Analysis of the Requirement for a pUB110 mob Region during Tn916-Dependent Mobilization

Tn916-dependent mobilization of nonconjugative plasmids pUB110 and its derivative pUB110Deltam was compared. Deleting a 787-bp fragment from the pUB110 mob region created plasmid pUB110Deltam. Deletion of the mob region of pUB110 rendered the plasmid nontransferable by the conjugative plasmids of Bacillus thuringiensis subsp. israelensis. During matings between Bacillus subtilis (Tn916) and B. thuringiensis subsp. israelensis, however, Tn916-dependent mobilization of plasmids pUB110 and pUB110Deltam was observed at a frequency of approximately 2 x 10(-6) transconjugants per donor. The results show that Tn916-mediated conjugal transfer of plasmids is a mob-independent event. Jaworski and Clewell (J. Bacteriol 177; 6644-6651) recently demonstrated the presence of an IncP-like nicking site in the oriT of Tn916. These data suggest that a IncP-like nickling site is essential for Tn916-mediated plasmid transfer.     

Genetic relationship between pUB110 and antibiotic-resistant plasmids obtained from thermophilic bacilli

The molecular relationship between pUB110 (Kmr, 4.4 kilobases (kb] and antibiotic-resistant plasmids from thermophilic bacilli, pTHT15 (Tcr, 4.5 kb) and pTHN1 (Kmr, 4.8 kb), were studied by blot hybridization. Extensive homology was observed between pUB110 and pTHT15 at the region which includes the replication origin. Incompatibility studies revealed that pTHT15 and pUB110 were slightly incompatible in Bacillus subtilis but that they were apparently compatible in B. stearothermophilus. This difference in incompatibility between pTHT15 and pUB110 in the two host cells might be due to a difference in the copy number of pTHT15 in the two organisms. From the results of blot hybridization, mode of kanamycin inactivation, and DNA sequencing, it was determined that pTHN1 encoded the identical gene for kanamycin nucleotidyl transferase as that of pUB110. All three plasmids pTHT15, pTHN1, and pUB110 shared a common DNA homology at the in vitro membrane-binding region.     

4',4' adenyltransferase activity on conjugative plasmids isolated from Staphylococcus aureus is encoded on an integrated copy of pUB110.

In staphylococci, linked resistance to the aminoglycosides kanamycin, neomycin, paromomycin, and tobramycin (KmNmPmTmr) is generally mediated by an aadD determinant which encodes production of an adenyltransferase aminoglycoside modifying enzyme, AAD(4',4'). The aadD resistance determinant is located on small multicopy plasmids such as pUB110, and has also been found on large multiresistance plasmids and on the chromosome in some strains. Examination of two conjugative plasmids from strains of Staphylococcus aureus isolated in North America indicated that the aadD determinant on these plasmids is located on an integrated copy of pUB110. The integrated pUB110 is flanked by direct repeats of the staphylococcal insertion sequence IS257. Analysis of the conjugative plasmid pSK41 showed an 8-bp duplication of the pUB110 sequence immediately adjacent to flanking IS257 elements, suggesting that integration of pUB110 was mediated by IS257.     

Replication origins of single-stranded-DNA plasmid pUB110.

The two replication origins of plasmid pUB110 have been characterized. The site of initiation of DNA replication at the plus origin was mapped to within an 8-base-pair sequence. DNA synthesis initiated at the origin was made to terminate precociously in an inserted sequence of 18 base pairs that is homologous to a sequence in the origin. This suggests that pUB110 replicates as a rolling circle. The minus origin of plasmid pUB110 has been characterized, and the minimal sequence required for function has been determined. As with other minus origins, activity is orientation specific with respect to the direction of replication. Its activity is sensitive to rifampin in vivo, suggesting that RNA polymerase catalyzes single-strand to double-strand conversion. Unlike all other plasmids of gram-positive bacteria thus far described, the pUB110 minus origin is functional in more than one host.     

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.     

A Conjugative Transfer System for the Rumen Bacterium, Butyrivibrio fibrisolvens, Based on Tn916-Mediated Transfer of the Staphylococcus aureus Plasmid pUB110

A limitation of genetic studies of the rumen bacterium, Butyrivibrio fibrisolvens, has been the availability of suitable vectors and transfer systems. Using the conjugative tetracycline resistant transposon, Tn916, the Staphylococcus aureus plasmid, pUB110, and the pUB110-based shuttle vector, pUBLRS, a conjugative transfer system was developed for B. fibrisolvens. B. fibrisolvens donor strains H17c2 and H17c12, containing Tn916 and pUB110 or pUBLRS, respectively, were used in mating experiments with selected B. fibrisolvens strains. Kanamycin resistant transconjugants, containing pUB110, of strains 193, 194, and 195 were detected at a combined average frequency of 7.78 × 10^-7 per donor and 1.11 × 10^-5 per recipient. Transconjugants of strains 193 and 194, containing pUBLRS, were detected at an average frequency of 1.22 × 10^-6 per donor and 4.70 × 10^-8 per recipient. Southern hybridization analysis confirmed the presence of pUB110 and pUBLRS in transconjugants. Results indicated that Tn916 was necessary for mobilization of pUB110 as transconjugants were not detected when the transposon was absent from the donor strains. The ability to mobilize pUB110 and pUBLRS between B. fibrisolvens strains provides a conjugative transfer system that circumvents problems encountered with electroporation.     

Hybrid vectors for the cyanobacterium Anacystis nidulans R2, containing the plasmid pUB110 from Staphylococcus aureus

The recombinant vector plasmids were constructed having the DNA of pUB110 plasmid (4,5 kb, KmR) from Staphylococcus aureus inserted into the cryptic plasmids pANS (8 Kb) and pANL (48,5 kb) of cyanobacterium Anacystis nidulans R2. The hybrid plasmids transform cyanobacterial cells to Km-resistance with high efficiency. The plasmid pBS20, containing the complete sequence of pANS and pUB110 DNA, transforms Bacillus subtilis rec E4 protoplasts being, however, unstable in bacilli cells and disintegrates deriving a parent pUB110 plasmid.     

Tn916-dependent conjugal transfer of PC194 and PUB110 from Bacillus subtilis into Bacillus thuringiensis subsp. israelensis

The Staphylococcus aureus plasmids pC194 and pUB110 were introduced into Bacillus thuringiensis subsp. israelensis by using the Streptococcus faecalis transposon Tn916 as a mobilizing agent. Plasmid transfer occurred only when B. thuringiensis subsp. israelensis was mated with a B. subtilis donor that contained both pC194 and pUB110 and Tn916; plasmid transfer was not observed in the absence of the transposon. B. thuringiensis transconjugants resistant to chloramphenicol (Cmr) and tetracycline (Tetr) were detected at a frequency of 1.96 x 10(-6) per recipient cell, whereas the Tetr phenotype, but not the Cmr, was observed at a frequency of 1.09 x 10(-4). The converse, Cmr but not Tetr, was observed at a frequency of 2.94 X 10(-5). The transfer of pUB110 from B. subtilis to B. thuringiensis subsp. israelensis was observed at a frequency of 3.0 x 10(-6) per recipient cell but concomitant transfer of pUB110 and Tn916 was not observed. Mobilization of plasmid pE194 was not observed under these conditions. Transconjugants were detected in filter matings only, not in broth. The Tn916 phenotype was maintained during serial passage of B. thuringiensis without selection, whereas the pC194 phenotype was not. Unlike pC194, however, pUB110 remained stable in B. thuringiensis during several passages through nonselective medium. Southern hybridization analysis demonstrated that Tn916 had inserted into several different sites on the B. thuringiensis chromosome and that pC194 and pUB110 were maintained as an autonomous plasmid.     

Nucleotide sequence and physical map of kanamycin-resistant plasmid pUB110 from Staphylococcus aureus

The complete nucleotide sequence of Staphylococcus aureus plasmid pUB10 was determined. The sequence consists of 4545 b.p. and contains 64% A-T and 36% G-C pairs. pUB110 was found to contain four open reading frames, capable of coding for polypeptides having more than 80 amino acids. All the putative polypeptides are coded for by one DNA strand. The molecular weights of four putative polypeptides are (in kilodaltons): A-49.5; B-38.8; C-28.8 and D-9.5. Polypeptide C is involved in kanamycin resistance. Polypeptide B is, possibly, involved in pUB110 replication. No role has yet been established for polypeptides A and D, since deletions in their coding sequences have no detectable effect on any properties of pUB110 plasmid.     

Transformation of Bacillus licheniformis by plasmid DNA

A system for obtaining regenerating protoplasts of highly active Bacillus licheniformis 1001 strain was developed. Transformation of protoplasts by pUB110 and pminiKC plasmids (constructed from plasmids pUB110 and pC194) leading to the expression of kanamycine resistance, was demonstrated. It is supposed that in Bac licheniformis, the pminiKC plasmid is integrated into cellular chromosome, in contrast to pUB110 and parental Bac subtilis (pminiKC) strain. Still, the integrated plasmid seems to be not completely under control of the host chromosome. As a result of such integration, the plasmid conversion takes place, resulting in alteration of cytokinesis (filament formation) and sporulation, but not interfering with the ability to produce antibiotic bacitracin.     

Enrichment of plasmid DNA in cell membranes of Bacillus subtilis

Abstract The discrepancy between previously reported copy numbers for the plasmid pUB110 in Bacillus subtilis and the copy number determined by nucleic acid sandwich hybridization of a pUB110-derivative, pKTH10, was studied. The bulk of plasmid DNA was found to be enriched in the cell membranes in a non-covalently closed circular (ccc) form. The binding was strong enough to resist standard solubilization procedures. The conventional methods for copy number determination fail to detect plasmid DNA in this form, which explains the discrepancy we encountered. The copy number of the parental plasmid, pUB110, was also determined by the sandwich hybridization method and found to be of the same order of magnitude as that of pKTH10.     

Instability of recombinant pUB110 plasmids in Bacillus subtilis: Plasmid-encoded stability function and effects of DNA inserts

Two series of pUB110-derived plasmids were constructed to study segregational stability in Bacillus subtilis. pEB plasmids were based on the entire pUB110, whereas pLB plasmids lack the membrane-binding areas BA3 and BA4. Two kinds of stability defects were observed. The first was characterized by a strong size dependency and occurred with different inserts at various positions in pLB and pEB plasmids. Size-dependent reductions in plasmid copy numbers appeared to underly this phenomenon. This may render pUB110 unsuitable for the cloning of inserts larger than about 3 kb, in particular if no selective conditions can be applied. The second defect, observed with pLB plasmids, was caused by the absence of the membrane-binding areas BA3 and BA4. Deletion of BA3 resulted in the accumulation of single-stranded plasmid DNA, suggesting that BA3 contains the initiation signal for complementary strand synthesis. The BA3 region is very rich in hyphenated dyad symmetry which, in single-stranded DNA, could result in several stable alternative secondary structures. It is speculated that the activity of the BA3-associated initiation signal contributes to the segregational stability of pUB110-derived plasmids in B. subtilis. The absence of the BA3 stability function could not account for all stability defects observed. Additional stability functions seemed to be located on the BA4 fragment.     

枯草芽孢杆菌分子克隆系统受体的改造

实验证明枯草芽孢杆菌(Bacillus subtilis)BR151和MI112中的pUB110质粒,在液体中传代要比固体斜面传代稳定。通过NTG诱变获得了对pUB110稳定的受体BSG.来自大肠杆菌的穿梭质粒在BSG菌中转化频率较亲本高。小的外源DNA片段插入pBE2后在BSG菌中转管传代30次是稳定的。     

Site-specific in vitro binding of plasmid pUB110 to Bacillus subtilis membrane fraction.

The in vitro membrane binding of pSL103, a composite plasmid consisting of Staphylococcus aureus plasmid pUB110 and a Bacillus pumilus trpC+ DNA fragment, to the Bacillus subtilis membrane fraction was studied with a total lysate of B. subtilis cells. The binding reaction required a heat treatment at 45 degrees C and had an optimum KCl concentration of 60 mM. Nonradioactive pSL103, but not Escherichia coli plasmid pACYC184, competed with 3H-labeled pSL103 for binding to the membrane. By the use of 32P-labeled restriction fragments of pSL103 and pUB110, it has been found that only the pUB110 portion of pSL103 binds to the membrane and that there are four specific regions in pUB110 which bind to the membrane. Two of the four binding regions flank the replication origin. This in vitro binding was high-salt sensitive and apparently independent of the configurations of the plasmid. We have previously shown that the functional product of the initiation gene dna-1 is required in vivo both for replication initiation and the binding of a DNA region near the replication origin to the membrane. Unlike in vivo binding, which is high-salt resistant and dependent on the product of dna-1 gene (type-I binding), the in vitro binding reported in this paper was high-salt sensitive and independent of the dna-1 gene product (type-II binding).     

增加复制起始区对基因表达的影响

我们在质粒ｐｕＢ１１０的基础上组建了ｐＤＲ质粒,它们具有双复制起始区而只有一个抗卡那霉素基因。携带了这些质粒的宿主细胞对卡那霉素的抗性明显高于亲本株（Ｂ,ｓｕｂｔｉｌｉｓ １５０（ｐｕＢ１１０））。经限制性酶切图谱分析新获得的转化株具有二个复制起始区及一个 Ｋｍ＆４基因。说明增加复制起始区是提高重组子表达能力的途径之一。     

Enzymatic and nucleotide sequence studies of a kanamycin-inactivating enzyme encoded by a plasmid from thermophilic bacilli in comparison with that encoded by plasmid pUB110

The product of a kanamycin resistance gene encoded by plasmid pTB913 isolated from a thermophilic bacillus was identified as a kanamycin nucleotidyltransferase which is similar to that encoded by plasmid pUB110 from a mesophile, Staphylococcus aureus. The enzyme encoded by pTB913 was more thermostable than that encoded by pUB110. In view of a close resemblance of restriction endonuclease cleavage maps around the BglII site in the structural genes of both enzymes, ca. 1,200 base pairs were sequenced, followed by amino-terminal amino acid sequencing of the enzyme. The two nucleotide sequences were found to be identical to each other except for only one base in the midst of the structural gene. Each structural gene, initiating from a GUG codon as methionine, was composed of 759 base pairs and 253 amino acid residues (molecular weight, ca. 29,000). The sole difference was transversion from a cytosine (pUB110) to an adenine (pTB913) at a position + 389, counting the first base of the initiation codon as + 1. That is, a threonine at position 130 for the pUB110-coded kanamycin nucleotidyltransferase was replaced by a lysine for the pTB913-coded enzyme. The difference in thermostability between the two enzymes caused by a single amino acid replacement is discussed in light of electrostatic effects.     

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.