Autonomously replicating plasmids and chromosome rearrangement during transformation of Nectria haematococca.

A previously described, autonomously replicating plasmid was examined for its ability to replicate in the plant pathogenic fungus, Nectria haematococca (Nh). The plasmid, pFOLT4R4, replicates as a linear molecule, contains a subterminal inverted repeat, as well as the repeated hexanucleotide telomere consensus sequence, TTAGGG, at both ends, and increases frequency of fungal transformation approximately 100-fold compared to a similar integrative plasmid, pHRC. Transformation of Nh occurs by way of autonomous replication; the transformed, hygromycin B-resistant (HyR) phenotype is unstable without selection and in most cases pFOLT4R4 is maintained in the fungus, separate from chromosome-sized DNAs. Surprisingly, a non-autonomously replicating derivative of pFOLT4R4 (called pLD), lacking the subterminal inverted repeat and having the 5'-TTAGGG repeat in only one direction on the plasmid, transformed Nh at a rate as high as pFOLT4R4. Therefore, autonomous replication and high-frequency transformation are separable phenomena in Nh. In pLD transformants, plasmid sequences are integrated into chromosome-sized DNAs of Nh and these cultures generally have a stable HyR phenotype. Treatments involving ligation of Nh genomic DNA to pLD result in a lower frequency of transformation. In many cultures transformed with pLD plus genomic DNA, one wild-type chromosome-sized band is not visible, but another smaller chromosome-sized band is found. Mobility changes in some cases are consistent with deletions of over 1000 kb. Some HyS revertants of transformants appear to lack the entire chromosome into which integration had occurred. These results indicate that the Nh genome is extremely malleable and large portions may be non-essential for growth in culture.     

A cryptic plasmid from Shigella sonnei

pNZ500 is a 1.5 kb cryptic plasmid from a Shigella sonnei isolate. It was introduced into Escherichia coli by cotransformation, where it is maintained at about 30 copies per chromosome equivalent. Hybridization studies show that pNZ500 exhibits a high level of sequence similarity to other 1.5 kb plasmids found in different S. sonnei isolates but shares no homology with larger S. sonnei plasmids. pNZ500 shares a small degree of sequence homology with pBR322 and with pAC184. The homology with pBR322 is restricted to sequences close to the ori-bom region of this plasmid. Nevertheless, pNZ500 maintenance in E. coli is not dependent on DNA polymerase I activity, and does depend on continuing protein synthesis. pNZ500 encodes two polypeptide gene products whose monomer molecular weights are 24500 and 18000. The examination of host cells for the expression of possible plasmid phenotypes revealed no differences between cells bearing pNZ500 and plasmidless cells.     

Fate in soil of a recombinant plasmid carrying aDrosophila gene

A recombinant plasmid (C357; 3.5 Mdal) containing heterologous DNA (pBR322 [2.6 Mdal] with cDNA for an egg yolk protein fromDrosophila grimshawi) inEscherichia coli strain HB101 survived in and was recovered on selective media from sterile and nonsterile soil during 27 days at frequencies similar to those of theE. coli(pBR322) system. In sterile saline, the numbers of all cells decreased during 34 days, but the numbers of the plasmidless host declined less. There was no selective loss of the heterologous DNA in either soil or saline, as determined by colony hybridization with a³²P-labeled DNA probe for the cDNA, but the HB101(C357) appeared to be less able than HB101(pBR322) to cope with conditions of starvation. These results suggested that nonessential eucaryotic DNA inserted into plasmid DNA has little effect on the survival in soil or saline of the bacterial host and the maintenance of the vector.     

New yeast vectors containing the autonomously replicating sequences from Candida maltosa genome

Two different DNA sequences from the yeast Candida maltosa confer the ability to replicate autonomously to the yeast integrative vector pLD700 on which they are cloned. The recombinant plasmids pLD701 and pLD702 with autonomously replicating sequences (ARS) from Candida maltosa and LEU2 gene from Saccharomyces cerevisiae transform the auxotrophic strain S. cerevisiae DC5 with the efficiency 3-5 x 10(3) per microgram of DNA. Like other yeast vectors harbouring ARS, these plasmids are not stable in yeast cells. Restriction and hybridization analyses have revealed the pLD701 plasmid to contain ARS from chromosomal DNA of C. maltosa. Plasmid pLD701 appears to be a useful vector for yeast transformation.     

Inhibition of macrophage-mediated antigen presentation by hemolysin-producing Listeria monocytogenes

T lymphocytes and macrophages from Listeria-infected mice were used to evaluate the processing and presentation of live Listeria monocytogenes in vitro. Antigen presentation to T cells was quantitated by interleukin-2 production. In contrast to inert antigens such as heat-killed Listeria, live bacteria were processed and presented poorly. To evaluate the role of hemolysin (Hly), we used isogenic pairs of Hly+ and Hly- Listeria as antigens. In contrast to live Hly- bacteria, which were presented as well as heat-killed Listeria, live Hly+ bacteria were presented poorly. Hly+ bacteria also inhibited the presentation of heat-killed Listeria. This effect was apparent with as few as 10 bacteria/macrophage and was not due to loss of macrophage viability or decreased Ia expression after exposure to the live bacteria. With respect to murine listeriosis, the LD50 values for the Hly- strains were at least 1000 times higher than those for the Hly+ strains. These results suggest that the ability of Hly+ bacteria to inhibit antigen processing and presentation may be an important determining factor in Listeria infection and immunity.     

Bacillus licheniformis as an object for the propagaton of heterologous genetic material from bacilli

Bacillus licheniformis was transformed with plasmids pUB110 and pJJ10 (pUB110 - pBR322) isolated from Bac. subtilis and Escherichia coli, respectively. It was revealed that the structure and genetic properties of the plasmids did not change during the transformation process. pJJ101 (pJJ10-rib) DNA isolated from E. coli and containing helper pJJ10 plasmid was used, as a recipient. It was shown that pJJ101 rib markers were "rescued" by the resident plasmid during transformation of Bac. licheniformis (pJJ10). Plasmid pLP1 containing ribB, ribD, Kmr genes and the pUB110 replicator, was isolated from the transformants. pLP1 plasmid might be considered as a detected derivative of the parental pJJ101 plasmid. The deletion is presented by 3,9 MD segment that contains the pBR322 replicator. pLP1 DNA is capable of transforming plasmidless strains of Bac. licheniformis and Bac. subtilis.     

Acceptance and transfer of plasmid Rts1 by bacteria of the genus Erwinia]

The ability of 13 Erwinia strains to accept, to inherit and to transmit the Rts1 factor by conjugation was studied. 11 strains accepted the Rts1 factor from Escherichia coli K-12 CSH-2 with the frequency of about 10(-7)--10(-3). The Rts1 factor was genetically stable in the Erwinia cells and was not eliminated by acriflavine and under the temperature of 37 and 42 degrees C. All the R+ exconjugants were characterized with more high degree of the resistance of kanamycin than E. coli cells harbouring the same R factor. Erwinia strains harbouring the Rts1 plasmid transferred it by conjugation into homologic (Erwinia) and heterologic (E. coli) bacteria. The study of kinetics of the transfer of the Rts1 factor in different mating systems showed that the transfer of this plasmid from R+ Erwinia into R- Erwinia and R- E. coli--in the liquid medium. It is concluded that Erwinia can be the host and the donor of the Rts1 factor.     

Mathematical modeling of population dynamics of unstable plasmid-containing bacteria during continuous cultivation in a chemostat

A structural approach to studying the regularities of the population dynamics of unstable recombinant bacterial strains in a chemostat was elaborated. The approach is based on the mathematical modeling of cell distribution in a population with different numbers of plasmid copies. The effect of decreased selective preference of plasmidless variants of the recombinant strain in the chemostat, which is related to a decrease in the number of plasmid copies in cells upon long-term incubation was analyzed. It is shown that the time of half-elimination of plasmids from the bacterial population in the steady state in the chemostat T1/2 does not depend on the maximum number of plasmid copies in cells N but is determined only by the mean time of generation g and the probability of the loss of one plasmid copy tau. The dependence of the preference of bacterial plasmidless variants on the efficiency of expression of genes cloned into plasmids in chemostat was analyzed using the recombinant strain E. coli Z905, whose plasmids pPHL-7 contain cloned genes for the luminescence system of marine luminescing bacteria Photobacterium leiognathi.     

Escherichia coli growth and plasmid copy numbers in continuous cultivations

Summary AnEscherichia coli K-12 strain harbouring either the plasmid pBR322, or the recombinant plasmid pKTH1220, a 14 kb derivative of pBR322, or no plasmid was grown in a chemostat. The cultivations were continued for 300–400 bacterial generations.E. coli hosts harbouring pBR322 or no plasmid grew in a similar way, but the growth of the host containing the big recombinant plasmid was slower. The plasmid copy numbers increased up to 2–3 fold as the dilution rate was increased from 0 to ca. 1 h^–1. After this point the increase in dilution rate seemed to induce a rapid decrease in the plasmid copy numbers. High copy numbers could be maintained using dilution rates resulting in good productivity of the cell mass.     

Plasmid contents of lactic acid bacteria isolated from different types of sour doughs

The plasmid contents of 13 lactic acid bacteria isolated from different types of sour doughs were examined and compared with the plasmid contents of 11 culture collection strains and one commercial pure starter culture for sour doughs. In addition, plasmid analysis was used as a tool to study the stability of a starter culture during sour dough fermentation in a bakery.The tested strains varied in plasmid content from no plasmid up to six plasmids, with molecular weights from 1.5 to 43 MDal. In most cases, the wild-type strains contained a higher number of plasmids than the culture collection strains. The ability of the strains to ferment different carbohydrates was also investigated, but no obvious correlations between the fermentation patterns and the plasmid patterns could be observed. During the fermentation of the bakery sour dough, strains other than the inoculated starter culture gradually became dominant in the microflora. These new strains contained 1–3 plasmids, contrary to the plasmidless starter culture, and they also fermented more carbohydrates than the starter culture.     

Selective activation of human intestinal mast cells by Escherichia coli hemolysin

Mast cells (MCs) are recognized to play an important role in bacterial host defense in the murine system. In this study, we studied the interaction of human MCs, isolated from the intestine and purified to homogeneity, with different Escherichia coli and Shigella flexneri strains. We show that alpha-hemolysin (Hly)-producing E. coli strains induce the release of histamine, leukotrienes, and proinflammatory cytokines in intestinal MCs. In contrast, MCs were virtually unresponsive to S. flexneri and several Hly-negative E. coli strains, including the isogenic Hly-deficient mutants of Hly(+) strains. Hly(+) E. coli but not Hly(-) E. coli caused an increase in intracellular Ca(2+) levels. Blocking of extracellular Ca(2+) and of the calmodulin/calcineurin pathway by cyclosporin A inhibited the response to Hly(+) E. coli. Furthermore, inhibition of MAPKs p38 and ERK reduces activation of MCs by Hly(+) E. coli. In addition, using an ex vivo system, we directly record the histamine release by MCs located in the lamina propria after infection with Hly(+) E. coli. Our data indicate that human intestinal mast cells interact with selected Gram-negative bacteria, establish E. coli Hly as a factor regulating MC effector functions, and argue further for a role of human MCs in innate immunity.     

Cytosine-specific DNA modification interferes with plasmid establishment in Escherichia coli K12: involvement of rglB

Summary Several chimeric pBR322/328 derivatives containing genes for cytosine-specific DNA methyltransferases (Mtases) can be transformed into the Escherichia coli K12/E. coli B hybrid strains HB101 and RR1 but not into other commonly used E. coli K12 strains. In vitro methylation of cytosine residues in pBR328 and other unrelated plasmids also reduces their potential to transform such methylation sensitive strains, albeit to a lesser degree than observed with plasmids containing Mtase genes. The extent of reduced transformability depends on the target specificity of the enzyme used for in vitro modification. The role of a host function in the discrimination against methylated plasmids was verified by the isolation of K12 mutants which tolerate cytosine methylated DNA. The mutations map in the vicinity of the serB locus. This and other data indicate that the host rglB function is involved in the discrimination against modified DNA.     

Plasmidless, photosynthetically incompetent mutants of Rhodospirillum rubrum

Ethyl methanesulfonate rendered a high percentage of Rhodospirillum rubrum cells plasmidless and photosynthetically incompetent (Kuhl et al., J. Bacteriol. 156:737-742, 1983). By probing restriction endonuclease-digested chromosomal DNA from these plasmidless strains with 32P-labeled R. rubrum plasmid DNA, we showed that no homology exists between the plasmid and the chromosomal DNA of the mutant. Loss of the plasmid in all the nonphotosynthetic isolates was accompanied by the synthesis of spirilloxanthin under aerobic growth conditions, resistance to cycloserine and HgCl2, and loss of ability to grow fermentatively on fructose. Changes in both the protein and lipid composition of the membranes and the impaired uptake of 203HgCl2 in the plasmidless strains (compared with the wild type) suggest either that membrane modification occurs as a result of plasmid loss, accounting for several of the acquired phenotype characteristics of the cured strains, or that both membrane modification and plasmid loss are part of the same pleiotropic mutation.     

Cloning of the adenylate cyclase gene of Escherichia coli K-12

The adenylate cyclase gene of Escherichia coli has been cloned on the plasmid vector pBR325. The hybrid plasmid pTH4 obtained has a molecular weight of 6,4 megadalton and represents pBR325 plasmid with the insertion of 2,8 megadalton in the Pst1 site. The cya mutant bacteria carrying pTH4 recover their ability to utilize mannitol, lactose and other carbohydrates as carbon sources, and lose this ability again in the case of rare spontaneous excision of the DNA insert from the Pst1 site. The phenotypical effect of pTH4 in cya mutants can be only seen in the crp+ genome. The strains carrying pTH4 are also characterized by the ability of beta-galactosidase induction under conditions of catabolite repression. Besides, the bacteria containing cya+ allele on the plasmid do not grow on glycerol, which seems to be caused by toxic concentrations of methylglyoxal formed as a result of the increased intracellular level of cyclic adenosine monophosphate.     

Multiple genes coding for octopine-degrading enzymes in Agrobacterium.

Most biotype 2 strains of Agrobacterium tumefaciens and A. radiobacter which utilize nopaline also degrade octopine. In all such strains studied, the ability to degrade octopine did not appear to be transferred to plasmidless recipient cells under conditions of plasmid transfer in which the ability to utilize nopaline was transferred. An octopine-degrading mutant was isolated in a strain cured of its plasmid, suggesting that genes of octopine degradation may have a chromosomal location in some strains. In strains in which octopine utilization is coded by plasmid genes, octopine degradation was always inducible, whereas in strains which degrade both octopine and nopaline, octopine utilization was constitutive although nopaline degradation was inducible. When plasmids coding for octopine-utilizing ability were transformed into a strain containing either a nopaline- or null-type plasmid, transformants able to degrade octopine were either not observed or were unstable upon purification. All of these data suggest that plasmids associated with virulence are incompatible with one another, and therefore imply that the major groups of plasmids associated with virulence have a common origin.     

Lethal and mutagenic effects of 8-methoxypsoralen-induced lesions on plasmid DNA

The genotoxic effect of 8-methoxypsoralen damages (monoadducts and crosslinks) on plasmid DNA was studied. pBR322 DNA was treated with several concentrations of 8-methoxypsoralen plus fixed UVA light irradiation. After transformation into E. coli cells with different repair capacities (uvrA, recA and wild-type), plasmid survival and mutagenesis in ampicillin- and tetracycline-resistant genes were analysed. Results showed that crosslinks were extremely lethal in all 3 strains; indeed, it seemed that they were not repaired even in proficient bacteria. Monoadducts were also found to be lethal although they were removed to some extent by the excision-repair pathway (uvrA-dependent). Damaged plasmid DNA appeared to induce mutagenic repair, but only in the wild-type strain. In order to study the influence of the SOS response on plasmid recovery, preirradiation of the host cells was also performed. Preirradiation of the uvrA or wild-type strains significantly increased plasmid recovery. Consistent with the expectations of SOS repair, no effect was observed in preirradiated recA cells. Plasmid recovery in the excision-deficient strain was mainly achieved by the mutagenic repair of some fraction of the lesions, probably monoadducts. The greatest increase in plasmid recovery was found in the wild-type strain. This likely involved the repair of monoadducts and some fraction of the crosslinks. We conclude that repair in preirradiated repair-proficient cells is carried out mainly by an error-free pathway, suggesting enhancement of the excision repair promoted by the induction of SOS functions.     

Design of a species-specific DNA probe based on the pFra plasmid of the plague pathogen]

The recombinant plasmid pBS1 carrying a 2 kb SalGI fragment of Yersinia pestis pFra plasmid was constructed by insertion of the fragment into a vector plasmid pBR327. SalGI-BspRI 400 bp subfragment was recloned into a pBR322 vector plasmid. Open reading frame was found in the fragment by DNA sequencing technique. The subfragment designated F1-probe permits one to identify specifically the Yersinia pestis strains harbouring pFra plasmid, thus, differing them from closely related Yersiniea and other representatives of Enterobacteriaceae family.     

Helper plasmid cloning in Streptococcus sanguis: cloning of a tetracycline resistance determinant from the Streptococcus mutans chromosome

A model system for testing the helper plasmid cloning system of Gryczan et al. (Mol. Gen. Genet. 177:459-467, 1980) was devised for the Streptococcus sanguis (Challis) host-vector system. In this system, linearized pVA736 plasmid efficiently transformed an S. sanguis (Challis) host containing a homologous plasmid, pVA380-1, but did not transform a plasmidless host or a host containing a nonhomologous plasmid, pVA380. In addition, whereas monomeric circular pVA736 transformed a plasmidless host with two-hit kinetics, it transformed a pVA380-1-containing host with one-hit kinetics. This helper plasmid cloning system was used to isolate two HindIII fragments (5.0 megadaltons [Mdal] and 1.9 Mdal in size) from the chromosome of Streptococcus mutans V825 which conferred high-level tetracycline resistance. One tetracycline-resistant clone was examined and found to contain three plasmids which were sized and designated pVA868 (9.0 Mdal), pVA869 (9.5 Mdal), and pVA870 (9.8 Mdal). Results of Southern blot hybridization and restriction endonuclease digestion confirmed that all three chimeras were composed of two HindIII fragments of the S. mutans V825 chromosome, as well as a large portion, varying in size for each chimera, of the 2.8 Mdal cloning vector, pVA380-1. Incompatibility observed between pVA380-1 and each of the chimeras indicated that replication of the chimeras was governed by the pVA380-1 replicative origin. Southern blotting experiments revealed that the chimeras hybridized to Tn916, providing the first evidence that transposon-related genes of enteric streptococcal origin are disseminated among oral streptococci.     

Bacteriophages F0lac h, SR, SF: phages which adsorb to pili encoded by plasmids of the S-complex

Phage F0lac is an RNA-containing phage which plates only on strains carrying the plasmid EDP208, a pilus derepressed derivative of the unique incompatibility plasmid F0lac. A host range mutant, phage F0lac h, was selected which plated on strains carrying the ungrouped plasmid pPLS::Tn5 and lysed strains carrying another ungrouped plasmid TP224::Tn10 or the Com9 plasmid R71. An RNA-containing phage, SR, was isolated from sewage on bacteria harbouring plasmid pPLS::Tn5. It was antigenically distinct from the above two phages but had the same host range as phage F0lac h. Phages F0lac h and SR adsorbed unevenly to the shafts of the conjugative pili. Another phage, SF, was filamentous and plated or propagated on strains carrying any of the above plasmids as well as on strains harbouring IncD or F-complex plasmids. Plasmids TP224::Tn10 and pPLS::Tn5 were compatible with representative plasmids of all Inc groups also encoding thick flexible pili. The four plasmids EDP208, R71, TP224::Tn10 and pPLS::Tn5 were compatible with one another except for the reaction of TP224::Tn10 in the presence of pPLS::Tn5 which was slightly ambiguous. The host ranges of the bacteriophages, together with the serological relatedness of the thick flexible pili determined by these four compatible plasmids, suggested that they constitute a new complex, here designated S.     

Introduction of protein or DNA delivered via recombinant Salmonella typhimurium into the major histocompatibility complex class I presentation pathway of macrophages

Recombinant (r) Salmonella typhimurium aroA strains which display the hen egg ovalbumin OVA(257-264) peptide SIINFEKL in secreted form were constructed. In addition, attenuated rS. typhimurium pcDNA-OVA constructs harbouring a eukaryotic expression plasmid encoding complete OVA were used to introduce the immunodominant OVA(257-264) epitope into the major histocompatibility complex (MHC) class I presentation pathway. Both modes of antigen delivery (DNA and protein) by Salmonella vaccine carriers stimulated OVA(257-264)-specific CD8 T-cell hybridomas. An in vitro infection system was established that allowed both rSalmonella carrier devices to facilitate MHC class I delivery of OVA(257-264) by coexpression of listeriolysin (Hly) or by coinfection with rS. typhimurium Hlys (Hess J., Gentschev I., Miko D., Welzel M., Ladel C., Goebel W., Kaufmann S.H.E., Proc. Natl. Acad. Sci. USA 93 (1996) 1458-1463). Coexpression of Hly and coinfection with rS. typhimurium Hlys slightly improved MHC class I processing of OVA. Our data provide further evidence for the feasibility of attenuated, Hly-expressing rS. typhimurium carriers secreting heterologous antigens or harbouring heterologous DNA as effective vaccines for stimulating CD8 T cells in addition to CD4 T cells.