Efficiency of homologous DNA recombination varies along the Bacillus subtilis chromosome

Abstract

Structures consisting of a genetic marker (erythromycin or kanamycin resistance, thymidylate synthetase) flanked by 3.4-kilobase direct repeats (pBR322 sequences) were inserted in 12 different locations of the Bacilus subtilis chromosome. Recombination between the repeats was followed by the loss of the genetic marker. Recombination frequencies found in different locations varied from 1.2 x 10-5 to 40 x 10-5 per cell generation. Such differences were highly significant (P < 0.001).Direct repeats, single and multiple, are maintained rather stably in the thyB region of the Bacillus subtilis chromosome, which 'indicates that recombination occurs infrequently at this location (10,17). In contrast, multiple direct repeats are not maintained stably in the pheA region of the chromosome (28). Some instability of multiple repeats was also reported in the amyE region (8). The last two observations, in line with previous reports of unstable maintenance of multiple genes in the Escherichia coli chromosome (5,20), raised the possibility that in the thyB region of the B. subtilis chromosome recombination occurs with an unusually low frequency and that elsewhere in the chromosome the frequency is higher. To test this hypothesis, we measured the recombination frequency between identical duplications generated at 12 different sites of the B. subtilis chromosome. At all sites the recombination frequency was low and generally close to that measured previously in the thyB region. Significant differences between certain sites were nevertheless detected. MATERIALS AND METHODSBacterial strains and plasmids are listed in Table 1. Enzymes were commercial preparations and were used as specified by the suppliers. Plasmid and chromosomal DNA were prepared by previously described methods (2, 7). Competence induction and transformation of E. coli and B. subtilis cells was described before (3, 15). To select resistant E. coli cells, ampicillin (Ap), 50 ,ug/ml, and erythromycin (Em), 250 ,ug/ml, were used. Chloramphenicol (Cm), 3 to 5 ,ug/ml, kanamycin (Km), 5 pLg/ml, erythromycin, 0.3 pug/ml and trimethoprim (Tp), 30 ,ug/ml (in the presence of thymine [50 ,ug/ml]) were used for selecting resistant B. subtilis cells.Labeled probes were prepared by nick translation (21) and used as described before (23).E. coli HVC45 and HVC1001 and B. subtilis SB202 and HVS496 were used as hosts for plasmid constructions. HVS496 was obtained as follows. First, plasmid pHV438 (16), which is composed of pBR322, the Cmr gene from pC194, the B. subtilis thyB gene, and a B. subtilis chromosomal segment of unknown genetic content (named X) was cleaved with PvuII, ligated, and introduced into E. coli cells.This treatment deleted 700 base pairs (bp) from the thyB gene and gave the plasmid named pHV482. This plasmid was cleaved at its unique Sall site and used to transform B. RESULTSExperimental strategy and strain construction. To measure recombination frequencies in the B. subtilis chromosome, we generated a structure consisting of direct repeats flanking a genetic ...

Use of integrational plasmid excision to identify cellular localization of gene expression during sporulation in Bacillus subtilis

“…Selection for heat-resistant spores was used to measure the frequency of plasmid excision. Frequency ranged from 2.4 x 10-5 to 3.3 X 1O-4 heat-resistant cells per viable cell, which is consistent with recent reports on the stability of reiterated sequences in the B. subtilis chromosome (31,32), and appeared to be related to the size of the insert in the integrational plasmid.…”

Use of integrational plasmid excision to identify cellular localization of gene expression during sporulation in Bacillus subtilis

“…Selection for heat-resistant spores was used to measure the frequency of plasmid excision. Frequency ranged from 2.4 x 10-5 to 3.3 X 1O-4 heat-resistant cells per viable cell, which is consistent with recent reports on the stability of reiterated sequences in the B. subtilis chromosome (31,32), and appeared to be related to the size of the insert in the integrational plasmid.…”

Single-crossover integration in the Lactobacillus sake chromosome and insertional inactivation of the ptsI and lacL genes

“…This variation could be due to the presence of specific signals on the homologous segment or to the target location, creating mutations and new phenotypes or making important modifications in the local conformation surrounding the segment. Such location-specific effects are expected from random insertions and were previously reported for Lactococcus lactis (4) as well as for other bacteria (18,29).…”

A Method to Invert DNA Segments of theBacillus subtilis168 Genome by Recombination between Two Homologous Sequences

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Use of integrational plasmid excision to identify cellular localization of gene expression during sporulation in Bacillus subtilis

“…Selection for heat-resistant spores was used to measure the frequency of plasmid excision. Frequency ranged from 2.4 x 10-5 to 3.3 X 1O-4 heat-resistant cells per viable cell, which is consistent with recent reports on the stability of reiterated sequences in the B. subtilis chromosome (31,32), and appeared to be related to the size of the insert in the integrational plasmid.…”

Single-crossover integration in the Lactobacillus sake chromosome and insertional inactivation of the ptsI and lacL genes

“…This variation could be due to the presence of specific signals on the homologous segment or to the target location, creating mutations and new phenotypes or making important modifications in the local conformation surrounding the segment. Such location-specific effects are expected from random insertions and were previously reported for Lactococcus lactis (4) as well as for other bacteria (18,29).…”

A Method to Invert DNA Segments of theBacillus subtilis168 Genome by Recombination between Two Homologous Sequences

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Use of integrational plasmid excision to identify cellular localization of gene expression during sporulation in Bacillus subtilis

“…Selection for heat-resistant spores was used to measure the frequency of plasmid excision. Frequency ranged from 2.4 x 10-5 to 3.3 X 1O-4 heat-resistant cells per viable cell, which is consistent with recent reports on the stability of reiterated sequences in the B. subtilis chromosome (31,32), and appeared to be related to the size of the insert in the integrational plasmid.…”

Single-crossover integration in the Lactobacillus sake chromosome and insertional inactivation of the ptsI and lacL genes

“…This variation could be due to the presence of specific signals on the homologous segment or to the target location, creating mutations and new phenotypes or making important modifications in the local conformation surrounding the segment. Such location-specific effects are expected from random insertions and were previously reported for Lactococcus lactis (4) as well as for other bacteria (18,29).…”

A Method to Invert DNA Segments of theBacillus subtilis168 Genome by Recombination between Two Homologous Sequences

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