BIO 143 Week 4 Part 2 - Fall 2018 PDF

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Week 4 part 2 lecture notes...

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BIO 143 - SEA PHAGES Laboratory II Week 4 Part 2 Further Discovery An Overview: Characterizing Patterns of Phage Sensitivity Repressors ● Immunity repressors are highly specific for their operator and stoperator sequences. ● Even a single base change in the operator/stoperator sequence can alter or prevent binding to the target DNA. ○ Therefore, phages that cannot infect a lysogen, because of actions of the immunity repressor, share at least some genomic similarity to the prophage’s operator/stoperator sequences. ■ Frequently, the inability of two different actinobacteriophages to infect each other’s lysogens, called homoimmunity, is a property shared by members of the same subcluster. ● It is important to note that lysogens are only considered “immune” to superinfection if the immunity repressor is the mechanism by which infection is prevented.

Exclusion ● In addition to the immunity repressor protein, some phages express proteins that can alter the surface of the bacterial cell. ○ When these proteins or carbohydrates appear on the bacterial cell surface, the prophage can prevent other phages from binding to the cell and injecting their DNA. ● This ability is called surface exclusion.

○ Unlike immunity, which is generally restricted to genomically related phages ■

exclusion depends on interactions between the tail fiber(s) of the infecting phage, which is the primary means of attachment to the cell, and the receptor on the surface of the lysogen. ●

Tail fibers are highly mosaic and modular ○ and they may or may not be shared among phages that are otherwise very similar at the genomic level.

Other Factors ● Finally, it is important to note that not that every phage infection of a bacterial population will yield intrinsically insensitive host cells. ○ There have been cases of transient insensitivity owing to the stage of a bacterial cell cycle as well as permanent resistance caused by mutation. ■ A liquid culture of bacteria in stationary phase likely has 108–109 cells per milliliter! ■ A mutant present in the culture at only a few tenths of a percent will rapidly become the dominant strain when the bulk of the cells in the culture are destroyed by phage infection. ● So how do you tell the difference between a true lysogen and a naturally resistant bacterial cell in the lab? ○

Lysogens will be insensitive to superinfection from all subsequent attempts at infection with the initial phage— ■ no matter how many times the lysogen is streak-purified or regrown from a liquid stock. ● Also, liquid cultures of lysogens will release phage into the supernatant of the culture.

● This is because some of the cells in the population are undergoing spontaneous induction of the lytic cycle at all times. ○ And finally, the gold standard for lysogen confirmation is to demonstrate that the prophage is in the cell. ■ This can be done using a technique called polymerase chain reaction (PCR) and amplifying the new junction between the bacterial and phage genomes. ■ Or confirmation can be done by carefully repeating rounds of purification and then sequencing of all ● of the DNA in the cell to look for the presence of the prophage genome. ● It is possible to isolate lysogens by carefully purifying them away from exogenous infecting phage. ○ Once isolated, lysogens can be used to create bacterial lawns to test the ability of other phages to infect the new lysogen. ■

This immunity data can be helpful in characterizing a whole collection of sequenced or unsequenced phages. ● It is likely that temperate phages carry a variety of genes that are expressed in lysogens and influence phage sensitivity. Therefore, exploring these patterns can provide valuable insights into phage mechanisms of bacterial sensitivity and insensitivity to bacteriophage....