Transformation of Bacillus stearothermophilus with plasmid DNA and characterization of shuttle vector plasmids between Bacillus stearothermophilus and Bacillus subtilis.

A thermophilic bacterium Bacillus stearothermophilus IFO 12550 (ATCC 12980) was transformed with each of the following plasmids, pUB110 (kanamycin resistance, Kmr), pTB19 (Kmr and tetracycline resistance [Tcr]), and its derivative pTB90 (Kmr Tcr), by the protoplast procedure in the presence of polyethylene glycol at 48 degrees C. The transformation frequencies per regenerant for pUB110, pTB19, and pTB90 were 5.9 x 10(-3), 5.5 x 10(-3), and 2.0 x 10(-1), respectively. Among these plasmids, pTB90 was newly derived, and the restriction endonuclease cleavage map was constructed. When tetracycline (5 micrograms/ml) was added into the culture medium, the copy number of pTB90 in B. stearothermophilus was about fourfold higher than that when kanamycin (5 micrograms/ml) was added instead of tetracycline. Bacillus subtilis could also be transformed with the plasmids extracted from B. stearothermophilus and vice versa. Accordingly, pUB110, pTB19, and pTB90 served as shuttle vectors between B. stearothermophilus and B. subtilis. The requirements for replication of pTB19 in B. subtilis and B. stearothermophilus appear to be different, because some deletion plasmids (pTB51, pTB52, and pTB53) derived from pTB19 could replicate only in B. subtilis, whereas another deletion plasmid pTB92 could replicate solely in B. stearothermophilus. Plasmids pTB19 and pTB90 could be maintained and expressed in B. stearothermophilus up to 65 degrees C, whereas the expression of pUB110 in the same strain was up to 55 degrees C.     

Isolation and characterization of antibiotic-resistance plasmids in thermophilic bacilli

Four antibiotic-resistance plasmids isolated from thermophilic bacilli were characterized in detail. Three tetracycline-resistance (Tc1) plasmids were designated as pTHT9 (7.7 kilobases (kb], pTHT15 (4.5 kb) and pTHT22 (8.4 kb). From the results of restriction endonuclease analysis and the subsequent Southern hybridization, these were found to possess extensive genetic homology in the regions that include the replication origin and the Tcr gene. Detailed restriction maps of the smallest Tcr plasmid pTHT15 and a kanamycin-resistance (Kmr) plasmid pTHN1 (4.8 kb) were constructed. The positions of antibiotic-resistance loci and regions essential for plasmid replication were determined by cloning plasmid fragments in Bacillus subtilis. These four plasmids were found to replicate and express the resistance genes stably in both B. subtilis and B. stearothermophilus.     

Recombination between compatible plasmids containing homologous segments requires the Bacillus subtilis recE gene product.

Plasmid pSL103 was previously constructed by cloning a Trp fragment (approximately 2.3 X 10(6) daltons) from restriction endonuclease EcoRI-digested chromosome DNA of Bacillus pumilus using the neomycin-resistance plasmid pUB110 (approximately 2.8 X 10(6) daltons) as vector and B. subtilis as transformation recipient. In the present study the EcoRI Trp fragment from pSL103 was transferred in vitro to EcoRI fragments of the Bacillus plasmid pPL576 to determine the ability of the plasmid fragments to replicate in B. subtilis. Endonuclease EcoRI digestion of pPL576 (approximately 28 X 10(6) daltons) generated three fragments having molecular weights of about 13 X 13(6) (the A fragment), 9.5 X 10(6) (B fragment, and 6.5 X 10(6) (C fragment). Trp derivatives of pPL576 fragments capable of autonomous replication in B. subtilis contained the B fragment (e.g., pSL107) or both the B and C fragments (e.g., pSL108). Accordingly, the B fragment of pPL576 contains information essential for autonomous replication. pSL107 and pSL108 are compatible with pUB110. Constructed derivatives of the compatible plasmids pPL576 and pUB110, harboring genetically distinguishable EcoRI-generated Trp fragments cloned from the DNA of a B. pumilus strain, exhibited relatively high frequency recombination for a trpC marker when the plasmid pairs were present in a recombination-proficient strain of B. subtilis. No recombination was detected when the host carried the chromosome mutation recE4. Therefore, the recE4 mutation suppresses recombination between compatible plasmids that contain homologous segments.     

Two replication determinants of an antibiotic-resistance plasmid, pTB19, from a thermophilic bacillus

Two different replication determinants were found on an antibiotic resistance plasmid, pTB19, from a thermophilic bacillus. One replication determinant (designated RepA) was functional only in Bacillus subtilis, whereas the other (designated RepB) functioned in both B. subtilis and Bacillus stearothermophilus. A deletion plasmid, pTB90, carrying the RepB derived from pTB19 coincidentally contained the specific 1.0 MDal EcoRI fragment of a cryptic plasmid pBSO2 from B. stearothermophilus. The presence of this 1.0 MDal EcoRI fragment in various deletion plasmids from pTB90 increased transformation frequencies for B. stearothermophilus 10(3) to 10(4) times and lowered plasmid copy numbers in the host strain to about one-tenth of those found for plasmids lacking this fragment.     

The large Bacillus plasmid pTB19 contains two integrated rolling-circle plasmids carrying mobilization functions

Plasmid pTB19 is a 27-kb plasmid originating from a thermophilic Bacillus species. It was shown previously that pTB19 contains an integrated copy of the rolling-circle type plasmid pTB913. Here we describe the analysis of a 4324-bp region of pTB19 conferring resistance to tetracycline. The nucleotide sequence of this region revealed all the characteristics of a second plasmid replicating via the rolling-circle mechanism. This sequence contained (i) the tetracycline resistance marker of pTB19, which is highly similar to other tetL-genes of gram-positive bacteria; (ii) a hybrid mob gene, which bears relatedness to both the mob-genes of pUB110 and pTB913; (iii) a palU type minus origin identical to those of pUB110 and pTB913; and (iv) a plus origin of replication similar to that of pTB913. A repB-type replication initiation gene sequence identical to that of pTB913 was present, which lacked the middle part (492 bp), thus preventing autonomous replication of this region. The hybrid mob gene was functional in conjugative mobilization of plasmids between strains of Bacillus subtilis.     

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.     

Drug resistance and R plasmids in Pasteurella multocida isolates from swine

A total of 163 strains of Pasteurella multocida isolated from swine were examined for drug resistance and R plasmids. Strains resistant to sulfadimethoxine (Sar), ampicillin (Apr), streptomycin (Smr), kanamycin (Kmr), and chloramphenicol (Cpr) were found in 93.9, 1.8, 16.6, 1.2, and 10.4%, respectively. There were two patterns of drug resistance (Sar and SarCpr) in isolates from nasal cavities, and five patterns (Sar, SarSmr, SarSmrCpr, SarSmrApr, and SarSmrKmrCpr) in isolates from pneumonic lung specimens. Two isolates studied were proved to carry a nonconjugative R plasmid pJY2 or pJY8 with other unidentified plasmids, respectively. pJY2 (3.6 megadaltons) encoding resistance to SarSmr had one cleavage site for EcoRI or HindIII endonuclease and two sites for PstI endonuclease. pJY8 (5.5 megadaltons) encoding resistance to Sar SmrKmrCpr had one EcoRI site and two PstI sites.     

Isolation and partial characterization of four plasmids from antibiotic-resistant thermophilic bacilli.

Twenty-nine antibiotic-resistant isolates of thermophilic bacilli were examined for the presence of covalently closed circular duplex DNA molecules by agarose-gel electrophoresis and caesium chloride-ethidium bromide density gradient centrifugation. Five of the 29 strains tested contained covalently closed circular molecules. Two of the streptomycin-resistant strains contained the same two plasmids: pAB118A of molecular weight 4.9 X 10(6) (7.0 kilobases) and pAB118B of molecular weight 3.0 X 10(6) (4.3 kilobases). Two of the tetracycline-resistant strains each contained a plasmid (pAB124) of molecular weight 2.9 X 10(6) (4.14 kilobases), while a third harboured a small plasmid (pAB128) of molecular weight 2.5 X 10(6) (3.57 kilobases). These plasmids were digested with 19 different restriction endonucleases and the numbers of cleavage sites were determined. Transformation of Bacillus subtilis (168 (Trp-) with purified plasmid DNA indicated that pAB124 conferred tetracycline resistance on the host.     

Drug resistance and R plasmids in Bordetella bronchiseptica isolates from pigs

A total of 207 strains of Bordetella bronchiseptica isolated from pigs in 1978 and 1979 were tested for drug resistance and for the properties of their R plasmids. Apart from intrinsic resistance to spectinomycin, single (sulfadimethoxine), double (sulfadimethoxine and streptomycin), andt riple (sulfadimethoxine, streptomycin, and ampicillin) resistance were found in 54.1%, 1.0%, and 15.9% of the strains, respectively. All of the triple-resistance determinants were associated with mercury resistance and were conjugative. pBB1, one of these R plasmids, was identified as Fi- (F) and Spp- (no suppression of phage multiplication) type, and as a member of incompatability group IncP. The single- and double-resistance determinants were nonconjugative. pBB2, one of the double-resistance determinants, was mobilized by an R plasmid, RP4, with the high efficiency of 80% and at a frequency of 3.3% without cotransfer of RP4. The molecular weight of pBB1 and pBB2 was estimated at 36 X 10(6) and 13 X 10(6) daltons, respectively, by electron microscopy and agarose gel electrophoresis. pBB1 had five cleavage sites for EcoRI endonuclease, and four sites for HindIII. pBB2 had two EcoRI sites, one HindIII, and one BamHI site. Cells carrying pBB1 or pBB2 produced enzymic activity tha inactivated streptomycin in the presence of ATP.     

Genetic, molecular, and functional analysis of Streptococcus faecalis R plasmid pJH1.

Streptococcus faecalis JH1 contains two conjugative plasmids, pJH1, an R plasmid that codes for resistance to kanamycin, streptomycin, erythromycin, and tetracycline, and pJH2, a hemolysin-bacteriocin plasmid. Strain JH1 was used as an antibiotic resistance donor in conjugation experiments with two plasmid-free S. faecalis recipient strains, JH2-2 and OG1-RF1. Plasmid pJH1 was purified from one transconjugant, DL77, and subjected to restriction endonuclease analyses. Five restriction enzymes, EcoRI, XbaI, BamHI, SalI, and XhoI, yielding 10, 9, 3, 2, and 2 fragments, respectively, were used to determine the size (80.7 kilobases) of pJH1 and to construct a restriction endonuclease map of the plasmid. Twenty-eight percent of the antibiotic-resistant transconjugants examined expressed only part of the resistance pattern (Kmr Smr Emr Tcr) associated with pJH1, that is, they were resistant to kanamycin, streptomycin, and erythromycin; to erythromycin and tetracycline; or to erythromycin or to tetracycline only. Most of these strains also produced hemolysin and bacteriocin, and several contained a hybrid plasmid consisting of pJH2 and specific segments of pJH1 DNA. Several of these hybrid plasmids, as well as a deletion derivative of pJH1 that coded for resistance to tetracycline but not to kanamycin, streptomycin, or erythromycin, were purified and used to confirm the arrangement of restriction endonuclease fragments on the pJH1 map and to locate the resistance determinants on this map.