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.     

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.     

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.     

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).     

Similarity of minus origins of replication and flanking open reading frames of plasmids pUB110, pTB913 and pMV158.

Plasmids pMV158 and pTB913, originating from Streptococcus agalactiae and a thermophilic Bacillus respectively, were sequenced to completion. Both contained a BA3-type minus origin of replication and an RSA-site, believed to constitute a site-specific recombination site. These two regions were more than 99% homologous to the corresponding regions of the Staphylococcus aureus plasmid pUB110. Deleting the BA3-type minus origin resulted in the accumulation of a considerable amount of single-stranded DNA, both in L. lactis subsp. lactis and B. subtilis, indicating that this minus origin was functional in both bacterial species. Like pUB110, both plasmids contained an open reading frame encoding a putative plasmid recombination enzyme (Pre protein), which was located downstream of the RSA-site. On the basis of sequence comparisons between pUB110, pMV158, pTB913, pT181, pE194, pNE131 and pT48 two distinct families of RSA-sites and Pre proteins could be distinguished.     

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.     

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.     

Improvement of the electro-transformation efficiency of facultatively alkaliphilic Bacillus pseudofirmus OF4 by high osmolarity and glycine treatment

An electro-transformation method was established for facultatively alkaliphilic Bacillus pseudofirmus OF4. A high osmolarity electroporation medium with a high electric field strength was effective. Transformation efficiency improved 110-fold in glycine-treated cells compared to non-glycine-treated cells. Under optimum conditions, the transformation efficiency was 1.69 x 10(6) transformants per microg of pUB110.     

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.     

The nucleotide sequence of pUB110: some salient features in relation to replication and its regulation

For the study of DNA-membrane interaction and the regulation of replication initiation we have determined the total nucleotide sequence of pUB110. As previously reported, this plasmid replicates in B. subtilis at a copy number of 30-50 per cell, with a majority of plasmids (60-80%) bound to the membrane (type-I binding). The type-I membrane binding is apparently necessary for pUB110 initiation of replication in vivo, but the membrane binding site is not known. Furthermore, four areas of the plasmid specifically bind to Bacillus subtilis membrane in an in vitro binding reaction (type II binding). These two types of membrane binding of pUB110 are different in that the in vivo binding (type-I) requires one (dnaBI) of the host initiation genes and is high-salt resistant, whereas the in vitro binding (type-II) does not require the dnaBI gene product and is high-salt sensitive. 7-mer double-strand sequence, TCAGCAA/AGTCGTT, or one-base derivatives of this sequence are frequently (17 of 23 of the 7-mer sequences) found in or close to the type-II binding areas. One of them is found at a restriction enzyme recognition site of a binding area that destroys the type-II membrane binding. These sequences may or may not have significance in type-II membrane binding. In addition to the neomycin resistance gene, the sequence data indicate two sizable open reading frames, ORF alpha and ORF beta, and two small ORF, gamma, and delta. All of these reading frames are in the same direction, which coincides with the direction of the replication. The open reading frame alpha (ORF alpha) corresponding to 334 amino acids close to the replication origin may be essential for the initiation of replication of PUB110. The putative protein alpha corresponding to this open reading frame contains a consensus sequence of the DNA binding sites which are found in a number of known DNA-binding proteins. The consensus DNA binding site of protein alpha is flanked by two hydrophobic areas. These two observations suggest that the corresponding protein may have both an affinity to a specific site in pUB110, and an affinity to the membrane.     

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.     

Transformation and expression of an anaerobic fungal xylanase in several strains of the rumen bacterium Butyrivibrio fibrisolvens

AIMS: To obtain reliable transformation of a range of Butyrivibrio fibrisolvens strains and to express a Neocallimastix patriciarum xylanase gene in the recipients. METHODS AND RESULTS: Eight strains (H17c, E14, LP1309, LP1028, AR11a, OB156, LP210B and LP461A) of Bu. fibrisolvens were transformed by the Gram-positive vector pUB110. A xylanase expression/secretion cassette containing Bu. fibrisolvens promoter and signal peptide elements fused to catalytic domain II of the N. patriciarum xylanase A cDNA (xynANp) was inserted into pUB110 to create the plasmid pUBxynA. pUBxynA was used to transform seven of the Bu. fibrisolvens strains transformed by pUB110. In strain H17c pUBxynA, which produced native xylanase, 2.46 U mg-1 total xylanase activity was produced with 45% extracellular xylanase. In strain H17c pUMSX, 0.74 U mg-1 total xylanase activity was produced with 98% extracellular xylanase. H17c pUBxynA exhibited increased (28.7%) degradation of neutral detergent fibre compared with unmodified H17c; however, progressive loss of pUBxynA was observed in long-term cultivation. CONCLUSIONS: A stable transformation system was developed that was applicable for a range of Bu. fibrisolvens strains and high levels of expression of a recombinant xylanase were obtained in H17c which lead to increased fibre digestion. SIGNIFICANCE AND IMPACT OF THE STUDY: This stable transformation system with the accompanying recombinant plasmids will be a useful tool for further investigation aimed at improving ruminal fibre digestion.     

Transformation of Heat-Treated Clostridium acetobutylicum Protoplasts with pUB110 Plasmid DNA

Heat treatment of Clostridium acetobutylicum SA-1 protoplasts at 55°C for 15 min before transformation resulted in expression in this microorganism of the kanamycin resistance determinant associated with plasmid pUB110. No heat treatment, or heat treatment at 65 or 44°C for various time intervals, resulted in no kanamycin resistance transformants being recovered on selective kanamycin-containing regeneration medium. DNase plate assay indicated that treatment at 55°C for 15 min completely inactivated the DNase activity associated with SA-1 protoplasts. Treatment of protoplasts at 65 or 55°C for various periods under simulated transformation conditions had an inhibitory effect, although prolonged treatment at 55 or 44°C appeared to stimulate DNase activity. Inactivation of protoplast-associated DNase activity by heat treatment at 55°C for 15 min correlated with successful expression of kanamycin resistance and suggests that an extremely active, heatsensitive, protoplast-associated DNase may be a factor in the polyethylene glycol-induced transformation of C. acetobutylicum SA-1 protoplasts. Plasmid pUB110 DNA was isolated from C. acetobutylicum SA-1 kanamycin-resistant (Km^r) transformant cultures by a modification of the procedure used for C. perfringens plasmids. Detection of pUB110 DNA was possible only when diethyl pyrocarbonate was incorporated into isolation protocols to inactivate DNase activity. Restriction studies further verified the presence of pUB110 DNA in C. acetobutylicum SA-1 Km^r transformants.     

短小芽孢杆菌作为芽孢杆菌属基因工程受体菌的研究

以质粒pUB110 DNA转化B. pumilus 289原生质体,转化频率为10~(-3)—10~(-9)与B.tubtilis 168系统相当;但B.pumilus 289原生质体的再生频率(0.3—12.0％)略低于B.subtilis 168(1.53—24.16％);在无选择压力条件下质粒pUB110在B.pumilus 289中经过45个世代周期,自发丢失率小于3％,同于B.subtilis 168系统。外源基因在B.pumilus 289中经25个世代周期丢失率低于5％,而在B.subtilis 168系统中则高达24％;外源基因的表达水平亦高于B.subtilis 168系统。因此,B.pumilus 289是一个值得进一步开发的基因工程受体系统。     

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.     

Stability of Integrated Plasmids in the Chromosome of Lactococcus lactis

Derivatives of plasmids pBR322, pUB110, pSC101, and pTB19, all containing an identical fragment of lactococcal chromosomal DNA, were integrated via a Campbell-like mechanism into the same chromosomal site of Lactococcus lactis MG1363, and the transformants were analyzed for the stability of the integrated plasmids. In all cases the erythromycin resistance gene of pE194 was used as a selectable marker. Transformants obtained by integration of the pBR322 derivatives contained a head-to-tail arrangement of several plasmid copies, which most likely was caused by integration of plasmid multimers. Single-copy integrations were obtained with the pSC101 and pTB19 derivatives. In all of these transformants no loss of the erythromycin gene was detected during growth for 100 generations in the absence of the antibiotic. In contrast, transformants containing integrated amplified plasmid copies of pUB110 derivatives were unstable under these conditions. Since pUB110 appeared to have replicative activity in L. lactis, we suggest that this activity destabilized the amplified structures in L. lactis.     

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.     

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.