MUTATIONS Stable, heritable, alterations in the gene sequence which usually produce phenotypic changes (ie mutant) Morphological mutations-change microorganisms appearance, colony or cellular morphology Lethal mutations-result in death of microorganism Conditional mutations-expressed under certain environmental conditions eg. not expressed at a low temperature (permissive temp), but expressed at high temperature (non-permissive temp) Biochemical mutations-alter a biosynthetic pathway and the organisms ability to grow on minimal media *AUXOTROPH- requires nutrient supplement cannot grow on minimal media *PROTOTROPH- wild-type, not a mutant thus can grow on minimal media Resistant mutations- a mutant that is now resistant to an antibiotic or virus

How Do Mutations Occur? Spontaneous mutation-occurs randomly, may result from errors during DNA replication Transition-changing a purine for the other purine or a Pyrimidine for the other pyrimidine Transversion-purine is substituted for a pyrimidine Frameshift- deletion or addition of a base causes an altered Codon reading frame and the synthesis of a non functional protein

Occurs after exposure to a physical or chemical agent called Induced Mutations Occurs after exposure to a physical or chemical agent called a mutagen Modes of action Incorporation of base analogs-structurally similar to the normal bases that get incorporated into the growing chain during replication. Typically they base pair with different bases and thus replace the normal base pair. eg. 5-Bromouracil Specific mispairing-mutagen changes a base structure eg. Methylnitrosoguanidine puts a CH3 group on guanine causing it to mispair with thymine 3. Intercalating agents-distort DNA to induce single nucleotide pair insertions and deletions UV radiation-generates thymine dimers between adjacent pyrimidines. Usually this triggers repair mechanisms, but repair may not always occur.

Types of Mutations Wild-type = No mutation Forward mutation = a mutation that alters phenotype Reverse mutation = a second mutation which may make the mutant appear wt (in same gene) Suppressor mutation = a mutation in a different gene which overcomes the effect of the 1st mutation resulting in a wt phenotype Point mutation = affects only 1 bp at a single lociation Silent mutation = a point mutation that has no visible effect because of code degeneracy Missense mutation = a single base substitution in the DNA that changes a codon from one amino acid to another Nonsense mutation = converts a sense codon to a nonsense or stop codon, results in shortened polypeptide Frameshift mutation = arise from insertion or deletion of one or two bp within coding region of gene, results in the synthesis of nonfunctional protein

Screening for auxotrophic mutants Mutant Detection In order to study microbial mutants, one must be able to detect them and isolate them from the wt organisms Detection Systems in bacteria are straightforward because there is only 1 copy of the gene therefore any new allele should be seen immediately Screening vs Selection Systems Screening for auxotrophic mutants A lysine auxotroph will only grow on media that is supplemented with lysine because it cannot synthesize the amino acid Use of replica plating to find mutant Selection for antibiotic resistant mutants Technique uses conditions under which only the mutant will grow

Bacterial Viruses Just like we have viruses that infect us causing the common cold to meningitis, bacteria also have viruses that infect them called: Bacteriophages Bacteriophage cannot reproduce and survive on it’s own, it must take over it’s host cell Virulent bacteriophages = lytic life cycle (eg T4) Adsorption of phage to host Penetration of virus genetic material Replication, synthesis of virus DNA and proteins Cell lysis and release of phage particles Temperate bacteriophages = lysogenic life cycle (eg l) Adsorption of phage to host Penetration of virus genetic material Integration of virus into host chromosome = prophage Replication along with host chromosome, maintained as a prophage

Temperate Bacteriophages Instead of destroying host to produce virus progeny, the viral genome remains within the host cell and replicates with the bacterial chromosome. This relationship between phage and host is called lysogeny Lambda (l), family Siphoviridae, is the best understood Temperate phage DNA is double stranded with cohesive ends (cos sites) which are ss stretches of DNA that are complementary to each other so it circularizes immediately after injection into the host Once a closed circle is formed transcription by host RNA polymerase is initiated The BIG Decision: Lytic or Lysogenic life cycle? Battle between two repressors, cI or cro which compete for The same binding sites (operators) if cI binds represses synthesis of all genes = Lysogeny If cro binds represses synthesis of cI = Lytic

l is smarter than we think! Temperate phage l If cI repressor wins the circular DNA is inserted into the chromosome via a process called integration At this stage it is called a Prophage The l prophage can decide to leave the host genome and begin the the production of new phage This is triggered by a drop in cI levels Environmental factors such as UV light or chemical Mutagens that damage host DNA causes a host protein, recA, to act as a protease and cleave the cI repressor Now the balance will be shifted to cro and the lytic cycle l is smarter than we think!

Recombination Process in which nucleic acid molecules are rearranged or combined to produce new sequences This occurs in a process called crossing-over In bacteria General Recombination involves exchange between a pair of homologous DNA sequences Since bacteria don’t have sex (reproduce via meiosis) Recombination takes places in several ways following Horizontal Gene Transfer Direct transfer between 2 bacteria temporarily in physical contact [conjugation] Transfer of naked DNA fragments [transformation] Transport of bacterial DNA by a bacteriophage [transduction]

Plasmids Plasmids are small ds DNA molecules, usually circular that can exisit independently of the host chromosome. They have their own replication origin so can replicate automonously (Replicon) and have relatively few genes (<30) that are not essential to the host. Conjugative plasmids have genes for pili and can transfer copies of themselves to other bacteria during conjugation Fertility factor or F factor - These plasmids can also intergrate into the host chromosome or be maintained as an episome (independent of chromosome) R factor - Also conjugative plasmids which have genes that code for antibiotic resistence for the bacteria harboring them. These do not integrate into the host chromosome. Col Plasmids - harbor Bacteriocins which are proteins that destroy other bacteria (eg cloacins kill Enterobacter species) Virulent plasmids - have genes which make bacteria more pathogenic because the bacteria is better able to resist host defenses or produce toxins/invasins

Transposable Elements Transposons - DNA segments that carry genes that allow them to move about the chromosome (transposition) Behave somewhat like a lysogenic phage Unlike plasmids or phages, they are unable to reproduce or exist apart from the host chromosome Insertion sequences - IS elements- short sequence of DNA Containing only genes required for transposition Flanked by inverted repeats (IR) - identical or similar sequences 15-25 bp in reversed orientation Transposase - enzyme that recognizes the IR and promotes transposition Composite transposon (Tn)- contains other genes in addition to transposase like antibiotic resistance genes or toxins Importance Can insert within a gene to cause a mutation or stimulate DNA rearrangement leading to deletions of genetic material Can have stop codons or termination sequences to block translation or transcription Can have promoters which activate genes near pt of insertion Can be on plasmids to aid in insertion of F plasmids into host chromosome Some bear transfer genes (Tn916) and can move between bacteria through conjugation (conjugative transpososn)

Conjugation Conjugation - transfer of genetic material by direct cell contact. First evidence by Lederberg and Tatum 1946 - mixed two auxotrophic strains for several hours in rich medium than plated on minimal medium. Only recombinants which are now Prototrophic will be able to grow Second evidence- U tube experiment proved direct cell contact was required During this process there is a definite donor strain , F+ (F factor) and a recipient strain, F- (does not have the F factor) The F factor codes for pilus formation which joins the donor and recipient and for genes which direct the replication and transfer of a copy of the F factor to the recipient The F factor can remain as an episome or it can integrate into the bacterial chromosome via IS sequences. This type of donor is called and Hfr strain (High frequency recombination) F’- When the F factor in an Hfr strain leaves the chromosome, sometimes is makes an error in excision and picks up some bacterial genes

Transformation Transformation- uptake by a cell or a naked DNA molecule or fragment from medium and incorporation of this DNA into the recipient chromosome. This process is random and any portion of the genome may be transferred. For this process to occur the bacterial recipient must be able to take up the DNA, called competency Competent bacteria must be in a certain stage of growth (usually exponential) and secrete a small protein (competency factor) that stimulates production of new protein required for DNA uptake Gene transfer by this process occurs in soils and marine environments so it is an important route of genetic exchange in nature Artificial transformation- carried out in laboratory to transfer plasmid DNA, a common method for introducing recombinant DNA into bacterial cells. eg CaCl2 or electroporation

Transduction Transduction is the transfer of bacterial genes by phage. Bacterial genes are incorporated into a phage capsid due to errors made during the virus life cycle. The virus containing these genes then injects them into another bacteria *most common mechanism for gene exchange and recombination in bacteria. Generalized Transduction- occurs during the lytic cycle of virulent or temperate phages. When the viral chromosomes are being packaged into phage heads, random fragments of partially degraded bacterial chromosome may be packaged by mistake. Transducing particle- the phage which injects bacterial DNA into a new recipient. Simply a carrier of genetic information Specialized Transduction- occurs when an error is made during the lysogenic life cycle. When a prophage in induced to leave the chromosome inaccurate excision results in specific bacterial genes packaged into the phage head and subsequently transferred

Antibiotic Resistance Resistance to antibiotics can be acquired as genes are transferred between bacteria (eg R plasmids or transposons) Many infections are treated with antibiotics and the increasing number of drug resistant pathogens is a serious public problem Antimicrobial agents kill or stop growth of various pathogens Inhibit cell wall synthesis Affect protein or nucleic acid synthesis Disrupt membrane structure and function Block metabolic pathways Mechanisms by which bacteria resist antibiotic treatment Inactivation of antibiotic through chemical modification Change in target of antibiotic to inhibit action Change in permeability by reducing uptake How do we break the cycle of antibiotic resistance?

Recombinant DNA technology Genetic Engineering - the deliberate modification of an organisms genome. The methods used to accomplish this are know as recombinant DNA technology Recombinant DNA - DNA with a new sequence formed by joining fragments from different sources Restriction enzymes - bacterial enzymes (endonucleases) that recognize and cleave specific sequences (4-8 bp long). Normally bacteria use them to destroy foreign DNA like incoming phage DNA Type II Restriction enzymes cleave DNA at specific recognition site which is a palendrome (read same forward and backward) and cleavage leaves single stranded or “sticky” ends. Methodology used to construct a genomic library (bank of all genes in a given organism) or express foreign genes in bacterial cells