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.     

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.     

TPW22, a lactococcal temperate phage with a site-specific integrase closely related to Streptococcus thermophilus phage integrases

The temperate phage TPW22, induced from Lactococcus lactis subsp. cremoris W22, and the evolutionarily interesting integrase of this phage were characterized. Phage TPW22 was propagated lytically on L. lactis subsp. cremoris 3107, which could also be lysogenized by site-specific integration. The attachment site (attP), 5'-TAAGGCGACGGTCG-3', of phage TPW22 was present on a 7.5-kb EcoRI fragment, a 3.4-kb EcoRI-HindIII fragment of which was sequenced. Sequence information revealed the presence of an integrase gene (int). The deduced amino acid sequence showed 42 and 28% identity with integrases of streptococcal and lactococcal phages, respectively. The identities with these integrase-encoding genes were 52 and 45%, respectively, at the nucleotide level. This could indicate horizontal gene transfer. A stable integration vector containing attP and int was constructed, and integration in L. lactis subsp. cremoris MG1363 was obtained. The existence of an exchangeable lactococcal phage integration module was suggested. The proposed module covers the phage attachment site, the integrase gene, and surrounding factor-independent terminator structures. The phages phiLC3, TP901-1, and TPW22 all have different versions of this module. Phylogenetically, the TPW22 Int links the phiLC3 lactococcal integrase with known Streptococcus thermophilus integrases.     

Cloning and DNA sequence analysis of two abortive infection phage resistance determinants from the lactococcal plasmid pNP40.

The lactococcal plasmid pNP40, from Lactococcus lactis subsp. lactis biovar diacetylactis DRC3, confers complete resistance to the prolate-headed phage phi c2 and the small isometric-headed phage phi 712 in L. lactis subsp. lactis MG1614. A 6.0-kb NcoI fragment of pNP40 cloned in the lactococcal Escherichia coli shuttle vector pAM401 was found to confer partial resistance to phi 712. Subcloning and deletion analysis of the recombinant plasmid pPG01 defined a 2.5-kb ScaIHpaI fragment as conferring phage insensitivity. Sequence analysis of this region confirmed the presence of two overlapping open reading frames (ORFs). Further subcloning of pNP40 to characterize the resistance determinant active against phi c2 identified a 5.6-kb EcoRV fragment of pNP40 which, when cloned in pAM401, conferred partial resistance to both phi c2 and phi 712. Subcloning and deletion analysis of the recombinant plasmid pCG1 defined a 3.7-kb EcoRV-XbaI fragment as encoding phage insensitivity. DNA sequence analysis of this region revealed the presence of a single complete ORF. The introduction of a frameshift mutation at the unique BglII site within this ORF disrupted the phage resistance phenotype, confirming that this ORF is responsible for the observed phage insensitivity. The mechanisms encoded by pPG01 and pCG1 in L. lactis subsp. lactis MG1614 conformed to the criteria defining abortive infection and were designated AbiE and AbiF, respectively. Analysis of the phage DNA content of phi 712-infected hosts containing AbiF demonstrated that it inhibited the rate of phage DNA replication, while AbiE had little effect on phage DNA replication, suggesting a later target of inhibition. The predicted protein product of abiF shows significant homology to the products of two other lactococcal abortive infection genes, abiD and abiD1.     

Complete genome sequence of the prototype lactic acid bacterium Lactococcus lactis subsp. cremoris MG1363

Lactococcus lactis is of great importance for the nutrition of hundreds of millions of people worldwide. This paper describes the genome sequence of Lactococcus lactis subsp. cremoris MG1363, the lactococcal strain most intensively studied throughout the world. The 2,529,478-bp genome contains 81 pseudogenes and encodes 2,436 proteins. Of the 530 unique proteins, 47 belong to the COG (clusters of orthologous groups) functional category "carbohydrate metabolism and transport," by far the largest category of novel proteins in comparison with L. lactis subsp. lactis IL1403. Nearly one-fifth of the 71 insertion elements are concentrated in a specific 56-kb region. This integration hot-spot region carries genes that are typically associated with lactococcal plasmids and a repeat sequence specifically found on plasmids and in the "lateral gene transfer hot spot" in the genome of Streptococcus thermophilus. Although the parent of L. lactis MG1363 was used to demonstrate lysogeny in Lactococcus, L. lactis MG1363 carries four remnant/satellite phages and two apparently complete prophages. The availability of the L. lactis MG1363 genome sequence will reinforce its status as the prototype among lactic acid bacteria through facilitation of further applied and fundamental research.     

Phenotypic and genetic characterization of the bacteriophage abortive infection mechanism AbiK from Lactococcus lactis.

The natural plasmid pSRQ800 isolated from Lactococcus lactis subsp. lactis W1 conferred strong phage resistance against small isometric phages of the 936 and P335 species when introduced into phage-sensitive L. lactis strains. It had very limited effect on prolate phages of the c2 species. The phage resistance mechanism encoded on pSRQ800 is a temperature-sensitive abortive infection system (Abi). Plasmid pSRQ800 was mapped, and the Abi genetic determinant was localized on a 4.5-kb EcoRI fragment. Cloning and sequencing of the 4.5-kb fragment allowed the identification of two large open reading frames. Deletion mutants showed that only orf1 was needed to produce the Abi phenotype. orf1 (renamed abiK) coded for a predicted protein of 599 amino acids (AbiK) with an estimated molecular size of 71.4 kDa and a pI of 7.98. DNA and protein sequence alignment programs found no significant homology with databases. However, a database query based on amino acid composition suggested that AbiK might be in the same protein family as AbiA. No phage DNA replication nor phage structural protein production was detected in infected AbiK+ L. lactis cells. This system is believed to act at or prior to phage DNA replication. WHen cloned into a high-copy vector, AbiK efficiency increased 100-fold. AbiK provides another powerful tool that can be useful in controlling phages during lactococcal fermentations.     

Lactococcal plasmid pWV01 as an integration vector for lactococci.

A Bacillus subtilis strain was constructed that contained the repA gene of the lactococcal plasmid pWVO1 in its chromosome. This strain was used to construct the pWVO1-based integration vector pINT1, which lacked the repA gene. The 3.6-kb plasmid pINT1 was not able to replicate in Lactococcus lactis MG1363 but integrated into the chromosome via a Campbell-like mechanism when a lactococcal chromosomal DNA fragment was incorporated in the plasmid. Transformants were obtained that carried between one and four plasmid copies, in stable tandem arrangement on the chromosome. The results indicate that pWVO1 can be used for the development of a Campbell-like integration system fully derived of lactococcal DNA, with which stable multiple copies of any gene of interest can be generated in the lactococcal chromosome.     

Molecular characterization of plasmids pS7a and pS7b from Lactococcus lactis subsp. lactis bv. diacetylactis S50 as a base for the construction of mobilizable cloning vectors

Aims:  Strain Lactococcus lactis subsp. lactis bv. diacetylactis S50 harbours five theta-replicating plasmids (pS6, pS7a, pS7b, pS80 and pS140). The aim of this study was to characterize domains involved in the replication and conjugative mobilization of the small plasmids pS7a and pS7b, which are structurally very similar.
Methods and Results:  Complete nucleotide sequences of pS7a and pS7b were determined by cloning DNA fragments of different sizes into Escherichia coli vectors. Linearized plasmids and four Eco RI fragments of the pS7a and pS7b were cloned into an origin probe vector. Constructed plasmids (pSEV10, pSK10, pISE1a and pISE1b) were able to replicate in the strain L. lactis subsp. cremoris MG1363. In addition, experiments showed that plasmids pS7a and pS7b contained oriT sequences and their conjugative transfer directly depended on the presence of pS80 in donor cells.
Conclusions:  Plasmids pS7a and pS7b contained typical lactococcal theta replication origin and repB gene that enable them to replicate in the strain L. lactis subsp. cremoris MG1363. Plasmid pS80 plays a key role in the conjugative transfer of small plasmids.
Significance and Impact of the Study:  Plasmids pS7a and pS7b-based derivatives could be valuable tools for genetic manipulation, studying processes of plasmid maintenance and horizontal gene transfer in lactococci.     

Lactococcal plasmid pWV01 as an integration vector for lactococci.

A Bacillus subtilis strain was constructed that contained the repA gene of the lactococcal plasmid pWVO1 in its chromosome. This strain was used to construct the pWVO1-based integration vector pINT1, which lacked the repA gene. The 3.6-kb plasmid pINT1 was not able to replicate in Lactococcus lactis MG1363 but integrated into the chromosome via a Campbell-like mechanism when a lactococcal chromosomal DNA fragment was incorporated in the plasmid. Transformants were obtained that carried between one and four plasmid copies, in stable tandem arrangement on the chromosome. The results indicate that pWVO1 can be used for the development of a Campbell-like integration system fully derived of lactococcal DNA, with which stable multiple copies of any gene of interest can be generated in the lactococcal chromosome.     

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.     

Plasmid-encoded copper resistance in Lactococcus lactis

A 54-kb plasmid (pND306) from Lactococcus lactis subsp. lactis 1252D encoded resistance to both Cu and Sn . The copper resistance determinant was subcloned on a 12.8-kb PvuII DNA fragment and mapped using a number of restriction endonucleases. Six other copper resistant lactococcal strains were also identified and all contained multiple plasmids. Plasmids in five of these strains showed strong hybridization with a probe made using the 12.8-kb DNA fragment, however no chromosomal homologs were detected. The copper resistance determinant was further isolated as a 10.6-kb SphI fragment and used to construct pND968 that expresses resistance to both copper and nisin.     

Molecular characterization of a theta replication plasmid and its use for development of a two-component food-grade cloning system for Lactococcus lactis

pCD4, a small, highly stable theta-replicating lactococcal plasmid, was used to develop a food-grade cloning system. Sequence analysis revealed five open reading frames and two putative cis-acting regions. None appears to code for undesirable phenotypes with regard to food applications. Functional analysis of the replication module showed that only the cis-acting ori region and the repB gene coding for the replication initiator protein were needed for the stable replication and maintenance of pCD4 derivatives in Lactococcus lactis. A two-component food-grade cloning system was derived from the pCD4 replicon. The vector pVEC1, which carries the functional pCD4 replicon, is entirely made up of L. lactis DNA and has no selection marker. The companion pCOM1 is a repB-deficient pCD4 derivative that carries an erythromycin resistance gene as a dominant selection marker. The pCOM1 construct can only replicate in L. lactis if trans complemented by the RepB initiator provided by pVEC1. Since only the cotransformants that carry both pVEC1 and pCOM1 can survive on plates containing erythromycin, pCOM1 can be used transiently to select cells that have acquired pVEC1. Due to the intrinsic incompatibility between these plasmids, pCOM1 can be readily cured from the cells grown on an antibiotic-free medium after the selection step. The system was used to introduce a phage resistance mechanism into the laboratory strain MG1363 of L. lactis and two industrial strains. The introduction of the antiphage barrier did not alter the wild-type plasmid profile of the industrial strains. The phenotype was stable after 100 generations and conferred an effective resistance phenotype against phages of the 936 and c2 species.     

Development of a lactococcal integration vector by using IS981 and a temperature-sensitive lactococcal replication region.

A vector (pKMP10) capable of Campbell-like integration into the Lactococcus lactis subsp. lactis LM0230 chromosome via homologous recombination with chromosomal IS981 sequences was constructed from the replication region of lactococcal plasmid pSK11L, an internal fragment of IS981, and the erythromycin resistance gene and Escherichia coli replication origin of pVA891. The pSK11L replication region is temperature sensitive for maintenance in L. lactis subsp. lactis LM0230, resulting in loss of unintegrated pKMP10 during growth at greater than 37 degrees C. pKMP10 integrants made up 8 to 75% of LM0230(pKMP10) erythromycin-resistant cells following successive growth at 25 degrees C with selection, 39 degrees C without selection, and 39 degrees C with selection. pKMP10 integrants were also isolated from L. lactis subsp. lactis MG1363(pKMP10) but at a 10-fold-lower frequency (4%). No integrants were isolated form L. lactis subsp. lactis MMS368(pKMP10) (a Rec-deficient strain) or LM0230(pKMP1-E) (the corresponding plasmid lacking the IS981 fragment). Examination of 17 LM0230 integrants by Southern hybridization revealed pKMP10 integration into five different chromosomal sites. Four of the integration sites appeared to be chromosomal IS981 sequences, while one was an uncharacterized chromosomal sequence. The four IS981 integrants seemed to have pKMP10 integrated in a tandem repeat structure of undetermined length. Integrated pKMP10 was more stable (0 to 2% plasmid loss) than unintegrated pKMP10 (100% plasmid loss) when grown for 100 generations at 32 degrees C without selection.     

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     

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.     

Molecular organization of the minimal replicon of novel, narrow-host-range, lactococcal plasmid pCI305

Plasmid pCI305 is an 8.7-kb, narrow-host-range, cryptic plasmid originating from Lactococcus lactis subsp. lactis UC317. The nucleotide sequence of the pCI305 replication region was determined. A single open reading frame of 1158 bp was identified in the trans-active domain repB. The size of the predicted repB protein (46 kDa) is in close agreement with the size of the repB product visualized in vivo in Escherichia coli when repB was placed under control of the inducible phi T7 RNA polymerase promoter. In vivo substitution of the native repB promoter sequence with a Tn5-derived promoter sequence was demonstrated. repA, a 344-bp cis-acting region which is the probable pCI305 replication origin region, was noncoding, was AT-rich, and possessed a unique set of inverted and direct repeat sequences. No significant homology between repA or repB and other gram-positive replication regions was evident. Combined with the absence of a detectable single-stranded DNA intermediate during replication, these results indicate that the pCI305 replication region differs markedly from most gram-positive replicons examined to date. The presence on other lactococcal plasmids of replication regions related to that of pCI305 was demonstrated.     

High-frequency, site-specific recombination between lactococcal and pAM beta 1 plasmid DNAs.

In vivo recombination events involving the 75-kilobase lactose proteinase plasmid pCI301 of Lactococcus lactis subsp. lactis UC317 and the conjugative enterococcal plasmid pAM beta 1 were analyzed. A fragment, identified as containing the pCI301 recombination site, mediated greatly elevated levels of mobilization and recombination with pAM beta 1 when cloned in a nonmobilizable L. lactis-Escherichia coli shuttle vector. This latter recombination event was site and orientation specific on both plasmids. Recombination on pAM beta 1 was within the region associated with plasmid replication, but no effect on pAM beta 1 replication functions was detected. Resolution of recombinant plasmids generated derivatives indistinguishable from the parental plasmids.     

A system to generate chromosomal mutations in Lactococcus lactis which allows fast analysis of targeted genes.

A system for generating chromosomal insertions in lactococci is described. It is based on the conditional replication of lactococcal pWV01-derived Ori+ RepA- vector pORI19, containing lacZ alpha and the multiple cloning site of pUC19. Chromosomal AluI fragments of Lactococcus lactis were cloned in pORI19 in RepA+ helper strain Escherichia coli EC101. The frequency of Campbell-type recombinants, following introduction of this plasmid bank into L. lactis (RepA-), was increased by combining the system with temperature-sensitive pWV01 derivative pVE6007. Transformation of L. lactis MG1363 (pVE6007) with the pORI19 bank of lactococcal chromosomal fragments at the permissive temperature allowed replication of several copies of a recombinant plasmid from the bank within a cell because of the provision in trans of RepA-Ts from pVE6007. A temperature shift to 37 degrees C resulted in loss of pVE6007 and integration of the pORI19 derivatives at high frequencies. A bank of lactococcal mutants was made in this way and successfully screened for the presence of two mutations: one in the monocistronic 1.3-kb peptidoglycan hydrolase gene (acmA) and one in the hitherto uncharacterized maltose fermentation pathway. Reintroduction of pVE6007 into the Mal- mutant at 30 degrees C resulted in excision of the integrated plasmid and restoration of the ability of ferment maltose. The integration plasmid (pMAL) was rescued by using the isolated plasmid content of a restored Mal+ colony to transform E. coli EC101. Nucleotide sequencing of the 564-bp chromosomal fragment in pMAL revealed an internal part of an open reading frame of which the translated product showed significant homology with ATP-binding proteins MalK of E. coli, Salmonella typhimurium, and Enterobacter aerogenes and MsmK of Streptococcus mutans. This combined use of two types of conditional replicating pWV01-derived vectors represents a novel, powerful tool for chromosomal gene inactivation, targeting, cloning, and sequencing of the labelled gene.     

Isolation of a replication region of a large lactococcal plasmid and use in cloning of a nisin resistance determinant

The replication region of a 28-kilobase-pair (kbp) cryptic plasmid from Lactococcus lactis subsp. lactis biovar diacetylactis SSD207 was cloned in L. lactis subsp. lactis MG1614 by using the chloramphenicol resistance gene from the streptococcal plasmid pGB301 as a selectable marker. The resulting 8.1-kbp plasmid, designated pVS34, was characterized further with respect to host range, potential cloning sites, and location of replication gene(s). In addition to lactococci, pVS34 transformed Lactobacillus plantarum and, at a very low frequency, Staphylococcus aureus but not Escherichia coli or Bacillus subtilis. The 4.1-kbp ClaI fragment representing lactococcal DNA in pVS34 contained unique restriction sites for HindIII, EcoRI, XhoII, and HpaII, of which the last three could be used for molecular cloning. A region necessary for replication was located within a 2.5-kbp fragment flanked by the EcoRI and ClaI restriction sites. A 3.8-kbp EcoRI fragment derived from a nisin resistance plasmid, pSF01, was cloned into the EcoRI site of pVS34 to obtain a nisin-chloramphenicol double-resistance plasmid, pVS39. From this plasmid, the streptococcal chloramphenicol resistance region was subsequently eliminated. The resulting plasmid, pVS40, contains only lactococcal DNA. Potential uses for this type of a nisin resistance plasmid are discussed.