MICR5846 L6: Core and Accessory Genome II 8/31/25 P22/39 UNFINISHED

What is this?

-Viruses infecting bacteria

-Protein Coat, Nucleic Acid genome, Tail

Bacteriophages

What components belong to the protein coat of a Bacteriophage?

-Head

-Capsid

What is this?

-Lytic phage

-Lyse and kill host cells

-Virulent phages

What is this?

-Lysogenic

-Remains in host cell for period without killing it

-Temperate phage

What options do Lytic phages have?

1) Multiply within the host cell, released via lysis

What options do lysogenic phages have?

1) Multiply like virulent phages and kill the host cell

2) Remain in the host cell without destroying it

What type of phage is T4?

Lytic phage

What type of phage is Lambda?

Lysogenic phage

What is the first step of the Lytic Cycle in T4 phage of E. coli?

T4 adsorption and DNA injection mediated by tail fibers and base plate

What components of the phage protein coat mediate T4 adsorption/DNA injection during the Lytic Cycle?

-Tail fibers

-Base plate

What happens during the Lytic Cycle of T4 phage after this?

-T4 adsorption and DNA injection mediated by tail fibers and base plate

Tail fibers attach to LPS, Outer Membrane proteins of E. coli

What do the E. coli tail fibers attach to during the Lytic Cycle?

-LPS

-Outer Membrane proteins

What happens during the Lytic Cycle of T4 phage after this?

-Tail fibers attach to LPS, Outer Membrane proteins of E. coli

-Sheath contracts

-Pushes central tube through cell wall

What happens during the Lytic Cycle of T4 phage after this?

-Sheath contracts and pushes central tube through cell wall

DNA injected into cell via a pore through cytoplasmic membrane

What happens during the Lytic Cycle of T4 phage after this?

-DNA injected into cell via a pore through cytoplasmic membrane

-Phage chromosome is replicated by the host

-Bacterial chromosome is broken down

What happens during the Lytic Cycle of T4 phage after this?

-Phage chromosome is replicated by the host and bacterial chromosome is broken down

Phage genes encoding structural proteins are expressed in large numbers

What happens during the Lytic Cycle of T4 phage after this?

-Phage genes encoding structural proteins are expressed in large numbers

Phage progeny assembly within the host cell

What happens during the Lytic Cycle of T4 phage after this?

-Phage progeny assembly within the host cell

Host lysis releases progeny into the environment

What is this?

-Assembly

-Phage uses the bacterial cell as a factory, to produce copies of itself

-Items made separately, then joined together

True or False: ATP is not necessary for DNA to enter the phage head

False

Where does the ATP energy for DNA entering the phage head come from?

Host cell metabolism

What happens once a lysogenic phage genome enters a host cell and circularizes?

1) Enter lytic cycle

2) Integrate into genome, become prophage

What is this?

-Lysogen

Host cell with a lysogenic phage

What influences the "decision point" at which a phage either enters the lysogenic or lytic cycle?

-Environmental signals

-Abundance of nutrient

What happens first during the Lysogenic Cycle with a Lambda phage?

-Prophage may remain dormant for a very long time

-Genes will be repressed and not expressed

What happens during the Lysogenic Cycle with a Lambda phage after this?

-Prophage may remain dormant for a very long time, genes are not expressed

Phage will be passed to all daughter cells of the host through genome replication

What happens during the Lysogenic Cycle with a Lambda phage after this?

-Phage will be passed to all daughter cells of the host through genome replication

-Expression of prophage genes is induced

-Phage genome is now excised through recombination

-Can reenter lytic cycle again

What are some conditions that would promote induction of the Lysogenic cycle?

Host DNA damage, antibiotics, host stress

How can we find evidence of phages in bacterial genomes?

-Find homologs of known phage genes

-Multiple prophages are usually present

-70% bacterial genomes contain prophages

Name two reasons why prophages accumulate in bacterial genomes

1) Bacterial evolution

2) Phages often encode virulence factors

True or False: Prophages are molecular time bombs because induction will inevitably lead to lysis/cell death

True

How do prophages contribute to fitness of host population?

-Contributes to HGT

-Transduction allows host to acquire new genes

-Genes can provide evolutionary advantage

What is this?

-Transfer of bacterial genes inside a phage head due to an error in prophage excision

Transduction

What happens as a result of phage infection via Transduction?

-Bacterial DNA is packaged inside phage capsids

-Bacterial genes are transferred to recipient

-Recombination adds bacterial DNA to recipient chromosome

True or False: The phage particle after transduction will only contain bacterial DNA and no phage DNA is transferred

True

What happens as a result of errors made while cutting the prophage out of the host (Specialized Transduction)?

-Phage genome now carries adjacent host DNA

-Packaged into head to create transducing particles

-Prophage with both viral/donor DNA

What bacterial genes can be transferred during Specialized Transduction?

Adjacent bacterial genes

What happens after Specialized Transduction when recombination integrates the bacterial genes into the recipient genome?

Leaves behind intact copy of phage genome

Which prophage toxins are the best studied?

-Botulinum

-Cholera

-Diphtheria

-Shigella

Which phages contribute to biofilm formation?

-B. anthracis: Sigma factors

-P. aeruginosa: Prophages

-Neisseria: MDA phage

How do most species induce prophages and release biofilm component?

Release eDNA

What is the advantage of a bacterial host keeping its virulence genes on a phage genome?

-Rapid spread via phage infection of new host cells, lysogenic conversion

-Better environmental resistance and survival inside a phage head

How do bacteria evade phage infection?

1) Altering phage binding site on bacterial surface

2) Restriction-modification

3) CRISPR-Cas

How do bacteria alter the phage binding site on the bacterial surface to evade phage infection?

-Mutation

-Capsule switching

What bacterial species use capsule switching to alter their phage binding site?

-Acinetobacter

-Klebsiella

What is this?

-Restriction-Modification

-Methylase methylates specific motifs to protect bacterial DNA

-Incoming foreign DNA is not methylated, gets cleaved by RE

True or False: Restriction enzymes that cleave foreign DNA in Restriction-Modification systems will target the same bacterial motifs as the methylase

True

What is this?

-CRISPR Cas Systems

-Adaptive defense mechanism to protect bacteria against invaders

-Target and destroy foreign nucleic acids

What are some foreign nucleic acids that CRISPR-Cas might target and destroy?

Phage genomes, plasmids, Tns and other bacterial DNA

What is this?

-CRISPRs (Clustered regularly interspaced short palindromic repeats)

oh god what a mouthful

-DNA loci with sequences and spacer DNA

-Cas genes

-Leader sequence

What sequences are found in the CRISPR DNA loci?

-Short repetitive sequences

-Short non-repeating spacer DNA

What is the function of the 4-20 Cas genes of CRISPR?

-Helicase activity

-Unwind DNA so nuclease can cut it

What is the purpose of the CRISPR leader sequence?

-Contains promoter

-Induces transcription of CRISPR array

What happens first when Adaptation incorporates foreign DNA into the CRISPR locus?

Foreign DNA enters the cell

What happens during Adaptation for CRISPR after this step?

-Foreign DNA enters the cell

-Cas complex scans the incoming DNA

-Finds sequence similar to PAM

-Cas1-Cas2 complex binds the protospacer region

What is this?

-Protospacer Associated MOTIF (PAM)

-Short, specific DNA sequence located upstream of target DNA

-If sequence is similar to it, Cas1-Cas2 will bind protospacer region

What complex will bind the protospacer region if a similar sequence to PAM is found?

Cas1-Cas2

What happens during Adaptation for CRISPR after this step?

-Cas complex scans the incoming DNA for similar sequences to PAM, and Cas1-Cas2 binds protospacer

-New spacer DNA is copied from the foreign DNA

-Added to repeat-spacer array with new repeat unit

What is the first step of CRISPR-Cas Maturation, allowing CRISPR to function in immunity?

Entire array locus is transcribed as single pre-crRNA

What happens during CRISPR-Cas Maturation after this step?

-Entire array locus is transcribed as single pre-crRNA

-Each repeat unit is cleaved from the pre-crRNA

-Forms mature crRNA

What is a mature crRNA called after a repeat unit is cleaved from pre-crRNA?

Guide RNA

What happens during CRISPR-Cas Maturation after this step?

-Each repeat unit is cleaved from pre-crRNA to form a mature crRNA (guide RNA)

-crRNA is bound by Cas proteins

-Forms ribonucleoproteins (RNPs)

What is this?

-Interference

CRISPR-Cas RNP prevents entry by foreign DNA

What is the first step of CRISPR-Cas Interference?

-crRNAs act as antisense guides

-Direct the RNP to incoming DNA

What proteins act as antisense guides to direct RNP towards incoming/foreign DNA?

crRNAs

What happens during CRISPR-Cas Interference after this step?

-crRNAs act as antisense guides which direct the RNP to incoming DNA

Nuclease domain of the RNP cleaves the incoming DNA

What domain of the RNP is responsible of cleaving the incoming DNA?

Nuclease domain

What happens during CRISPR-Cas Interference after this step?

-Nuclease domain of the RNP cleaves the incoming DNA

-Target DNA is degraded

-Prevents infection by phages, plasmids, etc

What is the most well-known of the 6 CRISPR-Cas system types?

Type II (CRISPR-Cas 9)

What proteins are used to guide the Cas9 nuclease in T2SS?

-TracrRNA

-crRNA

What relationship do TracrRNA and pre-crRNA have to each other in T2SS?

TracrRNA is complementary to the repeat regions of pre-crRNA

What protein is responsible for binding the tracrRNA-crRNA complex and initiating cleavage in T2SS?

Cas9

What enzyme is responsible for initiating cleavage and generating crRNA guides in T2SS?

RNase

What sequences does Cas9 recognize on target DNA, allowing the tracrRNA/crRNA complex to pair with cDNA?

PAM sequences

What happens after the tracrRNA/crRNA complex pairs with cDNA in T2SS?

Cas9 nuclease cleaves target DNA