Illegitimate recombination in Bacillus subtilis: nucleotide sequences at recombinant DNA junctions

Summary The illegitimate integration of plasmid pGG20 (the hybrid between Staphylococcus aureus plasmid pE194 and Escherichia coli plasmid pBR322) into the Bacillus subtilis chromosome was studied. It was found that nucleotide sequences of both parental plasmids could be involved in this process. The recombinant DNA junctions between plasmid pGG20 and the chromosome were cloned and their nucleotide sequences were determined. The site of recombination located on the pBR322 moiety carried a short region (8 bp) homologous with the site on the chromosome. The nucleotide sequences of the pE194 recombination sites did not share homology with chromosomal sequences involved in the integration process. Two different pathways of illegitimate recombination in B. subtilis are suggested.     

Interplasmidic illegitimate recombination in Bacillus subtilis

Summary The illegitimate recombination between Staphylococcus aureus plasmids pE194 (or pGG20, the hybrid between pE194 and Escherichia coli plasmid pBR322) and pBD17 (plasmid pUB110 without HpaII C-fragment) was studied in Bacillus subtilis. Cointegrates were generated with the frequency of 1–3x10^-8. Among 22 hybrids analysed 9 types of recombinants were found. Nucleotide sequences of all three parental plasmids were involved in intermolecular recombination. Nucleotide sequencing of recombinant DNA junctions revealed that in 8 cases recombination occurred between short homologous regions (9–15 bp). One recombinant was formed using nonhomologous sites. The similarity was demonstrated between nucleotide sequences of the recombination sites of two types of cointegrates and those used for pE194 integration into the B. subtilis chromosome. Possible mechanisms of illegitimate recombination are discussed.     

Integration of various plasmids into the Bacillus subtilis chromosome

Some features of integration of temperature-sensitive pE194, pGG10 and pGG20 plasmids into the Bacillus subtilis chromosome were studied. Several auxotrophic mutations were obtained using insertion of these plasmids into the chromosome. The sites of plasmids for illegitimate recombination were determined. It was shown that the integration into the Bac. subtilis chromosome is characteristic not only for the plasmid pE194 but is the property of Staphylococcus aureus plasmid pC194 and Escherichia coli pBR322 plasmid. The influence of different Bac. subtilis rec mutations on the frequency of integration was studied.     

Construction of direct selection vectors for use inBacillus sphaericus

Genetic manipulation in the mosquito larvicidal organismBacillus sphaericus has been hampered by a lack of effective molecular biological techniques and tools. Currently available vectors forBacillus subtilis are inappropriate as they are selected with antibiotics to whichB. sphaericus is naturally resistant. Construction of chimeras between theStaphylococcus aureus plasmid pE194 and theB. subtilis direct selection vector pBD214 permitted selection of plasmid transformants with erythromycin. As with pBD214, direct selection of recombinants is accomplished by insertional inactivation of the plasmid-borne thymidylate synthetase gene and subsequent selection with trimethoprim.     

recE-Dependent Gene Amplification Induced by a Tunicamycin-resistant Mutation (tmr A7) in Bacillus subtilis

A recombinant Bacillus subtilis phage, ρ11-AA248, contains the tmr A7-amy R2-amy E⁺-tmr B⁺-aroI⁺ region of the B. subtilis N7 chromosome on a 22.4 kb DNA fragment. The amy E⁺-tmr B⁺ gene region in the phage genome of the B. subtilis 207-21 transductants by ρ11-AA248 was amplified to approximately 10 copies after cultivation in the presence of tunicamycin (10 μg/ml) and to two copies without tunicamycin. The amplification of the gene region caused hyper-production of extracellular α-amylase. In contrast, no amplification of the gene region was detected in the transductants of B. subtilis 207-25, a recE-deficient derivative of 207-21 strain.     

Insertion and amplification of foreign genes in the Lactococcus lactis subsp. lactis chromosome

The plasmid pE194 is unable to replicate in Lactococcus lactis subsp. lactis (formerly Streptococcus lactis). When linked to resident bacteriophage sequences, pE194 was able to integrate into the L. lactis subsp. lactis chromosome either by Campbell-like recombination or by double crossing over with deletion. Integration occurred into the DNA of the prophage and prevented its multiplication. When a selective pressure was applied to an integrant in which pE194 was flanked by two direct repeats of prophage fragment, amplification of pE194 and the prophage fragment was observed. The pE194 copy number was assessed at six to nine, and amplification was stable upon growth under nonselective conditions.     

Insertion and amplification of foreign genes in the Lactococcus lactis subsp. lactis chromosome.

The plasmid pE194 is unable to replicate in Lactococcus lactis subsp. lactis (formerly Streptococcus lactis). When linked to resident bacteriophage sequences, pE194 was able to integrate into the L. lactis subsp. lactis chromosome either by Campbell-like recombination or by double crossing over with deletion. Integration occurred into the DNA of the prophage and prevented its multiplication. When a selective pressure was applied to an integrant in which pE194 was flanked by two direct repeats of prophage fragment, amplification of pE194 and the prophage fragment was observed. The pE194 copy number was assessed at six to nine, and amplification was stable upon growth under nonselective conditions.     

Interplasmid illegitimate recombination in Bacillus subtilis cells

The illegitimate recombination between S. aureus plasmids pE194 (or pGG20-the hybrid between pE194 and E. coli plasmid pBR322) and pBD17 (plasmid pUB110 without Hpa-II-C-fragment) in B. subtilis was studied. Plasmid cointegrates were generated with the frequency of 1-3.10(-8). Among the 22 hybrids analysed 9 types of recombinants were found. Nucleotide sequences of all the parental plasmids were involved in intermolecular recombination. Nucleotide sequencing of recombinant DNA junctions has revealed that in 8 cases recombination occurred between short homologous regions (9-15 b.p.). One of the recombinants resulted from nonhomologous recombination. The similarity between nucleotide sequences of recombination sites of two types of contegrates and those used for pE194 integration into the B. subtilis chromosome (Bashkirov et al. 1987) was demonstrated. Possible mechanisms of illegitimate recombination are discussed.     

Studies on the synthesis of plasmid-coded proteins and their control in Bacillus subtilis minicells.

The minicell system of Bacillus subtilis has been used to study the expression of plasmid genes using several R plasmids derived from Staphylococcus aureus. pE194, pC194, and pUB110 as well as several mutant and in vitro recombinant derivatives of these plasmids segregate into minicells. A copy control mutant of pE194 was used to show that the extent of segregation is proportional to the copy number. The polypeptides specified by these plasmids were examined by SDS-polyacrylamide gel electrophoresis. Six proteins specified by pE194, an erythromycin resistance plasmid, were identified using cop mutants. These comprise about 90% of the potential coding capacity of the 2.4-Mdal pE194 plasmid. One of these proteins (29,000 daltons) is inducible by erythromycin in the wild type pE194 but is synthesized constitutively in a mutant derivative which also expresses antibiotic resistance constitutively. Several other proteins are detected only in copy control mutants. pUB110, a kanamycin resistance plasmid, expresses three major proteins which comprise 50% of the coding capacity of this 3.0-Mdal plasmid. Two additional minor proteins are occasionally observed. pC194 (2.0 Mdal), which confers chloramphenicol resistance, expresses two polypeptides comprising about 25% of its coding capacity. One of these polypeptides (22,000 daltons) is inducible by chloramphenicol. pBD9, an in vitro composite of pUB110 and pE194, probably expresses all of the major parental plasmid proteins with the exception of one from pUB110 and one from pE194.     

Functional Comparison of Conjugative Transposons Tn916and Tn925

During interspecies matings betweenBacillus subtilisandBacillus thuringiensissubsp.israelensis,transfer of conjugative transposon Tn916was detected at a frequency of 1.1 × 10⁻⁴transconjugants per donor. Tn916-dependent transfer of plasmids pC194 and pE194 was detected at frequencies of 1.4 × 10⁻⁵and 3.2 × 10⁻⁷transconjugants per donor, respectively. Similar frequencies were obtained during parallel matings with otherwise isogenic strains that contain Tn925instead of Tn916. Tn916- or Tn925-dependent transfer of plasmids pC194 or pUB110 from the recipient to the donor (retrotransfer) was not observed during inter- or intraspecies matings. Transposon-mediated plasmid transfer by Tn916and Tn925is a Rec independent event. Thus, the data from studies in which otherwise isogenic donor and recipient strains were used indicated that Tn916and Tn925are, from a functional point of view, much more similar than previously suggested.     

A bifunctional plasmid carrying the recA gene of E. coli

Summary To investigate the effect of an active, plasmid-carried recA gene on the stability and/or the expression of plasmid genes in different genetic backgrounds, we have constructed a bifunctional plasmid (able to replicate in Escherichia coli and in Bacillus subtilis). Chimeric plasmids were obtained by inserting pC194 (Ehrlich 1977) into pDR1453 (Sancar and Rupp 1979). pDR1453 is a 12.9 Kbp plasmid constructed by inserting an E. coli chromosome fragment carrying the recA gene into pBR322. The expected bifunctional recombinant (pMR22/1) (15.7 Kbp) was easily obtained but surprisingly the Cm resistance was expressed only at a very low level in E. coli (as compared, for example, to pHV14, pHV15). We attribute this effect to the presence of multiple recA genes in the cell. On the contrary, Cm^r E. coli transformants bear a recombinant plasmid (pMR22/n) containing tandemly repeated copies of pC194 in equilibrium with excised free pC194. Such amplification has never been observed in a Rec^- background and is therefore mediated by the recA genes. Growth of these clones in the absence of Cm causes the loss of the extra copies, yielding a plasmid with a single copy of pC194, indistingishable from pMR22/1. Interestingly, we have observed that deletions occur at high frequency in pC194, which drastically increase Cm^r in E. coli containing plasmids with a single copy of pC194. Two types of such deletions were detected: (a) large 1050 bp deletions covering about onethird of pC194 and (b) small 120–150 bp deletions (near the MspI site) in the region containing the replicative functions of pC194 (Horinouchi and Weisblum 1982). Both types of deletion render the recombinant plasmid unable to replicate in B. subtilis. pM22/1 replicates, although with a low copy-number, and is stable in B. subtilis wild type; the recA gene of E. coli does not complement any of the rec ^- mutations of B. subtilis. A strong instability, mainly of the E. coli and pBR322 sequences, was observed in many dna and rec mutants of B. subtilis yielding smaller plasmid with a much higher copy-number.     

Organization of the pE194 genome

Summary pE194 is a 3.5 kilobase erythromycin resistance plasmid which was originally isolated from Staphylococcus aureus and has been introduced into Bacillus subtilis. This plasmid specifies at least five polypeptides, detectable in minicell extracts, one of which (E3) is inducible by erythromycin and is required for the expression of erythromycin resistance. We have constructed a detailed restriction endonuclease cleavage site map of pE194, and have localized the DNA sequences which code for the five polypeptides on the map. Four of the five polypeptides (E2, 3, 4 and 5) are specified by a region of the genome which has half the coding capacity required if these proteins were specified by contiguous genes. The determinant of E3 inducibility is located in the same segment. Based on the deficiency in coding capacity and on additional evidence including peptide mapping, we suggest that either the genes for these four polypeptides overlap, and are read in the same frame, or that some of these proteins represent degradation products. The latter alternative appears less likely since E3 is regulated independently of the other three proteins. The fifth protein (E1) is probably transcribed in the opposite direction. Strand separation and hybridization experiments confirm that both strands are transcribed. Hybridization of labeled RNA from a plasmid-carrying strain to restriction fragments of pE194 reveals that the expected plasmid sequences are transcribed in vivo, as is a region of the genome which is near the replication origin and which does not specify any known polypeptide. The map locations of 3–4 RNA polymerase binding sites are presented. Two of them are found in the segm ent which is predicted to contain the E1 and the E2-5 promoters, and a third is in the region of the replication origin.     

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

Summary To determine the minimal DNA sequence homology required for recombination in Bacillus subtilis, we developed a system capable of distinguishing between homologous and illegitimate recombination events during plasmid integration into the chromosome. In this system the recombination frequencies were measured between is pE194 derivatives carrying segments of the chromosomal -gluconase gene (bglS) of various lengths and the bacterial chromosome, using selection for erythromycin resistance at the non-permissive temperature. Homologous recombination events, resulting in disruption of the bglS gene, were easily detected by a colorimetric assay for -gluconase activity. A linear dependence of recombination frequency on homology length was observed over an interval of 77 bp. It was found that approximately 70 bp of homology is required for detectable homologous recombination. Homologous recombination was not detected when only 25 by of homology between plasmid and chromosome were provided. The data indicate that homology requirements for recombination in B. subtilis differ from those in Escherichia coli.     

Genetic analysis of a lactococcal plasmid replicon

Summary The sequence and genetic organization was determined of the 2508 by lactococcal portion of pFX2, which was derived from a crypticLactococcus lactis subsp.lactis plasmid and used as the basis for construction of a series of lactococcal vectors. A lactococcal plasmid plus origin and two replication protein-coding regions (repA andrepB) were located. RepA has a helix-turn-helix motif, a geometry typical of DNA-binding proteins. RepB shows a high degree of homology to the plasmid replication initiation proteins from other gram-positive bacteria andMycoplasma. The transcribed inverted repeat sequence betweenrepA andrepB could form an attenuator to regulate pFX2 replication. Upstream of theori site, and in a region which was non-essential for replication, a 215 by sequence identical to the staphylococcal plasmid pE194 and carrying the RS_A site was identified. The genetic organization of this lactococcal plasmid replicon shares significant similarity with pE194 group plasmids.     

Imprecise excision of plasmid pE194 from the chromosomes of Bacillus subtilis pE194 insertion strains.

Plasmid pE194 has been shown to be rescued by integration after cultivation of infected Bacillus subtilis recE4 cells at a restrictive high temperature. The plasmid is also spontaneously excised from the chromosome at a low frequency by precise or imprecise excision (J. Hofemeister, M. Israeli-Reches, and D. Dubnau, Mol. Gen. Genet. 189:58-68, 1983). We have investigated nine excision plasmids, carrying insert DNA 1 to 6 kbp in length, either in a complete pE194 or in a partially deleted pE194 copy. Type 1 (additive) excision plasmids have the left- and right-junction DNAs preserved as 13-bp direct repeats (5'-GGGGAGAAAACAT-3') corresponding to the region between positions 864 and 876 in pE194. In type 2 (substitutive) excision plasmids, a conserved 13-bp sequence remains only at the right junction while the left junction has been deleted during the excision process. The type 3 excision plasmid carries at each junction the tetranucleotide 5'-TCCC-3', present in pE194 between positions 1995 and 1998. Although we isolated the excision plasmids from different integration mutants, the insert DNAs of eight independently isolated plasmids showed striking sequence homology, suggesting that they originated from one distinct region of the B. subtilis chromosome. Thus, we postulate that imprecise excision of pE194 occurs most frequently after its translocation from the original insertion site into a preferred excision site within the host chromosome. The imprecise excision from this site occurs at excision breakpoints outside the pE194-chromosome junctions in a chromosomal region which remains to be investigated further.     

Recombination between repeated DNA sequences occurs more often in plasmids than in the chromosome of Bacillus subtilis

Summary Directly repeated pBR322 sequences 3.7–3.8 kb long recombine with a frequency of about 10% per generation when carried on plasmids related to pC194 and 0.01% per generation when carried on the Bacillus subtilis chromosome. Recombination is therefore 1,000 times more efficient in plasmids than in the chromosome of this organism.     

Sequencing and analysis of aMycoplasma gallisepticum A5969 chromosome region containing the S10 andrrn23-5 operons

A 20,115-nt region of theMycoplasma gallisepticum A5959 genome was sequenced (GenBank accession no. AF036708). The region contains therrn23-5 and S10 operons, the lactate dehydrogenase gene, and two open reading frames (ORF293 and ORF129/ORF171) coding for proteins of unknown function. Therrn23-5 operon includes genes for 23S and 5S rRNAs. The S10 operon includes genes for 20 ribosomal proteins, Sec Y transport protein, adenylate kinase, and methionine aminopeptidase, and lacks theinfA-rpl36-rps13-rpoA-rpl17 genes found in the S10 operon ofM. genitalium, M. pneumoniae, andBacillus subtilis. The product ofM. gallisepticum ldh is equally similar to the corresponding proteins of mycoplasmata andB. subtilis but contains only a part of the motif characteristic of the active center of lactate dehydrogenases. The chromosome region adjacent to the sequenced one containsuvrA,nrdE,nrdF, andptsI.     

Glycogen in Bacillus subtilis: molecular characterization of an operon encoding enzymes involved in glycogen biosynthesis and degradation

Although it has never been reported that Bacillus subtilis is capable of accumulating glycogen, we have isolated a region from the chromosome of B. subtilis containing a glycogen operon. The operon is located directly downstream from trnB, which maps at 275 on the B. subtilis chromosome, it encodes five poly-peptides with extensive similarity to enzymes involved in glycogen and starch metabolism in both prokaryotes and eukaryotes. The operon is presumably expressed by an Eσ^E-controlled promoter, which was previously identified downstream from trnB. We have observed glycogen biosynthesis in B. subtilis exclusively on media containing carbon sources that allow efficient sporulation. Sporulation-independent synthesis of glycogen occurred after integration of an Eσ^A controlled promoter upstream of the operon.     

In Vivo Random Mutagenesis of Bacillus subtilis by Use of TnYLB-1, a mariner-Based Transposon

This report describes the construction and characterization of a mariner-based transposon system designed to be used in Bacillus subtilis, but potentially applicable to other gram-positive bacteria. Two pUC19-derived plasmids were created that contain the mariner-Himar1 transposase gene, modified for expression in B. subtilis, under the control of either σ^A- or σ^B-dependent promoters. Both plasmids also contain a transposable element (TnYLB-1) consisting of a Kan^r cassette bracketed by the Himar1-recognized inverse terminal repeats, as well as the temperature-sensitive replicon and Erm^r gene of pE194ts. TnYLB-1 transposes into the B. subtilis chromosome with high frequency (10⁻²) from either plasmid. Southern hybridization analyses of 15 transposants and sequence analyses of the insertion sites of 10 of these are consistent with random transposition, requiring only a “TA” dinucleotide as the essential target in the recipient DNA. Two hundred transposants screened for sporulation proficiency and auxotrophy yielded five Spo⁻ clones, three with insertions in known sporulation genes (kinA, spoVT, and yqfD) and two in genes (ybaN and yubB) with unknown functions. Two auxotrophic mutants were identified among the 200 transposants, one with an insertion in lysA and another in a gene (yjzB) whose function is unknown.