Exam 2 Microbio

Lectures 8-15

“normal“ growth conditions

• sea level

• 20-40 C

• 0.9% salt

• ample nutrients

Environmental Limits on Microbial Growth

• temperature

• pH

• osmolarity

• oxygen

• pressure

Hyperthermophiles

Organisms that thrive in extremely high temperatures, above 80 degrees Celsius. They are found in geothermal areas like hot springs and hydrothermal vents.

Thermophiles

grow between 50-80 degrees celsius

Mesophile

grow between 15-45 C

Psychrophile

grow below 15 degrees C

Alkaliphile

growth above pH 9 (alkaline)

Neutralophile

grow between 5-8 pH

Acidophile

grow below 3 pH

Halophile

growth in high salt (>2M NaCl)

Aerobe

growth only in O2

Faculative

growth with or without O2

Microaerophile

growth only in small amounts of O2

Anaerobe

growth only without O2

Barophile

growth at high pressure (above 380 atm)

Barotolerant

growth between 10 and 495 atm (high pressure but not extremely high)

hypotonic solution effect

cause cell to swell and burst

hyperyonic solution effect

cause cell to shrivel (plasmolysis)

Microbes that grow at temperatures between 40°C and 80°C are called:

A. Psychrophiles

B. Mesophiles

C. Thermophiles

D. Extreme Thermophiles

Thermophiles

Bacteria cannot grow in solutions with very high concentrations of sugar because:

A. Bacteria cannot digest pure sugar

B. Sugar raises the solution’s osmolarity

C. Sugar alters the solution’s pH

Sugar raises the solution’s osmolarity

Weak acids can pass through membranes which:

Disrupts cell pH homeostasis, and kills cells. This makes them good food preservatives.

How do anaerobes survive?

Must use a terminal electron acceptor other than O2

Three oxygen-removing techniques are used today

1. Special reducing agents

   (thioglycolate) or enzyme

   systems (Oxyrase) can be

   added to ordinary liquid

   media

2. An anaerobe jar

   - O2 is removed by a reaction

   catalyzed by palladium

   - Newer methods ‘Gaspak’,

   don’t need palladium

   catalyst

3. An anaerobic chamber

   with glove ports

   - O2 is removed by vacuum

   and replaced with N2 and

   CO2

Eutrophication

the sudden infusion of large quantities of a formerly limiting nutrient, which can lead to a bloom of microbes

sterilization

killing of all living organisms

disinfectant

killing or removal of pathogens from inanimate objects

antisepsis

killing or removal of pathogens from a surface of living tissue

sanitation

reducing the microbial population to safe levels

Pasteurization vs Flash Pasteurization

63°C for 30 minutes vs. 72°C for 15 seconds

Ultra High Temperature

150°C for 3 seconds, sterilizes (all bacteria killed)

• Used for creamers

Autoclave

steaming (heat and moisture) under pressure (kills bacteria and endospores)

121°C, 15 psi (2 atm) for 20 min

A number of factors influence the efficacy of a given chemical agent, including:

- The presence of organic matter

- The kinds of organisms present

- Corrosiveness

- Stability, odor, and surface tension

Probiotics

“good bacteria” that displace disease causing organisms from tissues

Bacteriophage

Viruses that attack bacteria (do not harm eukaryotes)

which microbe is most resistant to killing by environmental stress agents?

prions

To sterilize heat-labile (sensitive) solutions, one should use

A. dry heat.

B. an autoclave.

C. membrane filtration.

D. Pasteurization.

Membrane filtration

Ethanol can be used as an effective bactericide when used at a concentration of

A. 70%.

B. 50%.

C. 40%.

D. 100%

70%

Generally, the simpler the organism

the smaller the genome

Purine

A and G

Pyrimidine

C, T and U

Nucleotides are connected by

a 5’-3’ phosphodiester bonds

why is GC pair is stronger than AT pair?

GC has 3 hydrogen bonds instead of the two that AT has

If the sequence of one strand of DNA is

5’ TCGATC 3’, what is the sequence of the complementary strand?

a) 5’ CTAGCT 3’

b) 5’ GCTAGC 3’

c) 5’ AGCTAG 3’

d) 5’ GATCGA 3’

C

what regulates supercoiling?

topoisomerases

Plasmids

extrachromosome DNA molecules.

- Much smaller than the chromosome

- Usually circular

- Need host proteins to replicate

- Low-copy-number plasmids

- High-copy-number plasmids

Plasmid replication

rolling circle (unidirectional) replication, complementary strand synthesized as unroling occurs

• Used by many bacteriophage viruses and mammalian DNA viruses

Analysis of the genome of a newly discovered bacterial strain reveals that it is composed of a double-stranded DNA molecule containing 16% thymine. Based on this information, what would you predict the percentage of cytosine to be?

A. 34%

B. 68%

C. 16%

D. 32%

34%

When the DNA replicates, how is the newly made strand related to its template strand?

A. The two strands have identical sequences and are antiparallel to each other

B. The two strands have complementary sequences and are parallel to each other

C. The two strands have complementary sequences and are antiparallel to each other.

D. The two strands have identical sequences and are parallel to each other.

E. The two strands have identical sequences and are antiparallel to each other, except that U replaces T.

C. The two strands have complementary sequences and are antiparallel to each other.

Replication, transcription, and translation all have 3 major steps

1. initiation

2. elongation

3. termination

RNA Polymerase

Enzyme responsible for transcription in cells. It synthesizes RNA molecules using a DNA template. Helps in the production of mRNA, tRNA, and rRNA.

• Core polymerase: a2, b, b’, w

• sigma subunit guides RNA Polymerase to target DNA sequence

Transcription initiation steps

1. sigma (as part of RNA holoenzyme) scans and binds starting sequence

2. Binding to the promoter sequence allows for the closed complex to form

3. RNA polymerase unwinds DNA and begins transription

4. sigma factor leaves the complex

The unwinding of DNA ahead of the moving complex forms a

17 base pair transcription bubble

Rho-dependent vs rho-independent termination

Termination of transcription in bacteria can be rho-dependent or rho-independent. Rho-dependent termination relies on a protein called Rho, which binds to the mRNA and causes the RNA polymerase to detach from the DNA template.

Rho-independent termination, on the other hand, occurs when a specific DNA sequence forms a hairpin loop followed by a string of uracil bases in the mRNA. This structure causes the RNA polymerase to pause and eventually dissociate from the DNA.

Antibiotics that Affect Transcription

• Rifamycin B

• Actinomycin D

What processes are common to replication, transcription, and translation?

A. Initiation, base-pairing, termination

B. Initiation, elongation, termination

C. Elongation, use of GTP for energy, use of accessory proteins

B

The sigma factor is directly required for

a) Transcription initiation

b) Transcription elongation

c) Translation initiation

d) Translation elongation

Transcription initiation

Number of possible codon sets

64 total

• 3 stop

• 61 amino acid coding sets (only 20 amino acids)

A tRNA molecule has two functional regions:

• Anticodon: hydrogen bonds with the mRNA codon specifying an amino acid

• 3′ (acceptor) end: binds the amino acid

The charging of tRNAs is carried out by a set of enzymes called

aminoacyl-tRNA synthetases

In prokaryotes, the subunits are

30S and 50S and combine to form the 70S ribosome

3 binding sites of 70s ribosomes:

1. A (acceptor) site: binds incoming aminoacyl-tRNA

2. P (peptidyl-tRNA) site: harbors the tRNA with the growing polypeptide chain

3. E (exit) site: binds a tRNA recently stripped of its polypeptide

Antibiotics That Affect Translation

• Streptomycin: inhibits 70S ribosome formation

• Tetracycline: inhibits aminoacyl-tRNA binding to the A site

• Chloramphenicol: inhibits peptidyltransferase

• Puromycin: triggers peptidyltransferase prematurely

• Erythromycin: causes abortive translocation

• Fusidic acid: prevents translocation

Protein Modification

Enzymes modify translated protein

• fMet removed from N-terminus

• Small groups added to amino acids

  • Phosphoryl groups added

  • Methyl groups added

  • Adenylate groups added

• Protein may be cleaved

• Protein may be refolded by helping enzymes

signal sequences

target proteins for transportation

• found on N terminus end of proteins

The types of RNA that physically catalyzes the formation of polypeptides is:

a. sRNA

b. tRNA

c. rRNA

d. mRNA

rRNA

Which of the following RNAs is used as templates for protein translation?

a) mRNA

b) tRNA

c) rRNA

d) tmRNA

mRNA

Chaperones are proteins that

a) Export other proteins out of the cell

b) Help other proteins fold properly

c) Degrade misfolded proteins

d) Bind other proteins to inactivate them

Help other proteins fold properly

Signal sequences are found

a) At the 3′-end of an mRNA molecule

b) At the 5′-end of an mRNA molecule

c) In the 50S ribosomal subunit

d) At the C-terminus of a protein

e) At the N-terminus of a protein

at the N-terminus of a protein

Shine-Dalgarno sequences

is complementary to a sequence at the 3′ end of 16S rRNA of the 30S subunit, it directs the start of translation

mutation

a heritable change in DNA

Point mutations

change of a single base (not deleted or inserted)

transition

purine→purine or pyrimidine→pyrimidine

transversion

purine↔pyrimidine

insertion or deletion

addition/subtraction of one or more bases

inversion

DNA is flipped in orientation

reversion

DNA mutates back to original sequence

silent mutation

mutation does not change the amino acid

missense mutation

mutation changes the coded amino acid

nonsense mutation

mutation changes to a stop codon

Frame-shift mutation

mutation changes the ORF of a gene

Mutations Arise in Diverse Ways

1. Tautomeric shifts in DNA bases that alter base-pairing properties

2. Oxidative deamination of bases

3. Formation of apurinic sites

4. Damage caused by reactive oxygen species

Ames Test

DNA repair

• Error-proof repair

• Error-prone repair

Error-proof repair

pathways, which prevent mutations

• Methyl mismatch repair

• photoreactivation

• nucleotide excision repair

• base excision repair

• recombinational repair

Error-prone repair

pathways, which risk introducing mutations

• Operate only when damage is so severe that the cell has no other choice but to die

Methyl mismatch repair

based on recognition of the methylation pattern in DNA bases.

• Uses methylation of the parental strand to discriminate from newly replicated DNA

Photoreactivation

The enzyme photolyase binds to the pyrimidine dimer and cleaves the cyclobutane ring

Nucleotide excision repair

An endonuclease removes a patch of single-stranded DNA containing certain types of damaged bases, including dimers.

Base excision repair

Specialized enzymes can recognize specific damaged bases and remove them without breaking the phosphodiester bonds.

• This AP site allows DNA pol I to synthesize a replacement strand containing the proper base

Recombinational repair

A single-stranded segment of the undamaged daughter strand can be used to replace a gap in the damaged daughter strand

SOS (“Save Our Ship”) repair

Induced by extensive DNA damage

- RecA coprotease activity stimulates autodigestion of the LexA repressor.

- Expression of many DNA repair enzymes

- Among them, 2 “sloppy” DNA polymerases that lack proofreading activity

- However, the cell has no other option but to “mutate or die.”

Taking up foreign DNA can be beneficial.

• Imported DNA can be used as an alternative food source.

• Repair damaged chromosomes

• Drive genome evolution

Why do species undergo natural transformation?

• Use indiscriminate DNA as food

• Use specific DNA to repair damaged genomes

• Acquire new genes through horizontal gene transfer

Conjugation

“bacterial sex”, transfers DNA through sex pilus

F’ factor

F plasmid contains extra genes (in addition to genes for pilus and transfer)

Transfers extra genes to recipient

Hfr conjugation

F factor integrates on bacterial chromosome, tries to transfer entire chromosome (Requires 100 minutes for E. coli). Transfers genes in order and can be used to determine order of genes on chromosome

transduction

the process in which bacteriophages carry host DNA from one cell to another

Which of the following sequences is not a restriction enzyme cut site?

A) GGATCC

B) GAATTC

C) CTTAAG

D) GGGCCC

E) CCCTTT

E) CCCTTT

CRISPR

consist of repeats and spacers that do not encode proteins, but near them lie CRISPR-associated gene families that do encode proteins

in an Ames Test, a very strong mutagen will cause the appearance of:

a. Many colonies over the plate, with a clear space close to the disk of mutagen.

b. A few colonies throughout the plate up to the edge of the disk.

c. Many colonies covering the whole plate, extending up to the disk.

a. Many colonies over the plate, with a clear space close to the disk of mutagen.