Walter R. Guild

Destruction of low efficiency markers is a slow process occurring at a heteroduplex stage of transformation.

SummaryDirect evidence is presented that the mechanism which discriminates against low efficiency markers in transformation of Diplococcus pneumoniae of genotype hex+ acts on them after the formation of donor-recipient heteroduplexes. This conclusion is based on assays of the transforming activity of donor markers in lysates made after various times of incubation of recipient cells following exposure to DNA. The activity of a low efficiency marker rises substantially, indicating formation of native-like heteroduplex structures, and then falls. At 37° C the process is essentially completed 10 minutes after entry, and the apparent half life of a susceptible heteroduplex is 1.5 to 2 minutes. Data from these and other experiments imply that about as many of the surviving low efficiency markers have simply escaped attack as have been inserted into both strands by the excision-repair process suggested by Ephrussi-Taylor.

DNase-resistant transfer of chromosomal cat and tet insertions by filter mating in pneumococcus

Abstract Genes for chloramphenicol resistance (Cmr) and tetracycline resistance (Tcr), which are present as heterologous insertions in the chromosomes of some clinical isolates of Streptococcus pneumoniae (pneumococcus) and derivative strains, were transferred at a low frequency to other pneumococci by a DNase-resistant filter mating process that resembles conjugation. Cotransfer of Cmr and Tcr was the most common event. Tetracycline resistance was transferred alone from one Tcr strain or rarely from CmrTcr donors, whereas Cmr was never transferred alone. Neither the donor strains nor the transconjugants contained detectable plasmids. Transconjugants acted as donors for transformation and for filter mating and had properties similar to those of the parent strain. The presence of the conjugative plasmid pIP501 in the donor did not appear to influence the transfer properties of the Cmr or Tcr determinants. No transfer of Cmr or Tcr toStreptococcus faecalis JH2-2 was observed.

Monomer plasmid DNA transforms Streptococcus pneumoniae

SummaryThe covalently closed (CC) monomer form of plasmid pMV158 was found to transform pneumococcus (Streptococcus pneumoniae) and to do so with two-hit kinetics. The evidence came from analysis of the behavior of the transforming activity in fractions from preparative gel electrophoresis. Activity in the first major peak to elute (i) co-eluted with monomer CC as detected on analytical gels, (ii) banded as CC in dye-buoyancy gradients, (iii) sedimented with the velocity expected for monomer CC, and (iv) gave two-hit kinetics as functions of both concentration and time of exposure of the cells to DNA. A second major peak of activity behaved physicall as though mostly due to dimer CC forms and gave single-hit response curves. Because almost no dimer was detectable optically on analytical gels of starting preparations, its specific activity was high relative to that of the monomers.

Breakage prior to entry of donor DNA in Pneumococcus transformation

Abstract Products of the binding and uptake of high molecular weight [ 3 H]DNA by competent cells of Diplococcus pneumoniae were examined. Bound DNA, still sensitive to DNAase, could be released by NaOH or by guanidine · HCl and then examined. Double-strand breaks were found in it. The strand length distribution of this DNA was not distinguishable from that of the DNA found inside the cells at the same time, and was that expected for insertion of new breaks outside the cell at a median spacing of 2.8 · 10 6 daltons/strand. Conversion of donor label to a DNAase-resistant state was accompanied by production of an equal weight of acid-soluble donor fragments in the extracellular medium. These results suggest that double-strand breaks precede entry, that the strand segments thus produced enter without further breakage, and that the complement of each entering segment probably is degraded with release of the products outside the cell.

Transfer of plasmids by conjugation in Streptococcus pneumoniae

Abstract Transfer of resistance plasmids occurred by conjugation in Streptococcus pneumoniae (pneumococcus) similarly to the process in other streptococcal groups. The 20-megadalton plasmid pIP501 mediated its own DNase-resistant transfer by filter mating and mobilized the 3.6-megadalton non-self-transmissible pMV158. Pneumococcal strains acted as donors or as recipients for intraspecies transfers and for interspecific transfers with Streptococcus faecalis . Transconjugants contained the plasmids expected from their phenotypes and acted as donors for further transfers. Deficiency in an endonuclease essential for entry of transforming DNA did not affect the frequency of transfer. Transfer-deficient mutants of pIP501 have been found.

Properties and Transforming Activities of Two Plasmids in Streptococcus pneumoniae

SummaryTwo plasmids from group B streptococcus were introduced into pneumococcus (Streptococcus pneumoniae) and examined for copy number, stability, and some features of the process by which they transform pneumococcal recipients. The 3.6 Mdal pMV158 (tet) was present at a minimum of 12 to 16 copies per chromosome and was never observed to be cured. The 20 Mdal pIP501 (cat erm) had a minimum copy number of 3 to 4 per chromosome and was lost spontaneously at a frequency near 0.03 per division. The presence of novobiocin increased this frequency 2 to 3-fold. Competence for chromosomal transformation and the membrane endonuclease needed for normal DNA entry were required for plasmid transformation. Plasmid transformants segregated transformed cells one generation ahead of chromosomal transformants. Both single and multiple hit components of the transformation reaction kinetics were observed, but the latter could not be seen in the presence of competing chromosomal DNA. The majority of the transforming activity behaved as covalently closed circular DNA in dye-buoyancy gradients. Although most of the activity for both plasmids sedimented in sucrose gradients more rapidly than did monomeric closed circular DNA, a significant fraction was found at a position suggesting that it may have been due to monomeric plasmids.

Irreversible effects of formaldehyde on DNA

Abstract We find that at least one reaction of formaldehyde with DNA occurs at 100°, pH 8, which is effectively irreversible at 20–37° and is only partially reversible on further heating at 100° for 20 min after dialysis. This is in addition to the well known reversible binding of formaldehyde to amino groups. Radioactive binding measurements show that 3 to 5 moles formaldehyde per 100 moles nucleotide remains with the DNA after exhaustive dialysis. This amount of formaldehyde is distributed widely enough through the DNA to destroy transforming activity, in a manner not reversible by renaturing conditions, and to affect the buoyant density patterns of denatured and annealed DNA in CsCl.

Intracellular competition for a mismatch recognition system and marker-specific rescue of transforming DNA from inactivation by ultraviolet irradiation

SummaryWe show that donor-recipient heterozygosity for unlinked genes allows low efficiency markers in transforming DNA to escape from attack by the discrimination system present in Diplococcus pneumoniae of genotype hex+. The observed efficiency of a marker is a function of donor DNA concentration, duration of exposure, and relatedness of the donor to the recipient strain. Efficiency can be increased at least six to seven fold by addition of excess homologous but nonisogenic DNA, which appears to compete for a limited amount of an enzyme system recognizing mismatched heteroduplexes. Low efficiency markers are often hypersensitive to ultraviolet irradiation of the donor DNA, and this effect also is reversed by the presence of excess nonisogenic homologous DNA, leading to a dramatic (for example, 260-fold) marker-specific rescue from inactivation.

The Pathway of Plasmid Transformation in Pneumococcus

Plasmids transform Streptococcus pneumoniae by a process involving low efficiency assembly of replicons from fragments of single strands that have entered the cell separately. Transformation of preexisting replicons is much more efficient. We have cloned the erm gene of pIP501 into pMV158, which so far as we know is the first example of cloning in a pneumococcus host-vector system.