Experimental basis for a stable plasmid, pLS30, to shuttle between Bacillus subtilis species by conjugational transfer

The use of Bacillus subtilis 168 as the initial host for molecular cloning and subsequent delivery of the engineered DNA to other Bacillus hosts appears attractive, and would lead to an efficient DNA manipulation system. However, methods of delivery to other Bacillus species are limited due to their inability to develop natural competence. An alternative, unexplored conjugational transfer method drew our attention and a B. subtilis native plasmid, pLS30, isolated from B. subtilis (natto) strain IAM1168 was characterized for this aim. The nucleotide sequence (6,610 bp) contained the mob gene and its recognition sequence, oriT, that features pLS30 as a mobile plasmid between Bacillus species on conjugational transfer. Plasmid pLS3001, a chimera with a pBR322-based plasmid prepared in Escherichia coli to confer an antibiotic resistance marker, showed apparent mobilizing activity in the pLS20-mediated conjugational transfer system recently established. The rep gene and associated palT1-like sequence common to all other pLS plasmids previously sequenced indicated that pLS30 is a typical rolling circle replicating (RCR) type plasmid. Due to the significant stability of pLS30 in IAM1168, application of a mobile plasmid would allow quick propagation to Bacillus species.     

Involvement of Tn4430 in transfer of Bacillus anthracis plasmids mediated by Bacillus thuringiensis plasmid pXO12.

The self-transmissible plasmid pXO12 (112.5 kilobases [kb]), originally isolated from strain 4042A of Bacillus thuringiensis subsp. thuringiensis, codes for production of the insecticidal crystal protein (Cry+). The mechanism of pXO12-mediated plasmid transfer was investigated by monitoring the cotransfer of the tetracycline resistance plasmid pBC16 (4.2 kb) and the Bacillus anthracis toxin and capsule plasmids, pXO1 (168 kb) and pXO2 (85.6 kb), respectively. In matings of B. anthracis donors with B. anthracis and Bacillus cereus recipients, the number of Tcr transcipients ranged from 4.8 x 10(4) to 3.9 x 10(6)/ml (frequencies ranged from 1.6 x 10(-4) to 7.1 x 10(-2), and 0.3 to 0.4% of them simultaneously inherited pXO1 or pXO2. Physical analysis of the transferred plasmids suggested that pBC16 was transferred by the process of donation and that the large B. anthracis plasmids were transferred by the process of conduction. The transfer of pXO1 and pXO2 involved the transposition of Tn4430 from pXO12 onto these plasmids. DNA-DNA hybridization experiments demonstrated that Tn4430 was located on a 16.0-kb AvaI fragment of pXO12. Examination of Tra- and Cry- derivatives of pXO12 showed that this fragment also harbored information involved in crystal formation and was adjacent to a restriction fragment containing DNA sequences carrying information required for conjugal transfer.     

Bacteriocin and antibiotic resistance plasmids in Bacillus cereus and Bacillus subtilis.

A number of plasmids have been isolated as covalently closed circular DNAs from strains of Bacillus cereus and B. subtilis. From 12 out of 15 strains of B. cereus, plasmids could be isolated. Most of the B. cereus strains contained two or more plasmids. Their molecular weights ranged from 1.6 X 10(6) to 105 X 10(6). Bacteriocin production could be attributed to a 45 X 10(6)-dalton plasmid (pBC7) from B. cereus DSM 336, and tetracycline resistance to a 2.8 X 10(6) plasmid (pBC16) from B. cereus GP7. Two streptomycin-resistant strains of B. subtilis harbored plasmids of 5.2 X 10(6) and 9 X 10(6), respectively, which were, however, not correlated with the antibiotic resistance. The plasmid carrying resistance to tetracycline, pBC16, which was originally isolated from B. cereus, could be subsequently transformed in B. subtilis, where it is stably maintained.     

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.     

A site-specific recE4-independent intramolecular recombination between Bacillus subtilis and Staphylococcus aureus DNAs in hybrid plasmids

A composite plasmid pLS253 was constructed from pLS103 [carrying the Bacillus subtilis leucine genes on B. subtilis (natto) plasmid pLS28] and pHV14 [a recombinant plasmid composed of pBR322 and the staphylococcal R-plasmid pC194] employing BamHI endonuclease, T4 DNA ligase, and B. subtilis transformation. All the Leu+ Cmr transformants tested harbored not only pLS253 but also two smaller plasmids designated as pLS251 and pLS252. pLS253 DNA, when purified on an agarose gel, retained both Leu+ and Cmr transforming activities; however, in all the Leu+ Cmr transformants, the two smaller plasmids reappeared. pLS251 and pLS252 exhibited Leu+- or Cm4-transforming activity, respectively, and must have been derived from the pLS253 parent by an intramolecular recombination event, since the sum of the pLS251 and pLS252 DNAs represent the entire pLS253 genome. The recombination occurred between specific sites on the B. subtilis (natto) and Staphylococcus aureus plasmids. When the composite plasmid, pLS254, was constructed by BamHI cleavage of pLS251 and pLS252 followed by ligation, Leu+ Cmr transformants segregated two smaller plasmids which were indistinguishable from the original plasmids pLS103 and pHV14, respectively. They must have been derived from pLS254 through a reversal of the original recombination event. No intermolecular recombination between pLS251 and pLS252 DNA was detected. The recombination process was independent of recE function of the host cells, and its mechanism is discussed.     

Insertion of Tn916 into Bacillus pumilus plasmid pMGD302 and evidence for plasmid transfer by conjugation.

As part of an effort to develop systems for genetic analysis of strains of Bacillus pumilus which are being used as a microbial hay preservative, we introduced the conjugative Enterococcus faecalis transposon Tn916 into B. pumilus ATCC 1 and two naturally occurring hay isolates of B. pumilus. B. pumilus transconjugants resistant to tetracycline were detected at a frequency of approximately 6.5 x 10(-7) per recipient after filter mating with E. faecalis CG110. Southern hybridization confirmed the insertion of Tn916 into several different sites in the B. pumilus chromosome. Transfer of Tn916 also was observed between strains of B. pumilus in filter matings, and one donor strain transferred tetracycline resistance to recipients in broth matings at high frequency (up to 3.4 x 10(-5) per recipient). Transfer from this donor strain in broth matings was DNase-resistant and was not mediated by culture filtrates. Transconjugants from these broth matings contained derivatives of a cryptic plasmid (pMGD302, approx 60 kb) from the donor strain with Tn916 inserted at various sites. The plasmids containing Tn916 insertions transferred to a B. pumilus recipient strain at frequencies of approx 5 x 10(-6) per recipient. This evidence suggests that pMGD302 can transfer by a process resembling conjugation between strains of B. pumilus.     

Construction of a recombinant plasmid composed of B. subtilis leucine genes and a B. subtilis (natto) plasmid: its use as cloning vehicle in B. subtilis 168.

Summary Recombinant plasmids composed of Bacillus subtilis 168 leucine genes and a B. subtilis (natto) plasmid have been constructed in a recombination deficient (recE4) mutant of Bacillus subtilis 168. The process involved EcoRI fragmentation and ligation of a B. subtilis (natto) plasmid and a composite plasmid RSF2124-B · leu in which B. subtilis 168 leucine genes are linked to the R-factor RSF2124. A constructed plasmid (pLS102) was found to be composed of an EcoRI fragment derived from the vector plasmid and two tandemly repeated EcoRI fragments carrying the leucine genes. A derivative plasmid (pLS101 or pLS103) consisting of one molecule each of the EcoRI fragments was obtained by in vivo intramolecular recombination between the repeated leucine gene fragments in pLS102. pLS103 was cleaved once with BamNI, SmaI and HpaI. Insertion of foreign DNA (Escherichia coli plasmid pBR322) into the BamNI site inactivated leuA but not the leuC function which thus can serve as selective marker if the plasmid is used as vector in molecular cloning. The penicillin resistance carried in pBR322 was not functionally expressed in B. subtilis cells. By partial digestion of pLS103 with HindIII followed by ligation with T4-induced ligase, pLS107 was obtained which contained only one EcoRI site. However, insertion of exogenous DNA (pBR322) into this EcoRI site inactivated both leuA and leuC functions.     

Protoplast fusion in Bacillus cereus

Polyethyleneglycol induced fusion of Bacillus cereus protoplasts and its genetic consequences have been investigated. The technique used allows the transfer of a small plasmid pBC16 between Bacillus cereus cells. Fusion has resulted in isolation of hybrid cells having acquired TcR phenotype (harbouring pBC16) with high frequencies (10(-2)-10(-3)). However, the genetic instability of hybrid cells and segregation effects have influenced dramatically the final results of plasmid transfer. Nevertheless, the fusion of protoplasts proves to be useful in construction of BAcillus cereus strains inheriting plasmid determinants.     

Construction and characterization of plasmid vectors for cloning in the entomocidal organism Bacillus sphaericus 1593

A plasmid vector for cloning in Bacillus sphaericus 1593 was constructed in B. subtilis from two parent plasmids, pBC16 and pBD64. When characterized, the 3.9-MDa chimeric plasmid pNN101 was found to consist of the MspI fragment containing the chloramphenicol acetyltransferase (CAT) gene from pBD64 inserted into an MspI site in pBC16. pNN101 was shown to replicate, express, and be stably maintained in B. sphaericus 1593 without affecting the mosquito larvicidal activity of this organism. A derivative of this plasmid, pNN302, was constructed in which a unique HindIII site was introduced into the CAT gene without loss of chloramphenicol resistance.     

Two conjugation systems associated with Streptococcus faecalis plasmid pCF10: identification of a conjugative transposon that transfers between S. faecalis and Bacillus subtilis.

The tetracycline resistance plasmid pCF10 (58 kilobases [kb]) of Streptococcus faecalis possesses two separate conjugation systems. A 25-kb region of the plasmid (designated TRA) was shown previously to determine pheromone response and conjugation functions required for transfer of pCF10 between S. faecalis cells (P. J. Christie and G. M. Dunny, Plasmid 15:230-241, 1986). When S. faecalis cells were mixed with Bacillus subtilis in broth, tetracycline resistance was transferred from S. faecalis. The tetracycline-resistant B. subtilis cells contained a 16-kb region of pCF10 (distinct from TRA) that carried the tetracycline resistance determinant (Tetr). This Tetr element was found to transfer between S. faecalis and B. subtilis strains in the absence of plasmids. Genetic and molecular techniques were used to establish locations of the element at several different sites on the B. subtilis chromosome. The Tetr element could be transferred in filter matings from B. subtilis to S. faecalis strains and between recombination-proficient and -deficient S. faecalis strains in the absence of any plasmid DNA. The transfer required direct cell-to-cell contact and was not inhibited by DNase. The Tetr element was shown to transpose from the S. faecalis chromosome to various locations within the hemolysin plasmid pAD1. Together, the data indicate that the Tetr element, termed transposon Tn925, is very similar to the conjugative transposon Tn916 in both structure and function. A derivative of Tn925, containing transposon Tn917 inserted into a site approximately 3 kb from one end, exhibited elevated transfer frequencies and may provide a useful means for delivering Tn917 by conjugation into various gram-positive species.     

Structural and genetic analyses of a par locus that regulates plasmid partition in Bacillus subtilis.

The Bacillus plasmid pLS11 partitions faithfully during cell division. Using a partition-deficient plasmid vector, we randomly cloned DNA fragments of plasmid pLS11 and identified the locus that regulates plasmid partition (par) by cis complementation in Bacillus subtilis. The cloned par gene conferred upon the vector plasmid a high degree of segregational stability. The par locus was mapped to a 167-base-pair segment on pLS11, and its nucleotide sequence was determined. The cloned par fragment regulated the partition of several different Bacillus replicons, and it only functioned in cis; it did not contain the replication function nor elevate the plasmid copy number in B. subtilis. The expression of par was orientation specific with respect to the replication origin on the same plasmid. We propose that the pLS11-derived par functions as a single-stranded site that interacts with other components involved in plasmid partition during cell division.     

Transformation of Bacillus polymyxa with plasmid DNA

A plasmid transformation system was developed for Bacillus polymyxa ATCC 12321 and derivatives of this strain. The method utilizes a penicillin-treated-cell technique to facilitate uptake of the plasmid DNA. Low-frequency transformation (10(-6) per recipient cell) of plasmids pC194, pBD64, and pBC16 was accomplished with this method. Selection for the transformants was accomplished on both hypertonic and nonhypertonic selective media, with the highest rates of recovery occurring on a peptone-glucose-yeast extract medium containing 0.25 M sucrose. Several additional plasmids were shown to be capable of transferring their antibiotic resistance phenotypes to B. polymyxa through the use of a protoplast transformation procedure which allowed for a more efficient transfer of the plasmid DNA. However, cell walls could not be regenerated on the transformed protoplasts, and the transformants could not be subcultured from the original selective media.     

Conjugational transfer system to shuttle giant DNA cloned by Bacillus subtilis genome (BGM) vector

The Bacillus subtilis GenoMe (BGM) vector was designed as a versatile vector for the cloning of giant DNA segments. Cloned DNA in the BGM can be retrieved to a plasmid using our Bacillus recombinational transfer (BReT) method that takes advantage of competent cell transformation. However, delivery of the plasmid to a different B. subtilis strain by the normal transformation method is hampered by DNA size-related inefficiency. Therefore, we designed a novel method, conjugational plasmid-mediated DNA retrieval and transfer (CReT) from the BGM vector, and investigated conjugational transmission to traverse DNA between cells to circumvent the transformation-induced size limitation. pLS20, a 65-kb plasmid capable of conjugational transfer between B. subtilis strains, was modified to retrieve DNA cloned in the BGM vector by homologous recombination during normal culture. As the plasmid copy number was estimated to be 3, the retrieval plasmid was selected using increased numbers of marker genes derived from the retrieved DNA. We applied this method to retrieve Synechocystis genome segments up to 90 kb in length. We observed retrieved plasmid transfers between B. subtilis strains by conjugation in the absence of structural alterations in the DNA fragment. Our observations extend DNA transfer protocols over previously exploited size ranges.     

Plasmid transformation of Bacillus cereus protoplasts

The process of polyethyleneglycol-induced plasmid transformation of Bacillus cereus protoplasts was studied. Plasmid transfer into Bacillus cereus strains was demonstrated with the frequencies 1.3.10(1)-1.6.10(2) transformants per 1 mkg of plasmid DNA. The plasmids transferred are stably inherited by Bacillus cereus cells causing tetracycline resistance (pBC16) or kanamycin resistance (pUB110 and pBD64). The proposed method can be used for construction of Bacillus cereus strains having the plasmid determined characteristics.     

Mobilization of closely related plasmids pUB110 and pBC16 by Bacillus plasmid pXO503 requires trans-acting open reading frame beta.

Genetic analysis of the closely related nonconjugative plasmids pUB110 and pBC16 has demonstrated that the open reading frame beta (ORF-beta) region in pUB110 and the corresponding homologous region in pBC16 are essential for mobilization of these plasmids by pLS20 or its derivatives. Deletions in this region or insertions that interrupted ORF-beta severely impaired or eliminated the mobilization of pUB110::pUC18 and pBC16::pUC18 hybrids. In contrast, a hybrid in which pUC18 was inserted into pBC16 at a point outside ORF-beta transferred at a frequency comparable to that of intact pUB110 or pBC16 (10(-4) transcipients per donor cell). The defect of most transfer-deficient (Mob-) hybrid plasmids could be complemented by an intact sister plasmid (i.e., pBC16 for pUB110::pUC18 Mob- hybrids). The inability to complement certain constructs suggested that the origin of transfer might be located in an area 5' to ORF-beta. Furthermore, cloning the region 5' to ORF-beta onto a nonmobilizable pC194::pUC18 construct resulted in a hybrid plasmid, pUCCoriTBC16, that could be mobilized with complementation. These results indicate that mobilization of pUB110 and pBC16 by conjugative helper plasmids requires ORF-beta in trans and at least one other region, including the RSA sequence, which presumably functions as an origin of transfer, in cis.     

Transformation of Bacillus polymyxa with plasmid DNA.

A plasmid transformation system was developed for Bacillus polymyxa ATCC 12321 and derivatives of this strain. The method utilizes a penicillin-treated-cell technique to facilitate uptake of the plasmid DNA. Low-frequency transformation (10(-6) per recipient cell) of plasmids pC194, pBD64, and pBC16 was accomplished with this method. Selection for the transformants was accomplished on both hypertonic and nonhypertonic selective media, with the highest rates of recovery occurring on a peptone-glucose-yeast extract medium containing 0.25 M sucrose. Several additional plasmids were shown to be capable of transferring their antibiotic resistance phenotypes to B. polymyxa through the use of a protoplast transformation procedure which allowed for a more efficient transfer of the plasmid DNA. However, cell walls could not be regenerated on the transformed protoplasts, and the transformants could not be subcultured from the original selective media.     

Comparative study of the symbiotic plasmid DNA in free living bacteria and bacteroids of Rhizobium leguminosarum

Plasmids pIM13, pT127 and pBC16 delta 1, introduced by transformation into Clostridium acetobutylicum N1-4081, were shown to replicate in, and to confer antibiotic resistance upon this new host. Recombinant plasmids were constructed by inserting erythromycin-resistant plasmid pIM13 into the unique ClaI site of pBR322 or by ligating a tetracycline-resistant determinant of plasmid pT127 to HindIII-linearized pIM13. The hybrid plasmids replicated and expressed erythromycin resistance in C. acetobutylicum strain N1-4081 and in Escherichia coli or Bacillus subtilis, indicating that they might be useful as shuttle vectors for transferring genes between these strains. The efficiency and stability of different replicons in C. acetobutylicum were compared.     

Transformation of Bacillus thuringiensis subsp. galleria protoplasts by plasmid pBC16.

Protoplasts of the entomopathogenic bacterium Bacillus thuringiensis subsp. galleria were transformed by plasmid pBC16. The frequency of transformation was much lower than that of Bacillus subtilis. All isolated B. thuringiensis transformants were characterized by increased sensitivity to lysozyme as compared with the original strain.     

Mating system for transfer of plasmids among Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis.

To facilitate the analysis of genetic determinants carried by large resident plasmids of Bacillus anthracis, a mating system was developed which promotes plasmid transfer among strains of B. anthracis, B. cereus, and B. thuringiensis. Transfer of the selectable tetracycline resistance plasmid pBC16 and other plasmids from B. thuringiensis to B. anthracis and B. cereus recipients occurred during mixed incubation in broth. Two plasmids, pXO11 and pXO12, found in B. thuringiensis were responsible for plasmid mobilization. B. anthracis and B. cereus transcipients inheriting either pXO11 or pXO12 were, in turn, effective donors. Transcipients harboring pXO12 were more efficient donors than those harboring pXO11; transfer frequencies ranged from 10(-4) to 10(-1) and from 10(-8) to 10(-5), respectively. Cell-to-cell contact was necessary for plasmid transfer, and the addition of DNase had no effect. The high frequencies of transfer, along with the fact that cell-free filtrates of donor cultures were ineffective, suggested that transfer was not phage mediated. B. anthracis and B. cereus transcipients which inherited pXO12 also acquired the ability to produce parasporal crystals (Cry+) resembling those produced by B. thuringiensis, indicating that pXO12 carries a gene(s) involved in crystal formation. Transcipients which inherited pXO11 were Cry-. This mating system provides an efficient method for interspecies transfer of a large range of Bacillus plasmids by a conjugation-like process.     

Identification of self-transmissible plasmids in four Bacillus thuringiensis subspecies.

The transfer of plasmids by mating from four Bacillus thuringiensis subspecies to Bacillus anthracis and Bacillus cereus recipients was monitored by selecting transcipients which acquired plasmid pBC16 (Tcr). Transcipients also inherited a specific large plasmid from each B. thuringiensis donor at a high frequency along with a random array of smaller plasmids. The large plasmids (ca. 50 to 120 megadaltons), pXO13, pXO14, pXO15, and pXO16, originating from B. thuringiensis subsp. morrisoni, B. thuringiensis subsp. toumanoffi, B. thuringiensis subsp. alesti, and B. thuringiensis subsp. israelensis, respectively, were demonstrated to be responsible for plasmid mobilization. Transcipients containing any of the above plasmids had donor capability, while B. thuringiensis strains cured of each of them were not fertile, indicating that the plasmids confer conjugation functions. Confirmation that pXO13, pXO14, and pXO16 were self-transmissible was obtained by the isolation of fertile B. anthracis and B. cereus transcipients that contained only pBC16 and one of these plasmids. pXO14 was efficient in mobilizing the toxin and capsule plasmids, pXO1 and pXO2, respectively, from B. anthracis transcipients to plasmid-cured B. anthracis or B. cereus recipients. DNA-DNA hybridization experiments suggested that DNA homology exists among pXO13, pXO14, and the B. thuringiensis subsp. thuringiensis conjugative plasmids pXO11 and pXO12. Matings performed between strains which each contained the same conjugative plasmid demonstrated reduced efficiency of pBC16 transfer. However, in many instances when donor and recipient strains contained different conjugative plasmids, the efficiency of pBC16 transfer appeared to be enhanced.