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.     

Investigation into the nature of a Bacillus promoter cloned into a promoter-probe plasmid

The alpha-amylase-coding gene (amy) of Bacillus amyloliquefaciens NCP1 was cloned into the Bacillus subtilis promoter probe vector pPL603b.1, using a BglII digest of chromosomal DNA. The resulting plasmid, pVC102, was shown to have a BglII site within the insert. It was determined that this was the result of the fortuitous co-cloning of 2.88-kb and 0.92-kb BglII fragments separated in NCP1 DNA by approx. 3 kb. Unexpectedly, this co-cloning was readily repeated. Subcloning showed that while the 2.88-kb amy-bearing fragment was sufficient for amylase production, it might not have been capable of promoting sufficient levels of chloramphenicol resistance under the conditions used in the cloning experiments. The promoter on the 0.92-kb BglII fragment was more efficient, although its sequence differed from the canonical promoter sequence recognised by B. subtilis RNA polymerase E.sigma 43. As other promoter-bearing fragments from NCP1 DNA operated equally efficiently when cloned into pPL603b.1, the reason for the repeated co-cloning of the 2.88-kb and 0.92-kb NCPI BglII fragments may well be due to structural parameters, whereby certain nucleotide sequences are more readily cloned than others.     

In vitro genetic labeling of Bacillus subtilis cryptic plasmid pHV400.

A DNA segment which encodes resistance to tetracycline, and cannot replicate autonomously, was excised by HindIII endonuclease from plasmid pT127 and joined to the cryptic Bacillus subtilis plasmid pHV400. The analysis of resulting chimerae has allowed us to identify a 1.8 × 10⁶ segment of pHV400 which carried the replication functions of the cryptic plasmid. Another DNA segment, designated pHV32, which can replicate in Escherichia coli but not in B. subtilis has also been used for genetic labeling of the replication region of pHV400. pHV32 is convenient for use in isolating cryptic replicons active in B. subtilis because (1) it can be prepared in large quantities, free from any interferring B. subtilis replicons, from an appropriate E. coli strain; (2) it carries unique sites for various restriction endonucleases; (3) the chloramphenicol resistance gene which it specifies can transform B. subtilis at a high efficiency (10⁶–10⁷ transformants/μg of DNA).     

Integration and amplification of plasmid DNA in the Bacillus subtilis chromosome

Some Bacillus subtilis strains demonstrate a high degree of pHV14 and pP1251 plasmid integration into the chromosome (1.10(-1]. According to the data of genetical analysis the plasmids are integrated into several regions of the same chromosome. A significant plasmid amplification in the composition of chromosome is observed when breeding on an increasing chloramphenicol concentration (20-300 micrograms/ml). Both tandem repetitions and single plasmid DNA copies and their fragments are found in the bacterial chromosomal DNA.     

Cloning of an unstable spoIIA-tyrA fragment from Bacillus subtilis

A recombinant cosmid clone was isolated from a library created from cosmid pQB79-1 and Bacillus subtilis DNA, and a 15 kb BamHI fragment derived from the cloned insert was transferred to the vector pHV33. The recombinant clone, pRC12, was capable of complementing eight auxotrophic markers in the spoIIA-tyrA region of the B. subtilis chromosome (map positions 205-210). It also complemented eight of nine markers in the spoIIA locus. The exception, spoIIA176, is the most distal marker from lysine. Although pRC12 failed to complement sporulation defects in spoVA or spoIVA (spoIIA+) strains, subclones of pRC12, lacking a functional spoIIA gene, did complement these mutations. pRC12 inhibited sporulation in a spo+ recE strain, possibly due to the presence of multiple functional spoIIA genes. Both the original cosmid and pRC12 were unstable in Escherichia coli and B. subtilis. Antibiotic selection of the vector resulted in extensive deletion of the insert, while selection for insert function in B. subtilis invariably led to loss of the chloramphenicol resistance vector function.     

Isolation and characterization of a recombinant plasmid carrying a functional part of the Bacillus subtilis spoIIA locus

Plasmid pHM2 consists of a 3.3 kb insert of Bacillus subtilis DNA in the chimeric plasmid pHV33, and can replicate in Escherichia coli and B. subtilis. In B. subtilis, pHM2 complements the defects resulting from mutations spo-42, spo-50, spo-69 and sas-1 in the spoIIA locus. This complementation can occur in recE4 strains where recombination of the plasmid with the chromosome is prevented, and the chromosome retains the mutant allele. Thus the plasmid carries a functional part of the spoIIA locus; it does not contain the complete locus as it cannot complement several other spoIIA mutations. It is likely that the locus is complex, containing at least two genes.     

Structural stability of heterologous genes cloned in Streptomyces plasmid pIJ702

A recombinant plasmid pWCL1 containing Streptomyces plasmid pIJ702, E. coli plasmid pUC12, and hepatitis B viral surface antigen (HBsAg) gene was stably maintained in E. coli, but exhibited structural instability in S. lividans 1326. The deletions were found ranging from 2.75 to 5.65 kilobases (kb) and most of them occurred within the melanin (mel) gene of pIJ702, resulting in the loss of part of the mel gene sequence plus the insert. The removal of the pUC12 sequence from pWCL1 eliminated the instability. However, pUC12 alone inserted in either orientation on pIJ702 also caused the deletion in S. lividans 1326. The results indicated that the structural instability of hybrid plasmid of pIJ702 depended on the interaction between the mel sequence and the inserted sequence.     

Effect of Bacillus subtilis BsuM restriction-modification on plasmid transfer by polyethylene glycol-induced protoplast fusion

Polyethylene glycol (PEG)-induced cell fusion is a promising method to transfer larger DNA from one cell to another than conventional genetic DNA transfer systems. The laboratory strain Bacillus subtilis 168 contains a restriction (R) and modification (M) system, BsuM, which recognizes the sequence 5'-CTCGAG-3'. To study whether the BsuM system affects DNA transfer by the PEG-induced cell fusion between R(+)M(+) and R(-)M(-) strains, we examined transfer of plasmids pHV33 and pLS32neo carrying no and eight BsuM sites, respectively. It was shown that although the transfer of pLS32neo but not pHV33 from the R(-)M(-) to R(+)M(+) cells was severely restricted, significant levels of transfer of both plasmids from the R(+)M(+) to R(-)M(-) cells were observed. The latter result shows that the chromosomal DNA in the R(-)M(-) cell used as the recipient partially survived restriction from the donor R(+)M(+) cell, indicating that the BsuM R(-)M(-) strain is useful as a host for accepting DNA from cells carrying a restriction system(s). Two such examples were manifested for plasmid transfer from Bacillus circulans and Bacillus stearothermophilus strains to a BsuM-deficient mutant, B. subtilis RM125.     

Restriction and modification in Bacillus subtilis Marburg 168: target sites and effects on plasmid transformation

Summary The effects of the restriction system of Bacillus subtilis strain M on plasmid transformation were studied. Plasmid pHV1401 DNA prepared from B. subtilis transformed the restriction-proficient M strain 100 times more efficiently than the DNA prepared from Escherichia coli, while the two DNA preparations transformed restriction-deficient derivatives of that strain with similar efficiencies. This indicates that transformation with pHV1401 is sensitive to the M restriction system. pHV1401 contains three CTCGAG (XhoI sites). Successive removal of these abolished the effect of restriction. This indicates that the XhoI sites are the targets for the M restriction system.Abbreviations used Ap^r resistance to ampicillin - Cm^r resistance to chloramphenicol - R/M restriction and modification - Tc^r resistance to tetracycline     

A strategy to construct vector-free amylolytic strains through nondisruptive homologous recombination: application to Enterococcus faecalis.

In order to direct chromosomal integration of the alpha-amylase-encoding gene from Bacillus licheniformis (amyL) under the control of expression and secretion signals from Enterococcus faecalis, the chromosomal fragment (named AB) from the pGIP3124 plasmid [Hols et al., Gene 118 (1992) 21-30] was chosen and split into two fragments (A and B). A translation fusion between the A fragment and 'amyL, deleted of its expression and secretion signals, was made and this fusion was flanked with the AB fragment at its right end. The A::'amyL:AB integration module was cloned into a thermosensitive pE194 replicon (chloramphenicol resistant; CmR) and electro-transformed into E. faecalis OG1X. After an overnight culture in selective liquid medium, the offspring from the amylolytic transformants obtained was shown to yield CmR colonies with two distinct halo sizes on iodine-stained starch plates. Southern analysis clearly showed that the smaller halos corresponded to descendants in which the plasmid had integrated into the chromosome through homologous recombination. One such Amy+ integrant in the AB site was further cultured under nonselective conditions at 42 degrees C for about 20 generations, and the offspring was screened for Amy+/CmS clones. Such revertants were indeed found, and Southern analysis clearly showed that the vector matrix had been excised through homologous recombination between the redundant A sites, leaving the integrated amy gene intact.     

Plasmid R1 in Salmonella typhimurium: Molecular instability and gene dosage effects

Plasmid R1 drd-19 and two of its copy mutants (pKN102 and pKN103) were transferred from Escherichia coli to Salmonella typhimurium, where the expression of the copy mutations was studied further. The copy number (ratio of plasmid DNA to chromosomal DNA) was the same in S. typhimurium and in E. coli. The activities of the plasmid-coded antibiotic-metabolizing enzymes β-lactamase, chloramphenicol acetyltransferase, and streptomycin adenylyltransferase as well as the resistances to ampicillin and streptomycin were proportional to the gene dosage up to at least a threefold increase in the steady state plasmid copy number, whereas resistance to chloramphenicol showed no increase with increased number of plasmid copies per chromosome equivalent. Also the resistance to rifampicin was affected since S. typhimurium cells became more sensitive the higher the copy number of the resident plasmid. Furthermore, plasmid R1 showed molecular instability in S. typhimurium cells since there was a tendency to dissociate into resistance transfer factors and resistance determinants and also to form miniplasmids. This tendency to instability was more pronounced the higher the plasmid copy number.     

Cloning in Streptococcus lactis of plasmid-mediated UV resistance and effect on prophage stability.

Plasmid pIL7 (33 kilobases) from Streptococcus lactis enhances UV resistance and prophage stability. A 5.4-kilobase pIL7 fragment carrying genes coding for both characters was cloned into S. lactis, using plasmid pHV1301 as the cloning vector. The recombinant plasmid was subsequently transferred to three other S. lactis strains by transformation or protoplast fusion. Cloned genes were expressed in all tested strains.     

Cloning in Streptococcus lactis of plasmid-mediated UV resistance and effect on prophage stability

Plasmid pIL7 (33 kilobases) from Streptococcus lactis enhances UV resistance and prophage stability. A 5.4-kilobase pIL7 fragment carrying genes coding for both characters was cloned into S. lactis, using plasmid pHV1301 as the cloning vector. The recombinant plasmid was subsequently transferred to three other S. lactis strains by transformation or protoplast fusion. Cloned genes were expressed in all tested strains.     

Are single-stranded circles intermediates in plasmid DNA replication?

Plasmid pC194 exists as circular double-stranded and single-stranded DNA in Bacillus subtilis and Staphylococcus aureus. We report here that the plasmid pHV33, composed of pBR322 and pC194, exists as double- and single-stranded DNA in Escherichia coli, provided that the replication functions of pC194 are intact. Single-stranded pHV33 DNA is converted to double-stranded DNA by complementary strand synthesis probably initiated at rriB, a primosome assembly site present on pBR322. The efficiency of complementary strand synthesis affects the double-stranded copy number, which suggests that single-stranded DNA is a plasmid replication intermediate.     

Identification of Pseudomonas alcaligenes chromosomal DNA in the plasmid DNA of the chlorobenzene-degrading recombinant Pseudomonas putida strain CB1-9.

The recombinant Pseudomonas putida strain CB1-9, which acquired the ability to grow on chlorobenzenes, contains a 33-kilobase (kb) plasmid (pKFL3) which lacked homology to an indigenous 15-kb plasmid (pKFL1) in Pseudomonas alcaligenes C-0 parent but was homologous to a 55-kb plasmid (pKFL2) from the P. putida R5-3 parent. Chromosomal DNA of P. alcaligenes C-0 hybridized to probes prepared from pKFL3 but not to probes prepared from pKFL2. A single clone from a genomic library of P. alcaligenes C-0 hybridized to EcoRI-digested pKFL3. Southern blot hybridization with the insert DNA from that clone identified homology with specific restriction enzyme fragments in pKFL3. The ability of the recombinant to utilize 3-chlorobenzoate, chlorobenzene, and 1,4-dichlorobenzene as well as its loss of utilization of xylenes and methylbenzoates appears to be associated with the transfer and integration of chromosomal DNA from P. alcaligenes into a Tol-like plasmid of P. putida R5-3.     

Replication functions of pC194 are necessary for efficient plasmid transduction by M13 phage.

Escherichia coli plasmids pBR313 and pBR322 were transduced by phage M13 with low efficiency (10(-8) transductants/phage). Hybrid plasmids pHV12 or pHV33, composed of Staphylococcus aureus plasmid pC194 and pBR313 or pBR322, respectively, were transduced much more efficiently (10(-4) transductants/phage). Inactivation of either of the two zones necessary for pC194 replication, one coding for a protein, the other not, reduced the transforming efficiency of hybrids to the level of pBR322. Activity of the pC194 replication region was not necessary for the formation of chimeras between M13 and the transduced plasmid in the donor cells, but rather for the establishment of the plasmid in the recipient cells.     

Characterization of chloramphenicol resistance inLactobacillus plantarum caTC2

A strain ofLactobacillus plantarum caTC2R isolated from a meat source was resistant to chloramphenicol (30 g/ml). Resistance was mediated through an inducible chloramphenicol acetyltransferase. Plasmid analysis of this strain showed three plasmids, of which the 8.5-kb plasmid apparently encodes the gene for chloramphenicol resistance. This plasmid was lost at high frequency (25%) when theLactobacillus was subcultured at a higher than optimal temperature (40°C).     

Identification of Pseudomonas alcaligenes chromosomal DNA in the plasmid DNA of the chlorobenzene-degrading recombinant Pseudomonas putida strain CB1-9

The recombinant Pseudomonas putida strain CB1-9, which acquired the ability to grow on chlorobenzenes, contains a 33-kilobase (kb) plasmid (pKFL3) which lacked homology to an indigenous 15-kb plasmid (pKFL1) in Pseudomonas alcaligenes C-0 parent but was homologous to a 55-kb plasmid (pKFL2) from the P. putida R5-3 parent. Chromosomal DNA of P. alcaligenes C-0 hybridized to probes prepared from pKFL3 but not to probes prepared from pKFL2. A single clone from a genomic library of P. alcaligenes C-0 hybridized to EcoRI-digested pKFL3. Southern blot hybridization with the insert DNA from that clone identified homology with specific restriction enzyme fragments in pKFL3. The ability of the recombinant to utilize 3-chlorobenzoate, chlorobenzene, and 1,4-dichlorobenzene as well as its loss of utilization of xylenes and methylbenzoates appears to be associated with the transfer and integration of chromosomal DNA from P. alcaligenes into a Tol-like plasmid of P. putida R5-3.