Micro - E4 Flashcards
Bacterial virus
Bacterial virus = phage = bacteriophage
- Genome can be double-stranded or single-stranded DNA or RNA
- Capsid: protein coat
- Envelope: lipid coat (some may have this)
Two Classes of bacteriophages
- Virulent or Lytic: results in cell lysis (cell death) and production of many progeny phage particles.
- Temperate or lysogenic: can either cause the cell to lyse and produce more progeny phage particles or can lead to maintenance of the virus genome within the living host cell resulting in a dormant state (lysogeny)
Lytic Growth Cycle
1) Adsorption (attach to receptors on bacterium)
2) Penetration (injection and membrane fusion – if have envelope)
3) Gene expression
4) Nucleic acid replication
5) Synthesis of structural proteins
6) Assembly of virons
7) Lysis of bacterium and release of progeny phage
Liberation of progeny bacteriophage
Lysis of the bacterial host and release of the newly synthesized bacteriophages coincides with activation of degrative enzymes that destroy the cytoplasmic membrane and the peptidoglycan cell wall.
- These enzymes are produced as “late proteins”
T7 early genes (timing?) (transcribed by which polymerase?)
code for proteins required for viral replication (e.g., a specific DNA polymerase or polymerase component) (1-12 mins after phage injects its genome into the cytoplasm)
E. coli “host” RNAP recognizes early promoter and transcribes early mRNA, but doesn’t recognize late promoters.
T7 late genes (timing?) (transcribed by which polymerase?)
code for the structural components (capsid, tail fibers) of the virus, and the lysis proteins (8minutes-end)
Transcribed by T7 polymerase
Why does it take 8 minutes before the T7 late transcripts start to be synthesized?
phage polymerase, “T7 polymerase”, is the product of an early gene, called gene 1. (specific for the phage late genes because it only recognizes the late promoters)
takes ~8 minutes to transcribe the gene 1 gene and translate the gene 1 mRNA to make functional T7 polymerase proteins.
Why does transcription of early T7 genes (and host genes) cease during late gene expression?
product of the late gene 2 (Gp2) binds to E. coli RNA polymerase holoenzyme and totally inhibits its activity.
–> shuts off all RNA synthesis from bacterial host and early phage promoters.
Kinds of transcription terminators at end of early T7 mRNA at TE?
Both factor-dependent (has rho-attached to RNAP and factor-independent)
Temperate vs. Lytic Phage
- Temperate: undergoes lytic or lysogenic life cycle (exp. Bacterial lambda)
- Lytic (virulent phages): always undergo lytic growth cycle, cannot “lysogenize” (a repressed viral state with viral genome inserted into host chromosome, ensuring stable inheritance & maintenance)
Inherited phage genome
“prophage” or “provirus”
Cell containing a prophage
lysogeny
N protein
associates with RNAP and causes it to be blind to terminators (a transcriptional “antiterminator”) allowing RNA polymerase to bypass the EARLY terminators
Q-protein
an antiterminator of transcription from Plate. Late genes encode phage structural components and the host lysis enzymes. (once you read through terminator, DESTINED for lysis)
Which gene was not expressed during bacteriophage lambda lytic growth?
cI –> repressor of lytic growth
Recall: protein that binds to DNA at a site within or downstream of a promoter site –> blocks transcription from that promoter by competing with RNA polymerase for interaction with the DNA.
To choose the lysogenic response, the phage needs to do two things quickly
o synthesize a high concentration of CI repressor
o synthesize the Int protein, a DNA recombination protein, that integrates the phage genomic DNA into the bacterial host chromosome at a specific site.
CI gene is transcribed from two promoters:
PRE (promoter for repressor establishment)
PRM (promoter for repressor maintenance)
CI protein
CI –> repressor that binds to PL and PR –> shuts off all early and middle gene expression of the phage (preventing lytic infection)
CII protein
CII –> transcriptional activator that binds to Pre (promoter for repressor establishment), acts as + regulator, leads to high levels of CI repressor (λ repressor) expression.
In order to maintain repression of the lytic cycle, phage λ has a second mechanism for synthesizing the repressor CI…
another promoter, PRM (promoter for repressor MAINTENENCE)
requires the λ CI repressor to act as a positive regulator for its own synthesis. (C1– negative reg of pL and pR but + regulator of its own synthesis)
Conditions which bacteriophage lambda undergoes lytic growth
RICH MEDIUM –> HIGH PROTEASE –> CII degraded–> phage undergoes LYTIC GROWTH
Conditions which bacteriophage lambda undergoes lysogeny
POOR MEDIUM –> LOW PROTEASE –-> CII activates Repressor Promoter –>Phage undergoes LYSOGENY
How prophages escape from lysogenic state
INDUCTION (Elicted by DNA damage through the “SOS response”)
-The return to the lytic cycle can only occur if the repressor protein self-destructs or autoproteolyzes, which occurs upon DNA damage.
How does SOS response affect a prophage?
DNA damage activates RecA.
Activated RecA triggers temperate phage repressors to self-destruct (autoproteolyze).
Loss of phage receptor –> initiation of prophage excision (imprecise) from bacterial chromosome and subsequent lytic growth.
- DNA damage “activates” the cellular protein RecA, which, in turn, interacts with certain bacterial repressors, like LexA (represses expression of binding of DNA repair genes) and causes them (LexA) to self-destruct. This is termed the “SOS” response.
- No LexA –> SOS DNA repair genes are expressed
- int and xis: carry out a recombination reaction that removes the phage DNA from the chromosome as a circle that is identical to the original phage DNA molecule that had previously integrate
imprecise excision or “misexcision” of the prophage
Int and Xis proteins recombine sequences within phage genes and nearby adjacent bacterial chromosome sequences (production of circles DNA containing mostly phage and some bacterial DNA.)
Specialized transduction
transfer of host bacterial chromosomal DNA limited to the flanking regions of a prophage integration site that was misexcised and packaged in phage particles and delivered to another bacterium. *ONLY TEMPERATE phages can do this
Lysogenic conversion
acquisition of a new property (phenotype) by a host bacterium due to establishment of lysogeny by a temperate phage
exp. Shiga-like dysentery toxins produced by E. coli O157:H7 and its relatives are encoded on λ-like temperate phages
Process of expression of Shiga-like dysentery toxins produced by E. coli O157:H7
Certain abx –> bacterial DNA damage –> activated RecA* protein –> phage repressor undergoes autoproteolysis –> phage induced (expression of toxin genes stx A and B, enhanced by Q protein and expressed upon induction of the prophage)
3 phage resistance mechanisms
- Alter phage receptor (alter surface proteins used by virus to adsorb)
- Restriction modification systems (Degradative endonucleases (restriction enzymes) cleave or “restrict” phage DNA genomes.)
- CRISPR system: bacterial mechanism of adaptive immunity against invading nucleic acids (bacteria cleaves foreign DNA, can prevent any attacks from bacteriophages that they have previously encountered).
Pseudomonas aeruginosa
Gram-negative, highly motile, rod-shaped bacterium.
Adheres tightly to lung epithelium.
Produces extracellular polysaccharide (mucoidy).
Primary cause of death in CF patients.
Pseudomonas infection associated with cystic fibrosis
Sx: Bronchiectasis (abnormally dilated airways)
tx: IV abx therapy was initiated with a cephalosporin (Ceftazidime) and an aminoglycoside (Tobramycin)
Gram-negative bacterial envelope structure
Inner and outer membrane with periplasmic space between (periplasmic space: Hydrolytic enzymes, Chemoreceptors, Transport proteins)
Gram negative outer membrane
Phospholipid in inner leaflet
-Barrier to hydrophilic compounds.
LPS in outer leaflet
- Negatively charged surface
- Resistant to detergents and other hydrophobic molecules
PORES - allow entry of SMALL (< 700 MW) hydrophilic nutrient molecules through outer membrane
How is LPS (endotoxin or O-Ag) a potent antigen
Innate response: cytokine production, septic shock
Adaptive response: serotyping of strains, e.g. E. coli O157:H7
Adhesins
- on microbial cell surface
- bind to sugars or proteins on the host cell surface
- can be very specific: some bind only to a single type of epithelial cell in a single animal species
- some areas (human mouth) cell receptors and the adhesins that bind them vary from one tiny area to another
Adhesive structures of bacteria
- pilli (fimbriae)
- flagella
- capsules (see in mucoid material)
*PA has flagelli and pilli
Why are flagella are critical for P. aeruginosa infection mucous layer
Flagella act as adhesins and allow the bacteria to swim through the mucous layer and attack the intestinal epithelium
Can be polar or all over cell (peritrichous)
P. aeruginosa has a single, polar flagellum.
Bacterial chemotaxis and motility are controlled
by…
“two-component” regulatory systems
(1) membrane-bound sensor histdine kinase detects specific molecules in envio and (2) a cytoplasmic response regulator that is activated by the kinase, causes the flagellar motor to turn clockwise (tumbling movement) or counterclockwise (directed movement).
The phosphorylated response regulator binds to the flagellar motor and determines its rotational mode.
Capsules
- Loose network of polymers surrounding cell
- Polysaccharide or protein or both
Functions:
Resists drying
Promotes adherence
Protects against phagocytosis
exp. mannuronate-guluronate co-polymer (alginate of Pseudomonas)
Important Encapsulated Pathogens
Streptococcus pneumoniae
Haemophilus influenzae
Neisseria meningitidis
Pseudomonas aeruginosa
Encapsulated bacteria often capable of causing septicemia and meningitis
Streptococcus pneumoniae
Haemophilus influenzae
Neisseria meningitidis
What contributes to intractable nature of Pseudomonas lung infection
Capsule- resistance of drying, promotion of adherence, protection against some environmental stresses.
viscous mucus of CF patients, biofilm formation
^contribute to intractable Pseudomonas lung infection.
Role of alginate production (mucoidy) in P. aeruginosa infection of CF patients
Alginate (mannuronate-guluronate co-polymer) – extracellular polysaccharide excreted by P. aeruginosa (there are non-mucoid phenotypes of P. a)
Bacteria that produce large amounts of alginate are said to be “mucoid”
Alginate coats the bacterial cells forming a capsule-like structure
Alginate capsule protects P. aeruginosa against host defenses and antibiotics
What controls production of alginate?
Alginate production depends on AlgU (alternative sigma factor) for transcription of alginate genes
MucA
MucA –> negative regulator of AlgU (sigma factor).
MucA normally holds Algu in an inactive state –> alginate genes not expressed
Cells sense cell wall stress –> MucA degraded by a protease in periplasm –> AlgU is freed and activated –> alginate genes expressed
Why is there so much alginate in the lungs of CF patients?
Lungs of CF patients accumulate mutant strains of P. aeruginosa that produce a truncated inactive MucA
AlgU –> always active –> high levels of alginate produced = mucoidy.
Biofilms
thick layers of the extracellular polysaccharide matrix in which the bacterial cells are embedded
Protected from abx, if burst, can release other bacteria –> spread infection
When ready for release, start to express Fla and Pil
How do bacteria know when to form a biofilm?
Intercellular Signaling (generally associated with population crowding)
- Within a species or across species lines
- Within populations of dispersed cells or in communities of cells
Quorum sensing signaling compounds
Acylated homoserine lactones (Gram-negatives)
- generated and sensed by bacteria, lipophilic so can diffuse out of cell
- if P. a mutants are unable to make homoserine lactone –> defective in biofilm formation
Short peptides (Gram-positives)
- produced intracellularly, longer precursors are then modified as they’re pumped out of cytoplasmic membrane
- signaling peptides are taken into producer cell or other cells in vicinity by peptide transporters
Two-Component signal transduction system mechanism of regulating transcription (P. aeruginosa)
In absence of phosphorylated response regulator, unstable recognition of promoter -> no transcription of biofilm genes
In the presence of phosphorylated response regulator –> binds to DNA just upstream of RNAP and stabilizes RNAP binding to the promoter.
Crowded vs sparsely populated environments
In crowded environments –> lots of peptide –> pathogenic pathway genes turned on
In sparely envio –> peptides away from bacterium –> will not turn on bacterial genes
quorum sensing
Cell is sending out a signal that it then monitors. The signal can also be sensed by other bacteria of the same or related species.
In some cases –> inducer must be acted upon by some environmental factor before it can be sensed by the cell.
Other cases –> signal sent out is sensed without alteration. Cell is simply monitoring the concentration of the compound in the environment, not whether or not the inducer has been modified.
Sensing the concentration of a compound that is secreted by cells is called “quorum sensing.”
What are two features that distinguish Gram-positive from Gram-negative bacteria?
Gram-negative bacteria have an outer membrane and a periplasmic space, neither of which are found in Gram-positive bacteria.
Why is LPS not considered a classical toxin? How does it trigger toxic reactions?
The outer membrane, known as LPS or endotoxin. It is not a classical toxin because it is not a protein and does not have enzymatic activity. It causes a toxic reaction because when administered in high doses, it causes an outpouring of inflammatory cytokines.
What is a biofilm? Does their formation at infected sites affect treatment efficacy?
A biofilm is an architecturally complicated structure consisting of thick layers of the extracellular polysaccharide matrix in which the bacterial cells are embedded. Biofilm infections are particularly difficult to treat and often require physical removal for cure.
Two component regulatory systems are ubiquitous in bacteria. What are the two components and what do they do?
The component that senses the environment is a sensor histidine kinase. The sensor domain of this kinase is in the extracellular space, where it can receive environmental signals. The kinase domain is in the intracellular space, where it can phosphorylate a response regulator (2nd component of the two component system). The response regulator controls the expression of many genes and its DNA binding activity is altered by phosphorylation.
RNA viruses
Picornaviruses and Myxoviruses
