Microbiology Exam 2

eukaryote vs prokaryote transcription

• Eukaryotes • Each gene is transcribed individually into a single mRNA. • Replication and transcription occur in nucleus. • RNAs must be exported outside nucleus for translation

Prokaryotes • Multiple genes may be transcribed in one mRNA. • Coupled transcription and translation occur (Figure 4.7), producing proteins at maximal rate.

Do all organisms follow the central Dogma

No, Some viruses can violate it

Do bacteria and archaea have circular or linear genomes

Most Bacteria and Archaea have single circular chromosome carrying all/most genes.

Do Eukaryotes have circular or linear chromosomes?

Two linear

Transcription is carried out by what

DNA polymerase

What does RNA polymerase use as a template

RNA polymerase uses DNA as template

RNA polymerase recognizes DNA sites called ___

Promotors

Promotors are recognized by what part of RNA polymerase

Recognized by sigma factor of RNA polymerase

Transcription stops at specific sites called ___

Transcription Terminates

Promoters

site of initiation of transcription

Sigma factor of RNA polymerase recognizes initiation sites on DNA called promoters. What are the bacterial promotors

Pribnow box (–10 region) and TTGACA (– 35).

Termination of RNA synthesis is governed by a specific DNA sequence. What are the two terminator sequences in bacteria

Intrinsic terminators: transcription is terminated without any additional factors

Rho-dependent termination: Rho protein recognizes specific DNA sequences and causes a pause in the RNA polymerase

Operons are transcribed into a single mRNA called a polycistronic mRNA (Figure 4.23) containing multiple open reading frames that encode amino acids. Say True.

True

Operon:

cluster of genes arranged in a linear fashion whose expression is under control of a single promoter

Structural genes

the genes that are co-regulated by the operon.

Negative control involves the binding of a repressor to the operator to prevent transcription. Say True

True

Positive control involves an activator protein stimulating transcription by binding to DNA (usually at a site other than the operator). Say False

False

Most important recognition sequence is ___.

“TATA” box

Splicing

removing introns and joining exons

What does splicing in Eukaryotes

Spliceosome

What does splicing in Archaea and is splicing rare or common

Rare and must be done by special ribonuclease

Translation is broken down into three main steps:

1. Initiation: two ribosomal subunits assemble with mRNA Begins at an AUG start codon

Elongation: amino acids are brought to the ribosome and are added to the growing polypeptide Occurs in the A and P sites of ribosome Translocation: movement of the tRNA holding the polypeptide from the A to the P site

3. Termination: occurs when ribosome reaches a stop codon Release factors (RF): recognize stop codon and cleave polypeptide from tRNA • Ribosome subunits then dissociate • Subunits free to form new initiation complex and repeat process

Polysomes

a complex formed by ribosomes simultaneously translating mRNA

Start codon

Starts Translation

Reading frame

triplet code requires translation to begin at the correct nucleotide

Shine–Dalgarno sequence

Just like TATA box ensures proper reading frame in bacteria

Anticodon

three bases of tRNA that recognize three complementary bases on mRNA

Gene expression

transcription of gene into mRNA followed by translation of mRNA into protein

What are the two major levels of regulation in the cell

One controls amount of an enzyme

One controls the activity of an enzyme

Homodimeric proteins

proteins composed of two identical polypeptides

Helix-turn-helix

First helix is the recognition helix

Second helix is the stabilizing helix

Zinc finger

Protein structure that binds a zinc ion • Typically two or three zinc fingers on proteins that use them for DNA binding

Leucine zipper

Leucine residues are spaced every seven amino acids • Does not interact directly with DNA

Multiple outcomes after DNA binding are possible what are three of these outcomes?

DNA-binding protein may catalyze a specific reaction on the DNA molecule (i.e., transcription by RNA polymerase) 2. The binding event can block transcription (negative regulation) 3. The binding event can activate transcription (positive regulation)

Negative control

involves the binding of a repressor to the operator to prevent transcription

Positive control

involves an activator protein stimulating transcription by binding to DNA (usually at a site other than the operator)

Transcription is physically blocked when repressor binds to operator Say true

true

Enzyme induction can also be controlled by a repressor

Addition of inducer can inactivates repressor and transcription can proceed

repression:

preventing the synthesis of an enzyme in response to sufficient amounts of a product

Inducer:

substance that induces enzyme synthesis

Corepressor:

Substance that helps to repress enzyme synthesis

Effectors

Collective term for inducers and corepressors

What do effectors do

Effectors affect transcription indirectly by binding to specific DNA-binding proteins

What do effectors bind to and what does that molecule in turn, bind to

Corepressor molecules bind to an allosteric repressor protein

Allosteric repressor becomes active and binds to the operator

When are enzymes synthesized

Enzymes are synthesized only when they are needed

Negative repressible operons:

The arg Operon of E. coli • The trp Operon of E. coli

Negative inducible operons:

The lac Operon of E. coli

Positive control:

regulator protein activates/triggers the binding of RNA poly

What do activator protiens bind to

Activator proteins bind specifically to certain DNA sequence

What doactivator protiens do

Activator protein helps RNA polymerase recognize promoter

Positive inducible operons

Operon is typically OFF, because activator proteins are normally unable to bind to the pertinent DNA. Binding of inducer to the activator protein, causes a change in conformation so that the activator proteins can bind to the DNA and activate transcription.

Positive repressible

Operon is typically ON, as activator proteins are normally bound to the pertinent DNA segment. If an inhibitor binds to the activator, it is prevented from binding the DNA. This stops activation and transcription of the system

Regulons also exist for negatively controlled systems Say true

true

What is a Global control system and what is one example

regulate expression of many different genes simultaneously the best example is the lac operon which determines the usage of different sugars if the lac operon is on there is nothing else there because nothing likes lactose due to its low energy quality; Catabolite repression

What is catabolite repression

an example of global control • Synthesis of unrelated catabolic enzymes is repressed if glucose is present in growth medium • lac operon is under control of catabolite repression • Ensures the “best” carbon and energy source is used first

Diauxic growth

two exponential growth phases

Cyclic AMP receptor protein is what in catabolite repression

The activator protien and a regulatory nucleotide

Flagellar genes are also controlled by ___

Catabolite repression

Archaea use DNA-binding proteins (repressor and activator proteins) to control transcription say True

True

Repressor proteins in Archaea

either block RNA polymerase binding or block binding of TATA binding protein (TBP) and transcription factor B (TFB), which are required for archaeal RNA polymerase to bind to its promoter

Some archaeal regulators act as both _____and _____ in a process known as dual functionality; THIS IS NOT POSSIBLE IN BACTERIA

Repressors and activators

Signal transduction

External signal detected by sensor and transmitted to regulatory machinery

Two-Component Regulatory Systems are made up of two different proteins what are they and what do they do:

Sensor kinase: (in cytoplasmic membrane) detects environmental signal and autophosphorylates Response regulator: (in cytoplasm) DNA binding protein that regulates transcription

Modified two-component system used in chemotaxis to

Sense temporal changes in attractants or repellents; Regulate flagellar rotation thus regulate activity of preexisting proteins instead of modifying transcription of genes

Three main steps of chemotaxis in dual regulatory systems

1. Response to signal 2. Controlling flagellar rotation 3. Adaptation

Is which way does the flagella move when running vs tumbling

counterclockwise rotation and runs

results in clockwise rotation and tumbling

What do feedback loops do after taxes? (movement towards or away from a stimulus)

Allows the system to reset itself to continue to sense the presence of a signal • Involves modification of MCPs

Phototaxis:

Movement toward light

Aerotaxis

Movement toward oxygen

Quorum sensing

mechanism by which bacteria assess their population density

Why does quorum sensing exist?

Ensures sufficient number of cells are present before initiating a response that requires a certain cell density to have an effect (e.g., toxin production in pathogenic bacterium)

What allows bacteria to do quorum sensing

Each species of bacterium produces a specific autoinducer molecule

\Diffuses freely across the cell envelope • Reaches high concentrations inside cell only if many cells are near • Binds to specific activator protein and triggers transcription of specific genes

What are some examples of quorum sensing

P. aeruginosa switches from free living to growing as a biofilm

Escherichia coli O157:H7 (Figure 6.22) • shiga toxin–producing strain • produces AHL AI-3 that induces virulence genes • Epinephrine plus norepinephrine plus AI-3 bind to sensor molecules in plasma membrane. • activates motility, toxin secretion, and production of lesion-forming proteins

Why are quorum sensing disruptors useful?

are potential drugs for dispersing biofilms and preventing virulence gene expression.

Stringent response:

used to survive nutrient deprivation, environmental stress, and antibiotics • shuts down macromolecule synthesis and activates stress survival pathways

Examples of stringent responses

voiding E. coli in feces reduces nutrients, initiates ppGpp synthesis, stringent response occurs

Example: Caulobacter stringent response triggered by carbon/ammonia starvation instead of amino acid limitation • ppGpp increases swarmer (motile) cell formation, may reach a niche with more nutrients (Figure 6.24b)

Example: Mycobacterium tuberculosis • lungs hypoxic and phosphate-limited, triggering stringent response • converts a subpopulation of dormant persister cells resistant to antibiotics that can revert back to infective cells

The phosphate (Pho) regulon

P essential for DNA, RNA, membrane synthesis, energy generation, ad cell signaling

inorganic phosphate (PO4 3– or Pi ) often limiting in environment

two-component regulatory system regulating extracellular enzymes, Pi transporters, and storage enzymes (Figure 6.25)

represses many genes and controls some aspects of pathogenesis

Heat shock response

Global network heat shock proteins: counteract damage of denatured proteins and help cell recover from temperature stress

Growth:

: increase in the number of cells

Binary fission:

cell division following enlargement of a cell to twice its minimum size

Generation time:

time required for microbial cells to double in number

FtsZ

forms ring around center of cell; related to tubulin

ZipA

anchor that connects FtsZ ring to cytoplasmic membrane

FtsA

helps connect FtsZ ring to membrane and also recruits other divisome proteins

MreB

major shape-determining factor in prokaryotes

probably Archaea • Forms spiral-shaped bands around the inside of the cell, underneath the cytoplasmic membrane • Not found in coccus-shaped bacteria

Crescentin

shape-determining protein produced by vibrio-shaped cells of Caulobacter crescentus

Why are archaea more physically/morphologically similar to bacteria?

Because most archaeal genomes contain FtsZ and MreB-like proteins, thus cell morphology is similar to that seen in Bacteria

How does cell wall division vary in Cocci vs Rod shaped cells

In cocci, cell walls grow in opposite directions outward from the FtsZ ring

In rod-shaped cells, growth occurs at several points along length of the cell

Describe the severing of preexisting peptidoglycan for new peptidoglycan

Beginning at the FtsZ ring, small openings in the wall are created by autolysins • New cell wall material is added across the openings • Wall band: junction between new and old peptidoglycan

Bactoprenol

carrier molecule that plays major role in insertion of peptidoglycan precursors

Glycolases

enzymes that interact with bactoprenol

Insert cell wall precursors into growing points of cell wall • Catalyze glycosidic bond formation

Transpeptidation

final step in cell wall synthesis • Forms the peptide cross-links between muramic acid residues in adjacent glycan chains • Inhibited by the antibiotic penicillin

Exponential growth:

growth of a microbial population in which cell numbers double within a specific time interval During exponential growth, the increase in cell number is initially slow but increases at a faster rate

Batch culture:

a closed-system microbial culture of fixed volume

In a batch culture, growth conditions are constantly changing; it is impossible to independently control both growth rate and growth yield parameters

Typical growth curve for population of cells grown in a closed system is characterized by four phases:

Lag phase • Exponential phase • Stationary phase • Death phase

Lag phase • Interval between when a culture is inoculated and when growth begins

Exponential phase • Cells in this phase are typically in the healthiest state

Stationary phase • Growth rate of population is zero • Either an essential nutrient is used up or waste product of the organism accumulates in

Death Phase the medium • If incubation continues after cells reach stationary phase, the cells will eventually die

Continuous culture:

an open-system microbial culture of fixed volume

Describe a chemostat and the importance of choosing the right dilution rate and concentration

most common type of continuous culture device Has a sterile pump for new media and a output for old media

Both growth rate and population density of culture can be controlled independently and simultaneously • Dilution rate: rate at which fresh medium is pumped in and spent medium is pumped out • Concentration of a limiting nutrient

What are the two broad classes of media

Defined/minimal media: exact chemical composition known. • complex media: composed of digests of microbial, animal, or plant products (e.g., yeast and meat extracts

Describe Enriched media, Selective media and, Differential media

Enriched media • contain complex media plus highly nutritious materials (e.g., serum or blood) • used to culture fastidious (nutritionally demanding) microbes • Selective media • contain compounds that selectively inhibit growth of some microbes but not others • Differential media • contain an indicator, usually a dye, that detects particular metabolic reactions during growth

Limitations of microscopic counts

Cannot distinguish between live and dead cells without special stains • Small cells can be overlooked • Precision is difficult to achieve • Phase-contrast microscope required if a stain is not used • Cell suspensions of low density (<106 cells/ml) hard to count • Motile cells need to immobilized • Debris in sample can be mistaken for cells