BIOL402: Bacteriophage Structure and Diversity, T4 Replication

E3

order of tailed viruses:

Caudiovirales

family of contractile tailed viruses;

myoviridae

families of noncrontractile tailed viruses;

siphoviridae

podoviridae

families of ss DNA viruses:

microviridae: groups microviridae and gokushoviridae

inoviridae: groups inoviridae and plectoviridae

virus family which enters a cell by endocytosis:

cystoviridae

what are crAssohage?

a core phage in the human gut microbiome which was discovered with cross-assembly metagenomic analysis

How does phage diversity in the human gut change over a lifetime?

the diversity decreases but the stability increases

How does IBD impact phage in the gut microbiome?

phage diversity decreases and variability between individuals increases

bacteriophage replicative cycle:

attachment

penetration

biosynthesis

maturation

lysis

T4 bacteriophage classification

Order: Caudiovirales

Family: myoviridae

How does T4 attach to E. coli?

O specific sidechains on the bacteriophage bind to O subunit of LPS reversibly

fibritin function:

causes long sidechains to retract up the tail to inhibit premature binding

short tail function:

interacts with core polysaccharide in LPS to irreversibly bind

How does the T4 bacteriophage penetrate the outer membrane?

the outer layer of the sheath moves to contract and the inner sheath penetrates the outer membrane

How does the T4 bacteriophage penetrate the peptidoglycan layer?

lysozyme is present at the tip of the inner sheath and breaks peptidoglycan, allowing the inner sheath to fully penetrate the inner membrane

When does biosynthesis occur?

5 minutes after penetration

when are early, middle, and late genes expressed in a T4 bacteriophage?

early: 1 min

middle: 7 min

late: 18 min

when does host cell degradation occur?

6-25 minutes after infection

function of T4dCTPase:

converts dCTP to dCMP to differentiate between host and bacteriophage DNA

function of HMase:

converts dCMP into HMdCMP

how is host DNA degraded?

endonucleases are cytosine specific and introduce nicks at these sites, allowing denB to digest DNA and create a pool of nucleotides to replicate phage genome

temporal gene expression:

time dependent DNA replication, translation, and expression

what type of promotors do T4 genomes have?

-35 box, -10 box, up elements

Alt protein function:

ADP ribosyltransferase which targets CTD of alpha subunits in RNAP to impair binding to bacterial promoters in the up element and enhance recognition of T4 early genes

ModA and ModB are ____ proteins with ADP ribosyltransferase activity

early

ModA function:

acts on both alpha subunits and is highly toxic to E. coli

ModB function:

alters host transcription factors

middle gene expression depends on products _______ and ________

AsiA/ModA

AsiA function:

binds sigma70 and converts it from a helix-turn-helix to a contunuous helix to prevent binding to the -35 box

how does ModA function during middle gene expression?

it binds helix 5 of sigma70 and is a transcription factor which recruits AsiA modified RNAP to phage middle promoters

proteins critical for late gene expression:

GP55 and GP33

GP55 function:

recognizes AT rich regions around the -10 box to recognize late promoters

GP33 function:

represses basal transcription by reducing binding to the promoter

concatomer-

a segment of DNA which contains multiple genomes due to rolling circle replication

R loops:

the initial loop formed in T4 replication between complementary bp in mRNA

D loops;

secondary stage of T4 replication when a 3rd DNA strand binds mRNA

when does maturation occur?

12 min after penetration

terminase complex function:

binds end of T4 genetic material and associates with portal ring to feed genome into a capsid

terminase complex structure:

TerL and TerS

TerL function:

ATP dependent motor which has terminase activity to cleave a DNA strand after the capsid contains one whole genome

TerS function:

increases TerL protein

release proteins:

E and T

release protein T function:

causes lesions in the cell membrane

release protein E function:

an endolysin

when does release occur?

30 min after penetration

how many bacteriophage are released per infected E. coli cell?

100-150