after interspecific mating between Streptomyces bambergiensis and S. lividans. Cloning experiments using the non-conjugative, circular Streptomyces vector pIJ702 allowed the identification of a 5.74 kb region from pBL1 which facilitates plasmid transfer. Insertion and deletion mutagenesis, gene disruptions, and sequence data suggest that at least five previously unknown genes of pBL1 are required for efficient plasmid transfer and its regulation.     

Nonidentity between plasmid and chromosomal copies of ISS1-like sequences in Lactococcus lactis subsp. lactis CNRZ270 and their possible role in chromosomal integration of plasmid genes.

The nucleotide sequence of an insertion sequence (IS) observed during mating experiments using the lactose-protease plasmid, pUCL22, of Lactococcus (Lc.) lactis subsp. lactis CNRZ270, was found to be similar to that of ISS1 from Lc. lactis subsp. lactis ML3. The IS was named ISS1RS. The chromosome of this strain contains several copies of ISS1-like IS as assessed by hybridization. One of these copies was cloned and named ISS1CH. Its sequence differs from that of the plasmid-borne copy, and appears to be more closely related to ISS1N from Lc. lactis subsp. cremoris SK11. This suggests independent introduction of both ISS1 elements. Moreover, the observation of plasmid genes integrated in the CNRZ270 chromosome near ISS1CH suggests that their presence is the result of integration by a Campbell mechanism using both IS homologies. ISS1-like sequences were also found on plasmids of numerous Lc. lactis strains, as well as one out of seven Lactobacillus (Lb.) casei and one out of three Lb. plantarum strains examined.     

Thermosensitive plasmid replication, temperature-sensitive host growth, and chromosomal plasmid integration conferred by Lactococcus lactis subsp. cremoris lactose plasmids in Lactococcus lactis subsp. lactis.

Evidence is presented that lactose-fermenting ability (Lac+) in Lactococcus lactis subsp. cremoris AM1, SK11, and ML1 is associated with plasmid DNA, even though these strains are difficult to cure of Lac plasmids. When the Lac plasmids from these strains were introduced into L. lactis subsp. lactis LM0230, they appeared to replicate in a thermosensitive manner; inheritance of the plasmid was less efficient at 32 to 40 degrees C than at 22 degrees C. The stability of the L. lactis subsp. cremoris Lac plasmids in lactococci appeared to be a combination of both host and plasmid functions. Stabilized variants were isolated by growing the cultures at 32 to 40 degrees C; these variants contained the Lac plasmids integrated into the L. lactis subsp. lactis LM0230 chromosome. In addition, the presence of the L. lactis subsp. cremoris Lac plasmids in L. lactis subsp. lactis resulted in a temperature-sensitive growth response; growth of L. lactis subsp. lactis transformants was significantly inhibited at 38 to 40 degrees C, thereby resembling some L. lactis subsp. cremoris strains with respect to temperature sensitivity of growth.     

Thermosensitive plasmid replication, temperature-sensitive host growth, and chromosomal plasmid integration conferred by Lactococcus lactis subsp. cremoris lactose plasmids in Lactococcus lactis subsp. lactis

Evidence is presented that lactose-fermenting ability (Lac+) in Lactococcus lactis subsp. cremoris AM1, SK11, and ML1 is associated with plasmid DNA, even though these strains are difficult to cure of Lac plasmids. When the Lac plasmids from these strains were introduced into L. lactis subsp. lactis LM0230, they appeared to replicate in a thermosensitive manner; inheritance of the plasmid was less efficient at 32 to 40 degrees C than at 22 degrees C. The stability of the L. lactis subsp. cremoris Lac plasmids in lactococci appeared to be a combination of both host and plasmid functions. Stabilized variants were isolated by growing the cultures at 32 to 40 degrees C; these variants contained the Lac plasmids integrated into the L. lactis subsp. lactis LM0230 chromosome. In addition, the presence of the L. lactis subsp. cremoris Lac plasmids in L. lactis subsp. lactis resulted in a temperature-sensitive growth response; growth of L. lactis subsp. lactis transformants was significantly inhibited at 38 to 40 degrees C, thereby resembling some L. lactis subsp. cremoris strains with respect to temperature sensitivity of growth.     

pUCL287 plasmid from Tetragenococcus halophila (Pediococcus halophilus) ATCC 33315 represents a new theta-type replicon family of lactic acid bacteria

Abstract A cryptic plasmid, pUCL287, was isolated from Tetragenococcus halophila (Pediococcus halophilus) ATCC 33315. It had a theta-type mechanism of replication in its natural host. Its minimal replicon, Rep 281, was isolated on a 1.6-kb Eco RI fragment. The Rep 287 host range included the genera Pediococcus, Enterococcus, Lactobacillus and Leuconostoc but not genus Lactococcus . Plasmids hybridizing to pUCL287 are rare among lactic acid bacteria. As assessed by hybridization, Rep2Sl is dissimilar to pAMβ1, pIP50l and pUCL22, representatives of the most common theta-type replicon groups in Gram-positive bacteria. Therefore, pUCL287 appears to represent a new theta-type replicon family from lactic acid bacteria.     

Molecular characterization of the integration of the lactose plasmid from Lactococcus lactis subsp. cremoris SK11 into the chromosome of L. lactis subsp. lactis.

When Lactococcus lactis subsp. lactis LM0230 is transformed by the lactose plasmid (pSK11L) from Lactococcus lactis subsp. cremoris SK11, variants with pSK11L in the integrated state can be derived (J. M. Feirtag, J. P. Petzel, E. Pasalodos, K. A. Baldwin, and L. L. McKay, Appl. Environ. Microbiol. 57:539-548, 1991). In the present study, a 1.65-kb XbaI-XhoI fragment of pSK11L was subcloned for use as a probe in Southern hybridization analyses of the mechanism of integration, which was shown to proceed via a Campbell-like, single-crossover event. Furthermore, the presence of the XbaI-XhoI fragment in a nonreplicating vector facilitated the stable, Rec-dependent integration of the vector into the chromosome of L. lactis subsp. lactis LM0230 and other lactococci. DNA sequence analysis of the fragment revealed an open reading frame of 885 bp with lactococcal expression sequences. The putative gene did not have significant homology with other genes in computer data bases. The XbaI-XhoI fragment is a naturally occurring piece of lactococcal DNA that can be used as a recombinogenic cassette in the construction of integration vectors for the industrially important lactococci.     

Molecular characterization of the integration of the lactose plasmid from Lactococcus lactis subsp. cremoris SK11 into the chromosome of L. lactis subsp. lactis.

When Lactococcus lactis subsp. lactis LM0230 is transformed by the lactose plasmid (pSK11L) from Lactococcus lactis subsp. cremoris SK11, variants with pSK11L in the integrated state can be derived (J. M. Feirtag, J. P. Petzel, E. Pasalodos, K. A. Baldwin, and L. L. McKay, Appl. Environ. Microbiol. 57:539-548, 1991). In the present study, a 1.65-kb XbaI-XhoI fragment of pSK11L was subcloned for use as a probe in Southern hybridization analyses of the mechanism of integration, which was shown to proceed via a Campbell-like, single-crossover event. Furthermore, the presence of the XbaI-XhoI fragment in a nonreplicating vector facilitated the stable, Rec-dependent integration of the vector into the chromosome of L. lactis subsp. lactis LM0230 and other lactococci. DNA sequence analysis of the fragment revealed an open reading frame of 885 bp with lactococcal expression sequences. The putative gene did not have significant homology with other genes in computer data bases. The XbaI-XhoI fragment is a naturally occurring piece of lactococcal DNA that can be used as a recombinogenic cassette in the construction of integration vectors for the industrially important lactococci.     

Cloning and sequence analysis of a plasmid replicon from Lactococcus lactis subsp. cremoris FG2

A replication region from one of the Lactococcus lactis subsp. cremoris FG2 plasmids was isolated by cloning of a 4.8-kb XbaI fragment into a replication probe vector and transformation into L. lactis LM0230. A 1.8-kb region within this fragment was sequenced and confirmed by PCR subcloning to encode a functional replicon in LM0230. The replicon consists of an open reading frame encoding a putative replication protein (Rep) of 386 amino acids and a non-coding region (ori) which features several structural motifs typical of other known replication origins, including a 22-bp iteron sequence tandemly repeated three and a half times, a 10-bp direct repeat and two sets of inverted repeats. The ori region could drive replication of its plasmid when supplied with the replication region in-trans. The lack of detectable single-stranded DNA during replication and the existence of extensive homology with other known lactococcal theta replicons strongly suggest that this region encodes a theta-replicating mechanism.     

Characterization of a plasmid involved with cointegrate formation and lactose metabolism in Lactococcus lactis subsp. lactis OZS1

A 55 kilobase (kb) plasmid (pOZS550) in the non-clumping Lactococcus lactis subsp. lactis strain OZS1 carrying genes for lactose metabolism was characterised. A mobilizable cointegrate plasmid which is formed between pOZS550 and pOZS448 carries the necessary information for conjugation and transfer. Cointegrate formation was found to involve an insertional element located on pOZS550. The insertion sequence was found to be identical to ISS1 located on pSK08 in the clumping L. lactis subsp. lactis strain ML3. Restriction maps of pOZS550 and pSK08 were similar suggesting a close ancestral relationship, although pSK08, in addition to the lactose metabolism genes, expressed genes for proteinase activity and cell clumping, which were not expressed by pOZS550, and carried two copies of ISS1 compared to one on pOZS550. Furthermore, hybridization of the 18 base pair inverted repeat, of the insertion sequence, with various L. lactis subsp. lactis strains and two L. lactis subsp. cremoris strains showed moderate to strong hybridization to one plasmid in each organism.     

Replication and temperature-sensitive maintenance functions of lactose plasmid pSK11L from Lactococcus lactis subsp. cremoris.

The replication region of pSK11L, the lactose plasmid of Lactococcus lactis subsp. cremoris (L. cremoris) SK11, was isolated on a 14.8-kbp PvuII fragment by shotgun cloning into an Escherichia coli vector encoding erythromycin resistance and selection for erythromycin-resistant transformants of L. lactis subsp. lactis (L. lactis) LM0230. Deletion analysis and Tn5 mutagenesis of the resulting plasmid (pKMP1) further localized the replication region to a 2.3-kbp ScaI-SpeI fragment. DNA sequence analysis of this 2.3-kbp fragment revealed a 1,155-bp open reading frame encoding the putative replication protein, Rep. The replication origin was located upstream of rep and consisted of an 11-bp imperfect direct repeat and a 22-bp sequence tandemly repeated three and one-half times. The overall organization of the pSK11L replicon was remarkably similar to that of pCI305, suggesting that pSK11L does not replicate by the rolling-circle mechanism. Like pSK11L, pKMP1 was unstable in L. lactis LM0230. Deletion analysis allowed identification of several regions which appeared to contribute to the maintenance of pKMP1 in L. lactis LM0230. pKMP1 was significantly more stable in L. cremoris EB5 than in L. lactis LM0230 at all of the temperatures compared. This stability was lost by deletion of a 3.1-kbp PvuII-XbaI fragment which had no effect on stability in L. lactis LM0230. Other regions affecting stability in L. cremoris EB5 but not in L. lactis LM0230 were also identified. Stability assays conducted at various temperatures showed that pKMP1 maintenance was temperature sensitive in both L. lactis LM0230 and L. cremoris EB5, although the plasmid was more unstable in L. lactis LM0230. The region responsible for the temperature sensitivity phenotype in L. lactis LM0230 was tentatively localized to a 1.2-kbp ClaI-HindIII fragment which was distinct from the replication region of pSK11L. Our results suggest that the closely related L. lactis and L. cremoris subspecies behave differently regarding maintenance of plasmids.     

Identification and nucleotide sequence of the minimal replicon of the low-copy-number plasmid pBS2

The plasmid pBS2 has a low copy number and is endogenous to Bacillus subtilis. The replication of this plasmid depends on the function of most of the host's dna genes including dnaB, which is unique to B. subtilis and is required for both the initiation of chromosome replication and the DNA-membrane association. We have identified the region that is essential for the replication of pBS2 and determined the complete 2279-bp nucleotide sequence of this region. In this region, there are two stretches of sequence homologous to the 18-bp consensus sequence which commonly appears at the origin of replication of plasmids pUB110 and pC194. The entire region contains six sizable open reading frames. Two of them are probably translated. One open reading frame, designated ORF A, coding for 269 amino acids, has significant homology, in terms of amino acid sequence, with the open reading frame of the gene for the Rep U protein of plasmid pUB110. The similarities between pBS2 and other plasmids suggest that the pBS2 may also replicate as a rolling circle, which appears to be the salient feature of a mechanism of replication that is common to small plasmids in gram-positive bacteria.     

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.     

Protoplast-induced curing of bacteriocin plasmid in Lactococcus lactis subsp. lactis 484

Plasmid-specified traits like lactose metabolism and bacteriocin production could be eliminated from Lactococcus lactis subsp. lactis 484 culture during production and regeneration of protoplasts with lysozyme at the concentration of 300 μg/ml after 3 h treatment. Plasmid-free strains and cured derivatives harbouring only a single plasmid (2 MDa) were also obtained. Loss of high molecular weight (65 MDa) low copy number Lac plasmid occurred more frequently compared with low molecular weight (2 MDa) high copy number plasmid. Treatment of L. lactis subsp. lactis 484 cells with lysozyme at concentrations of 1000 μg/ml could produce a large number of Lac⁻ Bac⁻ variants at a very high frequency (94%). The curing data confirmed the linkage of Lac and Bac phenotypes to 65 and 2 MDa plasmids, respectively.     

Segregational stability and copy number of the theta-type lactococcal replicon Rep22 in Lactococcus

Rep22 is the replication region of the lactococcal theta replicating pUCL22 plasmid. The copy number of Rep22-based plasmids in Lactococcus was determined by using a chromosomal DNA fragment from Lactococcus lactis subsp. lactis MMS368 as reference. Segregational behavior appeared to be linked to copy number and therefore indicated random distribution of copies to daughter cells. Nevertheless, an active partitioning system was detected in the parental plasmid pUCL22. A pUCL22 138-bp DNA restriction fragment bearing a perfect 18-bp inverted repeat was involved in the improvement of Rep22-based plasmid segregational stability during discontinuous exponential growth.     

The stability of the lactose and citrate plasmids in Lactococcus lactis subsp. lactis biovar. diacetylactis

We have studied the intraperoxisomal location of catalase in peroxisomes of methanol-grown Hansenula polymorpha by (immuno)cytochemical means. In completely crystalline peroxisomes, in which the crystalline matrix is composed of octameric alcohol oxidase (AO) molecules, most of the catalase protein is located in a narrow zone between the crystalloid and the peroxisomal membrane. In non-crystalline organelles the enzyme was present throughout the peroxisomal matrix. Other peroxisomal matrix enzymes studied for comparison, namely dihydroxyacetone synthase, amine oxidase and malate synthase, all were present throughout the AO crystalloid. The advantage of location of catalase at the edges of the AO crystalloids for growth of the organism on methanol is discussed.     

pH influences growth and plasmid stability of recombinant Lactococcus lactis subsp. lactis

Continuous cultures of a recombinant Lactococcus lactis subsp. lactis strain were performed to display the effect of the fermentation pH on the specific growth rate and plasmid stability. The proportion of plasmid pIL252 bearing cells decreased exponentially with the number of generations. The influence of the pH on the rate of loss of plasmid pIL252 and on the specific growth rate of L. lactis IL2682 was described by second order polynomial equations. Optimal pH for the growth and plasmid stability were 6.39 and 6.41 respectively. © Rapid Science Ltd. 1998     

Campbell-like integration of heterologous plasmid DNA into the chromosome of Lactococcus lactis subsp. lactis.

Integrable vectors were constructed based on the plasmid pHV60, which is essentially a pBR322 replicon carrying a chloramphenicol resistance marker, by inserting 1.3-kilobase chromosomal fragments of Lactococcus lactis subsp. lactis MG1363 into this plasmid. Three constructs as well as pHV60 were electroporated to strain MG1363. Transformants were obtained with all constructs, and also with pHV60 (albeit with low frequency). By using Southern hybridizations, it appeared that pHV60 showed homology with the chromosome of MG1363, and that it most probably uses this homology to integrate in a Campbell-like manner. The presence of chromosomal sequences in pHV60 stimulated insertion elsewhere in the chromosome by a factor of 5 to 100. In all cases the integrated plasmids were amplified, at a selective pressure of 5 micrograms of chloramphenicol per ml, to a level of approximately 15 copies per chromosome. Although the amplification was gradually lost under nonselective conditions, one copy remained stably integrated in the chromosome. The results show that a Campbell-like integration strategy can be used to improve the accessibility of the lactococcal chromosome for genetic analysis and is potentially useful in stabilizing unstable genes in lactococci.