Muth

• process of transporting free DNA into bacterial cells

• use indiscriminate DNA as food

• Use specific DNA to repair damaged genomes

• Acquire new genes through horizontal gene transfer

Induced by quorum sensing

• the transfer of DNA from one bacterium to another following cell to cell contact

• Typically (only in gram-) initiated by a special pilus production from the donor cell

• Recquires the presence of special transferable (“conjugative”) plasmids (e.g. F Plasmid): these usually contain all genes needed for the pilus formation & DNA transport

Using the relaxase as a pilot protein, a specialiced Type IV protein sectreion system (T4SS) secretes the relaxase with the covalently linkes single stranded plasmid molecule to the recipient

• The F-factor can accidentally integrate intot he chromosome

• The cell is now designated Hfr (high frequency recombination)

• An Hfr cell is capable of transferring chromosome parts into a recipient cell

• This process can be used to map genes

• Agrobacterium tunefaciens causes crown gall disease: contains a tumor inducing plasmid that transfers T-DNA via conjugation to plant cells

• Neisseria gonnorhoeaea contains a 685 bp fragment, identical to human L1 element

The process in which bacteriophages carry host DNA from one cell to another

-> occurs accidentally as an offshoot of the phages life cycle

1. Adsoption to phage receptor (cell surface)

2. Injection of phage DNA

3. Replication of phage DNA, synthesis pf phage proteins

4. Packaging of phage DNA, by mistake packaging of chromosomal DNA

5. Release of phages

6. Infection of new recipient cells

7. Injections of (chromosomal) DNA

8. Integration of donor DNA into the recipient genome by homologous recombination

Principle: Inadvertently packaging chromosomal DNA instead of phage DNA

-> each gene marker is transduced with the same frequence

1. Adsorption to phage receptor (cell surface)

2. Injection of phage DNA

3. Lysogenic life cycle, integration of the prophage

4. Spontaneous induction of lytic cycle, inadvernently excisin of the prophage (including the neighboring chromosomal DNA fragment)

5. Packaging of phage DNA, release of phages, infection of new recipient cells

6. Injection of DNA

7. Integration of donor DNA into the recipient genome by homologous recombination

Principle: Inadvertenly excision of the prophage, phage DNA plus a piece of chromosomal DNA (next to the prophage integration site) are packed

-> only certain genes are transduced

• enzymatic cleavage of alien DNA by restriction endonucelases

• There are three main types of restriction endonucleases: type II possesses only endoculease activity & generally recognizes palindromic DNA sequences and cleaves at those sites

• Host DNA is protected by methylation

CRISPR = clustered regularly interspaced short palindromic repeats

• CRISPR consits of repeats & spacers that are non-coding but near them lie CRISPR-associated gene families that do encode proteins (CAS)

• CRISPR is a primitive microbial immunne system: an organism that manages to survive a phage attack captures a piece of the invaders genome & uses it as defense against future attack

• The function can be devided into three stages:

1. spacer acquisition

2. cr RNA processing

3. Effector stage

Genetic recombination i the process by which genetic material is broken & jointed to other genetic material

• the central players are RecA & Rec BCD

• RecA molecules are also called synaptases, they are able to scan DNA molecules for homology & align the homologous regions forming a triplet DNA molecule or synapse

• RecBCD initiates recombinational repair drom double stranded breaks

• RecBCD enzyme is both a helicase that unwinds the strands of DNA & a nuclease that makes single stranded nicks in DNA

A holliday junction can be resolved in two ways

• site specific recombination does not utilize RecA

• It involves very short regions of homology (att sites) between donor & target DNA molecules

• These are recognized by dedicated enzyme systems (integrases) which catalyze a crossover betewen them to produce a cointegrate molecule

• E.g. the integration of phage lampda/flagellar phase variations in salmonella enterica

A heritable change in the DNA

Does not change the amino acid sequence

Changes the amino acid sequence to another

Changes the amino acid sequence to a stop codon

Changes the open reading frame of the gene

0,003 mutations per genome/generation

-> spontaneous mutations are rare because of the efficiency of DNA proofreading & DNA repair pathways

• tautomeric shifts in DNA bases that alter base-pairing properties (rare enol-/imino bases)

• Oxidative deamination of bases

• Formation of apurinic sites

• Mutagens: chemical agents (base analogs, base modifiers, intercalators)/electormagnetic radiation ( X-rays & gamma rays break the DNA, ultraviolet rays form pyrimidine dimers)

Test to identify mutagens

• relies on a mutant bacterial stain that is defective in his G (+mut): cannot grow on a medium lacking histidine

• The strian is incubated with the mutagen & plated on medium lacking histidine

• Colony number = mutation frequency

• the premise is that the parental strand will contain the proper DNA sequence

• The methyl-directed missmatch repair proteins are called Mut because a high mutation rate results in strains that are defective in one of these proteins

• Methy missmatch repair is based on recognition of the methylation pattern in DNA bases to discriminate the parental from the newly replicated DNA

• when DNA damage is too severe to be repaired by error proof pathways the cell relaxes fidelity of DNE replication to ensure circularity of the chromosome

• Involves the cooridnated induction of > 40 genes

• Extensive DNA damage causes single-stranded DNA

• RecA assembles at ssDNA & becomes activated

• RecA coprotease activity stiulates autodigesteion of the LexA repressor

• This induces expression of many DNA repair enzymes

• Among them two “sloppy” DNA polymerases that lack proofreading activity & error prone repair systems

• The cell has no other option but ti “mutate or die”

Simple transposable elements containing a tranposase gene flanked by short inverted repeat sequences

Transfer by replicative or non replicative transposition

A transposable element in a plasmid can bring about cointegration of the plasmid into a target DNA molecule during replicative transposition

• complex transposable elements that carry additiona genes (e.g. drug resistance)

• E.g. composite, complex or conjugative transposons

Classical molecular model

• very complex shape requiring 20 gene products for assembly

• Capsid (head) contains linear dsDNA genome

• Tail: consists of shearth plus interna tube

Phage T4 binds to E. Coli by contact between its tail fibers & the outer membrane: a confromational change causes sheath to contract & inject the genome into the cell

• first earl genes are transcribes by host RNA pol: proteins unclude DNase & a DNA pol

• Phage T4 genome is synthesized within the host cell by rolling circle replication

• Progeny genoms are linked in a concatemer: cut with an offset so that individual linera genomes have slight overlaps

• Ultimately the late genes are induced to produce the capsid & tail proteins tat assemble on the membran: a late gene encodes lysozyme which lyses the cell releasing the mature phaged