Lecture 3 Flashcards
What macronutrients are often used to buffer and maintain osmolarity?
Magnesium and potassium
Macronutrients
- carbon - 50% of cell
- nitrogen - used in amino acids (proteins), nucleotides, NH3, NO3
- phosphorus- nucleotides, phospholipids
- sulfur- amino acids, SO42-, S2-
- potassium (k)
- magnesium (Mg)
- calcium
- sodium
- iron
The macronutrients for Sporulating cells
Calcium
Macronutrient needed by marine orgs
Sodium
Macronutrient needed by magnetic bacteria for magentisomes
Iron
Macronutrients trace elements
Metals: Cr, Co, Cu, Zn, Mn, etc.
Macronutrient growth factors
Vitamins and amino acids - organisms that can’t make their own growth factors have to get these from the environment= fastidious
Culture media
- nutrient base to support the growth of M.O. Types: 1. Selective media 2. Differential media 3. Complex media 4. Defined media
Selective media
selects for growth of a particular M.O.
Differential media
differentiates among organisms growing on media eg. usually colony color formation
Can media be selective and differential?
Yes
Complex media
cannot chemically define the exact composition of medium eg. digested protein - can’t tell the exact AA composition
- works to grow most fastidious orgs.
Defined media
know the exact chemical composition (including concentration) of the medium eg. has 2g/L glucose and can add specific growth factors for the growth of fastidious M.O.
Can you have selective or differential defined medium?
Selective defined medium
Can you have selective or differential complex medium?
differential complex medium
What is the universal growth medium and universal set of growth conditions?
There aren’t any
Growth is…
an increase in cell number
Through what mechanism is cell growth?
Binary fission (bacterial cell division)
How old is a bacterium?
Bacteria don’t age
What happens in binary fission?
Each parent cell split into two identical daughter cells (may not be true b/c of replication, mutation could occur)
How is growth of cells tracked?
As a population response, impossible to track individual cells
The steps to binary fission are largely unknown but are:
- slight elongation and duplication of resources (chromosomes, ribosomes, enzymes, transport proteins)
- septum formation and resource partitioning
- septum elongation
- separation of daughter cells
What is the septum
cell membrane, cell wall, outer membrane if GN
Phases of the growth curve of a closed system
- lag phase: cells preparing to grow/divide under enviro. conditions but no division is occuring
- exponential: cell division occurs on regular basis; predictable growth
- stationary: cell division = cell death; growth slows, cell death begins to occur b/c of limited nutrients or buildup of waste; some cells better adapted to survive and ddivide; cellular cannibalism occurs
- Death phase: cell death > cell division; rarely drops to 0 because of cannibalism
Axes of growth curve
Y = log # of cells per mL X = Time
Growth curve of an open system
All over the place
Number of generations (N)
(LogN - Log No)/0.301 = N
Growth rate (k)
K= N/t
Generation time (g)
g = 1/k
Factors influencing growth
to control growth:
- temp. (refridgeration)
- pH (pickling)
- O2 availability
- antimicrobial chemicals
Disinfection is
the killing/inhibition/removal of M.O.s from a given system or enviro.
Chemical used in disinfection:
- disinfectants: chemicals used to disinfect inanimate objects (lab bench)
- antiseptic: chemicals used to disinfect tissues (iodine)
- chemotherapeutic agents: chemicals used inside the body
Disinfecting chemicals are either _____ or ____ agents and either ____ or ____.
cidal agents: kill M.O.s (penicillin kills bacteria by destroying cell wall)
static agents: inhibit growth of M.O.s - once agent is removed, growth can resume
antibiotic: produced by other M.O.s (penicillin)
synthetic: artificially made or synthesized chemical (sulfadrug)
What do antimicrobials usually target?
something structural in a cell eg. cell wall, cell membranes, enzymes, ribosomes, transport proteins, etc.
Resistance mechanisms
- modification of structural target so antimicrobial no longer recognizes its target (eg. bonding change with NAM and NAG for penicillin resistance)
- degradation enzymes: enzymes designed to be excreted by the cell for degradation of antimicrobial (penicillinase degrades penicillin)
- Use of ATP dependent efflux pumps - transport proteins that use ATP to drive the removal of antimicrobial from the cell (penicillin-specific efflux pumps)
Example of chemical control of microbial groups (viral)
- protein capsid designed to protect genome
- relies on use of nucleotide analogs
- AZT (azidothydimine) is the antiviral for HIV (RNA genome virus)
- RNA converted to DNA via reverse transcriptase (RNA dependent DNA polymerase) and then DNA goes to RNA via host cell transcriptional machinery
- AZT is a chain terminator - lacks nucelotide 3’OH to allow for DNA synthesis to continue
- reverse transcriptase shows a higher affinity for AZT over T
Fungal control
- challenging to design antifungal chemicals that can distinguish fungi from other euk. cells
- ergosteral inhibitors - chemicals that inhibit the synthesis of ergosterols in fungi for maintaining membrane fluidity
Protozoal control
- challenge to design chemical that can distinguish protozoan from human cell
- hydroxy chloroquine - used against Plasmodium (malaria), inhibits choline synthesis
How many chromosomes do prok. have?
1 circular one
Where does DNA replication start?
at the origin of replication (ORI) site
What happens after DNA replication starts, what forms?
theta structure - visual cue of replication of chromosome; forms as 2 forks move half way through circular chromosome
DNA always synthesized by adding onto the
3’ end
Single-stranded binding protiens
protect single-stranded DNA from destruction by nucleases
DNA synthesis is always from ___ to ___
5’ to 3’
On the leading strand, DNA is synthesized ____ of the fork moving
in order
On the lagging strand, DNA is synthesized ____ of fork moving
opposite
DNA helicase
dsDNA –> ssDNA
DNA (RNA) primase
put down RNA primers, RNA polymerase is specific to RNA primer production
DNA gyrase
removes supercoiling
ozaki fragment
short segments of DNA synthesized on lagging strand
How many primers are needed to start DNA synthesis on the leading strand? And on the lagging strand?
leading: 1
lagging: multiple
DNA polymerase I (DNA pol. I)
- removes the RNA primer and fills gap with DNA on ozaki fragments but nick remains
- has exonuclease activity for RNA
- replaces with DNA using 3’OH from previous ozaki fragment
DNA ligase
connects 3’OH and the 5’PO4 of neighboring bases to seal ozaki fragment nicks
What two proteins are used to seal ozaki fragments?
- DNA pol. I
- DNA ligase
Mistakes made during replication
- 1 error/ 10^8 - 10^11 base pairs
- DNA polymerases have ability to correct some “incorrect” bases during DNA synthesis = exonuclease activity
Transcription is
DNA —> RNA (mRNA)
How does RNA differ from DNA?
- RNA is single-stranded
- Has U instead of T
RNA polymerase
- DNA dependent RNA polymerase (reads DNA but makes RNA)
- adds nucleotides to 3’OH of growing RNA strand (5’ —> 3’)
- synthesizes RNA denovo (doesn’t need primer)
- needs to act on conjunction with sigma factor protein to initiate transcription
Sigma factor
- recognizes gene to be transcribed
- recognizes start site (=promoter) for transcription
Promoter region
- many types of sigma factors in cell
- each sigma factor is specific to given variable region within the promoter region
- each sigma factor is specific to specific gene(s)
- -35 region (conserved), variable region, -10 region (conserved)
- promoter region activates transcription of a given gene via binding gene’s sigma factor but also aligns transciption machinery so that mRNA synthesis begins at the start of coding region of gene
Pribnow box
-35 and -10 sequences in promoter region
How serious is it if mistakes are made during transcription?
not fatal, always making multiple mRNA
What happens to the sigma factor once RNA pol. starts synthesizing RNA?
it is released
Rho independent termination
- stops transcription
- destabilization comes from stem loop and run of Us (run of A’s in DNA)
Rho dependent termination
- stops transcription
- rho protein recognizes rut site in mRNA
- rho moves along mRNA towards RNA pol.
- rho catches up and collides with RNA pol. and destabilizes it
- rho is a hexomer (has 6 subunits)
- destabilization occurs via stem loop and rho protein
The genetic code
- 4 bases –> 64 possible codons
- 1 start codon (AUG) = Met
- 3 stop codons (UAA, UAG, UGA)
- code is degenerate: many codons per amino acid; 1st 2 bases important, 3rd doesnt matter as much = wobble effect
DNA pol. III
main DNA polymerase synthesizing leading and lagging strands
translation
mRNA —> protein
Prok. translation
- AUG = N-formyl methhion (fMet) precursor to Met
- ribosome recognizes 5’ end of mRNA via shine degarno sequence (SDseq.)
transfer RNA (tRNA)
- single-stranded molecule folded in on itself
- has anticodon that is sequence of bases complimentary to codon
- Charged tRNA = AA attached
- uncharged tRNA = AA not attached
aminoacyl tRNA synthetases
enzymes that “charge” tRNAs with their appropriate AA
Ribosome
- comprised of RNA and protein
- prok: 30s subunit and 50s subunit: 70s ribosome
- euk. 40s subunit and 60s subunit = 80s subunit
prok. 70s ribosome
- 30s subunit: 16s rRNA + 21 proteins
- 50s subunit: 23s rRNA + 34 proteins
