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.     

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.     

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.     

Divergence of Genomic Sequences between Lactococcus lactis subsp. lactis and Lactococcus lactis subsp. cremoris

Relatedness between Lactococcus lactis subsp. cremoris and L. lactis subsp. lactis was assessed by Southern hybridization analysis, with cloned chromosomal genes as probes. The results indicate that strains of the two subspecies form two distinct groups and that the DNA sequence divergence between L. lactis subsp. lactis and L. lactis subsp. cremoris is estimated to be between 20 and 30%. The previously used phenotypic criteria do not fully discriminate between the groups; therefore, we propose a new classification which is based on DNA homology. In agreement with this revised classification, the L. lactis subsp. lactis and L. lactis subsp. cremoris strains from our collection have distinct phage sensitivities.     

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.     

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 phi712 (936 phage species) and the prolate-headed phage phic2 (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.     

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     

Comparison of cell wall proteinases from Lactococcus lactis subsp. cremoris AC1 and Lactococcus lactis subsp. lactis NCDO 763

Summary The cell wall proteinases of Lactococcus lactis subsp. lactis NCDO 763 and L. lactis subsp. cremoris AC1 hydrolyse -casein with a similar specificity even though some quantitative differences can be observed for a few degradation products analysed by reverse phase HPLC and sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The main peptides soluble in 1.1% trifluoroacetic acid and liberated by the two proteinases were identified and have been found to be the same for the two enzymes. They are located in two areas of the -casein sequence (53–93 and the C-terminal part: 129–209) and they include bitter tasting or physiologically active fragments. No narrow specificity was observed for these proteinases. However, glutamine and serine residues are more frequently encountered in position P1 and P1 of the sensitive peptide bond and the close environment (position P2 to P4 and P2 to P4) of the cleaved bond is mainly hydrophobic.     

Comparison of cell wall proteinases from Lactococcus lactis subsp. cremoris AC1 and Lactococcus lactis subsp. lactis NCDO 763

Summary Cell wall-associated proteinases were isolated from Lactococcus lactis subsp. cremoris AC1 and subsp. lactis NCDO 763 in order to compare their specificities towards different caseins. Two purification strategies were applied. Cells grown in casein-free M17 medium were a suitable starting material for purification, since electrophoretic purity could be achieved after one chromatographic step. Both enzymes has an apparent molecular mass of about 145000 daltons as judged by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Electrophoresis and reversed phase HPLC patterns of hydrolysates of _s1-, _s2-, -, and K-caseins indicated that both proteinases had a similar specificity. The enzyme of L. lactis subsp. lactis split _s1- and _s2-caseins more extensively than that of L. lactis subsp. cremoris.     

Efficient plasmid mobilization by pIP501 in Lactococcus lactis subsp. lactis.

pIP501 is a streptococcal conjugative plasmid which can be transmitted among numerous gram-positive strains. To identify a minimal mobilization (mob) locus of pIP501, DNA fragments of pIP501 were cloned into nonconjugative target plasmids and tested for mobilization by pIP501. We show that nonmobilizable plasmids containing a specific fragment of pIP501 are transmitted at high frequencies between Lactococcus lactis subsp. lactis strains if transfer (tra) functions are provided in trans by a pIP501 derivative. Independent transfer of the mobilized plasmid was observed in up to 44% of transconjugants. A 2.2-kb segment containing mob was sequenced. This DNA segment is characterized by three palindromes (palI, palII, and palIII) and a 202-amino-acid open reading frame (ORFX) of unknown function. The smallest DNA fragment conferring high frequency mobilization was localized to a 1.0-kb region (extending from pIP501 coordinates 3.60 to 4.60 on the 30.2-kb map) which contains palI (delta G = -27 kcal/mol [ca. -110,000 J/mol]). A 26-bp sequence identical to palI is present on pIP501, upstream of the plasmid copy control region. Further homologies with the palI sequence are also found with the related Enterococcus faecalis conjugative plasmid pAM beta 1. The region containing mob maps outside the previously described segment mediating pIP501 conjugation. Our results with recA strains indicate that the mob site is a hot spot for cointegrate formation.     

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.     

PCR Amplification of the Gene acmA Differentiates Lactococcus lactis subsp. lactis and L. lactis subsp. cremoris

The occurrence of the acmA gene, encoding the lactococcal N-acetylmuramidase in new lactococcal isolates from raw milk cheeses, has been determined. Isolates were genotypically identified to the subspecies level with a PCR technique. On the basis of PCR amplification of the acmA gene, the presence or absence of an additional amplicon of approximately 700 bp correlated with Lactococcus lactis subspecies. L. lactis subsp. lactis exhibits both the expected 1,131-bp product and the additional amplicon, whereas L. lactis subsp. cremoris exhibits a single 1,131-bp fragment.     

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.     

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.     

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.     

Nucleotide sequence and characterization of the cell envelope proteinase plasmid in Lactococcus lactis subsp. cremoris HP

AIMS: The major cell envelope proteinase (lactocepin; EC 3.4.21.96) produced by Lactococcus lactis cheese starter bacteria is required for starter growth and acid production in milk. The aim of this study was to characterize a lactocepin plasmid from a L. lactis subsp. cremoris cheese starter strain. METHODS AND RESULTS: A restriction map of the lactocepin plasmid pHP003 from strain HP was constructed, fragments were cloned in Escherichia coli vectors, and the complete DNA sequence (13,433 bp) was determined. Among 120 industrial L. lactis starter strains screened, five contained the same specificity-type lactocepin as pHP003. The lactocepin gene in these strains was invariably linked with a partially-deleted abiB gene. CONCLUSION: The lactocepin specificity type of strain HP, conferred by a known configuration of key residues, is relatively uncommon. The gene is invariably linked with a partially deleted abiB gene on each lactocepin plasmid. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first complete sequence reported for a lactocepin plasmid, and provides the basis for better understanding, or manipulation, of lactocepin production.     

PCR amplification of the gene acmA differentiates Lactococcus lactis subsp. lactis and L. lactis subsp. cremoris.

The occurrence of the acmA gene, encoding the lactococcal N-acetylmuramidase in new lactococcal isolates from raw milk cheeses, has been determined. Isolates were genotypically identified to the subspecies level with a PCR technique. On the basis of PCR amplification of the acmA gene, the presence or absence of an additional amplicon of approximately 700 bp correlated with Lactococcus lactis subspecies. L. lactis subsp. lactis exhibits both the expected 1,131-bp product and the additional amplicon, whereas L. lactis subsp. cremoris exhibits a single 1,131-bp fragment.     

Regulation of the ribonucleotide reductases in Lactococcus lactis subsp. cremoris

Lactococcus lactis has two essential ribonucleotide reductases for DNA biosynthesis and repair which are affected in the presence or absence of oxygen. Expression of glutaredoxin like protein (NrdH), the hydrogen donor for ribonucleotide reductase, was found to be regulated by the FNR like proteins (FlpA and FlpB). Proteomics study demonstrated that expression level of NrdH significantly decreased in the flpA and flpAB deletion mutants. The nrdH gene is located in an nrdHIEF operon and encoding the NrdEF ribonucleotide reductase, which is active under aerobic and anaerobic conditions. Regulation of expression of the nrdHIEF operons was investigated using beta-galactosidase as a reporter gene. The 588 bp fragment containing the nrdH promoter and gene cloned into the pORI vector immediately upstream of a promoterless lacZ gene. Constructed plasmid was transferred into wild type (MG1363), single mutant (flpA orflpB) and double mutant (flpAB). Aerobically, nrdH promoter activity is 15-fold higher than anaerobic expression.