Cloning of the phospho-β-galactosidase gene in Escherichia coli from lactose-negative mutants of Streptococcus mutans isolated following random mutagenesis with plasmid pVA891 clone banks

Abstract In order to mutagenize Streptococcus mutans a marker rescue plasmid, pVA891, was employed. The plasmid was ligated with Sau 3AI digested chromosomal DNA fragments from S. mutans GS-5IS3 and the resultant plasmids were amplified in Escherichia coli . These plasmids were then randomly integrated into the chromosome of strain GS-5IS3 following transformation. Lactose-negative transformants were isolated as white colonies on lactose-BTR-Xgal agar plates containing erythromycin. Six lactose-negative mutants representing three different chromosomal sites of integration were isolated from about eight thousand transformants. Mutant chromosomal DNA fragments flanking the plasmids were recovered by a marker-rescue method in E. coli and exhibited phospho-β-galactosidase activity.     

Transformation of Streptococcus sanguis Challis with a plasmid of Streptococcus pneumoniae

Abstract The present work is concerned with plasmid transformation of Streptococcus sanguis strain Challis with derivatives of pDP1/pSMB1, the only plasmid found to occur naturally in Streptococcus pneumoniae . Two recombinant plasmids derived from the cryptic pSMB1 were used: pDP27 (4.5 kb) conferring resistance to chloramphenicol (Cm), and pDP28 (7.8 kb), a shuttle plasmid, conferring resistance to Cm in Escherichia coli , and resistance to erythromycin (Em) in pneumococcus. It could be shown that pSMB1 can replicate in S. sanguis ; in fact, Challis strain V288 was transformed to Cm-resistance and to Em-resistance by pDP27 and pDP28 respectively.
Shuttle plasmid pDP28 can transform S. sanguis both when isolated from pneumococcus and from E. coli , albeit with a different efficiency. The low frequency of transformation observed when pDP28 was isolated from E. coli DH1 ( recA ) was shown to be due to lack of multimeric forms of the plasmid in the DNA preparations obtained from this strain. When pDP28 was isolated from E. coli C600 (RecA⁺), multimeric forms were present, and transformations of S. sanguis was more efficiency Using pDP28 as vector in cloning experiments, where S. sanguis was the host of the recombinant DNA molecules, treatment of the vector with alkaline phosphatase inhibited the recovery of recombinant clones.     

Bacillus licheniformis as an object for the propagaton of heterologous genetic material from bacilli

Bacillus licheniformis was transformed with plasmids pUB110 and pJJ10 (pUB110 - pBR322) isolated from Bac. subtilis and Escherichia coli, respectively. It was revealed that the structure and genetic properties of the plasmids did not change during the transformation process. pJJ101 (pJJ10-rib) DNA isolated from E. coli and containing helper pJJ10 plasmid was used, as a recipient. It was shown that pJJ101 rib markers were "rescued" by the resident plasmid during transformation of Bac. licheniformis (pJJ10). Plasmid pLP1 containing ribB, ribD, Kmr genes and the pUB110 replicator, was isolated from the transformants. pLP1 plasmid might be considered as a detected derivative of the parental pJJ101 plasmid. The deletion is presented by 3,9 MD segment that contains the pBR322 replicator. pLP1 DNA is capable of transforming plasmidless strains of Bac. licheniformis and Bac. subtilis.     

Plasmid transformation in Agmenellum quadruplicatum PR-6: construction of biphasic plasmids and characterization of their transformation properties.

Biphasic, chimeric plasmids for the transformation of Agmenellum quadruplicatum PR-6 (Synechococcus sp. strain 7002) were constructed by splicing the 3.0-megadalton cryptic plasmid from strain PR-6 into plasmids pBR322 and pBR325 from Escherichia coli. Transformants of either E. coli or strain PR-6 by these plasmids could be detected on the basis of the drug resistance marker(s) carried by the chimeric plasmids. Plasmid DNA isolated from a PR-6 transformant transformed PR-6 much more efficiently than plasmid DNA prepared from E. coli. Plasmids from which the AvaI recognition site was deleted (AvaI is an isoschizomer of the AquI restriction endonuclease of strain PR-6) also transformed strain PR-6 much more efficiently than did plasmids containing the AvaI recognition site. These and other results suggest that AquI strongly effects plasmid transformation when the donor plasmid contains an unmodified AquI recognition site. Multimeric forms of the chimeric plasmids are also much more efficient at transforming strain PR-6 than are the analogous monomeric forms.     

Transforming activity of R- and Lac-plasmids of clinical strains of Gram-negative bacteria]

The isolated plasmid DNA of clinical strains of Gram-negative bacteria were shown to have transforming activity when E. coli strain 0600 and S. typhimurium strain LT-2 were used as recipients. The frequency of transformation depended on the recipient strain and the character of the plasmids. The presence of deletion mutants was revealed among the transformants. Such mutants occurred with varying frequency, most often in S. typhimurium strain LT-20; the reason for this phenomenon is at present under discussion. The transformation of plasmids controlling lactose splitting and their conjugation transfer into recipient S. typhimurium strain LT-2 is possible only under condition of using recipient (R+). The possibility of the formation of the cointegrate (R and lac plasmids) in recipient S. typhimurium strain LT-2 is discussed.     

Transformation of Saccharomyces cerevisiae with UV-irradiated single-stranded plasmid.

UV-irradiated single-stranded replicative plasmids were used to transform different yeast strains. The low doses of UV used in this study (10-75 J/m2) caused a significant decrease in the transforming efficiency of plasmid DNA in the Rad+ strain, while they had no effect on transformation with double-stranded plasmids of comparable size. Neither the rev3 mutation, nor the rad18 or rad52 mutations influenced the efficiency of transformation with irradiated single-stranded plasmid. However, it was found to be decreased in the double rev3 rad52 mutant. Extracellular irradiation of plasmid that contains both URA3 and LEU2 genes (psLU) gave rise to up to 5% Leu- transformants among selected Ura+ ones in the repair-proficient strain. Induction of Leu- transformants was dose-dependent and only partially depressed in the rev3 mutant. These results suggest that both mutagenic and recombinational repair processes operate on UV-damaged single-stranded DNA in yeast.     

Genetic transformation of Streptococcus pneumoniae by heterologous plasmid deoxyribonucleic acid.

A number of heterologous plasmid deoxyribonucleic acids (DNAs) coding for erythromycin, tylosin, lincomycin, tetracycline, or chloramphenicol resistance have been introduced into Streptococcus pneumoniae via genetic transformation with frequencies that varied between 10(-5) to as high as 5 x 10(-1) per colony-forming unit. Transformation with plasmid DNA required pneumococcal competence, was competed by chromosomal DNA, and showed a saturation at about 0.5 micrograms/ml (with a recipient population of 3 x 10(7) colony-forming units of competent cells per ml). Plasmid transformation did not occur with a recipient strain, 410, defective in endonuclease I activity and in chromosomal genetic transformation. All erythromycin-resistant transformants examined contained covalently closed circular DNA with the same electrophoretic mobility on agarose gels as the donor DNAs, and when examined in detail the plasmid reisolated from the transformants had the same restriction patterns and the same specific transforming activity as the donor DNA. In the cases of two plasmids examined in detail--pAM77 and pSA5700 Lc9--most of the transforming activity was associated with DNA monomers; DNA multimers present in pSA5700 Lc9 also had biological activity. An unexpected finding was the demonstration of transformation (2 x 10(-5) per colony-forming unit) with plasmid DNAs linearized by treatment with S1 nuclease or with restriction endonucleases.     

A functional lagging strand origin does not stabilize plasmid pMV158 inheritance in Escherichia coli

Plasmid rolling circle replication generates single-stranded DNA intermediates. The intracellular amount of these molecules depends upon the efficiency of the conversion of single-stranded into double-stranded plasmid forms, that is, the functionality of the lagging strand origin (sso). The broad-host-range streptococcal plasmid pMV158 harbors two different ssos, both of which function efficiently in Streptococcus pneumoniae but poorly in Escherichia coli. Plasmid pMV158 is stably inherited in the pneumococcal host, but it is unstable in E. coli. A pMV158 derivative lacking its two ssos is unstable in both strains. We have cloned into this derivative the coliphage f1 lagging strand origin. Whereas the f1 sso was fully functional in E. coli, it did not show any activity in S. pneumoniae, a bacteria closely related to the pMV158 natural host. The presence of the f1 sso did not stabilize pMV158 inheritance in either the gram-positive or the gram-negative host.     

Streptococcus pneumoniae polA gene is expressed in Escherichia coli and can functionally substitute for the E. coli polA gene.

The Streptococcus pneumoniae polA+ gene was introduced into Escherichia coli on the recombinant plasmid pSM31, which is based on the pSC101 replicon. Extracts of E. coli polA5 mutants containing pSM31 showed DNA polymerase activity, indicating that the pneumococcal DNA polymerase I was expressed in the heterospecific host. Complete complementation of the E. coli polA5 mutation by the pneumococcal polA+ gene was detected in excision repair of DNA damage.     

Construction of a new Streptococcus pneumoniae-Escherichia coli shuttle vector based on the replicon of an indigenous pneumococcal cryptic plasmid.

The nucleotide sequence of a cryptic plasmid (pRMG1) isolated from a type 1 Streptococcus pneumoniae has been determined and two recombinant plasmids, pRMGE1 and pRMGE2, bearing the pRMG1 replicon have been constructed. pRMGE2 is a shuttle vector for Escherichia coli and S. pneumoniae. The important characteristics of this cloning vector are: a size of 5.5 kb including a 1.4 kb fragment of pRMG1 (containing a double-stranded replication origin and an open reading frame encoding a putative replication initiation protein), a multicloning site, two antibiotic resistance markers for selection of plasmid containing cells, and blue-white colony screening in E. coli for identification of insert-containing plasmids.     

Genetic transformation of Saccharomyces cerevisiae with single-stranded circular DNA vectors

We asked if single-stranded vector DNA molecules could be used to reintroduce cloned DNA sequences into a eukaryotic cell and cause genetic transformation typical of that observed using double-stranded DNA vectors. DNA was presented to Saccharomyces cerevisiae following a standard transformation protocol, genetic transformants were isolated, and the physical state of the transforming DNA sequence was determined. We found that single-stranded DNA molecules transformed yeast cells 10- to 30-fold more efficiently than double-stranded molecules of identical sequence. More cells were competent for transformation by the single-stranded molecules. Single-stranded circular (ssc) DNA molecules carrying the yeast 2 μ plasmid-replicator sequence were converted to autonomously replicating double-stranded circular (dsc) molecules, suggesting their efficient utilization as templates for DNA synthesis in the cell. Single-stranded DNA molecules carrying 2 μ plasmid non-replicator sequences recombined with the endogenous multicopy 2 μ plasmid DNA. This recombination yielded either the simple molecular adduct expected from homologous recombination (40% of the transformants examined) or aberrant recombination products carrying incomplete transforming DNA sequences, endogenous 2 μ plasmid DNA sequences, or both (60% of the transformants examined). These aberrant recombination products suggest the frequent use of a recombination pathway that trims one or both of the substrate DNA molecules. Similar aberrant recombination products were detected in 30% of the transformants in cotransformation experiments employing single-stranded and double-stranded DNA molecules, one carrying the 2 μ plasmid replicator sequence and the other the selectable genetic marker. We conclude that single-stranded DNA molecules are useful vectors for the genetic transformation of a eukaryotic cell. They offer the advantage of high transformation efficiency, and yield the same intracellular DNA species obtained upon transformation with double-stranded DNA molecules. In addition, single-stranded DNA molecules can participate in a recombination pathway that trims one or both DNA recombination substrates, a pathway not detected, at least at the same frequency, when transforming with double-stranded DNA molecules     

Plasmid vector for cloning in Streptococcus pneumoniae and strategies for enrichment for recombinant plasmids

A new plasmid, pLS101, was constructed for use as a vector for cloning in Streptococcus pneumoniae. This plasmid carries two selectable genes, tet and malM, each of which contains two or more restriction sites for cloning. Insertional inactivation of the malM gene allowed direct selection of TcRMal- clones containing recombinant plasmids. Other means of enriching a recipient population for cells containing recombinant plasmids were examined. The effect of removing vector terminal phosphate in attempts to clone heterogeneous DNA fragments, such as those from chromosomal DNA, was to abolish recombinant plasmid establishment altogether, presumably because donor DNA processing during entry into the cell prevented establishment of the hemiligated molecule. However, with homogeneous DNA fragments, such as those from plasmid or viral DNA, vector phosphate removal allowed enrichment for recombinant plasmids. In the cloning of heterogeneous DNA that was homologous to the recipient chromosome (i.e. chromosomal DNA from S. pneumoniae), recovery of recombinant plasmids could be enriched tenfold (relative to the regenerated vector) by the process of chromosomal facilitation of plasmid establishment. This involved an additional passage of the mixed plasmids in which interaction with the chromosome of plasmids containing chromosomal DNA inserts (i.e. recombinant plasmids) increased their frequency of establishment relative to the vector plasmid. An overall strategy for cloning in S. pneumoniae, depending on the nature of the fragment to be cloned, is proposed.     

Natural transformation in Campylobacter species.

Growing cells of Campylobacter coli and C. jejuni were naturally transformed by naked DNA without the requirement for any special treatment. Transformation frequencies for homologous chromosomal DNA were approximately 10(-3) transformants per recipient cell in C. coli and 10(-4) in C. jejuni. Maximum competence was found in the early log phase of growth. Campylobacters preferentially took up their own DNA in comparison with Escherichia coli chromosomal DNA, which was taken up very poorly. Three new Campylobacter spp.-to-E. coli shuttle plasmids, which contained additional cloning sites and selectable markers, were constructed from the shuttle vector pILL550A. These plasmid DNAs were taken up by campylobacters much less efficiently than was homologous chromosomal DNA, and transformation into plasmid-free cells was very rare. However, with the use of recipients containing a homologous plasmid, approximately 10(-4) transformants per cell were obtained. The tetM determinant, originally obtained from Streptococcus spp. and not heretofore reported in Campylobacter spp., was isolated from an E. coli plasmid and was introduced, selecting for tetracycline resistance, by natural transformation into C. coli.     

Chromosomal transformation of Escherichia coli recD strains with linearized plasmids.

Wild-type Escherichia coli are resistant to genetic transformation by purified linear DNA, probably in part because of exonuclease activity. We demonstrate that E. coli containing a recD mutation could be easily transformed by linearized plasmids containing a selectable marker. The marker was transferred to the chromosome by homologous recombination, whereas plasmid markers not in the region of homology were lost.     

Organization and transfer of heterologous chloramphenicol and tetracycline resistance genes in pneumococcus.

The cat and tet genes of chloramphenicol- and tetracycline-resistant clinical isolates of Streptococcus pneumoniae from Paris and Japan were shown to be contained in adjacent heterologous insertions into the chromosome. The two insertions transformed laboratory strains at frequencies that were low, unequal, and, for tet, very sensitive to the length of the donor deoxyribonucleic acid strand. In contrast, the transforming activity of cat was relatively stable. There was an unusual asymmetric cotransfer, in that a majority of the tet transformants also acquired cat, whereas only a few of the cat transformants also acquired tet. The evidence for chromosomal insertion came from genetic data showing linkage of cat to a chromosomal gene and from cosedimentation of cat with chromosomal markers in both velocity and dye-buoyancy experiments. Genes on a known plasmid introduced into pneumococcus from Streptococcus faecalis showed very different physical behavior. Most of the transformation properties of these genes can be readily accounted for by analogy to transformation of deletions of normal genes. Whether transposition contributes any of the transfers remains to be determined. The presence of one of the genes in the recipient promoted the integration of the other, demonstrating enhanced accumulation of heterologous genes by a process that did not involve plasmids in the species of concern.     

Cloning and expression in Escherichia coli of a gene encoding proline oxidase of Serratia marcescens.

Proline plays a central role in the biosynthesis of prodigiosin by Serratia marcescens. Proline catabolism takes place by oxidation catalysed by the enzyme proline oxidase encoded by the gene putA. A gene bank of chromosomal DNA from S. marcescens was constructed using the plasmid vector pBR328, and then recombinant DNA was used in transformation experiments with Escherichia coli HB 101 as recipient strain. One of the recombinant plasmids, pSL001, was encoded for proline oxidase. Subcloning experiments led to a second plasmid pSL008 able to maintain proline oxidase activity.     

Genetic transformation of Geobacillus kaustophilus HTA426 by conjugative transfer of host-mimicking plasmids

We established an efficient transformation method for thermophile Geobacillus kaustophilus HTA426 using conjugative transfer from Escherichia coli of host-mimicking plasmids that imitate DNA methylation of strain HTA426 to circumvent its DNA restriction barriers. Two conjugative plasmids, pSTE33T and pUCG18T, capable of shuttling between E. coli and Geobacillus spp., were constructed. The plasmids were first introduced into E. coli BR408, which expressed one inherent DNA methylase gene (dam) and two heterologous methylase genes from strain HTA426 (GK1380-GK1381 and GK0343-GK0344). The plasmids were then directly transferred from E. coli cells to strain HTA426 by conjugative transfer using pUB307 or pRK2013 as a helper plasmid. pUCG18T was introduced very efficiently (transfer efficiency, 10(-5)-10(-3) recipient(-1)). pSTE33T showed lower efficiency (10(-7)-10(-6) recipient(-1)) but had a high copy number and high segregational stability. Methylase genes in the donor substantially affected the transfer efficiency, demonstrating that the host-mimicking strategy contributes to efficient transformation. The transformation method, along with the two distinguishing plasmids, increases the potential of G. kaustophilus HTA426 as a thermophilic host to be used in various applications and as a model for biological studies of this genus. Our results also demonstrate that conjugative transfer is a promising approach for introducing exogenous DNA into thermophiles.     

Rapid electroporation-mediated plasmid transfer between Lactococcus lactis and Escherichia coli without the need for plasmid preparation

A simple and rapid method for the transfer of plasmids between the Gram-positive species Lactococcus lactis and Escherichia coli without the need for plasmid preparation is described. The donor strain was subjected to an electroporation pulse which released plasmid DNA into the suspending buffer which was then centrifuged to remove cells and debris. The supernatant was mixed with the recipient strain and subjected to a second electroportion pulse, resulting in the transfer of plasmid from donor to recipient. In cases where a high transformation efficiency is not required, such as the transfer of a cloned construct from E. coli to Lactococcus or vice versa , this method has the advantages of convenience and rapidity.     

Analysis of Junction Sequences Resulting from Integration at Nonhomologous Loci in Neurospora Crassa

We have analyzed the junctions involved in two examples of ectopic integration of plasmids containing the am+ (glutamate dehydrogenase) gene into a strain of Neurospora crassa bearing a complete deletion of the am locus. In one transformed strain a single copy of plasmid DNA had been integrated into linkage group (LG) III DNA without the loss of chromosomal DNA. In contrast, 450 bp had been lost from plasmid sequences at the site of integration. The transforming DNA used was circular, so we postulate that the plasmid was linearized and truncated prior to its integration by end joining into a break in LG III DNA. There was no significant homology between the incoming DNA and DNA at the site of integration. The second transformed strain resulted from transformation with a linearized plasmid. It contained multiple integrated copies of plasmid DNA, one of which was recloned, together with adjacent chromosomal DNA, by plasmid rescue in Escherichia coli. Prior to integration into chromosomal DNA, the linear plasmid had been truncated by 64 bp on one end and 3.2 kbp on the other end. One end of the integrated DNA was adjacent to DNA from the right arm of LG I, while the other end was integrated into a copy of a repetitive sequence. Restriction fragment length polymerism mapping showed that integration was in a copy of the repetitive sequence that is linked to the previously unassigned telomere M11 and is distantly linked to the LG VI marker con-11. Genetic analysis revealed that a long segment of LG I containing all markers from un-1 to the right tip has been translocated to the right end of LG VI. Tetrad analysis showed that the integrated DNA was closely linked to the translocation. We conclude that the transforming DNA was truncated and joined to DNA from two different chromosomes by end joining during the formation of a quasiterminal translocation, T(IR----VIR) UK-T12. We also conclude that the previously unassigned telomere, M11, is the right end of LG VI.