Restriction endonuclease analysis of the lactose plasmid in Streptococcus lactis ML3 and two recombinant lactose plasmids.

We investigated the molecular relationship between the 60-megadalton (Mdal) recombinant lactose plasmids in ML 3 x LM2301 lactose-positive (Lac+) transconjugants and the genetic material of Streptococcus lactis ML3. Lactose metabolism is linked to the 33-Mdal plasmid pSK08 in ML3, and the recipient LM2301 is cured of plasmid DNA. The plasmids were analyzed with a series of restriction enzymes. We found that the 60-Mdal plasmids of Lac+ transconjugants contained pSK08 DNA, but were not simply dimers of pSK08. The 60-Mdal plasmids contained a segment of DNA not apparent in pSK08. The restriction patterns of the 60-Mdal plasmid in a Lac+ nonclumping transconjugant and that in a Lac+ clumping transconjugant were different. This suggested that there was a molecular differences between these two recombinant plasmids. We conclude that the segment of DNA in the 60-Mdal plasmids that was not present in pSK08 was the proposed transfer factor responsible for cell aggregation and high-frequency conjugation.     

Restriction endonuclease analysis of the lactose plasmid in Streptococcus lactis ML3 and two recombinant lactose plasmids

We investigated the molecular relationship between the 60-megadalton (Mdal) recombinant lactose plasmids in ML 3 x LM2301 lactose-positive (Lac+) transconjugants and the genetic material of Streptococcus lactis ML3. Lactose metabolism is linked to the 33-Mdal plasmid pSK08 in ML3, and the recipient LM2301 is cured of plasmid DNA. The plasmids were analyzed with a series of restriction enzymes. We found that the 60-Mdal plasmids of Lac+ transconjugants contained pSK08 DNA, but were not simply dimers of pSK08. The 60-Mdal plasmids contained a segment of DNA not apparent in pSK08. The restriction patterns of the 60-Mdal plasmid in a Lac+ nonclumping transconjugant and that in a Lac+ clumping transconjugant were different. This suggested that there was a molecular differences between these two recombinant plasmids. We conclude that the segment of DNA in the 60-Mdal plasmids that was not present in pSK08 was the proposed transfer factor responsible for cell aggregation and high-frequency conjugation.     

Identification and characterization of IS1381, a new insertion sequence in Streptococcus pneumoniae.

A new insertion sequence (IS1381) was identified in the genome of Streptococcus pneumoniae R6 as an 846-bp segment containing 20-bp terminal inverted repeats and flanked by 7-bp direct repeats. The three sequenced copies of this element have two overlapping open reading frame (ORF) genes named orfA and orfB. However, significant variations between individual copies were found, suggesting that inactivating mutations have occurred in an original single ORF. Accordingly, the consensus IS1381 element derived from the comparison of the three available copies should contain a single ORF sufficient to encode a basic protein of 267 amino acids which exhibited high similarity to the putative transposases of ISL2 from Lactobacillus helveticus and of IS702 from the cyanobacterium Calothrix sp. strain PCC 7601. A minimum of five to seven copies were detected by hybridization experiments in the R6 genome. In remarkable contrast with the two previously reported pneumococcal insertion sequences, several copies of IS1381 have been detected in all of the clinical isolates tested so far. Interestingly, Streptococcus oralis NCTC 11427 (type strain), a close relative of pneumococcus, does not contain this element, but its occurrence in the type strain of Streptococcus mitis (NCTC 12261) suggests that this species has exchanged DNA with S. pneumoniae directly or through an intermediate species.     

Influence of the lactose plasmid on the metabolism of galactose by Streptococcus lactis.

Streptococcus lactis strain DR1251 was capable of growth on lactose and galactose with generation times, at 30 degrees C, of 42 and 52 min, respectively. Phosphoenolpyruvate-dependent phosphotransferase activity for lactose and galactose was induced during growth on either substrate. This activity had an apparent K(m) of 5 x 10(-5) M for lactose and 2 x 10(-2) M for galactose. beta-d-Phosphogalactoside galactohydrolase activity was synthesized constitutively by these cells. Strain DR1251 lost the ability to grow on lactose at a high frequency when incubated at 37 degrees C with glucose as the growth substrate. Loss of ability to metabolize lactose was accompanied by the loss of a 32-megadalton plasmid, pDR(1), and Lac(-) isolates did not revert to a Lac(+) phenotype. Lac(-) strains were able to grow on galactose but with a longer generation time. Galactose-grown Lac(-) strains were deficient in beta-d-phosphogalactoside galactohydrolase activity and phosphoenolpyruvate phosphotransferase activity for both lactose and galactose. There was also a shift from a predominantly homolactic to a heterolactic fermentation and a fivefold increase in galactokinase activity, relative to the Lac(+) parent strain grown on galactose. These results suggest that S. lactis strain DR1251 metabolizes galactose primarily via the tagatose-6-phosphate pathway, using a lactose phosphoenolpyruvate phosphotransferase activity to transport this substrate into the cell. Lac(-) derivatives of strain DR1251, deficient in the lactose phosphoenolpyruvate phosphotransferase activity, appeared to utilize galactose via the Leloir pathway.     

Recombinant plasmid associated cell aggregation and high-frequency conjugation of Streptococcus lactis ML3

Lactose-positive (Lac+) transconjugants resulting from matings between Streptococcus lactic ML3 and S. lactis LM2301 possess a single plasmid of approximately 60 megadaltons (Mdal) which is nearly twice the size of the lactose plasmid of the donor. The majority of these Lac+ transconjugants aggregated in broth and were able to transfer lactose-fermenting ability at a frequency higher than 10(-1) per donor on milk agar plates or in broth. Lac+ transconjugants which did not clump conjugated at a much lower frequency. Lactose-negative derivatives of Lac+ clumping transconjugants did not aggregate in broth and were missing the 60-Mdal plasmid. The ability to aggregates in broth was very unstable. Strains could lose the ability to clump but retain lactose-fermenting ability. The majority of these Lac+ nonclumping derivatives of clumping transconjugants contained a plasmid of approximately 33 Mdal, the size of the lactose plasmid of the original donor ML3. These strains transferred lactose-fermenting ability at a frequency of approximately 10(-6) per donor, resulting in both Lac+ clumping transconjugants which contained a 60-Mdal plasmid and Lac+ nonclumping transconjugants which possessed a 33-Mdal plasmid. Our results suggest that the genes responsible for cell aggregation and high-frequency conjugation are on the segment of deoxyribonucleic acid which recombined with the 33-Mdal lactose plasmid in S. lactis ML3.     

IS431, a staphylococcal insertion sequence-like element related to IS26 from Proteus vulgaris

We present the nucleotide sequence of IS431, a new staphylococcal insertion sequence-like element flanking the mercury-resistance determinant of pI524 and associated with the methicillin-resistance determinant. IS431 left is 800 bp long and has a perfect terminal inverted repeat (IR) of 22 bp; IS431 right is 786 bp long and has a terminal IR homologous to the IR of IS431 left except that the terminal 8 bp are absent. Both IRs share a 10-bp homology with the IR of IS26 from Proteus vulgaris. No directly repeated sequences were detected immediately adjacent to the IRs. An open reading frame (ORF) of 675 bp spans most of the IS431 sequence. Its deduced amino acid (aa) sequence shows 40% homology to the 234-aa-long putative transposase coded by ORFI of IS26.     

Isolation of a recombination-deficient mutant of Streptococcus lactis ML3.

A recombination-deficient mutant of Streptococcus lactis ML3 designated MMS36 was isolated on the basis of its sensitivity to methyl methanesulfonate. This mutant also displayed sensitivity to UV irradiation. The inability of MMS36 to mediate homologous recombination was demonstrated by transduction of plasmid-linked lactose fermenting ability but not chromosomally mediated streptomycin resistance.     

Identification of a new genetic determinant for cell aggregation associated with lactose plasmid transfer in Lactococcus lactis.

Derivatives of the lactose miniplasmid pMG820 were constructed in which a staphylococcal erm gene was inserted and in which this was accompanied by subsequent deletion of the lactose genes. The resulting plasmids were thus marked with both erythromycin resistance and lactose utilization genes in pF1132 or solely erythromycin resistance in pF1133. These plasmids retained the normal conjugation properties characteristic of lactose plasmid pLP712, including the generation by intermolecular rearrangement of high-frequency-transfer Clu+ derivatives which exhibited cell aggregation. The use of such Clu+ plasmids in a variety of mating experiments between different lactococcal strains and the observation of cell aggregation when particular mating mixtures were made led to the discovery of a new component of this conjugation system named Agg. A chromosomal gene agg was postulated to be present in some but not all strains of lactococci. High-frequency conjugation and cell aggregation thus depend on the presence of both Agg and Clu, although in a mating pair these components can be in the same or in separate strains. The Agg and Clu components may be analogous to the binding substance and aggregation substance that are involved in the hemolysin plasmid transfer system of Enterococcus faecalis, although control of their expression is different.     

Nucleotide sequence of IS26, a new prokaryotic mobile genetic element.

The DNA sequence of a new IS element, the IS26, is 820 bp long and carries 14 bp perfect terminal inverted repeats. Upon integration, IS26 generates an 8 bp duplication of its target sequence. A large open reading frame within IS26 could code for a protein of 234 amino acids. On its reverse strand, IS26 also carries one large open reading frame, 591 bp long, which contains no stop codon within IS26.     

Identification of a new genetic determinant for cell aggregation associated with lactose plasmid transfer in Lactococcus lactis

Derivatives of the lactose miniplasmid pMG820 were constructed in which a staphylococcal erm gene was inserted and in which this was accompanied by subsequent deletion of the lactose genes. The resulting plasmids were thus marked with both erythromycin resistance and lactose utilization genes in pF1132 or solely erythromycin resistance in pF1133. These plasmids retained the normal conjugation properties characteristic of lactose plasmid pLP712, including the generation by intermolecular rearrangement of high-frequency-transfer Clu+ derivatives which exhibited cell aggregation. The use of such Clu+ plasmids in a variety of mating experiments between different lactococcal strains and the observation of cell aggregation when particular mating mixtures were made led to the discovery of a new component of this conjugation system named Agg. A chromosomal gene agg was postulated to be present in some but not all strains of lactococci. High-frequency conjugation and cell aggregation thus depend on the presence of both Agg and Clu, although in a mating pair these components can be in the same or in separate strains. The Agg and Clu components may be analogous to the binding substance and aggregation substance that are involved in the hemolysin plasmid transfer system of Enterococcus faecalis, although control of their expression is different.     

IS222, a new insertion element associated with the genome of Pseudomonas aeruginosa.

A new insertion element, IS222, was identified to be associated with the DNA of a mutant strain of the converting Pseudomonas aeruginosa bacteriophage D3. The insertion sequence was 1,350 base pairs in size and possessed terminal inverted repeats. The nucleotide sequence contained single cleavage sites for EcoRI and PvuI but none for BamHI, PstI, HindIII, SmaI, or SalI. By Southern hybridization analysis, no homology was found with genomic DNA from P. aeruginosa PAT or Escherichia coli. Genomic DNA from the phage host, P. aeruginosa PAO, contained two sequences homologous to IS222.     

Transposition of the Streptococcus lactis ssp. lactis Z270 lactose plasmid to pVA797: demonstration of an insertion sequence and its relationship to an inverted repeat sequence isolated by self-annealing

The lactose plasmid pUCL22 of the single plasmid strain Streptococcus lactis ssp. lactis Z270 was demonstrated to fuse with the heterologous conjugative plasmid pVA797. The fusion of pUCL22 with pVA797 occurred by recombination between a specific sequence of pUCL22 and different sites of pVA797. The cointegrates of pUCL22::pVA797 were unstable: in the absence of lactose selection, they segregated plasmids that corresponded to pVA797 enlarged by one sequence of 1.2 kb, common to all derivative plasmids. This resolution sequence (RS) was shown to originate in the 9.7 kb BstEII restriction fragment of pUCL22 and to duplicate during replicon fusion. In addition, after nuclease S1 treatment of pUCL22 DNA, a self-annealing sequence was isolated; the two copies of this inverted repeat (IR) sequence were located on the 18 kb BamHI segment of the plasmid. This latter sequence was distinct from the RS with which it hybridized weakly. The RS was responsible for the transposition of the entire lactose plasmid; the role of the IR remains to be elucidated.     

Identification of the theta-type minimal replicon of theLactococcus lactis subsp.lactis CNRZ270 lactose protease plasmid pUCL22

The replication region of pUCL22, the lactose-protease plasmid ofLactococcus lactis subsp.lactis (Lc. lactis) CNRZ270, was isolated on an 18-kbBamHI fragment, by cloning into pUCB300, anEscherichia coli vector encoding Em^r, and selecting for Em^r inLc. lactis MG1614. Subcloning and deletion analysis localized the replication region, namedRep22, on a 2.3-kb fragment. Replicons based on this region followed a theta-type mechanism of replication inLc. lactis. An internal 1251-bpDraI fragment ofRep22 used as a probe hybridized with numerous plasmids in bacteria from the generaLactococcus, Lactobacillus, andLeuconostoc. In some strains, two or three coresident plasmids hybridized with the probe in stringent conditions. It appears, therefore, that this family of theta-type replicons is widely distributed in lactic acid bacteria and contains several incompatibility groups.     

IS15, a new insertion sequence widely spread in R plasmids of gram-negative bacteria

We have shown that the IS15 element, first detected in Salmonella ordonez and previously designated IS1522 (Labigne-Roussel et al. 1981), could transpose, with an approximate frequency of 5 X 10(-5), to various sites of different replicons in an Escherichia coli host deficient for general homologous recombination. Physical mapping with restriction endonucleases of this 1,500 base pairs (bp) transposable module indicated the presence of two, possibly contiguous, directly repeated internal sequences, at least 480 bp in size. IS15 could generate in vivo, by intramolecular recombination between the two direct repeats, IS15-delta, which is 830 bp in size. The reverse transition, IS15-delta to IS15, was not observed. The two related structural forms of IS15 were detected, by Southern hybridization, on plasmids belonging to various incompatibility groups (Inc6-C, I1, 7-M, and Y) isolated from phylogenetically remote pathogenic bacterial genera (Escherichia coli, Salmonella panama, Enterobacter cloacae, and Acinetobacter calcoaceticus). Whereas IS15 could promote its own transposition and transposition of DNA fragments it flanked, IS15-delta resulting from the 670 bp 'clean' deletion and representing the most common natural deletion derivative could only induce replicon fusion. It appears, therefore, that the two structural configurations of IS15 have evolved to play, by transposition, distinct and complementary roles in bacterial evolution.     

Characterization of the genetic element coding for lactose metabolism in Lactococcus lactis subsp. lactis KP3

The Lactococcus lactis subsp. lactis KP3 Lac genetic element was investigated. KP3 is a lactose-positive (Lac+) transconjugant which contains no detectable plasmid DNA. The KP3 Lac genetic element was self-transmissible (Tra+) and encoded a reduced bacteriophage sensitivity (Rbs+) phenotype. Matings of KP3 with a recombination-deficient (Rec-) recipient resulted in Lac+ transconjugants which were phenotypically indistinguishable from KP3 and contained a 96-MDa plasmid (pJS96). Phenotypic and physical analyses of pJS96 indicated that it was a deletion derivative of a putative pKB32::pJS88 Lac+ Tra+ cointegrate. pKB32 is the Lac plasmid and pJS88 is the Tra+ Rbs+ plasmid in L. lactis subsp. lactis 11007, the donor used in obtaining KP3. The results presented suggest that pJS96 is an episome, since it appeared to replicate both as a plasmid and as an integrated part of the chromosome. Conjugal transfer of chromosomal DNA mediated by pJS96 was not observed. Conjugal transfer of pJS96 resulted in Lac+ transconjugants containing plasmids ranging in size from 21 to 90 MDa. Only in Rec+ recipients were transconjugants isolated which appeared to contain pJS96 integrated into the host chromosome. Restriction analysis of several plasmids in the 21 to 90 MDa range suggested the deletions were due to intramolecular transposition of a transposable element on pJS96. This report suggests that a self-transmissible episome exists in KP3 and provides an explanation of how plasmids which vary in size yet encode similar phenotypes may be formed and disseminated.     

Method for Growth and Purification of Bacteriophage 643 on Streptococcus lactis ML3

The yield of bacteriophage 643 was increased by infecting cultures of Streptococcus lactis ML3 in late-log phase growth, harvesting the infected cells, and suspending them in fresh, phosphate-buffered minimal medium. The cells lysed after this treatment and produced high titers of bacteriophage. The phage particles were dissociated from debris by 2 M NaCl and purified by differential and CsCl band centrifugation.     

Identification of IS 1206, a Corynebacterium glutamicum IS3-related insertion sequence and phylogenetic analysis

Integration of plasmid pCGL320 into a Corynebacterium glutamicum ATCC21086 derivative led to tandem amplification of the inserted plasmid (Labarre et a/., 1993). One amplification event was associated with integration of an insertion sequence that we have named IS 1206. Hybridizing sequences were only found in C. glutamicum strains and at various copy numbers. IS1206 is 1290bp long, carries 32 bp imperfect inverted repeats and generates a 3bp duplication of the target DNA upon insertion. IS1206 presents the features characteristic of the IS3 family and part of the DNA sequence centering on the putative transposase region (orfB) is similar to those of IS3 and some other related elements. Phylogenetic analysis of orfB deduced protein sequences from IS 1206 and IS3-related elements contradicts the phylogeny of the species, suggesting that evolution of these elements might be complex. Horizontal transfer could be invoked but other alternatives like ancestral polymorphism or/and different rates of evolution could also be involved.