Genetic exchange Mutations Genetic exchange: three mechanisms Transposons

Mutations and Adaptation In the course of DNA replication mutations can arise in the bacterial genome. These can be point mutations in which one base is substituted by another. Point mutations in a coding sequence can lead to, no change in the protein (silent mutation), a change in an amino acid or the conversion of an amino acid coden to a STOP codon. Deletion and insertion and insertion mutations normally have a deleterious effect. In summary point mutations can only be used to ”tinker” with existing genes and are not a viable mechanism for the aquisition of new genetic properties. In a given population of bacteria there will always be a certain level of mutants and these will only dominate if they can grow more quickly than the wild type. Point mutations and effect Insertions deletions indels effect Selection in a population

Genetic Exchange There are three different natural processes by which bacteria can gain new genetic material (DNA). Transformation in which DNA is taken up from the environment Conjugation in which a plasmid is transferred from one bacteria to another. Transduction in which the transfer of DNA from one bacteria to another is mediated by a bacteriophage.

Demonstration of transformation Transformation of avirulent Streptococcus pneumoniae to a virulent type. Avery, MacLeod and McCarty 1944

Demonstration of transformation Demonstration that the transforming factor is DNA.

Natural transformation competence Many different bacteria are naturally competent. Some are competent all the time, others only at specific stages in the bacteria’s growth phase. Examples are, Streptococcus pneumonia, Bacillus subtilis, Hemophilus influensa, Neisseria gonorrhoeae. Many other bacteria have been shown to contain the genes for natural competence but have never been observed to do so. The molecular mechanism of transformation has been studied in some detail in a few species. The subtrate is double stranded linear DNA but only one of the DNA strands enters the cell. Recombination of this single stranded DNA into the bacterial chromosome is necessary for expression. Normally only DNA from a closely related species can be taken up by transformation and integrated into the chromosome. Sketch on the blackboard of cell wall structures,

Details of homologous recombination

What are the consequences of transformation Existing genes can be extensively modified by exploiting existing variation within a population. It has been shown that this can be important for proteins associated with host interactions in pathogenic bacteria. Target genes for antibiotics can be quickly modified and antibiotic resistence can be established.

Artificial competence All bacteria can be treated (chemically / electroporation) such that they become competent and can take up DNA. The substrate here is normally a plasmid which can replicate in the bacteria cytoplasm. This is one of the corner stones of recombinant DNA technology. If linear DNA is artificially transformed into a bacteria then it must be incorporated into the bacterial chromosome by double recombination in order to be expressed. Note that we are now speaking about double stranded DNA.

Types of homologous recombination in bacteria

Double recombination Introduction of a mutation into a bacterial chromosome from a piece of DNA acquired by transformation. This is the basis for gene knock out.

Gene replacement Introduction of a new gene into a bacterial chromosome by transformation. General recombination occurs between homologous sequences that flank the gene.

Plasmids in general Some plasmids cannot be transferred by conjugation. Some plasmids cannot be transferred by conjugation but they can be helped to transfer if a conjugative plasmid is also present in the cell. Some plasmids contain all the necesary genes to mediate their transfer to another bacteria by conjugation. Some conjugative plasmids have a narrow host range , others are broad range.

Plasmids of pathogenic bacteria Genome maps of some plasmids found in pathogenic bacteria. (A) A widely distributed R plasmid, RK2. (B) Plasmid pCG86 of pathogenic E. coli. The gene product or function of the genes is indicated. β-Lactam antibiotics include ampicillin.

Ti plasmid Genome map of the Ti plasmid of Agrobacterium tumefaciens, showing the gene product or function of the genes. Phytohormones are responsible for the induction of plant tumors.

Genetic Exchange There are three different natural processes by which bacteria can gain new genetic material (DNA). Transformation in which DNA is taken up from the environment Conjugation in which a plasmid is transferred from one bacteria to another. Transduction in which the transfer of DNA from one bacteria to another is mediated by a bacteriophage.

Bacterial conjugation Transfer of F plasmid from donor to recipient cells by conjugation. Once transfer is complete, both cells have an intact copy of F plasmid and can act as donors. The F plasmid is large, ~100 kb and contains about 100 genes.

High-frequency recombinant cells

High-frequency recombinant cells Transfer of chromosomal genes into a recipient bacterium. Orientation of the inserted F plasmid in the opposite direction from that shown here would allow early transfer of genes e, b, and c and later transfer of d and a. Relative locations of genes in the bacterial chromosome can be mapped by mixing donor and recipient cells, interrupting the mating at various times, growing the cells on appropriate media, and identifying the transferred genes.

F' cells Formation of an F' cell from an Hfr cell, and transfer of a bacterial chromosome segment to a recipient cell.

Consequences of conjugation A bacteria cell can get many new genes and in turn new genetic properties when it gets a new plasmid. In some cases these can be incorporated into the genome by recombination and they thus become part of the genome (plasmids can be lost). Plasmids play an important roll in the transfer of antibiotic resistence between bacteria. A deadly combination if they are pathogenic.

Bacteriophage Lets take a general look at bacteriophages before we look at the roll of bacteriophages in genetic exchange.

Complex virus Graphic representation of T4 virus (phage).

Viral reproduction: the lytic cycle Generalized schematic for viral reproduction in a host bacterium, through the lytic cycle. In the lytic cycle, the virus (phage) multiplies in the host cell and the progeny viruses are released by lysis of cell.

Viral reproduction: the lysogenic cycle Generalized schematic for viral reproduction in a host bacterium, through the lysogenic cycle. In the lysogenic cycle, viral DNA is integrated into the host genome and replicates as the chromosome replicates, producing lysogenic progeny cells

Genetic Exchange There are three different natural processes by which bacteria can gain new genetic material (DNA). Transformation in which DNA is taken up from the environment Conjugation in which a plasmid is transferred from one bacteria to another. Transduction in which the transfer of DNA from one bacteria to another is mediated by a bacteriophage.

Generalized transduction: Lytic phage

Specialized transduction: Lysogenic phage

Consequences of transduction Specialized transduction can only transfer genes that flank the specific insertion site and as such do not contribute many new genes to the bacteria. Generalized transduction can be instrumental in the transfer of 50-100 new genes and make dramatic changes to the properties of the bacteria. Transduction plays an important roll in the transfer of, antibiotic resistence and pathogenicity factors. This can be a deadly combination.

Insertion elements and transposons Insertion sequences (IS) are short DNA sequences, about 700 to 5000 bp which can move from one location in a DNA sequence to another. They have short 16-41 bp inverted repeats on their ends. They encode a transposase which catalyses site-specific recombination. Simple transposons are mobile genetic elements in which a one or more genes are flanked by two insertion sequences.

Composite transposons Structures of some bacterial transposable elements. A composite transposon contains antibiotic genes flanked by two insertion sequences as direct or inverted repeats Shown here is the Tn5 transposon, with inverted repeats. The Tn3 transposon.