207 Exam 2

antibiotics

compounds produced by one species of microbe that can kill or inhibit the growth of other microbes (now typically chemically synthesized)

what complicates screening for new antibiotics

• some drugs only become antibiotics once partially metabolized in our bodies (e.g. sulfa drugs)

selective toxicity

• selectively kill or inhibit the pathogen but not the host

• target:

  • processes only occurring in target species (e.g. peptidoglycan bs)

  • structural differences shared protein/complexes (e.g.

    large subunit ribosome)

  • other physiological differences (e.g., sulfanilamide mechanism)

sulfanilamide mechanism

• inhibits biosynthesis of a vitamin key to nucleic acid biosynthesis in all life

• humans don’t synthesize this vitamin, instead uptake from diet, so are not affected

• pathogenic bacteria cannot take up the vitamin so have

  to synthesize it

antibiotics at high concentrations

• at high concentrations antibiotics with selective toxicity can have side effects

• allergies can be a real concern (antibiotics are foreign substances to our bodies, so immune system can overreact to them)

spectrum of activity

• broad vs narrow

bactericidal

kills bacteria

bacteriostatic

• inhibits growth

minimal inhibitory concentration (MIC)

• the lowest concentration of the drug that prevents growth

• Varies for different bacterial species

• Test by serial dilution of antibiotic

How do you determine if and and what concentration the antibiotic has bactericidal activity?

cell wall inhibitors

cell membrane inhibitors

metabolic inhibitors

DNA replication inhibitors

RNA polymerase inhibitors

protein synthesis inhibitors

gram postitive vs gram negative

beta lactam antibiotics

• Penicillin is an antibiotic derived from cysteine and valine

  • condensed by fungal enzymes to form a beta-lactam ring structure

• The beta- lactam ring chemically resembles the D-Ala-D-Ala peptide crosslink within peptidoglycan

• Allows penicillin to bind to and inhibit the transpeptidase enzyme that cross-links peptidoglycan chains.

ampicillin and amoxixillin

• versions of penicillin that can cross OM of gram negative

• more hydrophilic

Cephalosporins

• class of natural beta-lactams modified to combat penicillin-resistant pathogens

ways bacteria develop resistance to penicillin and beta lactams

• beta-lactamase enzyme that degrades beta-lactam ab

• altered transpeptidase that no longer binds beta-lactam ab

Methicillin-resistant Staphylococcus aureus (MRSA)

• Methicillin used initially as not sensitive to resistance due to beta-lactamase enzyme that degrades beta-lactam ab

• arose due to mutations leading to transpeptidase no longer binding to beta-lactam ab like methicillin

• Vancomycin now often last line of defense against MRSA

  • now also increasingly VRSA (still very rare, 16th case id’ed in 2021)

  • interacts w/ peptide bond instead of the enzyme

• New antibiotics like teixobactin may offer hope (targets multiple pathways at once, including cell wall biosynthesis, making resistance less likely)

Gramicidin

• Cyclic peptide produced by Bacillus brevis

• Inserts into membranes as a dimer and forms a leaky cation channel that disrupts ion concentration gradients

• Used only topically, as it also targets our cells’ plasma membrane

Why is a combined use of erythromycin, which inhibits protein synthesis, and penicillin counterproductive?

• E blocks growth of peptidoglycan, which P needs to stop new cross links

Why would a combination of erythromycin and penicillin sometimes be a good idea?

• when you need to suspend growth but not kill cells

• if they contain toxins (endo or exotoxins)

• E slows the growth, then P comes in

secondary metaboliotes

• often have no apparent primary use in the producing organism

• antibiotics

• only useful in certain conditions

• Not essential for survival under standard conditions

• May enhance ability to compete favorably with others

antibiotic resistance mechanisms

• Only finish synthesis when exported from cell

• Make enzymes to disable antibiotics

why do microbes make antibiotics

• Hypothesis 1: Biological warfare-gaining competitive advantage over other microbes or killing them to access cellular resources

• Hypothesis 2: At subinhibitory concentrations typically found in nature, they may act as signaling molecules regulating community interactions

how do bacteria keep antibiotics out of cell

• Destroy the antibiotic before it enters the cell.

  • The beta-lactamase enzyme specifically destroys penicillins

• Decrease membrane permeability across the outer membrane

  • Gram-negative bacteria can express outer membrane porins with pores too narrow to allow drug penetration.

• Pump the antibiotic out of the cell via specific transporterS

  • Membrane pumps bail drugs out of cell faster than they can enter

  • Multidrug resistance (MDR) efflux pumps can export

    many different kinds of antibiotics (work similarly to ABC export systems)

    • Can be problematic and contribute to drug resistance because they often work with little regard to structure.

    • resistance to many antibiotics

how do bacteria prevent antibiotics from binding to target

• Modify the target so that it no longer binds the antibiotic.

  • Mutations in key penicillin-binding proteins and ribosomal proteins confer resistance to methicillin and streptomycin, respectively.

• Add modifying groups that inactivate the antibiotic and make it less able to bind its target.

• This increases the MIC

how do bacteria dislodge an antibiotic already bound to its target

• Ribosome protection (or rescue)

  • Gram-positive organisms can produce proteins that bind to ribosomes and dislodge macrolide antibiotics bound near the peptidyltransferase site.

how did resistance start

• The presence of drug does not cause resistance, but it will kill off or inhibit the growth of competing bacteria that are sensitive, while resistant microbe grows to high numbers.

• De novo antibiotic resistance develops through gene duplication and/or mutations

• Antibiotic resistance also can be acquired via horizontal gene transfer (conjugation, transduction, and transformation).

• antibiotics in animal feed

• Collateral damage of antibiotics use: disturbing the microbial balance of power in the gut (C. diff, and links to IBD, vitamin deficiency, obesity, asthma)

antibiotics stweardships

• coordinated interventions that improve and measure antibiotic use

• Do not use antibiotics to treat viral infections

• Do not use an antibiotic if a patient’s microbiome includes a strain that is resistant to the drug. (avoid risk of HGT)

• Know which antibiotic resistant strains are prevalent in the community or hospital before prescribing.

• Consider how long the patient needs to take the antibiotic: leverage competition with sensitive bacteria that are more fit without ab present (trade-off)?

• De-escalate antibiotic usage whenever possible: transition from broad to narrow spectrum when possible

directly countering drug resistance

• Dummy target compounds overwhelm resistance enzymes

• Alter antibiotic’s structure so that it sterically hinders access of bacterial modifying enzymes

finding new antibiotics

• Screening of microbes, plants, and animals– incredible diversity remains untapped

• Chemical synthesis of new compounds

• Genome sequence analysis to identify potential bacterial molecular targets

• Interfering with quorum-sensing mechanisms

• CRISPR-based strategies for reversing antibiotic resistance

• quorum sensing

phage therapy and problems

• very narrow spectrum

• could gain resistance to this too

anti fungal agents

• Fungal infections are much more difficult to treat than bacterial infections.

• Fungi are eukaryotes, and so selective toxicity issues arise.

• Fungi have an efficient drug detoxification system that modifies and inactivates many drugs.

• Fungal infections can be divided into two main groups.

  • Superficial mycoses: treated topically

  • Systemic mycoses: treated internally

rapamycin

• Key drug in current medicine, initial as antifungal, but mostly as key drug as immunosuppressant in transplantation, heart stents, and in cancer treatment.

• Derived from Streptomyces hygroscopicus, originating from soil sample from Rapa Nui (Easter Island) taken by Canadian exhibition in 1970s

• Improbable path to becoming a billion-dollar drug

ways genomes differ

1. Gene content

   1. differences between different E. coli strains: pathogenic vs not, targeting GI vs urogenital systems

2. Sequence composition differences

   1. Between botulism toxin proteins affecting different animal

3. Genome organization differences

   1. between V. cholerae and closely related environmental

      Vibrio species

why is DNA ideal storage

• stable

• mutable

• replicable

genome

• All genetic information that defines an organism

Genes

Stretches of DNA information that can be “sent” out as RNA

structural gene

• produces a functional RNA, which usually encodes a protein

regulatory sequence

• regulates the expression of a structural gene.

• Does not encode an RNA or protein

• Includes promoters & binding sites for regulatory proteins

variation in genome organization

• Number, size, shape of chromosome/plasmids

• More DNA = trade-off between cost of replication/expression and additional functional capabilities.

non coding DNA

• It is typically > 90% of eukaryotic genomes, but < 15% of prokaryotic genomes

• Non-coding DNA of eukarya includes introns and pseudogenes (inactivated genes).

• Some archaeal genes have introns too, but rare

• introns not in bacteria

DNA functions

• DNA is more stable than RNA (2

• Nucleotides joined together via bonds between the 5ˈ phosphate group of one nucleotide and the 3ˈ OH group of another.

• Nucleotide base complementary between two strands of DNA: leveraged for DNA repair

• hydrostatic interactions (A:T, G:C) stable under physiological conditions (need for pH and ionic homeostasis inside cell)

• Extremophiles (pH, temperature) have additional DNA binding proteins or additional supercoiling to stabilize DNA)

• Antiparallel orientation to allow base-pairing → implications for replication process

how is DNA compacted

• Organized in domains, each domain supercoiled by topoisomerases

• anchored by histone-like proteins

where does bacterial replication begin

oriC

what happens after initiation of bacterial replication

• replication bubble

• continues until termination sequence (ter)

initiating bacterial replication steps

• precisely timed in function of cell growth (div and rep are linked)

• DnaA bound to ATP accumulates during growth, binds to region near oriC

• triggers the initiation of replication by looping and partially unwinding the DNA (replication bubble)

• allows replisome to assemble

• Within the replication bubble, DnaC (helicase loader) loads

  DnaB (helicase) onto each single stranded template.

• DnaB recruits DnaG (primase)

• primase synthesizes a short RNA primer against each template strand

• completes with DNA pol III and sliding clamp

difference in replication in archaea and eukaryotes

• archaea can have many Oris (not triggered by DNA A)

• eukaryotes have many Oris but they form in specific phases in the cell cycle (meiosis or mitosis, not continuously)

archaea DNA replication

• DNA polymerase unique to them

• the rest of their replication machinery related to the eukaryotic proteins, rather than to the bacterial versions.

DNA elongation

• The replisome ensures that the leading and lagging strands are synthesized simultaneously

• 5’ to 3’ direction

• the problem(lagging) strand loops out after passing through its polymerase.

filling in the gaps of leading and lagging strand

• RNA primers removed by RNase H or DNA pol I

• DNA pol I makes DNA patch

• DNA ligase repairs the remaining phosphodiester nicks

what is a benefit of bidirectional replication

• faster

• less exposed ssDNA at a time

plasmids

• found in archaea, bacteria, and eukaryotic microbes

• Typically much smaller than chromosomes

• Usually circular

• Copy number per cell varies widely

• Contain nonessential genes that often play critical roles in certain situations (e.g., antibiotic resistance)

• Can be transferred between cells

plasmid maintenance strategies

• Some plasmids ensure their inheritance by carrying genes whose functions benefit the host microbe under certain conditions (e.g., antibiotic resistance, pathogenesis factors, symbiosis proteins).

• High-copy-number plasmids flood the host cell cytoplasm with copies that give each daughter cell a very high likelihood of receiving at least one copy by chance alone.

• Low-copy-number plasmids evolved dedicated partitioning systems that ensure both daughter cells receive copies of the plasmid. (this requires use of ATP!)

what happens after translation

• each polypeptide is properly folded (this involves chaperone proteins)

• placed at the correct cellular or extracellular location (for example using the secretions systems we covered earlier)

making of a protein diagram

monocystronic

• RNA produced from a single gene

• all eukaryotic genes

polycistronic

• In bacteria and Archaea, genes may be organized in operons (multiple genes transcribed in single transcript), encoding multiple proteins

RNA polymerase holoenzyme

• Core polymerase (many subunits)

  • Required for the elongation phase

• Sigma factor

  • Required for the initiation phase: different

  • sigma factors allow for coordination of expression of different sets of genes

  • recognizes promoter

• euk & arc have core enzyme and no sigma factor, and

  instead use independent proteins called transcription factors

housekeeping sigma factor

• Recognizes, based on electrostatic interactions the promotor consensus sequences at the -10 and -35 positions

promoter consensus sequence

• Consensus sequence represents most common nucleotide at each position.

• Individual promotors diverge from this consensus sequence

• Some nucleotides more (yellow) and less (red) conserved across sequences.

A bacterium needs to transcribe a gene both in normal conditions as well as when undergoing heat shock. How is this possible?

• different promoters

initiation of transcription

• RNA pol holoenzyme binds to the promoter.

• This is followed by melting of the helix and synthesis of the first nucleotide of the RNA

termination of transcription

• RNA pol detaches from the DNA, after the transcript is made

rho dependent termination

• Relies on a protein called Rho and a strong pause site at the 3′ end of the gene

rho independent termination

• Requires a GC-rich region of RNA, as well as 4–8 consecutive U residues

antibiotics that affect transcription

• Kill or retard the growth of a pathogen

• Not harm the host

• Rifamycin B - Selectively binds bacterial RNA pol, Blocks RNA exit to inhibit initiation, Polymerase can still bind promoter

• Actinomycin D - Non-selectively intercalates between GC base pairs in DNA, Inhibits transcription elongation

classes of RNA and stability

codons

• Consists of nucleotide triplets

• There are 64 possible codons:

  • 61 specify amino acids

    • Includes the start codons

  • 3 are stop codons

• The code is degenerate or redundant.

• Multiple codons can encode the same amino acid.

• operates universally across species

  • very few exceptions

tRNA

• convert language of RNA into that of proteins.

• two functional regions:

  1. Anticodon: Hydrogen bonds with the mRNA codon specifying an amino acid

  2. 3’ (acceptor) end: binds the amino acid

• contain a large number of unusual, modified bases.

• same tRNA can recognize multiple codons

attaching AAs to tRNAs

• Each tRNA must be charged with the proper amino acid before it encounters ribosome.

• Ribosome cannot check if tRNA has correct amino acid on it.

• carried out by a set of enzymes called aminoacyl-tRNA synthetases

  • Each cell has 20 of these “match & attach” proteins, one for each amino acid.

• Each aminoacyl-tRNA synthetase must recognize its own tRNA but not bind to any other tRNA

  • each tRNA has its own set of interaction sites that match only the proper synthetase

prokaryotic ribosome

• the subunits are 30S and 50S and combine to form the 70S ribosome

• enzymatic activity - peptidyltransferase

  • ribozyme

  • part of 23S rRNA of large subunit

How does ribosome “know” which of 3 reading frames to use

• The upstream, untranslated leader RNA contains a purine-rich ribosome binding site with the consensus 5′-AGGAGGU-3′.

• This Shine-Dalgarno sequence is complementary to a sequence at the 3′ end of 16S rRNA of the 30S subunit

• only in Bacteria and Archaea, Eukarya have 5’ cap instead

initiation of translation

• brings the two ribosomal subunits together, placing the first amino acid in position

• requires protein factors and GTP

1. Dissociated ribosome units needed

2. 16S rRNA and mRNA interact at RBS

3. Met-tRNA binds the ribosome

4. Full ribosome assembles (GTP hydrolyzes)

elongation of translation

• sequentially adds amino acids as directed by mRNA transcript

• requires protein factors and GTP

1. tRNA-AA loads onto ribosome

2. Ribozyme activity (23S rRNA) binds new AA to existing peptide (GTP hydrolysis)

3. Empty tRNA removed upon addition of next tRNA-AA

termination of translation

• releases the completed protein and recycles ribosomal

  subunits

• requires protein factors and GTP

1. No tRNA with anticodon complementary to stop codons

2. Release factor proteins enters ribosome instead, eventually leading to dissociation of the two ribosome subunits at the expense of GTP hydrolysis

targets of antibiotics

• protein synthesis based on differences in structure of proteins and rRNA involved relative to eukarya

• additional proteins (initiation factors etc.)

• Archaea and Bacteria are more similar but differences in translation factors as well

protein folding and secretion

• Often, a protein must be modified after translation either to achieve an appropriate 3D structure or to regulate its activity.

• Post-translational modifications can affect activity of proteins-important in regulation (see next class)

• A healthy cell “cleans house” by degrading damaged or unneeded proteins

• Proteins destined for the bacterial cell membrane or envelope regions require special export systems.

• Proteins meant for the cell membrane are tagged with hydrophobic N-terminal signal sequences of 15-30 amino acids.

types of gene regulation

• Alteration of DNA sequence – flipping a DNA segment

• Control of transcription – Repressors, activators, sigma factors, sRNAs

• Control of mRNA stability – RNase activity, sRNAs

• Translational control – Hiding RBS sites, or other mRNA sequences

• Post-translational control – cleavage, phosphorylation, methylation, etc.

In general, ____ control is the most drastic and least reversible, whereas control at the ____ is the most rapid and most reversible

DNA sequence level, protein level

transcriptional control

• proteins bind at regulatory sequences (operators or repressors) to control initiation of transcription at promoters

• environmental changes in metabolites (ligands) or outside factors can affect activity

• ligands alter the DN binding affinity of regulatory proteins at the promoter

repressors

• prevent gene expression

• bind to operators

• inducer - binds in absence of ligand

• corepressor - binds in presence of ligand

activators

• stimulate gene expression

• contacts RNA polymerase positioned at a nearby promoter

• most are inducers - bind poorly to DNA sequences unless they are bound to their ligand

2 component signal transduction system

• helps the intracellular proteins interact with the external environment

1. Sensor kinase in cell membrane

   1. Binds to environmental signal-

   2. Activates itself via phosphorylation

2. Response regulator in cytoplasm

   1. Takes phosphate from sensor

   2. Binds chromosome

   3. Alters transcription rate for several genes

catabolism regulation

• often involves induction when substrate is present (activator or no repressor)

• catabolite repression

catabolite repression

• an operon enabling the catabolism of one nutrient is repressed by the presence of a more favorable nutrient.

• glucose and lactose

• diauxic growth (pic)

anabolism regulation

• different from cat. repression

• typically are regulated by inactive repressors (aporepressors)

  • bind the end product of the pathway (corepressor).

  • When an aporepressor binds its corepressor, the complex binds to an operator sequence upstream of a target gene or operon to turn transcription off (repression) by blocking access to the RNA polymerase

  • Negative feedback mechanism

  • trp operon

trp operon example

• repression of anabolic pathways

• When trp levels exceed cellular needs, excess Tryptophan (the corepressor) binds to inactive TrpR (the aporepressor).

• The complex then binds to an operator sequence upstream of the trp structural genes and represses expression by blocking RNA pol.

what are 2 different ways to control expression of multiple pathways at once (rewatch lecture)

• sigma factors → similar promoters

• same operon and operator

regulation after transcription

• Regulatory sequences in the mRNA can cause premature termination of transcription (e.g., attenuation).

• Some mRNA sequences prevent their own translation into protein (e.g., riboswitches).

• Other regulatory RNAs influence the fates of transcribed mRNAs (e.g., untranslated RNAs).

transcriptional attenuation

• translation of a leader peptide affects transcription of an operon’s downstream structural genes.

• At low levels of tryptophan, ribosome stalls, allowing a stem loop to form that allows downstream transcription to proceed.

• At high levels, different stem loops form that release the upstream RNApol and terminates transcription before it reaches trp genes

riboswitches

• usually found in the 5' untranslated region of mRNA that control gene expression by folding into three-dimensional structures that bind specific metabolites to sense their abundance in the cell.

Untranslated Small Regulatory RNAs

• Attenuators & riboswitches are part of mRNA transcript they control

• Untranslated Regulatory RNA molecules are transcribed independently and typically affect gene expression post-transcriptionally.

• Small RNAs (sRNAs) represent one of the most economical ways to regulate gene expression.

  • They do not require protein synthesis.

  • They diffuse rapidly.

  • They typically act on preexisting messages

quorum sensing

• The process where bacterial cells work together at high

  density.

• Discovered in Aliivibrio fischeri a bioluminescent bacterium that colonizes the light organ of the Hawaiian squid

• many pathogens use it to control expression of virulence genes

  • dont turn them on until there are many others around

• Pseudomonas aeruginosa (CF), Staphylococcus, Yersinia pestis, Vibrio cholerae,

autoindicator

• At a certain extracellular concentration, the secreted autoinducer reenters cells.

• Binds to a regulatory molecule. In the case of vibrio fischeri is LuxR

• LuxR-autoinducer complex then activates transcription of the luciferase target genes

circadian clocks

• can anticipate based on pattern

• photocynthetic bacteria

• Controlled by KaiABC proteins, that together cycle KaiC between P and NP state on a 24 hr cycle.