Transfer of plasmids and chromosomal genes amongst subspecies ofBacillus thuringiensis

Summary 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.     

Mobilization functions of the bacteriocinogenic plasmid pRJ6 of <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

Plasmid pRJ6 is the first known bacteriocinogenic mobilizable (Mob) plasmid of Staphylococcus aureus. Its Mob region is composed of four mob genes (mobCDAB) arranged as an operon, a genetic organization uncommon among S. aureus Mob plasmids. oriT _pRJ6 was detected in a region of 431 bp, positioned immediately upstream of mobC. This region, when cloned into pCN37, was able to confer mobilization to the re-combinant plasmid only in the presence of pRJ6. The entire Mob region, including oriT _pRJ6, is much more similar to Mob regions from several coagulase-negative staphylococci plasmids, although some remarkable similarities with S. aureus Mob plasmids can also be noted. These similarities include the presence within oriT _pRJ6 of the three mcb (MobC binding sites), firstly described in pC221 and pC223, an identical nick site also found in these same plasmids, and a nearly identical sra _pC223 site (sequence recognized by MobA). pRJ6 was successfully transferred to S. epidermidis by conjugation in the presence of the conjugative plasmid pGOl. Altogether these findings suggest that pRJ6 might have been originally a coagulase-negative staphylococci plasmid that had been transferred successfully to S. aureus.     

A highly efficient electroporation system for transformation of Bacillus licheniformis

Summary 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 &#x00D7; 10⁶, 1.2 &#x00D7; 10⁶ , and 5.2 &#x00D7; 10⁴ transformants per &#x03BC;g 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.     

Transformation of protoplasts ofCellulomonas flavigena

Summary Protoplasts ofCellulomonas flavigena (Cm^s) were transformed with plasmid pC194. Transformation frequency was 2.72×10^–3 in MR-1 regeneration medium with 2 g/ml chloramphenicol. Transformation conditions are described.     

Batch cultures of recombinant Lactococcus lactis subsp. lactis in a stirred fermentor

Summary The transfer of plasmids was studied in a stirred fermentor in the course of mixed batch cultures combining recombinant strains of Lactococcus lactis subsp. lactis (donor strains) with L. lactis subsp. lactis CNRZ 268M3 (recipient strain). Donor strains contained one or two of the following plasmids (coding for erythromycin or chloramphenicol resistance): pIL205 (self-transmissible), pIL252, pIL253 (non-transmissible but mobilizable by pIL205, respectively small and large copy number) and pE194 (inserted in the chromosome). Only self-transmissible plasmid pIL205 was transferred, with frequencies ranging from 10^–7 to 10^–8 after 12 h of fermentation. These frequencies were 60–400 times lower than in unstirred M17 broth and 100 000 times lower than on agar medium. In the latter case, non-transmissible plasmids pIL252 and pIL253 were mobilized by pIL205 with a frequency of about 10^–5–10^–6. Correspondence to: C.-Y. Boquien     

Interplasmidic illegitimate recombination in Bacillus subtilis

Summary The illegitimate recombination between Staphylococcus aureus plasmids pE194 (or pGG20, the hybrid between pE194 and Escherichia coli plasmid pBR322) and pBD17 (plasmid pUB110 without HpaII C-fragment) was studied in Bacillus subtilis. Cointegrates were generated with the frequency of 1–3x10^-8. Among 22 hybrids analysed 9 types of recombinants were found. Nucleotide sequences of all three parental plasmids were involved in intermolecular recombination. Nucleotide sequencing of recombinant DNA junctions revealed that in 8 cases recombination occurred between short homologous regions (9–15 bp). One recombinant was formed using nonhomologous sites. The similarity was demonstrated between nucleotide sequences of the recombination sites of two types of cointegrates and those used for pE194 integration into the B. subtilis chromosome. Possible mechanisms of illegitimate recombination are discussed.     

DNA sequence analysis of a putative primary replicon from a cryptic plasmid pNM214 of a hydrocarbon utilizing marine <Emphasis Type="Italic">Bacillus</Emphasis> strain NM21

Marine Bacillus strain NM21 isolated from hydrocarbon-contaminated site at Naval Harbour, Mumbai grows on high-speed diesel as a source of carbon and energy. This bacterium harbours four plasmids in it. The smallest plasmid, pNM214 was digested with EcoRI enzyme and cloned in pUC19 vector. The clone Om4 containing largest insert of >3.5 kb was sequenced by primer walking. DNA sequence analysis showed this fragment to be homologous to replication initiation protein (rep) gene and dso (double strand origin) of different plasmids from Bacillus subtilis and Bacillus pumilus species. The putative rep gene sequence of pNM214 showed 74.3–91.6% DNA identity to B. subtilis plasmids (pTA1015, pTA1060 and pTA1040) and 86.3% to 88.9% DNA identity to B. pumilus plasmids (pPL7065, pPL10 and pSH1452). The translated amino acid sequence of rep shows that it contains all the three conserved motifs present in the Rep protein of pC194 family of plasmids. DNA sequence comparison of putative dso of pNM214 with other bacillus plasmids belonging to pC194 group shows that it contains highly conserved nick site sequence 5′-TCTTTTCTTATCTTGATA-3′ and surrounding inverted repeats. Thus, it indicates that pNM214 to be a rolling circle replicating plasmid belonging to the pC194 group. The presence of rep and dso like sequences in the sequenced EcoRI fragment indicate that the cloned fragment contain putative primary replicon of pNM214.     

Functional Comparison of Conjugative Transposons Tn916and Tn925

During interspecies matings betweenBacillus subtilisandBacillus thuringiensissubsp.israelensis,transfer of conjugative transposon Tn916was detected at a frequency of 1.1 × 10⁻⁴transconjugants per donor. Tn916-dependent transfer of plasmids pC194 and pE194 was detected at frequencies of 1.4 × 10⁻⁵and 3.2 × 10⁻⁷transconjugants per donor, respectively. Similar frequencies were obtained during parallel matings with otherwise isogenic strains that contain Tn925instead of Tn916. Tn916- or Tn925-dependent transfer of plasmids pC194 or pUB110 from the recipient to the donor (retrotransfer) was not observed during inter- or intraspecies matings. Transposon-mediated plasmid transfer by Tn916and Tn925is a Rec independent event. Thus, the data from studies in which otherwise isogenic donor and recipient strains were used indicated that Tn916and Tn925are, from a functional point of view, much more similar than previously suggested.     

Characterization of a cryptic plasmid pPZZ84 from Bacillus pumilus

The complete nucleotide sequence of a cryptic plasmid pPZZ84 from Bacillus pumilus strain ZZ84 was determined. Plasmid pPZZ84 is 6817 bp long with GC content of 36.7%. Seven putative open reading frames were identified. ORF7 shows 91% and 90% amino acid identity with rep proteins of pSH1452 and pPL1, respectively, members of rolling-circle replication (RCR) pC194-family. A typical pC194-family double strand origin (dso), a single-stranded origin (sso) and rap (regulator aspartate phosphatase) proteins were also identified in the plasmid. These results imply that pPZZ84 belongs to the Bacillus subtilis species group of small rolling circle (BsSRC) replicating plasmids. The plasmid copy number of pPZZ84 in B. pumilus ZZ84 was estimated to be 46 per cell, more than that of other BsSRC plasmids in their hosts.     

Interspecific Transfer of Streptomyces Giant Linear Plasmids in Sterile Amended Soil Microcosms

The interspecific transfer of two giant linear plasmids was investigated in sterile soil microcosms. Plasmids pRJ3L (322 kb) and pRJ28 (330 kb), both encoding mercury resistance, were successfully transferred in amended soil microcosms from their streptomycete hosts, the isolates CHR3 and CHR28, respectively, to a plasmidless and mercury-sensitive strain, Streptomyces lividans TK24. Transconjugants of S. lividans TK24 were first observed after 2 to 3 days of incubation at 30°C, which corresponded to the time taken for the formation of mycelia in soil. Transfer frequencies were 4.8 × 10⁻⁴ and 3.6 × 10⁻⁵ CFU/donor genome for pRJ3L and pRJ28, respectively. Transconjugants were analyzed by pulsed-field gel electrophoresis for the presence of plasmids, and plasmid identity was confirmed by restriction digests. Total genomic DNA digests confirmed that transconjugants were S. lividans TK24. The mercury resistance genes were shown to be on the plasmid in the transconjugants by hybridization analysis and were still functional. This is the first demonstration of transfer of giant linear plasmids in sterile soil microcosms. Giant linear plasmids were detected in many Streptomyces spp. isolated from mercury-contaminated sediments from Boston Harbor (United States), Townsville Harbor (Australia), and the Sali River (Tucuman, Argentina). Mercury resistance genes were shown to be present on some of these plasmids. Our findings that giant linear plasmids can be transferred between Streptomyces spp. and are common in environmental Streptomyces isolates suggest that these plasmids are important in gene transfer between streptomycetes in the environment.     

Recombination between repeated DNA sequences occurs more often in plasmids than in the chromosome of Bacillus subtilis

Summary Directly repeated pBR322 sequences 3.7–3.8 kb long recombine with a frequency of about 10% per generation when carried on plasmids related to pC194 and 0.01% per generation when carried on the Bacillus subtilis chromosome. Recombination is therefore 1,000 times more efficient in plasmids than in the chromosome of this organism.     

Cloning and mapping of the genetic determinants for microcin C51 production and immunity

Microcin C51 is a small peptide antibiotic produced by Escherichia coli cells harbouring the 38 kb low copy number plasmid pC51, which codes for microcin production and immunity. The genetic determinants for microcin synthesis and immunity were cloned into the vectors pBR325, pUC19 and pACYC184. Physical and phenotypic analysis of deletion derivatives and mutant plasmids bearing insertions of transposon Tn5 showed that a DNA fragment of about 5 kb is required for microcin C51 synthesis and expression of complete immunity to microcin. Partial immunity can be provided by a 2 kb DNA fragment. Mutant plasmids were tested for their ability to complement Mic^– mutations. Results of these experiments indicate that at least three plasmid genes are required for microcin production. The host OmpR function is also necessary for microcin C51 synthesis.     

Incompatibility and molecular relationships between small bacteriocinogenic plasmids of Staphylococcus aureus

The sequence relations between small bacteriocinogenic plasmids (pRJ6, pRJ9, pRJ10 and pRJ11) of Staphylococcus aureus were investigated by comparing restriction maps and by hybridization. Plasmids pRJ10 and pRJ11 showed identical restriction maps, similar to that of pRJ9. The restriction map of pRJ6 differed from those of pRJ9 and pRJ10/pRJ11. Both groups of plasmids were shown to share a region of homology of at least 2.6 kb. The incompatibility relationships between them were also investigated by using plasmid derivatives tagged with transposon Tn551. Plasmids pRJ6 and pRJ9 proved to belong to different incompatibility groups.     

A bifunctional plasmid carrying the recA gene of E. coli

Summary To investigate the effect of an active, plasmid-carried recA gene on the stability and/or the expression of plasmid genes in different genetic backgrounds, we have constructed a bifunctional plasmid (able to replicate in Escherichia coli and in Bacillus subtilis). Chimeric plasmids were obtained by inserting pC194 (Ehrlich 1977) into pDR1453 (Sancar and Rupp 1979). pDR1453 is a 12.9 Kbp plasmid constructed by inserting an E. coli chromosome fragment carrying the recA gene into pBR322. The expected bifunctional recombinant (pMR22/1) (15.7 Kbp) was easily obtained but surprisingly the Cm resistance was expressed only at a very low level in E. coli (as compared, for example, to pHV14, pHV15). We attribute this effect to the presence of multiple recA genes in the cell. On the contrary, Cm^r E. coli transformants bear a recombinant plasmid (pMR22/n) containing tandemly repeated copies of pC194 in equilibrium with excised free pC194. Such amplification has never been observed in a Rec^- background and is therefore mediated by the recA genes. Growth of these clones in the absence of Cm causes the loss of the extra copies, yielding a plasmid with a single copy of pC194, indistingishable from pMR22/1. Interestingly, we have observed that deletions occur at high frequency in pC194, which drastically increase Cm^r in E. coli containing plasmids with a single copy of pC194. Two types of such deletions were detected: (a) large 1050 bp deletions covering about onethird of pC194 and (b) small 120–150 bp deletions (near the MspI site) in the region containing the replicative functions of pC194 (Horinouchi and Weisblum 1982). Both types of deletion render the recombinant plasmid unable to replicate in B. subtilis. pM22/1 replicates, although with a low copy-number, and is stable in B. subtilis wild type; the recA gene of E. coli does not complement any of the rec ^- mutations of B. subtilis. A strong instability, mainly of the E. coli and pBR322 sequences, was observed in many dna and rec mutants of B. subtilis yielding smaller plasmid with a much higher copy-number.     

Specific detection of an oil-degrading bacterium, Corynebacterium sp. IC10, in sand microcosms by PCR using species-specific primers based on 16S rRNA gene sequences

A species-specific PCR technique to detect an oil-degrading bacterium, Corynebacterium sp. IC10, released into sand microcosms is described. PCR primers, specific to strain IC10, were designed based on 16S rRNA gene sequences and tested against both closely and distantly related bacterial strains using four primer combinations involving two forward and two reverse primers. Two sets of them were specific to the strain IC10 and Corynebacterium variabilis and one set was selected for further analysis. The PCR amplification was able to detect 1 pg template DNA of strain IC10 and 1.2×10⁴ c.f.u. of IC10 ml wet sand^–1 in the presence of 3×10⁸ Escherichia coli cells. In non-sterile sand microcosms seeded with the strain IC10, the sensitivity of detection decreased to 9.6×10⁵ c.f.u. ml wet sand^–1. The detection sensitivity thus depends on the complexity of background heterogeneous DNA of environmental samples. The assay is suitable for detection of Corynebacterium sp. IC10 in laboratory microcosms, however, cross reaction with non-oil degrading coryneforms may prohibit its use in uncharacterized systems.     

Integrative transformation by homologous recombination in the zygomycete Mucor circinelloides

Summary Transformation of a Mucor circinelloides Leu^– strain with the plasmid pAD45, harbouring the wild-type allele (leuA⁺) and a chymosin gene, led to the identification of mitotically stable transformants after one to three vegetative growth cycles on non-selective medium. Southern analysis of the stable transformed strains demonstrated that the vector is integrated, as an intact molecule, into the resident Mucor leuA locus. Retransformation of Escherichia coli with genomic DNA restricted with enzymes having no or only a single recognition site within the inserted sequence did not permit isolation of plasmids or fragments carrying the leuA or chymosin gene.     

Role of lysogenizing phages in the spread of drug-resistance plasmids in a staphylococcal population

The frequency of the transduction of plasmids rms5, rms7, pT127, pC194, pS194 and pUB101 by phages belonging to serological group B (80, 52, 52A, 53, 85, phi 11, S2) in two systems was compared. In system 1 phages for transduction were obtained from plasmid-containing lysogenic donors in the process of induction with mitomycin C; in system 2 phages for transduction were obtained by their multiplication in plasmid-containing nonlysogenic donors. In system 1 the transduction of plasmids rms5, rms7, pT127, pS194 by phage 52A was found to occur with a greater (by 3-5 orders) frequency than in system 2 (the frequency of transduction was 10(-2) to 10(-4), and 10(-6) to 10(-8) respectively). A similar situation was observed with plasmids rms5 and rms7 and phage 52; plasmid pT127 and phage 53; but not observed with plasmids rms5 and rms7 and phages 80, phi 11 and S2; plasmids pC194 and pS194 and phage 53; plasmid pUB101 and phages 52A, 80 and phi 11; plasmids pC194, pS194 and pT127 and phage 85.     

Introduction of plasmid pC194 into Bacillus thuringiensis by protoplast transformation and plasmid transfer

The Staphylococcus aureus plasmid pC194 which codes for resistance to chloramphenicol was introduced into six Bacillus thuringiensis strains representing five varieties by protoplast transformation. Six other varieties could not be transformed. pC194 could be identified in transformed strains as autonomous plasmid. The transformed clones contained in addition a new extrachromosomal element of somewhat lower electrophoretic mobility hybridizing with pC194, and pC194 in multimeric forms. pC194 was also transferred from one B. thuringiensis variety to another and from Bacillus thuringiensis to Bacillus subtilis and vice versa by a conjugation-like process, requiring close cell-to-cell contact.Non-standard abbreviations BSA bovine serum albumin - CAT chloramphenicol acetyltransferase - Cm^R chloramphenicol resistant - PAB Penassay broth - SDS sodiumdodecylsulfate - Tc^R tetracycline resistant     

Molecular studies of FIme resistance plasmids, particularly in epidemic Salmonella typhimurium.

Summary The molecular sizes of F₁ me resistance plasmids from strains of Salmonella typhimurium, S. wien and S. typhi were within the range 87.9–102.6×10⁶ daltons. DNA reassociation studies indicated that the plasmids from these hosts had at least 80% of their nucleotide sequences in common. A high proportion of F₁ me plasmids cannot mediate their own transfer. The non-autotransferring property of such plasmids is the result of DNA deletion; a non-autotransferring F₁ me plasmid was about 10×10⁶ daltons shorter than autotransferring representatives of the group, and its DNA showed 100% homology with them. Plasmids of the F₁ me group are incompatible with the F factor and with F₁R factors. F₁ me plasmids are incompatible with the fi ⁺ MP10 plasmid of S. typhimurium, whereas F and F₁ factors are compatible with MP10 (Anderson et al., 1977). There was no significant DNA homology between members of the F₁ me group and MP10, and these plasmids may share only a small region of DNA responsible for their incompatibility. The F₁ me R factors examined had 29–37% DNA homology with the F factor, and 50–58% homology with the F₁ resistance plasmid, R162. Molecular examination therefore supports the genetic differentiation of members of the F₁ me group from other F-like plasmids. Both types of investigation can thus be used in epidemiological studies of bacterial strains carrying resistance or other plasmids.     

Ars region in TL-DNA on octopine type Ti plasmids

Summary In the TL-DNA region of the octopine type Ti plasmids, an ars region was assigned as the DNA segment conferring the replicational ability to YIp5 in Saccharomyces cerevisiae. T-DNA:YIp5 hybrid plasmids containing a particular T-DNA region could transform yeast cells at a frequency of 10³–10⁴ transformants per g plasmid DNA and they were rescued in Escherichia coli, although the transformed phenotype was mitotically unstable. The instability was inferred to be caused by segregation of the plasmids due to their low efficiency of replication. The ars region was mapped on the noncoding region between the coding regions corresponding to no. 5 and no. 7 mRNA, and its minimal length determined in this experiment was about 150 bp.Abbreviations Ti plasmid tumor inducing plasmid - T-DNA transferred DNA or tumor DNA - TL-DNA left T-DNA - ars autonomously replicating sequences