Gene Movement: Transformation and Conjugation
NATURAL TRANSFORMATIONFirst identified as phenomenon with Griffith experiment, using Streptococcus
pneumoniae (Fig. 11.15). While some types of bacteria are easily transformed in natural
environments, many bacteria are not naturally transformable (e.g., E. coli).
Steps common to all natural transformation systems- (1) development of
competence, (2) binding of DNA, (3) processing and uptake of DNA, and (4) integration
of DNA into the chromosome by recombination and expression.
Gram-positive transformation (Strep. pneumoniae and Bacillus; Fig. 11.16)competence, DNA binding, processing and uptake. dsDNA fragments are bound to cell
,generally about 10-15 kb in length. On external surface of cell, a nuclease converts
dsDNA to ssDNA before DNA entry into cell (before passage through the cytoplasmic
membrane). It is thought that the DNA passes through a transformation-specific
cytoplasmic membrane channel, similar to one seen in Gram negative organisms. No
specific DNA sequence that affects the ability of the DNA to bind to the cell surface prior
to transformation in Gram positive bacteria.
In Bacillus subtilis, the acquisition of competence is regulated by quorum sensing
mechanism at entry into stationary phase. The quorum sensing molecule is a small
peptide which is sensed in the environment by a 2-component regulatory system. The
activated response regulator induces expression of competence (com) genes.
Gram-negative transformation (Haemophilus influenzae,Neisseriae gonorrhoeae)dsDNA binds to membraneous transformasome structure forms, which can bind
sequences of up to 40 kb in length. Specific recognition sequences within the DNA are
required for DNA binding and uptake in at least some Gram negative bacteria. In
Haemophilius, this DNA binding sequence is 11bp in length. This specific DNA
sequence occurs approximately 1400 times in the Haemophilus chromosome (instead of
the 10 times predicted if present by random distribution in the sequence).
The linear dsDNA crosses the outer membrane and is converted to ssDNA as it
enters into the cytoplasm.
Artifical transformation
Requires treatment of cells with a variety of chemicals or with powerful electrical
pulse to cause DNA to enter the cell.
PLASMIDS & CONJUGATION (Reading assignment: 279-285; 301-308)
1. Plasmid biology: plasmids are extrachromosomal elements that can replicate
independently of the host cell chromosome (generally do not contain any essential
genes for the host cell’s survival)
plasmids are distinguished from one another in several ways, noted below.
-size: from 1 to >100 kb
-copy number: from 1 to >30/cell (size usually inversely proportional to copy #)
-host range: the species/genera of bacteria in which a plasmid can stably replicate
-incompatibilty group: that is, can two plasmids be stably maintained in same cell.
If 2 different plasmids can co-exist in the same cell, they are in different
incompatibility groups
-other traits: includes whether a plasmid is conjugal, carries antibiotic resistance
genes, confers phage sensitivity, encodes bacteriocin genes, etc (see Table 11.1)
2. Conjugation: Fig. 11.21; gene transfer from donor to recipient requiring cell to cell
contact which is mediated by the sex-pilus. Amount of genetic information
transferred can be significantly larger than in transformation or trandsduction.
During conjugation, generally a plasmid transferred to recipient. (Can also
mediate transfer of other non-conjugal (mobilizable) plasmids that may reside in same
cell)
Conjugation mechanism for F plasmid (and other Gram neg. plasmids)

initial contact mediated by conjugal pilus (Fig. 11.20)

establishment of mating pairs after retraction of pilus

conjugal DNA processing—plasmid nicked in site- and strand-specific
way at oriT (origin of transfer). Single strand of plasmid DNA transferred
in 5’ to 3’ direction to recipient cell, followed by re-ligation of nicked
DNA.

replacement DNA strand synthesis occurs in both the donor (leading
strand) and the recipient (lagging strand) cell, concurrently with DNA
transfer

after mating pair separate, both the donor and the recipient contain the
conjugal plasmid
3. Some plasmids can integrate into the chromosome of host cell via transposable
elements (either insertion sequences-IS- or transposons-Tn; Fig. 11.22). This can
happen at low but detectable frequency. If an F plasmid remains in the E. coli
chromosome, the cell is called an Hfr.
If the F plasmid inaccurately excises from the chromosome after formation of an Hfr,
it can take a portion of the chromosome with it, which then becomes part of the
plasmid itself. This form of the F plasmid is called an F’ (F prime).
4. Transfer of chromosomal DNA from a donor to a recipient can occur by transfer
mediated by an Hfr (where the chromosomal DNA transferred dependent on the site
of the F plasmid insertion into the chromosome; Fig. 11.23) or by an F’ (where only a
restricted amount of chromosome carried on the plasmid is transferred).