Increased efficiency of transformation ofBacillus stearothermophilus by a plasmid carrying a thermostable kanamycin resistance marker

Recombinant plasmids carrying either the wildtype kanamycin nucleotidyltransferase gene encoded originally by the mesophilic plasmid pUB110 or the gene encoding the thermostable TK101 mutant were constructed and introduced intoBacillus stearothermophilus by a protoplast transformation procedure. When kanamycin-resistant transformants were selected at 47°C, the transformation efficiency of the plasmid bearing the TK101 gene was nine times higher than that of the plasmid encoding the wildtype enzyme. The difference in transformation efficiencies between the two plasmids was increased when transformants were selected at higher temperatures, reflecting the difference in thermostabilities of the respective kanamycin nucleotidyltransferases. We conclude that, even though the pUB110 enzyme is sufficiently active at 47°C to confer kanamycin resistance toB. stearothermophilus, the additional stability of the TK101 mutant is advantageous in transformation ofB. stearothermophilus. The TK101 gene may also have broad utility as a marker for cloning vectors in other thermophiles.     

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.     

Conjugative Mobilization of the Rolling-Circle Plasmid pIP823 from Listeria monocytogenes BM4293 among Gram-Positive and Gram-Negative Bacteria

We determined the sequence and genetic organization of plasmid pIP823, which contains the dfrD gene; dfrD confers high-level trimethoprim resistance to Listeria monocytogenes BM4293 by synthesis of dihydrofolate reductase type S2. pIP823 possessed all the features of the pUB110/pC194 plasmid family, whose members replicate by the rolling-circle mechanism. The rep gene encoded a protein identical to RepU, the protein required for initiation of the replication of plasmids pTB913 from a thermophilic Bacillus sp. and pUB110 from Staphylococcus aureus. The mob gene encoded a protein with a high degree of amino acid identity with the Mob proteins involved in conjugative mobilization and interplasmidic recombination of pTB913 and pUB110. The host range of pIP823 was broad and included L. monocytogenes, Enterococcus faecalis, S. aureus, Bacillus subtilis, and Escherichia coli. In all these species, pIP823 replicated by generating single-stranded DNA and was stable. Conjugative mobilization of pIP823 was obtained by self-transferable plasmids between L. monocytogenes and E. faecalis, between L. monocytogenes and E. coli, and between strains of E. coli, and by the streptococcal conjugative transposon Tn1545 from L. monocytogenes to E. faecalis, and from L. monocytogenes and E. faecalis to E. coli. These data indicate that the gene flux observed in nature from gram-positive to gram-negative bacteria can occur by conjugative mobilization. Our results suggest that dissemination of trimethoprim resistance in Listeria spp. and acquisition of other antibiotic resistance determinants in this species can be anticipated.     

Screening for thermostable mutant of kanamycin nucleotidyltransferase by the use of a transformation system for a thermophile, Bacillus stearothermophilus

A structural gene of kanamycin nucleotidyltransferase cloned into a single-stranded bacteriophage M13 was subjected to mutagenesis with hydroxylamine. Having recloned the mutagenized gene of the enzyme in a vector plasmid pTB922, the recombinant plasmid was used to transform Bacillus stearothermophilus with a purpose of screening for the more thermostable enzyme than the wild type. Out of greater than 8 X 10(3) transformants, 12 clones that were suspected to harbor the mutant gene encoding the more thermostable enzyme were isolated by shifting from a permissive (55 degrees C) to a nonpermissive (61 degrees C) temperature that inactivates the wild-type enzyme. DNA sequence analysis of the mutant genes revealed two types of mutation of single base substitution and hence a single amino acid replacement. The first type was the replacement of an aspartate by a tyrosine at position 80 of the wild-type enzyme, while the second was that of a threonine by a lysine at position 130. Purified enzymes from the two mutant genes were confirmed to be substantially more thermostable than the wild type in vitro. The method of screening for a thermostable kanamycin nucleotidyltransferase presented here could be applied to any other enzyme, if a transformation system of a thermophile were available. Indeed, thermostable mutants with a subtle amino acid change would be of value for better understanding of forces and interactions that contribute to the stability of a protein.     

Purification and characterization of a kanamycin nucleotidyltransferase from plasmid pUB110-carrying cells of Bacillus subtilis.

The nucleotidyltransferase encoded by plasmid pUB110 was purified to greater than 95% purity with a 33% yield. The enzyme is a monomeric protein with a molecular weight of 34,000. The optimum pH for activity is 5, and the optimum MgCl2 concentration for activity is 18 mM. The enzyme, which is synthesized constitutively, is stable for several weeks at 4 degrees C. This enzyme would appear to be a good model gene product for the development of a pUB110 deoxyribonucleic acid-dependent in vitro protein-synthesizing system from Bacillus subtilis.     

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.     

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.     

Molecular cloning, DNA nucleotide sequencing, and expression in Bacillus subtilis cells of the Bacillus macerans cyclodextrin glucanotransferase gene.

The gene for cyclodextrin glucanotransferase from Bacillus macerans was cloned in an Escherichia coli bacteriophage, lambda D69, and was recloned in a Bacillus subtilis plasmid, pUB110. Starting from an ATG initiation codon, a unique reading frame was shown to extend for 2,142 base pairs (714 amino acids). The nucleotide sequence revealed that the enzyme is composed of two identical subunits.     

Complete nucleotide sequences of Bacillus plasmids pUB110dB, pRBH1 and its copy mutants

Summary The deletion plasmids, pRBH1 (1.5 MDa, kanamycin resistance, Km^r) and pUB110dB (1.5 MDa, Km^r), were obtained from pTB913 (2.9 MDa, Km^r, isolated from a thermophilic bacillus) and pUB110 (3.0 MDa, Km^r, from Staphylococcus aureus), respectively. All the nucleotide sequences of these deletion plasmids were determined. Replication origin regions of pRBH1 and pUB110dB contained, respectively, 63 base-pair inverted repeat and a large open reading frame, encoding RepB protein (235 amino acid residues). The nucleotide sequences were identical to each other except for one base in the center of the inverted repeat. Two copy number mutant plasmids, pRBHC3 and pRBHC7, were obtained from pRBH1. The mutation points were located at different positions in the RepB protein coding region (Gly to Asp for pRBHC3 and Gly to Glu for pRBHC7). RepB protein was shown to be involved in the copy number control of these plasmids.     

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.