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Exam 2… Mean = 72 (after adding 7 points to all tests) Bacterial Genetics - we will only discuss mechanisms of genetic exchange (gene transfer) in bacteria…. Read pages 323-334 and 203-215. Q. 19 - answers were messed up (real answer is c) so part of the 7 points was for that question. Spend a little time going over the test….. Transduction….. Transfer of host DNA via bacteriophage 2 types: Specialized & Generalized Transduction Generalized results from the lytic cycle of certain phage (see Fig. 8.16) Specialized results from lysogeny followed by the lytic cycle (Fig. 13.11) Three modes of genetic exchange: 1. Transduction (figs. 8.16 and 13.11) 2. Transformation (persp. 8.1, pg. 205) 3. Conjugation (Figs. 8.18 - 8.22) In order to understand transduction we need to know…..Viruses of Bacteria (bacteriophage or phage) Always are naked viruses (no membrane) why? Huge variety - One species of bacteria can have many different phage… Table 13.2 Important for: 1. Genetic transfer (transduction) 2. Control of Bacteria in Nature(read Persp. 13.1 pg. 330) 3. Some can cause bacteria to become pathogens (e.g. diphtheria - see Table 13.3) Table 13.2. Note the variety of shapes, nucleic acid content, and “life cycle”… See Fig. 13.1c Host ranges of phage: Phage are usually very specific to the species they infect - they attach to specific receptors on the outer layers of the bacterium e.g. some phage of E. coli attach specifically to the proteins of the flagellum… see Figures 13.12 and 8.17 Phage for a particular bacterium also have DNA methylation patterns like their host and thus avoid having their DNA cleaved by restriction enzymes when the DNA enters the cell…. See Figure 13.13 Bacteriophage attached to pilus of E. coli Fig. 13.12 Virus interactions with host cells or Replication Cycles of phage….. See Figs. 13.4 - 13.7 Phage tail fibers entwined around flagellum Phage T4 attached to specific receptors (cell wall proteins) of E. coli. Fig. 13.4 Fig. 13.04 Fig. 13.5. Example of the lytic cycle: T4 of E. col Fig. 13.6. Phage Lambda can undergo a lytic or the lysogenic cycle Latent state = Lysogenic state Fig. 13.7. Insertion of Lambda into a specific spot the bacterial chromosome…. Summary of lytic and lysogenic cycles Consequences of the lysogenic cycle: Cells are immune to further infection by that phage Can lead to specialized transduction (later) Can cause “lysogenic conversion” = viral genes that change the phenotype of the host cell - e.g. some phage have genes for toxin production and can convert a non-pathogenic bacterium to a pathogenic one…. See Table 13.3 for examples…. Table 13.3. Lysogenic conversion of bacteria conferring pathogenic properties…. Back to Bacterial Genetics Transduction….. Transfer of host DNA via bacteriophage 2 types: Prophage = the lysogenic phage incorporated into the bacterial chromosome….. Specialized & Generalized Transduction Generalized results from the lytic cycle of certain phage (see Fig. 8.16) Specialized results from lysogeny followed by the lytic cycle (Fig. 13.11) Fig. 8.16 Bacterial Genetics Generalized transduction results from the lytic cycle. Chromosome is digested into small pieces - some of which end up being packaged in virus particles… 2. Transformation… uptake and Fig. 13.11. Specialized transduction by a temperate phage. Results from the lysogenic cycle… Specific pieces of DNA (near insertion sites) are transferred… Fig. 13.11b. Specialized transduction by a temperate phage Persp. 8.1, pg. 205. Experiment carried out by Griffin, showing that something was transferred from dead, pathogenic, S. pneumoniae to live nonpathogenic cells transforming them into pathogens… It was later (1944) shown that the “transforming principle” was DNA (thus studies of transformation led to identification of DNA as the genetic material in cells). incorporation (into the chromosome) of “naked” DNA from the environment…. Expression of this new DNA can alter the phenotype of the organism, e.g. converting a non-pathogen into a pathogen…. e.g. Streptococcus pneumoniae… Fig. 19.10. Streptococcus pneumoniae is pathogenic only when it produces a capsule (which helps it avoid detection by antibodies and phagocytes) Fig. 8.14. Transformation of a cell from nonresistant (StrS) to resistant to streptomycin (StrR). Fig. 8.15. There are many artificial ways to make bacterial cells “competent” for transformation, e.g. electroporation, and many chemical treatments that make temporary holes in the cell wall and membrane Transformation can take place between even unrelated bacteria (but it is rarer than between related because most foreign DNA is degraded before it can be methylated - see figure 8.26) There are also genetic platforms (integrons) within bacterial chromosomes that can mobilize large pieces of DNA and integrate large pieces of foreign DNA behind a promoter so that they are easily transcribed… Bacterial Genetics Conjugation (bacterial sex) Usually occurs between a + strain (has a conjugative plasmid) and a - strain (no plasmid)…. Fig. 8.26 Quick review of Plasmids - Extrachromosomal, circular, double-stranded DNA molecules. Plasmids divide and copies go to both daughter cells during asexual reproduction. Many plasmids can be transmitted between closely related species but some are not limited to close relatives. Not usually essential for a given organism, rather they allow the organism to adapt to specific environmental conditions. Therefore, plasmids are often unstable in a host bacterium due to the increased metabolic load. Also found in Archaea, Fungi and other euks……… Fig. 8.17. Conjugation between F+ and and F- E. coli… All sorts of traits can be transferred via conjugative plasmids - the most relevant traits for us are antibiotic resistance genes…. But other important genes are also found on plasmids.. (Table 8.4) Antibiotic resistance - usually by coding for an enzyme that renders the antibiotic non-functional. Beta-lactamase (inactivates Beta-lactams) is one example. Penicillin is a Beta-lactam. Special metabolic properties - some plasmids allow bacteria to take advantage of situations that might be otherwise harmful. Breakdown of complex organic molecules is often plasmid encoded Table 08.04 The real scary thing about plasmids is that many of them have the above traits AND are conjugative as well Conjugation is brought about via information stored on fertility plasmids (= conjugative plasmids)… which contain genes for: 1. The F pilus 2. Genes to mobilize the plasmid (Transfer factors) 3. An origin of replication See figure 8.22 (Later in lecture…..) Virulence Plasmids - there are a number of ways that a plasmid can confer virulence in a bacterium. 1) The production of one or more toxins that can be directed toward the host or towards other bacteria (bacteriocins). 2) The ability to form a capsule. The recent anthrax scare is an example: Virulent B. anthracis have 2 plasmids that encode for toxin production and capsule formation. An avirulent strain used for veterinary vaccination lacks the capsule forming plasmid. Other virulence factors on plasmids: The production of siderophores that enable the bacterium to scavenge iron in the body (e.g. S. aureus). Adhesins - proteins or glycoproteins that are usually a component of capsules or fimbriae that allow the bacterium to adhere to specific cells. Fig. 8.18. Conjugation - transfer of the F plasmid. Note that both cells are F+ after the mating takes place….. Fig. 8.19. Hfr formation via integration of the F-plasmid into the host chromosome at specific insertion sequences (ISs) Fig. 8.20. Conjugation involving an Hfr cell. Basically the whole chromosome is now a giant F plasmid - but the whole thing is rarely transferred.. Fig. 8.21. An F’ plasmid results if some chromosomal DNA is excised when the plasmid pops back out…. Fig. 8.22. The regions of an R Plasmid. RTF = resistance transfer factors R= resistance genes Plasmids and a type of conjugation are also involved in the transfer of genetic information from bacteria to some Eucaryotes. Remember Agrobacterium tumefaciens (a Gm-, alphaproteobacterium related to Rhizobium Fig. 11.2) has a Ti plasmid (tumor inducing plasmid) that codes for: 1. A pilus 2. Transfer factors 3. Transferred DNA (T-DNA) that codes for plant hormone production and the production of opines 4. Metabolic genes that allows A. tumefaciens to eat opines Persp. 8.2, pg. 211 Movement of DNA within cells e.g. transposons Table 8.3 Transposition involves small segments of DNA (transposons, "jumping genes") that can move around chromosomes or plasmids. e.g. F-plasmids have insertion sequences that allow the plasmid to integrate into the chromosome => Hfr cell. Insertion sequences are very simple and typically contain only the information needed for insertion (see figure 8.24). Also used in experiments to disrupt specific genes. Transposons (Composite Transposon in Figure 8.24) are more complex and may contain a number of genes that confer e.g. antibiotic resistance and/or toxin production. Fig. 8.23. Movement of a transposon through a bacterial community. Fig. 8.24 a. Insertion sequence, b. Composite transposon containing an antibiotic resistance gene…. Fig. 08.24