Bacterial DNA Transfer and Recombination

• The image shows a bacterial cell's structure components including: Periplasmic space and cell wall, the nucleoid, the inner (plasma) membrane, and outer membrane.
• The scale indicates 0.5µm.
• E. coli cells containing double stranded circular DNA which can be genomic, or plasmid DNA can rupture.
• Genomic DNA is double stranded, circular, containing one molecule per cell until division, replicating via a mechanism learned in class; it occurs right before cell division from one origin of replication, and carries most genetic information.
• Plasmids refer to much smaller, double-stranded circular DNAs.
• There are usually multiple copies within a single bacterial cell.
• They replicate independently from replication of cell division and of genomic DNA.
• Plasmids replicate through different mechanisms, not the same as genomic DNA replication.
• Plasmids contain a small amount of genetic information that may be important for bacterial characteristics.
• Bacteria serve as good model systems for genetics.
• Bacteria are easy to grow and maintain and divide quickly by binary fission.
• They produce many cells from one original cell (clone or colony), and have lots of genetic variants or mutants available for study.
• Media for growing E. coli can be minimal or rich.

Minimal Medium

• Contains minimal compounds bacteria need to grow.
• Essential components include water, inorganic salts, and glucose as a source of C for energy and synthesis other organic compounds.

Rich Medium

• Includes extra compounds to enable bacteria to grow and divide faster than on minimal medium.
• Luria-Bertani (LB) broth is an example
• LB broth includes water, tryptone (peptides from casein digestion with trypsin), yeast extract (a mix of peptides, amino acids, vitamins, and carbohydrates from yeast cells obtained by autolysis), and sodium chloride.
• Aseptic techniques are important to avoid contamination by other microorganisms.

Sterilization Methods

• Autoclaving kills microorganisms using heating at 121°C for 20 minutes with steam and high pressure to prevent boiling of aqueous solutions at 121°C
• Pipet tips, microcentrifuge tubes and media need it
• Filtration is also important, the filters of 0.2 µm prevent microorganisms from passing through.
• Ampicillin solution for example
• Irradiation kills microorganisms by damaging biological molecules.

Bacterial Mutants

• Conditional mutants die under specific growth conditions but can survive under others.

Auxotrophic mutants

• Unlike wild type, these mutants cannot synthesize all required organic compounds.
• They cannot synthesize an essential compound, and therefore cannot survive unless that compound is added to the growth medium.
• Mutant phenotypes are labeled with an abbreviation of the compound, (normally amino acid, vitamin of nitrogenous base in DNA) usually a first capital letter, and a minus superscript.
• Bacteria are unable to synthesize the organic compound due to a mutation in genes encoding enzymes needed for its synthesis.
• The genotype of the auxotrophic mutant is indicated by the name of the gene with the mutation (italics, small case), and a minus superscript.
• The wild type gene would have a plus superscript.
• For example: leu2- is mutant, leu2+ would be wild type.

Carbon Source Mutants

• Unlike wild type bacteria which can use any sugar for carbon, carbon source mutants cannot use a particular sugar.
• Their mutant phenotype is indicated by the abbreviation of the sugar and a minus superscript.
• As example: cannot use Lactose → Lac-
• Bacteria are unable to use the sugar as a carbon source usually due to mutation in a gene encoding a protein needed to take it in from the medium/encoding an enzyme to convert it to another form.
• The genotype of the mutant is indicated by the name of the gene with the mutation (in italics and small case), and a minus superscript.
• The wild type gene has a plus superscript; E. coli needs β-galactosidase to break lactose into glucose and galactose, its encoded by the lacZ gene.

Antibiotic Resistant Mutants

• Wild type bacteria are sensitive to antibiotics but mutant bacteria are resistant to the antibiotic because they make an enzyme that breaks it down, or they make a variant of a protein/target molecule that no longer binds to the antibiotic.
• Mutants are labeled with the abbreviation of the antibiotic and an 'r' superscript.
• For example, resistant to streptomycin is Strr.
• Mutations in several genes resist the antibiotic; mutant genotypes shown by name of gene along with the mutations (in italics and small case) with a minus superscript.

Temperature Sensitive Mutants

• Wild type bacteria handle temperatures between 37°C (permissive temperature), and 42°C (restrictive temperature), but mutants may not show this tolerance.
• A temperature sensitive mutant will grow at 37 degrees and not at 42.
• Mutants cannot handle high temperatures because of a mutation in a gene so the protein folds incorrectly, is unable to carry out essential functions at higher temps.
• At higher temperatures, broken bonds in the polypeptide result in altered structure/function.
• Mutant bacteria can be selected for on semisolid medium with agar by spreading a diluted culture of bacteria, such that cells are separated.
• Where a single cell is on the plate, it will divide many times and produce a clone or colony of bacteria if the medium allows it.
• Minimal medium with leucine allows both Leu+ and Leu- to grow, minimal medium only lets Leu+ grow.
• Selection of carbon source mutants includes a minimal medium with glucose, allowing with both Lac+ and Lac⁻ strains.
• Minimal medium without glucose and with Lactose, lets only Lac + strains grow.
• Selection of antibiotic resistant mutants occurs with Strr strains needing a minimal medium with streptomycin to grow.
• Selection of temperature sensitive mutants can occur with minimal medium permissive T, allowing both wt+mutants to grow at this T, meaning that a minimal medium restrictive T only allows the wild type bacteria to grow at this T.
• Identification and recovery of all the types of mutants need two plates, with the exception of antibiotic-resistant mutants. This method is called replica plating.
• Replica plating uses complete (rich) media with amino acids/sugars.
• Replica plating uses minimal medium that is lacking key nutrients.
• Colonies are determined to be prototroph if they grow on complete and minimal Replica plates.
• Colonies are determined to be auxotroph if they grow only on complete Replica plates not grown on minimal Replica plates.
• When comparing the comparison of complete to minimal medium with proline.
• If comparing minimal medium with alanine, the conclusion of colony is ala⁻.
• When comparing minimal Replica plates with proline, the conclusion is pro- .
• Colony of bacteria can be a combination of auxotroph, or a unknown auxotroph.

Using Replica Plating

• Compare minimal medium and minimal medium plus alanine and proline plates.
• Colony 2 is ala⁻, pro⁻, lac⁺; colony 3 is ala⁻, lac⁻; colony 6 is pro⁻, lac⁻.
• Compare to minimal medium, Colonies 1, 5, 7, 9 are lactose, 4 and 10 are lact⁺.
• Auxotrophic colonies 2, 3, 6, and 8 cannot grow without supplementation.
• The results of comparing the lactose containing media plates to previous results identifies the other prototrophic lact⁻ colonies with with Replica plating..
• Table shows that it grows on lactose medium: those are Prototrophs.
• Table shows that it does not grow on minimal medium supplemented with alanine and proline.
• Colony auxotroph results: that it does not grow on minimal medium.
• To distinguish certain strains of bacteria from a wild-type strain a table can be used to determine selective media via writing 'S' for selective; and 'N' for non-selective if it cannot be used to distinguish for non-selective..
• Genetic information of bacteria, and thus bacterial traits, change via methods of how foreign DNA which can be introduced from bacterial plasmids.
• If it is just a random fragment of bacterial DNA, and for it to remain in the cell so that it will be inherited by the daughter cells once the cells divides the only method is for it to get integrated into the cells.
• This method is known as recombination and it does happen
• During recombination, the foreign DNA will replace the fragment of the genomic DNA if it is homologous to the strand.
• Lederberg and Tatum discovered that there is recombination
• Through recombination, they were able to discover that there needs to be DNA transferred from one type of bacterial strain to another bacterial strain.
• Once the DNA is in the genome, the bacterial cell gets transferred with the wild type genes and replaces the original mutant genes.
• If foreign bacterial DNA comes in as plasmid, it replicates independently and contains multiple copies of the plasmid, due to definition having to have a location of definition.
• Daughter cells will inherit the plasmid sequence.
• Transformation is uptake of DNA directly from the environment.
• Conjugation is DNA transferred from one bacterial cell to another.
• Transduction is DNA transferred from one bacterial cell to another through a bacteriophage.
• Transformation occurs where Some bacteria, like Streptococcus pneumoniae, take up are competent, taking in DNA automatically.
• Griffith's experiment discovered this
• Other bacterial cultures like E. Coli needs to be treat to be able to use.
• Transformation needs linear DNA or plasmid DNA
• Frequency of cotransformation provides information about the relative difference of two genes in the bacterial genome origin.
• Plate different types of medium to check if the wild type ile gene or wild type met gets taken up by the bacteria.
• To make competent, needs to be treated
• If the two genes are far apart, in terms of probabilities: those chances gets lowered from being in the same fragment as the genes are on the same DNA strand.
• If the two genes are relatively close and cotransformation only needs one fragment with the gene together, in terms of actual probability the number would be around 0.25%.
• In this case if true the frequency would be around at least a thousand times higher that it would be safe to infer that leu and met must be relatively close to each other.
• Conjugation requires constant physical contact and direct integration of donor/recipient.
• The F⁻pilus cell has donor/recipient bacteria strains.
• Alternating suction and pressure.
• No Donor Bacterial Genes Transferred to Exconjugant.

Rolling circle

• The F(fertility ) plasmid replicates through the rolling circle method of replication.
• The F plasmid replicates through the conjugate bridge via the rolling circle as the DNA is being replicated.
• Both the cell and the recipient will get a fully copied DNA.
• High frequency of recombination(Hfr) cell, integrates, and is also known as episome, also can replicate itself.

Hfr Conjugation

• When an Hfr cell integrates, the genomic DNA replicates rolling circle mode.
• The donor bacteria cell cannot give all of its DNA to a newly copied DNA daughter cell due to the process's transfer is only on the outer loop of the copy.
• Transfer of the sequences will recombine at both bacterial sequence the frequency High.
• Recipient bacteria are unable to become the copy since F- is a F-pilus.
• Time -of-entry can get mapped back to the bacterial gene, because it will enter a recombinant strain phenotypics.
• Genotypes will be different, the more that a gene is transferred it will continue to add on to recipient.
• High gene transfer equals new phenotype

Genetic Mapping

• Donor (Hfr) and a recipient (F-) are mixed and allowed to conjugate which is a selective gene donor.
• Aliquot points will plat and determined to have growth and are determined to grow together or at single cell division
• Hfr markers for genes can get more than two bacteria cell colony The time -of entry is the first time new genes can grow in an recombinant DNA.
• For table example, it shows mapping of E. coli and the genotypes/times.
• With the two donors, they have different distances, which is caused by integrations' in any of the orientations.
• Complete maps happen when two different Hfr strains contain two of the of same gene
• Circular maps indicate circular bacterial DNA of the genome.
• The Donor bacterial genomic DNA is broken in generalized cells and parts of the strand get into viral
• The viral particles carry genomic material into the first cell and its transforms DNA into the second cell: Transduction
• The virus (lysogenic) transfers is called generalized,
• Specialized transduction involves excision and is non-precise, of the genomic DNA and is segment DNA site next to the viral
• The Prophage is DNA. next/ always transfers.

Questions covering DNA transfer mechanisms in bacteria, including homologous recombination, conjugation, transduction, and DNA uptake. Key concepts involve plasmid integration, differences between conjugation and transduction, and the consequences of imprecise prophage excision.