Plasmid pC194 Research Articles

Survival of bacillus subtilis NB22, an antifungal-antibiotic iturin producer, and its transformant in soil-systems

Bacillus subtilis NB22 is an antifungal-antibiotic iturin producer that expresses broad suppressibility against phytopathogenic microorganisms. The survival of B. subtilis NB22-1, which is a spontaneous streptomycin-resistance mutant of NB22 was investigated in four different soils. After a gradual decline, the bacterial viable cell number stabilized at a level of 10 4–10 5 colony forming units/g-dry soil irrespective of soil differences. A similar decline and stabilization pattern was observed in the case of the transformant of B. subtilis NB22-1 with the plasmid pC194 in nonsterile soil. The transformant reached a much higher stabilized level in sterile soil than in nonsterile soil. However, significant loss of the plasmid was observed in both the soil systems after 10 to 20 d incubation. Plasmid pC194 was stable over a hundred generations in the strain when cultivated in a liquid complex medium, but unstable in minimal medium, indicating that the plasmid stability in soil does not necessarily reflect that in liquid culture.

Characteristics of plasmid stability in Bacillus subtilis NB22, an antifungal-antibiotic iturin producer

The stability of plasmids pC194 and pUB110 was investigated in an antifungal antibiotic iturin producer, Bacillus subtilis NB22. Although plasmid pC194 was maintained stably over a hundred generations in five successive cultivations in iturin-production no. 3 medium, significant curing was observed when the cultivation was prolonged for 10 d in the same medium. When the transformant of pC194 was cultivated in Schaeffer's sporulation medium, drastic curing took place in accordance with the occurrence of sporulation, even in the presence of an antibiotic for selective pressure. In the case of pUB110, in sharp contrast to the result with pC194, high stability was observed in the sporulation medium, but significant curing was observed during prolonged incubation in no. 3 medium.

Intramolecular homologous recombination in Bacillus subtilis 168.

Plasmid resolution from a phage::plasmid chimera was used to measure directly intramolecular recombination in Bacillus subtilis. The system is based on a sigma-replicating plasmid (pC194) cloned into a dispensable region of the lytic bacteriophage SPP1. The plasmid, which confers chloramphenicol resistance, is resolved when SPP1::pC194 phages infect B. subtilis cells, provided the chimera carries a functional, intact copy of the plasmid repH gene. Intramolecular homologous recombination was independent of the RecA and RecL-RecR functions, but dependent on RecF, RecB, RecG, RecP, RecH and AddAB functions. These results are consistent with the hypothesis that B. subtilis has multiple pathways for genetic recombination and allow us to tentatively place the recB and recG genes into a new epistatic group epsilon.

Transformation of a psychrotrophic bacterium with a plasmid from a mesophile

The psychrotrophic bacteriumBacillus psychrophilus was transformed with the broadhost-range plasmid pC194. The ability of the transformant to express chloramphenicol (CAM) resistance and the possible effects of such expression on the physiology of the psychrotroph were examined. The transformant exhibited growth rates, filament formation at elevated temperatures, synthesis of cold shock proteins and cold acclimation proteins, similar to the parentalB. psychrophilus.

Nucleotide sequence of the chloramphenicol resistance determinant of the streptococcal plasmid pIP501

We have sequenced the chloramphenicol resistance determinant ( cat) of plasmid pIP501 from Streptococcus agalactiae to investigate its relationship with other cognate cat determinants. Sequence analysis revealed that it exhibits a high degree of similarity with the cat genes of plasmids pC221 and pUB112 from Staphylococcus aureus and pSCS1 from Staphylococcus intermedius. These genes, however, display several differences in their regulatory and coding regions. These results demonstrate that the cat determinant of plasmid pIP501 belongs to the pC221 subgroup of CAT variants.

Shuttle plasmid vectors for Lactobacillus casei and Escherichia coli with a minus origin

Recombinant plasmids which can be used as shuttle vectors between Escherichia coli and the industrially used strains of Lactobacillus casei were constructed. They have replication regions closely related to those of pUB110 and are likely to replicate by a rolling-circle mechanism via a plus-strand-specific DNA intermediate in L. casei. Both orientations of palA from the staphylococcal plasmid pC194 and those of the intergenic region from coliphage M13 are identified as active minus origins in L. casei, in contrast to the pAM alpha 1 delta 1-derived BA3 minus origin which does not function in L. casei. Stability of the plasmids increased in L. casei when one of these two active minus origins was inserted. All the DNA sequences of the constructed vectors were known.

Transformation of protoplasts ofCellulomonas flavigena

Protoplasts ofCellulomonas flavigena (Cms) were transformed with plasmid pC194. Transformation frequency was 2.72×10−3 in MR-1 regeneration medium with 2 μg/ml chloramphenicol. Transformation conditions are described.

Cloning and sequence analysis of a plasmid-encoded chloramphenicol acetyltransferase gene from Staphylococcus intermedius

The chloramphenicol acetyltransferase gene (cat) of a 3.9 kb chloramphenicol resistance (CmR) plasmid from Staphylococcus intermedius, designated pSCS1, was cloned into an Escherichia coli plasmid vector. Sequence analysis revealed a high degree of base similarity with the cat gene of the S. aureus CmR plasmid pC221 but there were several differences in the regulatory region. A lesser degree of similarity was observed between the cat gene of the S. intermedius plasmid and the cat gene of the S. aureus plasmid pC194.

A highly efficient electroporation system for transformation of Bacillus licheniformis

Transformation of intact cells of Bacillus licheniformis 5A24 with plasmids pLM6 (2.8 kb), pC194 (2.9 kb) and pCP49 (7.1 kb) by electroporation resulted in 1.5 × 106, 1.2 × 106 , and 5.2 × 104 transformants per μg DNA, respectively. Transformation was possible with plasmid DNA, which was religated after restriction endonuclease digestions. In addition, evidence is presented that indicates that B. licheniformis possesses DNA restriction and modification systems.

Characterization of Gram-positive broad host-range plasmids carrying a thermophilic replicon

The cryptic plasmid pBC1 (1.6 kb) isolated from Bacillus coagulans Zu1961 was genetically marked with the genes for chloramphenicol and ampicillin resistance (Cm R and Ap R) from the Escherichia coli plasmid pJH101. The recombinant vector obtained (pCP49, 7.0 kb) replicated and expressed Cm R in B. subtilis and Cm R and Ap R in E. coli. Different shuttle vectors for Gram + bacteria were also constructed by inserting pBC1 into the Staphylococcus aureus plasmid pC194. The smallest of these, pLM6 (2.8 kb), containing essentially pBC1 and the chloramphenicol acetyl transferase gene from pC194, replicated in B. subtilis at a copy number of 60. By electroporation, these plasmids were introduced and stably maintained in B. subtilis, B. amyloliquefaciens, S. aureus, S. carnosus and Lactobacillus reuteri.

Transfer of plasmids and chromosomal genes amongst subspecies ofBacillus thuringiensis

The plasmids pBC16 and pC194 fromBacilus thuringiensis subsp.israelensis strains A084-16-194 were transferred to 25 subspecies ofB. thuringiensis by a conjugation-like process using broth mating technique. The frequencies of transfer varied considerably between different mating pairs, ranging from 1.1×10−9 to 9.8×10−5. Additionally, chromosomal transfer could also be demonstrated in tenB. thuringiensis subspecies with very low frequencies (4.3×10−9 to 3.7×10−7). The intersubspecies matings within a group of eight subspecies strains gave higher frequencies of transfer than the matings between the subspecies. Furthermore, the results indicated that the capability to transfer plasmids among these various subspecies did not depend on the presence of large plasmid.

Lactobacillus hilgardii plasmid pLAB1000 consists of two functional cassettes commonly found in other gram-positive organisms

A Lactobacillus hilgardii plasmid, pLAB1000, was studied to understand the organization of autonomous replicons from lactobacilli. Two cassettes could be identified. First, the replication region consisted of a sequence coding for a replication protein (Rep) and its corresponding target site, similar to those from plasmids pUB110, pC194 (Staphylococcus aureus), pFTB14, pBAA1 (Bacillus sp.), and pLP1 (Lactobacillus sp.). Sequence analysis indicated the possible synthesis of an antisense RNA that might regulate Rep production. The results also suggested that pLAB1000 replicates via a single-stranded DNA intermediate, and a putative lagging-strand initiation site was found that had similarities to those of alpha 3, St-1, and G4 isometric bacteriophages. The second cassette of pLAB1000 consisted of a sequence coding for a putative mobilization protein (Mob) and its corresponding RSA site. This cassette was similar to those found in pT181, pUB110, pE194 (S. aureus), and pG12 (Bacillus sp.), and it was found to be conserved among different Lactobacillus plasmid replicons. The origin and evolution of these functional cassettes are also discussed.

Use of a novel cassette to label phenotypically a cryptic plasmid of Bacillus subtilis and map loci involved in its stable maintenance

In order to facilitate studies on the maintenance of cryptic plasmids from Gram-positive bacteria we have constructed a novel cassette cAPG1000 (5.0 kb) which carries both a selectable marker (chloramphenicol resistance from Staphylococcus aureus plasmid pC194) and a screenable marker (the xylE gene from the TOL plasmid of Pseudomonas putida expressed from a cloned promoter of Bacillus phage SPO2) and which is flanked by terminators to prevent transcription from the cassette activating or inhibiting loci adjacent to the site of insertion. To demonstrate the usefulness of this cassette we have mapped loci required for stable maintenance of an 8.6 kb cryptic plasmid endogenous to Bacillus subtilis (pPOD2000) from the properties of cAPG1000 insertion and insertion/deletion derivatives. We have identified the replication region as well as separate regions required for segregational and structural stability. The segregational mechanism is very efficient since it allows no detectable loss despite the fact that bacteria carrying the plasmid have a greatly increased mean generation time.

In vitro studies of the initiation of staphylococcal plasmid replication. Specificity of RepD for its origin (oriD) and characterization of the Rep-ori tyrosyl ester intermediate.

Several staphylococcal plasmids from different incompatibility (inc) groups which replicate by a rolling circle mechanism each specify a replication initiator protein (Rep) which is homologous with that of the inc3 tetracycline resistance plasmid pT181. The rep gene sequences of six pT181-like plasmids are known, each encoding proteins of molecular mass 38 kDa with 62% overall amino acid sequence identity. The initiation of replication in vivo by each of the Rep proteins is plasmid specific, acting in trans only at the cognate replication origin (ori) of the encoding plasmid. Previous studies in vitro of the RepC protein of pT181 demonstrated replication initiator, topoisomerase-like, and DNA binding activities, which appeared to be specific for the origin (oriC) of pT181 when compared with unrelated staphylococcal plasmids. Although RepD, specified by the inc4 chloramphenicol resistance plasmid pC221, has a range of activities similar to those noted previously for RepC, manipulation of in vitro conditions has revealed discrete steps in the overall reaction of RepD with oriD. In addition, factors have been identified which are necessary not only for sequence-dependent discrimination in vitro by Rep proteins for all pT181-like plasmids but also for the absolute specificity of RepD for its cognate pC221 replication origin (oriD), the latter occurring in vivo and a function of the topological state of the ori-containing target DNA. Here we also demonstrate the presence of a covalent phosphoryl-tyrosine linkage between the RepD protein of plasmid pC221 and an oligonucleotide substrate corresponding to its replication origin (oriD). The reactive tyrosine (Tyr-188) was identified from amino acid sequences of 32P-labeled peptide-oligonucleotide fragments. Substitution of Tyr-188 with phenylalanine confirms the importance of the tyrosyl hydroxyl group since the Y188F protein retains the sequence-specific DNA-binding capabilities of wild-type RepD but is unable to attach covalently to the replication origin or participate in the nicking-closing reaction in vitro.

Specificity of RepC protein in plasmid pT181 DNA replication.

The plasmid pT181 of Staphylococcus aureus consists of 4437 base pairs and encodes resistance to tetracycline. Initiation of pT181 DNA replication specifically requires the plasmid-encoded initiator protein, RepC. The initiator protein binds specifically to a 32-base pair sequence within the pT181 origin of replication. RepC protein also has a nicking-closing activity that is specific for the pT181 origin. Replication of pT181 initiates by covalent extension of the nick and proceeds by a rolling circle mechanism. Two other small, multicopy plasmids pC221 and pS194 belong to the pT181 family and have common structural organization and replication properties. The replication proteins and replication origins of these plasmids have extensive sequence homologies, although they belong to different incompatibility groups. In spite of this homology, the replication proteins and replication origins of these three plasmids do not show any cross-reactivity in vivo. We have carried out a series of in vitro experiments to determine the specificity of pT181-encoded initiator protein, RepC. DNA binding experiments showed that although the binding of RepC to the pT181 origin was very efficient, little or no binding was seen with pC221 and pS194 origins. The nicking-closing activity of RepC was found to be equally efficient with the pC221 and pS194 plasmids. The plasmids pC221 and pS194 replicated efficiently in a RepC-dependent in vitro system. However, replication of these plasmids was greatly reduced in the presence of a competing pT181 origin. The results presented here suggest that nicking-closing by RepC at the origin is not sufficient for maximal replication and that tight binding of RepC to the origin plays an important role in the initiation of DNA replication.

Comparative expression of the pC194 cat gene in Streptococcus pneumoniae, Bacillus subtilis and Escherichia coli

The expression of the cat gene of the staphylococcal plasmid pC194 present in the pLS1-pC194 composite plasmid pJS37 was lower in Streptococcus pneumoniae and Escherichia coli than in Bacillus subtilis. Different transcription start points (and, by inference, different promoter utilization) of the cat mRNA synthesized in S. pneumoniae or B. subtilis were detected. Plasmid pJS37 is prone to deletion formation when host cells are grown in the presence of chloramphenicol (Cm). The analysis of the expression of the cat gene carried by the deleted derivatives of pJS37 has shown that a new promoter for the synthesis of cat mRNA is involved in the selective advantage conferred to the host by those deleted plasmids. Characterization of either in vivo or in vitro deleted plasmids has shown that the nucleotide sequence that could encode for a putative leader peptide is required for the Cm-induced pC194 cat gene expression.

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.

Transformation of Bacillus polymyxa with plasmid DNA.

A plasmid transformation system was developed for Bacillus polymyxa ATCC 12321 and derivatives of this strain. The method utilizes a penicillin-treated-cell technique to facilitate uptake of the plasmid DNA. Low-frequency transformation (10(-6) per recipient cell) of plasmids pC194, pBD64, and pBC16 was accomplished with this method. Selection for the transformants was accomplished on both hypertonic and nonhypertonic selective media, with the highest rates of recovery occurring on a peptone-glucose-yeast extract medium containing 0.25 M sucrose. Several additional plasmids were shown to be capable of transferring their antibiotic resistance phenotypes to B. polymyxa through the use of a protoplast transformation procedure which allowed for a more efficient transfer of the plasmid DNA. However, cell walls could not be regenerated on the transformed protoplasts, and the transformants could not be subcultured from the original selective media.

Replication origin of a single-stranded DNA plasmid pC194.

The replication of the single-stranded (ss) DNA plasmid pC194 by the rolling circle mechanism was investigated using chimeric plasmids that possess two pC194 replication origins. One of the origins was intact, whereas the other was either intact or mutated. The origins were activated by inducing synthesis of the pC194 replication protein, under the control of lambda phage pL promoter. Initiation of pC194 replication at one origin and termination at the other generated circular ssDNA molecules smaller than the parental chimeric plasmid. From the nature and the amount of ssDNA circles, the activity of an origin could be assessed. Our results show that (i) the signal for initiation of pC194 replication is more stringent than that for termination; (ii) the sequence and structure of the origin are important for its activity and (iii) successful termination of one replication cycle is not followed by reinitiation of another. This last observation differentiates a ssDNA plasmid (pC194) from a ssDNA phage (phi X174).

Transformation of Bacillus thuringiensis vegetative cells by electroporation.

A highly efficient procedure for the transformation of Bacillus thuringiensis and Bacillus subtilis using covalently closed circular plasmid DNA was developed by using the small Staphylococcus aureus plasmid pC194 and electroporation. We have achieved transformation efficiencies in B. thuringiensis subsp. kurstaki (HD-73) greater than 5 x 10(6) transformants/micrograms plasmid DNA. The electro-transformation (or electroporation) procedure also worked with B. subtilis 168 although at a 200-fold less level of efficiency. The results indicated that the plasmid exists in double and single-stranded forms both in B. subtilis and B. thuringiensis. A second single-stranded species was also observed in both species. This technique may prove to be applicable to other members of the genus Bacillus.