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Notani and Setlow (1974) have described the mechanism of bacterial transformation. Moreover, in S. pneumoniae the competent state is transient and persists only for a short period. The competent state is induced by the competence activator protein of molecular weight of 1,000 Dalton.
It binds to the plasma membrane of receptor and triggers the synthesis of 10 new proteins within 10 minutes. The competence factor (CF) accelerates the process of transport or leakage of autolysin molecules into the periplasmic space. Moreover, in H. influenzae no competence factors have been reported.
Only Changes in cell envelope accompany the development of competence state. The cell envelope of competent cells contains increased level of polysacccharide as compared to the cells of log phase.
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Structural changes in competent cells induce numerous vesicles called transformosome buds on the surface that contains protein and mediates the uptake of transforming DNA. Transformation is accomplished in the following steps (Fig.8.4).
Fig. 8.4 : Diagrammatic presentation of transformation in streptococci.
(a) DNA binding:
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As a result of random collision, DNA comes first in the contact of cell surface of competent bacteria (Figs. 8.4 and 8.5 A-B). First the DNA binding is reversible and lasts for about 4-5 seconds. Thereafter, it becomes irreversible permanently. For about 2 minutes it remains in non-transforming state. Thereafter, before 5 minutes it is converted into the transforming state.
The period (about 10 minutes) during which no transformation occurs in competent recipient cells is called eclipse. Both types of DNA, transforming and non-transforming, bind to the cell surface where the receptor sites are located. In B. subtilis membrane vesicles in competent cells are found that bind to 20 mg of dsDNA/mg of membrane protein. The competent cells show six fold more DNA binding sites than the non-competent cells.
In H influenzae transformosome bud forms the surface and contains proteins that mediate DNA uptake. It binds with conserved sequence (5’AAGTGCGGTCA 3′) present at 4 kb interval on DNA. The DNA uptake site contains two proteins of 28 and 52 kilo-Daltons. After binding, the receptor proteins present the donor DNA to the membrane associated uptake sites.
In S. pneumoniae the CF induces the ability to bind DNA molecules.
(b) Penetration:
The DNA molecules that bind permanently enter the competent recipient cells. DNA is also resistant to DNase degradation. The nucleolytic enzymes located at the surface of competent recipient cells act upon the donor DNA molecule when it binds the cell membrane.
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The endonuclease-1 of the recipient cells which is associated with cell membrane acts as DNA translocase by attacking and degrading one strand of the dsDNA. Consequently only complementary single strand of DNA enters into the recipient cells (Figs. 8.4 and 8.5A).
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It has been confirmed by performing the experiments with radiolabelling of donor DNA. The mutant cells of S. pneumoniae lack endonuclease – 1, therefore, transformation does not occur. Interestingly in B. subtilis degradation of one strand is being delayed. Hence, both the strands enter the recipient cell. The upper limit of peneterating DNA into the recipient cell is about 750 base pairs.
The size of donor DNA affects transformation. Successful transformation occurs with the donor DN.A of molecular weight between 30,00,000 and 8 million Dalton. With increasing the concentration of donor DNA the number of competent cells increases. DNA uptake process is the energy requiring mechanism because it can be inhibited by the energy requiring inhibitors.
After penetration the donor DNA migrates from periphery of cell to the bacterial DNA. This movement in different bacteria differs. For example, in B. subtilis this movement occurs for about 16-60 minutes. During this movement, DNA is associated with mesosomes which possibly transport it to the bacterial DNA.
(c) Synapsis formation:
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The single stranded DNA is coated with SSB proteins, which maintain, the single stranded region in a replication fork (Fig. 8.5B). The single strand of the donor DNA or portion of it is linearly inserted into the recipient DNA (Fig. 8.5 C-D). The bacterial protein like E. coli RecA protein probably facilitates the DNA pairing during recombination. It causes the local unwinding of dsDNA of the recipient cell from the 5′ end.
How the displaced single strand is cut, still not known? Base pairing i.e. synapsis occurs between the homologous donor ssDNA and the recipient DNA. Unwinding of the recipient DNA continues at the end of assimilated DNA and allows the fraction of invading DNA to increase base pairs. This process is called branch migration (F).
(d) Integration:
The endonuclease cuts the unpaired free end of donor DNA or the recipient DNA. This process is called trimming (Fig. 8.5E-F). The nick is sealed by DNA ligase (G). Consequently, a heteroduplex region containing a mismatched base pairs is formed (H). Furthermore, in the progenies whether the donor marker is or is not recovered, depends on the occurrence of mismatch repair.
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If the mismatch repair occurs again, it depends whether the unpaired base in the donor or recipient strand is removed. After replication the heteroduplex forms the homo-duplexes, one of these is of normal type and the second is transformed duplex. The normal duplex is from the recipient cell in origin, whereas the transformed duplex is from the donor genome.
The efficiency of integration of genetic markers into the genome of recipient cell varies with different genes that the recipient cell possesses. This genetic trait is called hex (high efficiency of integration). The hex system eliminates a large fraction of low efficiency (LE) markers and permits high efficiency (HE) markers to be integrated. Therefore, the hex function is a mismatch-base correction system.
The donor genes differing from the recipient genes by a single base pair create a mismatch when integrated initially. The hex mismatch repair system (with LE markers) can correct either of donor strands. Therefore, there is fifty-fifty chance for a given marker to be retained. The HE markers correct only the recipient strand.
For the LE markers, hex mismatch repair system unusually removes the mismatched bases of the donor DNA and the cell retains the recipient genotype, whereas for HE markers the same system removes the mismatched bases of recipient DNA and the cell consists of donor genotype.
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In the later case, after replication of chromosome and cell division the one progeny cell contains the donor genotype and the other has the recipient genotype. These two types of cells can be differentiated through plating method by using the antibiotic markers.
However, for pneumococci it is a general feature that all the strains discriminate between LH and HE markers when transformation has occurred with homologous DNA. The hex– cells (mutant in hex function) fail to discriminate between the two markers and, therefore, integrate all markers with high efficiency, because one of the two daughter cells after cell division contains the genotype.
