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.     

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.     

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.     

Functional analysis of the leading strand replication origin of plasmid pUB110 in Bacillus subtilis.

Supercoiled plasmid DNA is the substrate for initiation of pUB110 replication, and - by inference - for binding of its initiator protein (RepU) to the plasmid replication origin (oriU) in vivo. No hairpin structure is required for RepU-oriU recognition. RepH (the pC194 replication initiation protein) failed to initiate replication in trans at oriU. The nucleotides that determine the specificity of the replication initiation process are located within oriU but termination is unefficient. Therefore the segment that forms the full recognition signal for termination is probably located 3' of the oriU recognition sequence. Two overlapping domains, one for initiation and one required for termination, compose the leading strand replication origin of plasmid pUB110.     

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.     

Identification and nucleotide sequence of the minimal replicon of the low-copy-number plasmid pBS2

The plasmid pBS2 has a low copy number and is endogenous to Bacillus subtilis. The replication of this plasmid depends on the function of most of the host's dna genes including dnaB, which is unique to B. subtilis and is required for both the initiation of chromosome replication and the DNA-membrane association. We have identified the region that is essential for the replication of pBS2 and determined the complete 2279-bp nucleotide sequence of this region. In this region, there are two stretches of sequence homologous to the 18-bp consensus sequence which commonly appears at the origin of replication of plasmids pUB110 and pC194. The entire region contains six sizable open reading frames. Two of them are probably translated. One open reading frame, designated ORF A, coding for 269 amino acids, has significant homology, in terms of amino acid sequence, with the open reading frame of the gene for the Rep U protein of plasmid pUB110. The similarities between pBS2 and other plasmids suggest that the pBS2 may also replicate as a rolling circle, which appears to be the salient feature of a mechanism of replication that is common to small plasmids in gram-positive bacteria.     

Rolling circle replication of single-stranded DNA plasmid pC194.

A group of small Staphylococcus aureus/Bacillus subtilis plasmids was recently found to replicate via a circular single-stranded DNA intermediate (te Riele et al., 1986a). We show here that a 55 bp region of one such plasmid, pC194, has origin activity when complemented in trans by the plasmid replication protein. This region contains two palindromes, 5 and 14 bp long, and a site nicked by the replication protein. DNA synthesis presumably initiated at the nick in the replication origin can be terminated at an 18 bp sequence homologous to the site of initiation, deriving from another plasmid, pUB110, or synthesized in vitro. This result suggests that, similar to the Escherichia coli single-stranded DNA phages, pC194 replicates as a rolling circle. Interestingly, there is homology between replication origins and replication proteins of pC194 and the phage phi mX174.     

Sequence analysis of replication origin of plasmid pLS11 of Bacillus subtilis IFO 3022.

The structure of a 1.6-kb SphI-HindIII DNA sequence necessary and sufficient for the replication of a 8.6-kb plasmid pLS11 of Bacillus subtilis IFO 3022, which is responsible for gamma-polyglutamate production, has been characterized by using a trimethoprim (Tmp)-resistance gene derived form B. subtilis TTK24 chromosomal DNA as a selective marker. The 1.6-kb DNA sequence contains a rep gene encoding the protein (333 amino acids) essential for initiation of replication and a possible origin of replication. The predicted REP protein of pLS11 has an overall homology with the REP proteins of pUH1 (74.8% identity), pBAA1 (92.8%), and pFTB14 (78.7%) in Bacillus spp., pLP1 (42.1%) and pLAB1000 (36.3%) in Lactobacillus spp., and pUB110 (35.3%) and pC194 (37.4%) in Staphylococcus aureus, but has not any similarity with the REP protein of the staphylococcal plasmid pT181.     

Heat-inducible translational coupling in Bacillus subtilis.

Bacillus subtilis plasmid pGR71 is a promoter-probe shuttle vector derived from pUB110. The expression of the cat gene on pGR71 in B. subtilis requires the insertion of a Bacillus promoter and a ribosomal binding site (RBS) into the HindIII cloning site immediately upstream from the cat gene. A recombinant plasmid of pGR71, named pGR71-369, was obtained by a spontaneous deletion of a fragment containing most of the inserted HindIII fragment and the replication origin necessary for multiplication in Escherichia coli. The expression of the cat gene in B. subtilis cells carrying this plasmid was inducible by heat. Nucleotide sequence analysis of the upstream region of the cat gene, deletion analysis, and dot blot hybridization analysis of mRNA in various conditions revealed that the cat gene was expressed by heat-inducible translational coupling and that the regulatory region of heat inducibility was present in the upstream region of the cat gene.     

Identification of a low-copy-number mutation within the pUB110 replicon and its effect on plasmid stability in Bacillus subtilis

A mutation (cop1) within the minimal replicon of plasmid pUB110, reducing its copy number from 48 +/- 2 to 11 +/- 2 in a Bacillus subtilis host, was isolated. This mutation is a G----T transversion within the translation control region of the replication initiation gene (repU). The cop1 mutation, when present in the recombinant plasmid, increases both its structural and segregational stability.     

In vitro expression of a Tn9-derived chloramphenicol acetyltransferase gene fusion by using a Bacillus subtilis system.

A coupled in vitro protein-synthesizing system has been developed with components derived totally from Bacillus subtilis. The system synthesized specific gene products from various exogenous DNA templates, including B. subtilis phage phi 29, plasmid pUB110, and a heterologous B. subtilis-Escherichia coli gene fusion containing the transposon Tn9-derived chloramphenicol acetyltransferase (cat) gene. The gene fusion product was able to show CAT activity, bind specifically to a Sephacryl-chloramphenicol column, and react immunologically against anti-CAT antiserum. The fidelity of this in vitro system was demonstrated by the synthesis of gene products identical to that made in vivo. We suggest that this system may be used to study the regulation of gene expression in vitro.     

Initiation of DNA replication in Bacillus subtilis. IV. The effect of an intercalating dye, ethidium bromide

Summary The effects of an intercalating dye, ethidium bromide (EtBr), on the initiation of chromosome replication in Bacillus subtilis were studied. Spores of a thymine requiring mutant acquired the ability to initiate one round of replication in the absence of RNA and protein synthesis (initiation potential) during germination in a thymine starved medium. When EtBr was added after the initiation potential was fully established, initiation of replication was completely inhibited. This inhibition was reversible, and initiation was resumed when the drug was removed. The recovery of initiation occurred in the absence of protein synthesis but did require RNA synthesis and an active dna gene product.During germination both a DNA-protein complex and a DNA-membrane complex were formed at the replication origin in parallel with the establishment of initiation potential. EtBr destroyed both of these complexes at the concentration which inhibited initiation.The first round of replication of a plasmid DNA, pSL103, during spore germination was also prevented by EtBr. However a higher concentration was required to inhibit plasmid replication. It was found that the plasmid formed two complexes identical to the S- and M-complex of the chromosome origin. Compared to the chromosome complexes the plasmid complexes were less sensitive to EtBr. The loss of sensitivity was equivalent to that for the initiation of the plasmid compared to the chromosome. These results indicate that the target of EtBr is the DNA in the S- and M-complexes whose conformation is essential for the initiation of chromosome and plasmid replication.III of this series is Murakami et al. 1976     

Identification of the product of dnaB gene in Bacillus subtilis

In order to detect the product of dnaB gene in B. subtilis, a gene which is involved in the initiation of DNA replication and the formation of the DNA-membrane complex, we synthesized an origopeptide of 15 amino acids which corresponds to a region near the carboxyl-terminal of the gene product, and raised antibody against the synthetic peptide. We have also employed a filter binding assay to measure the predicted DNA binding activity of the product of the dnaB gene, using the plasmid pUB110. The binding activity was detected after fractionation of cell lysates of B. subtilis on sucrose-density gradients. When the active fraction was prepared from a mutant which was temperature-sensitive for the dnaB gene, the DNA binding activity in the fraction showed significant thermolability. Furthermore, the binding activity was inhibited by the purified antibody raised against the synthetic peptide. These results suggest that the product of the dnaB gene does indeed have DNA binding activity, and that the filter binding assay and the antibody can be used for the detection and characterization of the gene product.     

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.     

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.     

Mutation in the plasmid pUB110 Rep protein affects termination of rolling circle replication.

We isolated a mutant of plasmid pUB110 that has the following properties in Bacillus subtilis: (i) it is toxic for recA and add cells, particularly at elevated temperature; (ii) it has a copy number threefold higher than that of the parental plasmid, and the extra copies are present as multimers; and (iii) it can efficiently complement replication of a cmp- satellite plasmid, despite being cmp+. All these properties are due to a single change in the plasmid replication protein, i.e., Gly at position 148 to Glu. These properties of the mutant Rep protein reflect a diminished ability to terminate rolling circle replication. We propose that the Rep protein may have a diminished affinity for the plasmid origin; alternatively, it may be impaired for recognition of the plasmid conformations which distinguish initiation and termination.     

Dramatic increase in negative superhelicity of plasmid DNA in the forespore compartment of sporulating cells of Bacillus subtilis.

Plasmid pUB110, isolated from vegetative cells of Bacillus subtilis, has an average of 34 negative supertwists (tau av = -34). This value falls to -30 early in sporulation, and the plasmid in the mother cell compartment maintains a tau av of -30. However, the plasmid within the developing forespore becomes much more negatively supercoiled, reaching a tau av of -47 in the dormant spore. This increased negative supercoiling in the forespore plasmid takes place in parallel with the synthesis of small, acid-soluble spore proteins, alpha and beta; and the plasmid from spores lacking small, acid-soluble proteins alpha and beta has a tau av of -40. The large increase in negative supercoiling of spore plasmid was also observed with Bacillus megaterium and in B. subtilis containing a plasmid with an origin different from that of pUB110. During spore germination plasmid pUB110 rapidly relaxed back to the tau av value characteristic of vegetative cells. It is possible that the observed changes in forespore plasmid topology are involved in modulating gene expression, DNA photochemistry, or both of these parameters in this compartment.     

Molecular structure of the replication origin of a Bacillus subtilis (natto) plasmid, pUH1.

The structure of a 2.0-kb BstEII DNA sequence necessary and sufficient for the replication of a 5.7-kb Natto plasmid, pUH1, which is responsible for gamma-polyglutamate production by Bacillus subtilis (natto), has been characterized by using a trimethoprim resistance gene derived from B. subtilis chromosomal DNA as a selective marker. The 2.0-kb DNA sequence contains an open reading frame, rep, stretching for 999 bp; a promoter region for rep expression; and a possible replication origin for the plasmid upstream of the promotor. The predicted Rep protein has highly homologous amino acid sequences with rep14 of pFTB14 in B. amyloliquefaciens, RepB of pUB110, and protein A, which is necessary for pC194 replication in staphylococci throughout the protein molecule, but is not homologous with RepC of staphylococcal plasmid pT181.     

Molecular structure of the replication origin of a Bacillus subtilis (natto) plasmid, pUH1.

The structure of a 2.0-kb BstEII DNA sequence necessary and sufficient for the replication of a 5.7-kb Natto plasmid, pUH1, which is responsible for gamma-polyglutamate production by Bacillus subtilis (natto), has been characterized by using a trimethoprim resistance gene derived from B. subtilis chromosomal DNA as a selective marker. The 2.0-kb DNA sequence contains an open reading frame, rep, stretching for 999 bp; a promoter region for rep expression; and a possible replication origin for the plasmid upstream of the promotor. The predicted Rep protein has highly homologous amino acid sequences with rep14 of pFTB14 in B. amyloliquefaciens, RepB of pUB110, and protein A, which is necessary for pC194 replication in staphylococci throughout the protein molecule, but is not homologous with RepC of staphylococcal plasmid pT181.     

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.