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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

1

What macronutrients are often used to buffer and maintain osmolarity?

Magnesium and potassium

2

The macronutrients for Sporulating cells

Calcium

3

Macronutrient needed by marine orgs

Sodium

4

Macronutrient needed by magnetic bacteria for magentisomes

Iron

5

Macronutrients trace elements

Metals: Cr, Co, Cu, Zn, Mn, etc.

6

Macronutrient growth factors

Vitamins and amino acids - organisms that can't make their own growth factors have to get these from the environment= fastidious

7

Culture media

- nutrient base to support the growth of M.O.
Types:
1. Selective media
2. Differential media
3. Complex media
4. Defined media

8

Selective media

selects for growth of a particular M.O.

9

Differential media

differentiates among organisms growing on media eg. usually colony color formation

10

Can media be selective and differential?

Yes

11

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.

12

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.

13

Can you have selective or differential defined medium?

Selective defined medium

14

Can you have selective or differential complex medium?

differential complex medium

15

What is the universal growth medium and universal set of growth conditions?

There aren't any

16

Growth is...

an increase in cell number

17

Through what mechanism is cell growth?

Binary fission (bacterial cell division)

18

How old is a bacterium?

Bacteria don't age

19

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)

20

How is growth of cells tracked?

As a population response, impossible to track individual cells

21

The steps to binary fission are largely unknown but are:

1. slight elongation and duplication of resources (chromosomes, ribosomes, enzymes, transport proteins)
2. septum formation and resource partitioning
3. septum elongation
4. separation of daughter cells

22

What is the septum

cell membrane, cell wall, outer membrane if GN

23

Phases of the growth curve of a closed system

1. lag phase: cells preparing to grow/divide under enviro. conditions but no division is occuring
2. exponential: cell division occurs on regular basis; predictable growth
3. 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
4. Death phase: cell death > cell division; rarely drops to 0 because of cannibalism

24

Axes of growth curve

Y = log # of cells per mL
X = Time

25

Growth curve of an open system

All over the place

26

Number of generations (N)

(LogN - Log No)/0.301 = N

27

Growth rate (k)

K= N/t

28

Generation time (g)

g = 1/k

29

Factors influencing growth

to control growth:
- temp. (refridgeration)
- pH (pickling)
- O2 availability
- antimicrobial chemicals

30

Disinfection is

the killing/inhibition/removal of M.O.s from a given system or enviro.

31

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

32

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)

33

What do antimicrobials usually target?

something structural in a cell eg. cell wall, cell membranes, enzymes, ribosomes, transport proteins, etc.

34

Resistance mechanisms

1. modification of structural target so antimicrobial no longer recognizes its target (eg. bonding change with NAM and NAG for penicillin resistance)
2. degradation enzymes: enzymes designed to be excreted by the cell for degradation of antimicrobial (penicillinase degrades penicillin)
3. Use of ATP dependent efflux pumps - transport proteins that use ATP to drive the removal of antimicrobial from the cell (penicillin-specific efflux pumps)

35

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

36

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

37

Protozoal control

- challenge to design chemical that can distinguish protozoan from human cell
- hydroxy chloroquine - used against Plasmodium (malaria), inhibits choline synthesis

38

How many chromosomes do prok. have?

1 circular one

39

Where does DNA replication start?

at the origin of replication (ORI) site

41

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

42

DNA always synthesized by adding onto the

3' end

43

Single-stranded binding protiens

protect single-stranded DNA from destruction by nucleases

44

DNA synthesis is always from ___ to ___

5' to 3'

45

On the leading strand, DNA is synthesized ____ of the fork moving

in order

46

On the lagging strand, DNA is synthesized ____ of fork moving

opposite

47

DNA helicase

dsDNA --> ssDNA

48

DNA (RNA) primase

put down RNA primers, RNA polymerase is specific to RNA primer production

49

DNA gyrase

removes supercoiling

50

ozaki fragment

short segments of DNA synthesized on lagging strand

51

How many primers are needed to start DNA synthesis on the leading strand? And on the lagging strand?

leading: 1
lagging: multiple

52

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

53

DNA ligase

connects 3'OH and the 5'PO4 of neighboring bases to seal ozaki fragment nicks

54

What two proteins are used to seal ozaki fragments?

- DNA pol. I
- DNA ligase

55

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

56

Transcription is

DNA ---> RNA (mRNA)

57

How does RNA differ from DNA?

- RNA is single-stranded
- Has U instead of T

58

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

59

Sigma factor

- recognizes gene to be transcribed
- recognizes start site (=promoter) for transcription

60

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

61

Pribnow box

-35 and -10 sequences in promoter region

62

How serious is it if mistakes are made during transcription?

not fatal, always making multiple mRNA

63

What happens to the sigma factor once RNA pol. starts synthesizing RNA?

it is released

64

Rho independent termination

- stops transcription
- destabilization comes from stem loop and run of Us (run of A's in DNA)

65

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

66

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

67

DNA pol. III

main DNA polymerase synthesizing leading and lagging strands

68

translation

mRNA ---> protein

69

Prok. translation

- AUG = N-formyl methhion (fMet) precursor to Met
- ribosome recognizes 5' end of mRNA via shine degarno sequence (SDseq.)

70

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

71

aminoacyl tRNA synthetases

enzymes that "charge" tRNAs with their appropriate AA

72

Ribosome

- comprised of RNA and protein
- prok: 30s subunit and 50s subunit: 70s ribosome
- euk. 40s subunit and 60s subunit = 80s subunit

73

prok. 70s ribosome

-30s subunit: 16s rRNA + 21 proteins
-50s subunit: 23s rRNA + 34 proteins