Bacterial Genetics Lecture Notes PDF

 Genomic DNA Double stranded circular DNA One molecule per cell until cell is ready to divide Replicates through the mechanism learned in class, right before cell division, from one origin of replication Contains most of the genetic information Plasmids Much smaller...

 Genomic DNA Double stranded circular DNA One molecule per cell until cell is ready to divide Replicates through the mechanism learned in class, right before cell division, from one origin of replication Contains most of the genetic information Plasmids Much smaller double stranded circular DNAs Usually multiple copies per bacterial cell Replicate independently from replication of the genomic DNA and cell division Different plasmids replicate by different mechanisms, not the same as the replication of genomic DNA Contain a small amount of genetic information that may be important for bacterial characteristics, Bacteria are good model systems for genetics Easy to grow and maintain Divide in a short amount of time by binary fission Produce a large number of cells from one original cell (clone or colony) There are a lot of genetic variants or mutants available for study How do we grow E. coli ? Medium Minimal medium (minimal compounds required for the bacteria to grow) ▪ Water ▪ Inorganic salts ▪ Glucose as a source of C (for energy and synthesis of all other organic compounds) Rich medium (extra compounds that will allow the bacteria to grow and divide faster than in minimal medium) For example Luria- Bertani (LB) broth ▪ Water ▪ Tryptone- peptides obtained by digestion of casein with trypsin ▪ Yeast extract- mixture of peptides, amino acids, vitamins and carbohydrates from yeast cells obtained by autolysis ▪ Sodium chloride  How do we grow E. coli ? We need to use aseptic techniques- methods to prevent contamination by other microorganisms Sterilization Autoclaving Kills microorganisms by heating at 121°C for 20 minutes Uses steam and high pressure which will prevent boiling of aqueous solutions at 121°C Example: Pipet tips, microcentrifuge tubes, LB medium Filtration Filters of 0.2 µm will prevent microorganisms from going through Example: ampicillin solution Irradiation Kills microorganisms by damaging biological molecules Bacterial mutants Conditional mutants- they will die under certain growth conditions but they can survive under other conditions 1. Auxotrophic mutants Unlike wild type bacteria, they cannot synthesize all their required organic compounds. They cannot synthesize an essential compound and therefore they cannot survive unless that compound is added to the growth medium. Nomenclature: Their mutant phenotype is indicated by theusually abbreviation of the first capital letter compound (normally amino acid, vitamin or nitrogenous base in DNA) and a minus superscript. Why are the bacteria unable Example: to synthesize auxotrophic the  for leucine organic Leu - compound? Because they have a mutation wild in atype geneisencoding an enzyme prototrophic needed Leu for leucine for+ the synthesis of that compound. If the pathway were The genotype of the auxotrophic mutant will be indicated by the name of the gene with the mutation (in italics and small case) and a minus superscript, while the wild type gene would have a plus superscript. For example: mutant would be leu2- and the Bacterial mutants 2. Carbon source mutants Unlike wild type bacteria, which can use any sugar as their source of carbon, these mutants cannot use a particular sugar. Nomenclature: Their mutant phenotype is indicated byusually the abbreviation of first capital letter the sugar and a minus superscript. Example: cannot use lactose Lac - Why are the bacteria unable to use the sugar as a carbon source? Because wild type can use lactose Lac + they have a mutation in a gene encoding a protein needed to take it in from the medium, or encoding an enzyme necessary to convert that sugar into another one. The genotype of the mutant will be indicated by the name of the gene with the mutation (in italics and small case) and a minus superscript, while the wild type gene would have a plus superscript. For example, E. coli requires the enzyme β-galactosidase to break down lactose into glucose and galactose. This enzyme is encoded by the lacZ gene. The genotype of the Bacterial mutants 3. Antibiotic resistant mutants Wild type bacteria are sensitive to antibiotics. Mutant bacteria can be resistant to an antibiotic because they make an enzyme that breaks down the antibiotic or they make a variant of a protein or target molecule that no longer binds to the antibiotic. Nomenclature: Their mutant phenotype is indicated by the abbreviation of the antibiotic and an r superscript. Example: resistant to streptomycin  Strr Mutations in a variety of genes wild type can cause is sensitive the resistanceStr to streptomycin to sthe antibiotic and the mutant genotypes will be shown by the name of the gene with the mutations (in italics and small case) with a minus superscript. Bacterial mutants 4. Temperature sensitive mutants Wild type bacteria tolerate a range of temperatures. Mutants may not show this tolerance. For example, wild type E. coli permissive optimal growth temperature isrestrictive temperature 37° C, but it can survive temperature at 42 ° C. 37° C 42° C wild type E. coli + + T sensitive mutant + - Mutants do not tolerate the higher temperature because they have a mutation in a gene that results in the encoded protein folding abnormally and not being able to carry out its essential functions at a higher than normal temperature. At higher than normal temperature there are broken bonds in the polypeptide that result in altered structure and function.  How do we select mutant bacteria? For example, let’s imagine that you have a culture of E. coli and use UV light in the laminar flow hoods in the classroom to induce mutations. How would you recover a Leu- mutant, or a Lac- mutant, or a Strr mutant or a T sensitive mutant? In the liquid culture you have the wild type and mutant bacteria mixed and it will not be possible to separate them. You have to use semisolid medium with agar. If you spread a diluted culture of bacteria, individual cells will be separated. Wherever a cell is in the plate, if the medium allows its growth, the cell will divide many times and produce a clone or colony of bacteria.  Selection of auxotrophic mutants, like Leu- Leu- minimal medium with leucine minimal medium Both Leu+ and Leu- will grow on this medium Only Leu+ bacteria will grow  Selection of carbon source mutants, like Lac- Lac- minimal medium (glucose) minimal medium w/o glucose with lactose Both Lac+ and Lac- will grow on this medium Only Lac+ bacteria will grow Selection of antibiotic resistant mutants, like Strr Strr minimal medium with streptomycin Only Strr will grow on this medium  Selection of temperature sensitive mutants Temperature sensitive mutants minimal medium permissive T minimal medium restrictive T Both mutants and wt will grow at this T Only wt bacteria will grow  Identification and recovery of all types of mutants except antibiotic resistant mutants require the use of two plates: replica plating Complete medium=Rich medium It has amino acids and several sugars x Colony 8 is an unknown auxotroph Lactose Assume that you wish to distinguish certain strains from a wild type strain. In the table write S for selective if the medium in each row can distinguish the strain indicated in each column from the wild type strain. If the medium cannot be used to distinguish the strain from the wild type strain write N for non-selective. Genotype lac+leu-strs lac-leu-strs lac-leu+strs lac+leu+strr lac-leu-strr Minimal S N Minimal +leucine Minimal - glucose +lactose Minimal +streptomycin Minimal +leucine +streptomycin S Minimal -glucose +lactose +streptomycin Minimal +leucine -glucose N +lactose +streptomycin How can genetic information of bacteria and therefore bacterial traits change? Foreign DNA can be introduced in bacteria. What happens to the foreign DNA once inside the bacterial cell? If it is a random fragment of DNA Can it remain in the cell? If it remains in the cell, would it be inherited by the daughter cells once the cells divide? The only way that it can be inherited is if it gets integrated into the genomic DNA. This integration is called recombination and it does happen. The foreign DNA may replace a fragment of the genomic DNA by homologous recombination if its sequence is homologous (similar) to a genomic DNA sequence. Lederberg and Tatum showed that there is recombination There had to be DNA transferred from one bacterial strain to the other bacterial strain. Once inside the bacterial cell the transferred DNA with the wild type genes replaced the DNA with the mutant genes and the bacteria became prototrophic. Daughter cells inherit the DNA and the traits determined by those genes  What happens to the foreign DNA once inside the bacterial cell? If it is plasmid DNA Plasmid DNA by definition has its origin of replication, so the DNA does not have to integrate into the genomic DNA. The plasmid will replicate and generally there will be multiple copies of the plasmid. The daughter cells will inherit the plasmid sequences.  Methods of uptake of DNA by bacteria Transformation Bacterial cells take DNA from the environment Conjugation Bacterial cells get DNA transferred directly from another bacterial cell Transduction Bacterial cells get DNA from another bacterial cell through a bacteriophage  Transformation Some bacteria, like Streptococcus pneumoniae are naturally competent and can take up DNA from the environment Griffith’s experiment Some bacteria, like E. coli are not naturally competent and need to be treated for the cells to take up DNA The DNA introduced in the bacteria may be a linear DNA fragment or plasmid DNA  The frequency of cotransformation provides information about the relative distance of the two genes in the bacterial genome of origin Strain 1 of Bacillus subtilis Strain 2 of Bacillus subtilis Ile- Met- Ile+ Met+ DNA isolated and treated to make competent fragmented randomly Plate in different types Test for of medium to check if cotransformation the wild type ile gene : transformation or the wild type met with both genes gene or both were taken up by the bacteria  Results obtained Ile+ Met- Ile- Met+ Ile+ Met+ 158 x 10-6 162 x 10-6 27 x 10-6 = 0.27 x 10-4 If the two genes are far apart, chances are they ile will not be in the same fragment, so cotransformation would require two fragments to met get into the bacterial cell. The expected frequency of cotransformation would be the product of the transformation frequencies: 158 x 10-6 x 162 x 10-6= 0.25 x 10-7. If the two genes are relatively close to each other, ile met they may be together in some of the fragments, so cotransformation would only require that one fragment with both genes gets into the bacterial cell. The actual frequency of cotransformation would be higher than 0.25 x 10-7. The actual frequency in this case is about a thousand times higher than expected if genes were always in separate fragments, so the genes leu and met must be relatively close to each other.  Conjugation requires physical contact between the donor and recipient bacterial cells Prototrophic If a filter colonies were prevents only obtained physical contact when there was between cells of physical contact both strains between the two there is no DNA strains of transfer bacteria  Rolling circle replication The F (fertility) plasmid replicates by a rolling circle mode of replication The F plasmid replicates by the rolling circle mode of replication and the DNA gets transferred through the conjugation bridge while the DNA is being replicated. Both donor and recipient cell have a copy of the plasmid after conjugation, so both are F+. Hfr cells The F plasmid is an episome. Although it can replicate on its own, it can also get integrated into the bacterial genome. The F plasmid can get integrated in different positions of the bacterial genome and in the two possible orientations. The bacterial cell with the F plasmid integrated into the bacterial genome is a specific type of F+ cell called Hfr for High Frequency of Recombination.  When an Hfr cell conjugates with a recipient cell, the whole genomic DNA replicates through the rolling circle mode. Part of the F plasmid sequences along with some of the bacterial genomic DNA is transferred to the recipient cell through the conjugation bridge. The conjugation bridge is fragile and does not last long enough to allow transfer of the complete genomic DNA and remaining F sequences. The sequences transferred have homology to sequences in the genome of the recipient cell and therefore there is High Frequency of Recombination between them. Transfer of the sequences and recombination may change the characteristics of the recipient bacteria. The recipient bacteria did not get the complete sequences of the F plasmid and therefore -  Time-of-entry mapping The closer a gene is to the origin of replication the sooner it will get transferred to the recipient cell. Genes can be mapped in the bacterial genome of the donor strain based on the time it takes them to enter a recipient strain and change the phenotype of this recipient strain. Therefore, in order to be able to tell that a gene got transferred from the donor into the recipient cell the genotypes of donor and recipient strains for that gene need to be different. We can tell the changes in phenotype in the recipient strain because after the gene transfer the strain will be able to grow in medium it could not grow in before the conjugation. In order to be able to tell that the recipient strain received a certain gene we need to use a selective medium that would only allow that recipient strain to grow after the gene transfer, and would not allow the donor strain to grow.  Time-of-entry mapping How is it done? Donor (Hfr) and a recipient (F-) strains are mixed and allowed to conjugate. At certain time points an aliquot of the mixed culture is taken out and plated on selective medium. The number or percentage of recombinants (recipient bacteria that now grow on selective medium) are determined. The time-of-entry is determined as the first time at which recombinants would appear in the selective medium (where the graph intersects the X- axis). Example: Mapping of genes in E. coli from conjugation of P678 and HfrH Selective medium to determine time-of-entry of thr gene Minimal Minimal + Minimal threonine +streptomycin F- P678 (Thr- Strr) - + - HfrH (Thr+ Strs) + + - Exconjugant F- P678 (after transfer of the + + + thr+ gene from HfrH) (Thr+ Strr) In order to maintain the F- strain resistant to an antibiotic, such as streptomycin for selection: The gene responsible for the resistance/sensitivity to streptomycin has to be far away from the origin of replication, so it does not get transferred to the recipient strain or The recipient strain has a gene for antibiotic resistance on a plasmid The time-of-entry is determined as the first time at which recombinants would appear in the selective medium (where the graph intersects the X-axis).  Different Hfr strains have the F plasmid integrated in different locations in the genomic DNA and the plasmid sequences may be integrated in any of the two possible orientations. Therefore, in different Hfr strains the times-of-entry of the same gene will be different. Since the conjugation bridge is fragile, only genes relatively close to the origin for any given Hfr strain can be mapped. Combining the results of time-of-entry mapping experiments performed with different Hfr strains it is possible to get a more complete map of the genome. If the maps obtained with two different Hfr strains contain the same two genes, the maps can be overlapped. If the complete genome is included in the map, this map will be circular since the bacterial genome is circular. Integration of the F plasmid can happen in one of two orientations or or If you have the times-of-entry in minutes then you need to keep track of distances between genes: Hfr map of E. coli  The F factor can get excised from the genomic DNA in an Hfr strain Excision may not be precise and an F plasmid carrying some sequences from the genomic DNA is produced: F’ plasmid A bacterial cell with an F’ plasmid can function as a donor in conjugation  The complete F’ plasmid is transferred to the recipient cell. If the F’ plasmid carries a wild type copy of a gene that has a mutation in the recipient cell there will be complementation. The transferred gene does not have to be integrated into the genomic DNA. It will get expressed from the F’ plasmid and complement the mutation. The recipient cell will behave as wild type. If the gene carried by the F’ plasmid is not the one with the mutation in the recipient cell, there will be no complementation. Transduction involves transfer of DNA by a bacteriophage Lytic mode of infection The phage injects its DNA into the bacterial cell. The phage DNA is replicated and transcribed. The phage uses the bacterial ribosomes to translate its RNAs and produce phage proteins, and new viral particles are assembled. The host DNA breaks down, and the cell is lysed as it releases more viruses.  Lysogenic cycle cycle Lysogneic The phage injects its DNA into the bacterial cell. The phage DNA is integrated into the bacterial genomic DNA, as what is known as a prophage. The phage DNA can remain in the genomic DNA of the bacteria and it will be copied every time the genomic DNA is replicated. The daughter cells will inherit the phage DNA integrated into the genome. Eventually the prophage can be excised from the bacterial genome and the phage can resume a lytic cycle. Transfer of DNA from one bacterial strain to another may not require physical contact between bacterial cells  Generalized transduction As the host genomic DNA is broken down, some fragments of this DNA may get packaged into the viral particles. When the phage particle carrying genomic DNA from one bacterial cell infects another cell it transfers that DNA into the second cell: transduction. Any fragment of genomic DNA can be transferred between bacterial cells by the virus, that’s why it is called generalized. https://www.youtube.com/watch? v=C44ymgwgA-o  Specialized transduction As the prophage is excised from the genomic DNA, the excision may not be precise and a segment of genomic DNA next to the site where the phage DNA was integrated may be excised too, and get packaged into viral particles along with the viral DNA. When the phage particle carrying genomic DNA from one bacterial cell infects another cell it transfers that DNA into the second cell: transduction. Only genes that are next to the site of integration of that particular phage, which is always the same, can be transferred between bacterial cells by the virus, that’s why it is https://www.youtube.com/watch? called specialized. v=ZxbPYekSTLg