Microbiology Test 2

• Duplicate of original molecule
• Bidirectional
• Semiconservative
• Begins at origin of replication

• From DNA to RNA
• dsDNA to ssRNA

• mRNA to Protein
• Genetic code
• Reading frames
• Ribosomes (30S and 50S)

• At origin of DNA replication
• Adds nucleotides in 5’ to 3’ direction onto growing strand

• At origin of DNA replication
• Helps polymerase begin new DNA strand

• Used in DNA replication
• Keeps fork open
• Breaks hydrogen bonds

• Used in DNA replication
• Prevents breakage of strand
• Will cut strands to help relieve tension for helicase

• Are added to 3' of new DNA strands
• Direction of synthesis from 3' to 5'

• Used in DNA replication
• Adds nucleotides 5’ to 3’
• Moves in opposite direction

• Used in DNA replication
• Short strands of DNA that form on lagging
• side

• Used in DNA replication
• Glues Okazaki fragments together

• Synthesized from minus strand
• Almost identical to the plus strand
• Monocistronic or polycistronic
• Thymine is replaced with Urecil

Can transcribe one gene in mRNA

Can transcribe one or more genes making a giant mRNA

• Promoter (the address can go in any direction) recognized by RNA polymerase (the pizza) and
• sigma factor (the driver)

• Synthesis in 5’ to 3’ direction
• Same as replication
• Will always pick up 3’ to 5

Terminator and hairpin loop

• DNA strand is going in opposite direction
• RNA Primase
• DNA polymerase III
• DNA polymerase I
• DNA ligase
• DNA polymerase III coils down to bind the DNA strand back together

• What if there was a mutation in helicase that
• prevented it from performing its function?
• What would happen?

Origin

Promoter

5' to 3'

DNA

• RNA polymerase
• Sigma factor

dsDNA

ssRNA to mRNA

• Three nucleotides = codon
• Degenerate for encoding amino acids

Which triplet of nucleotides to use

• Protein and rRNA
• tRNA with amino acid attached

• Binding of ribosome (30S) to mRNA
• Start codon AUG
• f-Met and 50S bind clamp onto mRNA to elongate

P-site; A-site; E-site

• Stop codon UAA, UAG, UGA
• Release factors

• Modified during transcription
• 5’ end capped, 3’ end with poly A tail
• After transcription precursor mRNA modified
• -Splicing occurs

Removing introns (non-encoding regions of mRNA)

• Constitutive
• Repressible
• Inducible

• Always on
• Glycolysis

• Turn off
• Amino acids
• Aren’t always needed

• Turn on
• Β-galactosidase for breakdown of lactose (alternate carbon/energy source)
• Not always needed

A component that helps to begin transcription

• Activator binding site, promoter and operator
• Gene or genes to be transcribed

• Prevents transcription by RNA polymerase
• Repressor

• Encoded by a separate gene from operon
• Binds to operator
• Can require binding of a corepressor
• Can be blocked by binding of an inducer

• Transcription by RNA polymerase is facilitated
• Transcription enhanced by an activator
• Activator-binding site upstream of promoter

• Sometimes requires an inducer to bind
• •Extra symbol to find

• Lac operon
• 3 genes for utilization of lactose :lacZ, lacY and lacA- break down lactose
• Activator: CAP and cAMP (inducer)
• Repressor that is induced by allolactose
• A lot of glucose= low cAMP= not enough inducers to bind to CAP (activator)
• If lactose is present, repressor doesn’t bind

• Transcription dependent upon presence/absence of glucose or lactose
• Glucose is preferred
• Must “turn on” genes to utilize lactose

• Goes from lag to log
• lag to log

• Change in DNA sequence (genotype) passed to daughter cells
• Vertical gene transfer

• Acquisition of genes from another organism
• Horizontal gene transfer

• Spontaneous
• Induced

• Base substitution
• Remove or add nucleotides
• Transposable elements
• Mistake in DNA synthesis

• Chemical mutagens
• Transposons
• Radiation

• Point mutation (one nucleotide)
• Silent mutation
• Missense mutation
• Nonsense mutation
• - Knockout mutation

Any mutation that renders the protein product dysfunctional

• Three nucleotides added/removed
• One or two nucleotides added/removed
• Frameshift mutation
• Shift in reading frame of codons
• Stop codon may be created

• Transposon
• -Short segment of DNA
• -Transposition: Moves itself into a new position
• Barbara McClintock

• Chemical mutagens
• Base analogs
• Intercalating agents

Alkylating agents change purine and pyrimidine structure

• 5-bromouracil pairs with wrong base
• Once incorporated into DNA, the analog can shift in structure and pair with a different base

• Ethidium bromide inserts into ds DNA
• Frameshift mutation occurs
• Once the agent inserts into dsDNA, DNA polymerase will recognize the extra space as a need to insert a new base pair

• In induced mutation
• Thymine dimers
• Mutation from SOS repair

• In induced mutation
• ds and ss breaks in DNA

• Proofreading
• Mismatch Repair

Immediately correcting nucleotide

• After DNA synthesis
• Endonucleases to cut and trim away mistake
• DNA polymerase to make new strand
• DNA ligase to seal

• Light repair
• -Enzyme requires light to work
• Excision (dark) repair

• Last ditch effort for too many dimers
• New DNA polymerases made
• Many mutations created to compensate dimers

• Having a combination of properties from two cells
• -Use a defined medium

Integration of DNA from an outside source only if there is a similar region in the recipient's genome

• Uptake of a "naked" DNA
• Natural competence

Not associated with a chromosome or plasmids

• Entry of single strand DNA- degrade one side of DNA
• Integration of donor DNA by homologous recombination
• Multiplication by selection

Based on "Bacterial Transformation," which of the following terms accurately describes the removal of the mismatched strand after transformation?

• Escherichia coli
• Single and double stranded DNA can be introduced
• Use plasmid DNA
• -Can force DNA into a cell

• Transfer of DNA by a bacteriophage
• -Phage chops up chromosome making the bacteria no longer functional
• Generalized transduction
• Specialized transduction

• Picks up bacteria DNA instead of phage DNA
• -If attached to new bacteria, homologous recombination can occur

• Phage randomly selects host’s genes
• After lysis infects new cell, iransfers previous host’s genes

• Homologous recombination
• Competence
• Artificial: plasmid, fragment
• Natural: fragment

• Homologous recombination
• Bacteriophage
• Generalized
• Specialized

• Sitting outside of chromosome
• Used to benefit cells
• -Degrade a type of sugar
• Double stranded DNA
• -Origin of replication
• Usually encode for properties that may increase cell viability
• Low copy vs. high copy
• Narrow vs. broad host range

10 cells

Multiple

Can only be used by one species/genus

Can be used in multiple species/genus

• Plasmid transfer by cell-to-cell contact
• Resistant plasmids are among those transferred
• -Contains one or more drug resistant genes

Conjugation Requires...

• F plasmid integrates into chromosome
• Hfr – high frequency recombination
• Donor cell has F+

Steps for Conjugation

• A DNA segment that carries the gene(s) required for excision from and insertion into DNA
• -Often can insert randomly
• -Can move from one location to another within the chromosome
• Insertional inactivation of genes
• Knock-out mutation

Transposons can Contain...

Encodes for transposase, enzyme which transfers the transposon

• If transposons only jump from one part of a genome to another, could they jump from one cell to another?
• What would they require if they could jump from one cell to another

A metabolic product of an organism or a chemical compound that is inhibitory to microorganisms

• German physician began systematic search for treatment for syphilis
• Noted that some dyes stain bacteria but not human tissue
• Identified Salvarsan- arsenic derivative

• Screened large numbers of chemicals against infectious disease for Bayer in Germany
• Discovered Prontosil kills streptococci in mice but not in the test tube
• Prontosil broken down in body to yield active ingredient sulfanilamide

• Scottish physician who had treated wounds during World War I
• Worked with Staphylococcus aureus
• Noted that there was no growth of the bacteria in a zone surrounding the fungal contamination

Picked up Fleming’s work on penicillin and develop mass production of drug

Changing the side chain “R” of penicillin, other types of antibiotics were created

• Majority from Streptomyces and Bacillus (bacteria), Penicillium and Cephalosporium (fungal)
• Bacteriocidal
• Bacteriostatic
• Selective Toxicity
• Broad and narrow spectrum
• Synergistic, antagonistic, additive
• Uptake
• Half life

Antimicrobials kill their targets

• Allows the body to kill infection, but can kill healthy cells/bacteria
• -Secondary infections can be worse, so eat yogurt
• Antimicrobials inhibit the growth but do not kill their targets
• Allows immune system to catch up
• May be a problem if immunodeficient

• Causes greater harm to microbes than human
• Therapeutic index

• Lowest toxic dose/dose required for treatment
• Example: 10/2= 5 (low)
• Higher numbers= better, body will take care of it

• Can treat almost everything, except possibly microbacteria
• Kills all bacteria, but kills good body bacteria
• Useful in acute life-threatening disease

• Specific to just gram positive or gram negative
• Targets just pathogens leaving body alone, but does take a lot of time
• Requires identification of pathogen

When Antibiotics are Combined…

Greater affect than the two added together, use less

Adding antibiotics together to double effect

How many times a day you have to take an antibiotic for it to be affective

• Allergic reactions
• Toxic effects (e.g. aplastic anemia with chloramphenicol)
• Suppression of normal flora
• -Antibiotic associated colitis
• •Clostridium difficile
• Resistance

• Innate
• Acquired

• Step 1- Inhibit cell wall synthesis
• Step 2-Disrupt cell membrane
• Step 3- Inhibit nucleic acid synthesis
• Step 4- Inhibit protein synthesis
• Step 5- Anti-metabolite (metabolic analog)

• Inhibit transpeptidation & activation of cell wall lytic enzymes
• -Penicillin
• -Cephalosporin
• Inhibit transpeptidation
• -Vancomycin
• Inhibit transport by carrier
• -Bacitracin stops NAG and NAM from entering cell wall
• Vary in ability to inhibit Gram + or Gram -

• Binds membrane & disrupts integrity
• Effective against gram negative
• -Polymyxin B targets and pops a hole in the cell wall/membrane

• Broad spectrum
• Interfere with DNA gyrase
• -Ciprofloxacin
• Interfere with transcription
• -Rifampin

• DNA gyrase
• Transcription
• Not making RNA

• Aminoglycosides and tetracyclines – 30S
• -Streptomycin
• -Tetracycline
• Macrolides, chloramphenicol, lincosamides, oxazolidinones, streptogramins - 50S subunit
• Varies in spectrum and action
• Attacking ribosome

If DNA gyrase is inhibited, how does DNA begin to unwind?

• Broad spectrum
• Inhibits folic acid synthesis (needed for nucleotide synthesis)
• -Sulfonamides- competitive inhibitor
• Make folic acid themselves
• Blocks tetrahydrofolate synthesis
• -Trimethoprim
• Protein, RNA, mRNA, enzyme

• Bacterial culture spread over petri plate
• Discs saturated with specific concentration of antibiotic added
• Measure areas of clearing where culture did not grow
• -Zone of Inhibition

• A simple case scenario is forgetting to finish your prescribed antibiotics
• Conjugation and mutation

• 1. Decreased Uptake
• 2. Inactivation
• 3. Alteration of target
• 4. Elimination

• Enzyme that cuts penicillin to make “resistant”
• Beta lactamase

Mutation of enzyme

Efflux pump finds antibiotic and pushes out of body before any affects

• Vertical
• Horizontal

Spontaneous mutation

• Gene transfer
• -Conjugative transfer of R plasmids
• -Plasmids contain more than one resistance
• -Resistance genes can be carried on transposons

• Most are strict aerobes
• -Some can grow if nitrate is available for e- acceptor
• Can metabolize 80 different substrates
• -Ability to use this variety of substrates are encoded on plasmids
• Can be found growing in water of respirators

Found in a biofilm, attached to some surface or substrate, or in a planktonic form, as a unicellular organism, actively swimming by means of its flagellum

• 1. The species' inherent resistance to many drug classes
• 2. Its ability to acquire resistance, via mutations, to all relevant treatments
• 3. Its high and increasing rates of resistance locally
• 4. Its frequent role in serious infections
• A few isolates are resistant to all reliable antibiotics, and this problem seems likely to grow

Pseudomonas are...

Urinary tract infections, respiratory system infections, dermatitis, soft tissue infections, bacteremia, bone and joint infections, gastrointestinal infections and a variety of systemic infections, particularly in patients with severe burns and in cancer and AIDS patients who are immunosuppressed

A serious problem in patients hospitalized with cancer, cystic fibrosis, and burns. The case fatality rate in these patients is near 50 percent

1/3 infected worldwide; 2 million die annually

Slight fever, weight loss, chronic cough with bloodied sputum

• Inhalation Mycobacterium tuberculosis
• Multiply within macrophages
• Granulomas form to wall off bacteria
• -Called tubercles
• Latent infection
• Active tuberculosis
• Reactivation tuberculosis

Asymptomatic, not contagious

Symptomatic, can transmit bacteria

• Renewed growth after latent period
• Takes years to reactiviate in the body

• Acid-fast
• Gram positive bacillus
• Slow growth (generation time = 12 hours)
• Cell wall contains layer of complex glycolipids
• Resistant to drying, disinfectants and low/high pH
• Takes 12 hours to go through 1 binary fission

• Inhaled droplets, bacteria are attacked in lungs by macrophages
• Macrophages release signals to recruit other white blood cells
• Tubercle forms with bacteria, macrophages and other immune cells
• Turns latent in healthy individuals with walled off tubercles

• Caseous lesion- cheese like tubercle
• Tuberculous cavities
• -Liquefied tubercle
• -Spread of bacteria into rest of lung
• -Cavity slowly enlarge
• -Transmission occurs by coughing, spitting

• Injection of bacterial protein
• Slow forming reaction shows either existing bacteria or previous exposure
• Both latent and active TB are treated

Rifampin and isoniazid for 6-9 months

• Limited antimicrobials used
• -Chronic nature of disease
• -Slow growth
• -Waxy cell wall (mycolic acids)
• First line drugs – five combined
• -Target cell wall
• -Isoniazid
• -Ethambutol
• -Pyrazinamide

Inhibit mycolic acid synthesis

Inhibits cell wall components synthesis

Unknown

• Can be drug resistant way before anti-biotics
• Increase of 13.3% in US
• Occurs due to preexisting mutants
• M. tuberculosis exhibits spontaneous chromosomal mutations
• Results from
• -Administering only one drug
• -Improperly administered drug

4 cases reported to CDC in 2008

What are some ways that TB could be treated that would ensure that the patient finished their regimen of antibiotics?

Doctor went to house or made patient come in to make sure they’re taking the anti-biotics

Patient comes in every week for more pills

Send patient home with all anti-biotics

What happened between 1980-1992?

What is one trend on the “U.S. vs foreign born” chart?

• China, India, the Russian Federation and South Africa account for 60% of the global number of MDR-TB cases and have increased their financing for TB control
• Only 3% of the half million MDR-TB cases estimated to emerge each year worldwide are known to be receiving treatment according to WHO guidelines

• Gram positive
• Releases coagulase
• Clumping factor
• Alpha toxin
• Can be found in nose or skin
• Opportunistic

• Pyrogenic with inflammation; if spread to blood, fever ensues
• -Toxin producing strains can cause toxic shock

• Penicillin or modified beta lactam drug (methicillin)
• 90% now resistant to first penicillin due to plasmid-encoded resistance

• Methicillin resistant S. aureus (MRSA)
• Vancomycin resistant S. aureus (VRSA)

• Produces a penicillinase (also found on plasmid)
• Cell targets for beta-lactam drugs are not affected
• Vancomycin used for treatment

Synercid is made of two substances that block protein synthesis

• Most resistant strains are traced to hospitals and clinics
• 30-100% of surgical infections due to patient’s own staphylococcus strain in their nose
• Over time, 20% of the population will almost always be colonized with S. aureus
• -Termed carriers
• 60% of the population will be colonized with S. aureus off and on
• 20% are almost never colonized with S. aureus