Homologous recombination between plasmid and chromosomal DNA in Bacillus subtilis requires approximately 70 bp of homology

Хасанов, Ф. К.; Žvingila, Donatas; Zainullin, Almaz A.; Prozorov, A. A.; Bashkirov, Vladimir I.

doi:10.1007/bf00538711

Cited by 63 publications

(47 citation statements)

References 19 publications

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“…However, as the experimental systems used (interplasmid recombination [24] and suicide [34] and Ts plasmid delivery vectors) were all different, direct comparisons are not meaningful. In a recent study of sco integration in B. subtilis with a Ts derivative of rc plasmid pE194, integration frequency showed a linear dependence on substrate length (14). However, the homologous lengths were shorter than in this study (fragments of 77 to 165 bp), and it is likely that the starting vector (an rc plasmid containing E. coli DNA) produced HMW (9).…”

Section: Resultscontrasting

confidence: 66%

High-efficiency gene inactivation and replacement system for gram-positive bacteria

et al. 1993

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A system for high-efficiency single-and double-crossover homologous integration in gram-positive bacteria has been developed, with Lactococcus lactis as a model system. The system is based on a thermosensitive broad-host-range rolling-circle plasmid, pG'host5, which contains a pBR322 replicon for propagation in Escherichia coli at 37°C. A nested set of L. lactis chromosomal fragments cloned onto pG'host5 were used to show that the single-crossover integration frequency was logarithmically proportional to the length of homology for DNA fragments between 0.35 and 2.5 kb. Using random chromosomal 1-kb fragments, we showed that homologous integration can occur along the entire chromosome. We made use of the reported stimulatory effect of rolling-circle replication on intramolecular recombination to develop a protocol for gene replacement. Cultures were first maintained at 37°C to select for a bacterial population enriched for plasmid integrants; activation of the integrated rolling-circle plasmid by a temperature shift to 28°C resulted in efficient plasmid excision by homologous recombination and replacement of a chromosomal gene by the plasmid-carried modified copy. More than 50% of cells underwent replacement recombination when selection was applied for the replacing gene. Between 1 and 40%o of cells underwent replacement recombination when no selection was applied. Chromosomal insertions and deletions were obtained in this way. These results show that gene replacement can be obtained at an extremely high efficiency by making use of the thermosensitive rolling-circle nature of the delivery vector. This procedure is applicable to numerous gram-positive bacteria.Numerous gram-positive bacteria are targets of study as biological models (e.g., Bacillus subtilis), industrially important fermenter strains (the lactic acid bacteria), or pathogens (e.g., clostridia, listeria, staphylococci, and streptococci). Many strains of industrial or medical importance have been characterized physiologically, but relatively few have been studied or modified genetically. The study or modification of strains could be facilitated by the use of delivery vectors for the introduction of directed or nonspecific insertions in the bacterial chromosome. Delivery systems that rely on nonreplicative vectors are limited to bacteria that can be transformed at a high frequency, and those with conditionally active replicons are often restricted in their host range. Thus, the construction of recombinant strains requires substantial effort and can be applied efficiently only to particular organisms.We previously described a broad-host-range thermosensitive (Ts) plasmid, pVE6002 (22), which was isolated from pGK12 (15). Such plasmids replicate by a rolling-circle (rc) mechanism (rc plasmids) (21). pVE6002 was shown to have potential use as a delivery vector in numerous gram-positive bacteria (22). Ts plasmids are nonreplicative at 37°C, making them particularly useful in bacteria with a low-temperature growth range or when a drastic thermal shock is un...

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Section: Resultscontrasting

confidence: 66%

High-efficiency gene inactivation and replacement system for gram-positive bacteria

et al. 1993

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“…Furthermore, the small sizes of the repeats distinguishes illegitimate recombination events from homologous recombination, which requires extensive regions of homology. The extent of homology necessary to promote homologous recombination has been estimated to be approximately 70 bp in B. subtilis (30) and 30 bp in E. coli (24).…”

Section: Discussionmentioning

confidence: 99%

Spiroplasma citri virus SpV1-derived cloning vector: deletion formation by illegitimate and homologous recombination in a spiroplasmal host strain which probably lacks a functional recA gene

Marais¹,

Bové²,

Renaudin³

1996

J Bacteriol

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Spiroplasmas are wall-free prokaryotes with helical morphology and motility. They belong to the class Mollicutes, a group of organisms which have derived by regressive evolution from ancestors of gram-positive bacteria with low-level guanineplus-cytosine DNA (66, 70). The high-level adenine-thymine content has resulted in a peculiar codon usage. For instance, in mycoplasmas, ureaplasmas, mesoplasmas, entomoplasmas, and spiroplasmas, UGA is read as tryptophan and not as a termination signal. As a result, expression of mollicute genes in Escherichia coli has been limited to genes without the UGA tryptophan codon (8,26,42) or has led to truncated translation products (43). Cloning in a mycoplasma or a spiroplasma host would overcome this difficulty and, in addition, would open the way to genetic analysis of these organisms. Gene function can be identified only by analysis of their mutations, and complementation of gene mutations requires that genes be transferred between mutants. For members of the class Mollicutes, genetic studies have been hampered by the lack of metabolic markers and the lack of suitable vectors for transferring genes between isolates. Several approaches have been examined for the development of gene transfer systems (5, 17). Recently, potential cloning vectors based on the pKMK1 plasmid of Mycoplasma mycoides have been constructed (31).Plasmids have also been isolated from spiroplasma cells (see reference 6 for a review). However, a combination of the Spiroplasma citri pMH1 plasmid and the chloramphenicol acetyl transferase (cat) gene of pBR328 was found to be highly unstable, as the recombinant plasmid could not be recovered from the spiroplasmal transformants (56). A more successful approach was the use of the replicative form (RF) of S. citri virus SpV1, a rod-shaped virus with a circular, single-stranded DNA genome. A peculiar feature of SpV1 is the presence of many viral sequences integrated into the S. citri host chromosome. The viral sequences consist of both full-length and deletion forms of the viral genome (49).In previous experiments, we have used the RF of SpV1 as a vector for the cloning and expression of the cat gene in spiroplasma cells (59, 60). The S. citri-SpV1 cloning system was also used to express an epitope carried on the G fragment of the cytadhesin P1 gene from Mycoplasma pneumoniae (37). However, we now report the structural instability of the recombinant viral DNA carrying the G fragment. The finding that, in addition to illegitimate recombination, homologous recombination is very probably involved in deletion formation led us to characterize the recA gene of the S. citri R8A2 host strain. MATERIALS AND METHODSBacterial strains, viruses, plasmids, and media. E. coli TG1 [hsd⌬5 FЈ (traD36 proAB ϩ lacZ␣M15) supE thi ⌬(lac-proAB)] was used as the host for amplification of plasmid vector pBS ϩ (Stratagene Cloning Systems, La Jolla, Calif.) and its derivatives and for propagation of the M13 recombinant viruses. E. coli cells were grown in Luria-Bertani medium (52). S. citri...

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“…Lines approximate linear extrapolation of higher frequencies to zero recombination. (3,12,14,25,26), no statistical support was seen for an MEPS in S. acidocaldarius ME. Furthermore, recombinant frequency remains approximately proportional to marker separation up to at least 2 kbp in bacteriophage T4, 20 kbp in E. coli, and 30 kbp in S. cerevisiae (16,18,26) but only to about 0.05 kbp for ME in S. acidocaldarius.…”

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confidence: 94%

Conjugational Genetic Exchange in the Hyperthermophilic Archaeon Sulfolobus acidocaldarius : Intragenic Recombination with Minimal Dependence on Marker Separation

2005

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In Sulfolobus acidocaldarius conjugation assays, recombinant frequency was relatively constant for marker separations from 1,154 bp down to about 50 bp and readily detectable at 10 bp. Three-factor crosses revealed little, if any, genetic linkage over distances of 500 to 600 bp, and large deletion mutants were good donors but poor recipients in matings. The results indicate that most intragenic recombination events occur at one of the mutations, not in the interval between them.The conjugational mechanism of marker exchange (ME) in Sulfolobus acidocaldarius (8) is so far the only system in which natural exchange and recombination of chromosomal DNA in a hyperthermophilic archaeon can be quantified by genetic selection. This phenomenon occurs between virtually isogenic strains, is not mediated by culture supernatants, is not inhibited by extracellular DNase, and may account for the infectivity of homing introns that have been introduced into S. acidocaldarius cells by electroporation (1,7,8). ME appears to differ from bacterial Hfr conjugation with respect to both the requirements of DNA transfer and the manifestation of genetic linkage. Most examples of bacterial conjugation require a selftransmissible plasmid in the donor and no plasmid in the recipient, but in S. acidocaldarius, as in the halophilic archaeon Haloferax volcanii (17), all strains appear equally capable of serving as donor and recipient (8). In addition, ME-promoted recombination yields readily detectable recombination (representing a level of about 5% of the maximal value, for example) for mutations separated by only 28 bp (21), whereas in bacterial Hfr conjugation, recombination at 5% of the maximal frequency usually requires about 25 kbp between markers (16). In the present study, we systematically analyzed the effect of marker separation on the frequency of recombination and related molecular properties of ME to gain insight into its mechanism.Genetic analyses. Double mutant strains DG228 and DG229 were spontaneous fluoroorotic acid-resistant derivatives of strain DG64 (9). All other mutants were isolated as spontaneous fluoroorotic acid-resistant derivatives of wild-type strain ATCC 33909. The identity of the pyrE mutation in each strain was determined by PCR amplification and dye terminator sequencing (10, 21). Recombination, reversion, and effects of ␥ and UV radiation were quantified by determining appropriate plate counts (7,8,21,22).Recombination frequency as a function of marker separation. The first set of experiments examined 52 strain pairs defining marker separations of 0 to 1,154 bp. Matings were performed in four to seven independent experiments for each pair, and the median frequencies of recombination were plotted as a function of marker separation (Fig. 1A). Nearly all of the median values fell within a factor of 3 of the average of all medians (Fig. 1A) and were thus more uniform than those for Escherichia coli lacI point mutations (4,19). Linear regression of the entire set of points yielded a large, negative x-axis intercep...

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Homologous recombination between plasmid and chromosomal DNA in Bacillus subtilis requires approximately 70 bp of homology

Cited by 63 publications

References 19 publications

High-efficiency gene inactivation and replacement system for gram-positive bacteria

High-efficiency gene inactivation and replacement system for gram-positive bacteria

Spiroplasma citri virus SpV1-derived cloning vector: deletion formation by illegitimate and homologous recombination in a spiroplasmal host strain which probably lacks a functional recA gene

Conjugational Genetic Exchange in the Hyperthermophilic Archaeon Sulfolobus acidocaldarius : Intragenic Recombination with Minimal Dependence on Marker Separation

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