MCB Ch. 7: Microbial Growth

eukaryotic reproduction

(fungi)

• haploid or diploid

• asexual or sexual (microorganisms)

prokaryotic reproduction

• haploid only (1 chromosome)

• asexual

prokaryotic replication methods

• binary fission

• budding

• filamentous

binary fission

• one cell grows and divides into two identical cells

• time of this process can differ

• bigger size/higher complexity → slower replication

budding

• yeast and bacteria

• one mother cell gives rise to multiple buds

filamentous

attached and hair-like

• ex: streptomyces - source of antibiotics

chromosome replication and partitioning

• origin of replication (oriC)

• terminus (ter)

• chromosome must replicate before replication continues (rate-limiting step) ???

• septum forms → cell divides

origin of replication (oriC)

• within DNA

• site where replication starts

terminus (ter)

site where replication ends

chromosome partitioning

• Replisome pushes, or leads to condensation of, daughter chromosomes to opposite ends

• this system exists in almost 70% of 400 sequenced bacterial genomes

• ParA/ParB proteins

ParA/ParB

• proteins in C. crescentus

• C. crescentus floats around and attaches to hard surface (stalk) when it is ready to replicate

ParA

• protein ~ polymerase

• polymerize and binds to ParB

• as it depolymerizes, it pulls the other chromosome to the right (away from the stalk)

ParB

• sequence-specific protein ~ binding

• binds to parS (DNA sequence)

• ParB binds to pole close to stalk and parS, holding chromosome to left side

Z ring formation

role in septation

several steps

1. selection of site for septum formation

2. assembly of Z ring

3. assembly of cell wall synthesizing machinery

4. constriction of cell and septum formation

what is the Z ring made of

FtsZ

where does the Z ring form?

in the absence of MinCDE (center of cell)

what type of membrane protein is the Z ring?

peripheral protein (not attached to membrane)

what happens when the Z ring depolymerizes?

• separation and septum of the two cells form

  • FtsZ polymers no longer holds Z ring together → 2 daughter cells separate

assembly of cell wall synthesizing machinery

• peptidoglycan synthesizing enzymes

• anchoring proteins

peptidoglycan synthesizing enzymes

• main function is to prevent cell from growing too much and exploding

• building more peptidoglycan allows for bacterial growth

anchoring proteins

FtsA and ZipA help attach FtsZ to membrane

constriction of cell and septum formation

• MinCDE system

  • polymerize at one pole and then depolymerize and re-polymerize at another pole

    • goes left and right at all times

  • dictates location

    • inhibits function of Z ring

penicillin binding proteins (PBPs)

synthesizes and maintains peptidoglycan (bacterial cell wall)

targeted by β-lactam antibiotics, like penicillin, which disrupt peptidoglycan synthesis, leading to bacterial cell death.

transpeptidases (TPases)

type of PBP -

• catalyzes the cross-linking between peptide chains in the peptidoglycan

• they form the peptide bridges that stabilize the bacterial cell wall by linking the 3rd AA on one NAM-pentapeptide to the 4th AA (D-Ala) on another NAM

• final step in cell wall synthesis

first step of peptidoglycan synthesis → cellular growth

1. UDP-NAM-pentapeptide is synthesized in cytoplasm

second step of peptidoglycan synthesis → cellular growth

2. bactoprenol (lipid carrier facing the cytoplasm) + UDP-NAM-pentapeptide → lipid I

- joined via pyrophosphate bond

- bacteria uses UTP instead of ATP to activate sugar

- UDP has 2 phosphates

third step of peptidoglycan synthesis → cellular growth

3. UDP-NAG (activated NAG) + lipid I → lipid II

fourth step of peptidoglycan synthesis → cellular growth

4. bactoprenol flips (flippase) lipid II across cytoplasmic membrane (in/cytoplasm → out/periplasmic space), bringing NAM/NAG (peptidoglycan building blocks) to periplasmic space to be added to peptidoglycan

- enzymes come in to move NAM/NAG

fifth step of peptidoglycan synthesis → cellular growth

bactoprenol is flipped back to cytoplasm

autolysins

enzyme breaks down peptidoglycan to allow for growth

- NAM and NAG adds where it is broken

goal of antibiotics

make peptidoglycan weak enough for cell to lyse

- penicillin

- vancomycin

- bacitracin

penicillin

Binds to transpeptidase and prevents bridge between 3rd AA to 4th AA → cell wall is weak

vancomycin

Doesn't allow removal of terminal alanine

bacitracin

Inhibits bactoprenol from flipping from the outside back inside, can't bring NAM/NAG again

E. coli vs S. aureus in peptidoglycan

E. coli:

- DAP

- gram-negatives have a direct link between 3rd and 4th AA

S. aureus:

- L-Lys

- gram-positives have a bridge between 3rd and 4th AA

- has a 5th lysine

Both:

- secondary amino group which allows for 3 peptide bonds

lysosomes can break

cell walls

why can't lysosomes break pseudomurein

bc of the β-1,3 glycosidic bond

vibroid cell wall growth and cell shape

FtsZ, MreB, CreS, CrvA

when division occurs, there is expression of FtsZ and formation of Z ring

CrvA

• gives vibrio shape in V. cholerae, V. parahaemolyticus, V. vulnificus

• V. fischeri is not a human pathogen (glows in squid)

• not a good antibacterial target

spherical bacteria

e.g., S. aureus

• elongation:

  • no elongation

• division:

  • FtsZ forms Z ring to initiate division

rod bacteria

e.g., B. subtillis and E. coli

• elongation:

  • MreB elongates bacteria into rod shape

• division:

  • FtsZ forms Z ring

  • MreB continues elongation

vibroid bacteria

e.g., C. crescentus

• elongation:

  • MreB elongates

  • crescentin localizes to inner curve and inhibits growth, causing bending

• division:

  • FtsZ initiates division

  • MreB elongates

  • crescentin provides curvature

growth

increase in cellular constituents

growth leads to

increase in cell number (divide)

• discontinuous

increase in cell size

• continuous, though temporary - it eventually divides

what does growth refer to

population growth rather than growth of individual cells

growth curve

plotted as logarithm number of viable cells vs. time

5 distinct phases:

- lag, log/exponential, stationary, death, long-term stationary

viable cells

can replicate

metabolically active

1. lag phase

exists for a short or long period of time, or is skipped

cell is preparing to grow/replicate

transcribing and translating peptidoglycan building enzymes (FtsZ, MinCDE, ZipA, FtsA)

when does lag phase occur?

From stationary or death phase into a fresh medium

From exponential phase into a fresh medium of different chemical composition

- cell has to adapt to new medium

- biphasic growth (glucose → lactose)

From a rich to a poor culture (medium shift)

- learning how to make stuff they were previously given for free

biphasic growth

2 phases

ex: media has both lactose and glucose

uses glucose first because it's a monosaccharide (lactose is a disaccharide)

- if you can use lactose, you can use glucose (since lactose = glucose + galactose), but a cell that uses glucose may not be able to use lactose

when does lag phase not occur?

Cells growing exponentially into a fresh medium of the same chemical composition

- ex: lactose → glucose (bc lactose has glucose)

2. log/exponential phase

Replicating as fast as possible

growing rate is maximal

- healthiest

- prokaryotes grow faster than eukaryotes (because they are smaller, simpler, and haploid)

- smaller cells grow faster than larger cells

population is most uniform

can be physically seen

3. stationary phase

Running out of nutrients and space, collects toxic material

population growth eventually ceases

- limit reached

- accumulation of waste (bacteria stops replicating when environment changes)

total number of viable cells remains constant, no net change

no building enzymes (FtsZ)

4. death phase

Exponential but not as fast as log

two alternative hypotheses

1. viable but not culturable (VBNC)

2. programmed cell death (PCD)

viable but not culturable (VBNC)

includes E. coli (HUS), V. cholerae (cholera), and L. monocytogenes (listeriosis)

cannot replicate on a petri dish

decreased in size and slowed metabolism, "sleeping beauty"

programmed cell death (PCD)

apoptosis

cell is no longer viable and kills itself to release content into environment for a neighboring cell to absorb

5. long-term stationary phase

evolution (strongest survives)

small percent may mutate and thrive in waste material

bacterial population continually evolves → natural selection (surviving mutant)

Mutations can lasts months to years

'Birth' and 'death' rates are balanced

Growth advantage in stationary-phase (GASP) phenotype

measurement of growth rate and generation time

only for binary fission during exponential growth (not budding because it produces more than 2)

if a number is not given, N0 = 1

generation time

(g)

varies from minutes to hours to days

growth rate constant

(k)

generations per hour

larger number = faster growth

ex:

60/10 mins = 6 generations

60/20 = 3 g

60/60 = 1 g

n

generation number

Nt

total final cell number

Nt = 2^n

.

solving for populations reproducing by binary fission

Nt = N0 x 2^n

solving for n (number of generations)

n = log Nt - log N0 / 0.301

solving for k (growth rate constant)

k = log Nt - log N0 / 0.301(t)

solving generation doubling time

Nt = 2N0

k=1/g

or g=1/k

what conditions are organisms grown in

fairly moderate environmental conditions

few places on earth are considered sterile

extremophiles (archaea)

- non-pathogenic because the environment they thrive in is not something humans have

microbes are adapting

what environment do microbes grow in

hypotonic (water goes in)

they have developed to prevent water from rushing in

reduce osmotic concentration

mechanosensitive (MS) channels ~ hypotonic

contractile vacuoles in protists (eukaryote)

mechanosensitive (MS) channels

pressure on membrane makes it tighter and causes places to open for water to exit

if the channels are closed, water cant get out and cell explodes

as they feel the pull on the membrane, they open

- tighter: channel opens

- cell gets smaller: channel closes

contractile vacuoles in protists

vacuoles push water out

Increase internal solute concentration

compatible solutes (in cytoplasm) ~ hypertonic (from increase in salt)

osmophiles

compatible solutes (in cytoplasm)

• Equate inside and outside solute concentration

  • By increasing inside solute concentration

• Cannot inhibit enzymatic activities

  • Prolene: can increase conc of prolene which increases solute conc to be equal to outside, no net loss

  • Must be increasing concentration of something that does not cause harm to them

• Helps protect since they lack peptidoglycan

Nonhalophile

Not salt lovers

Halotolerant

tolerates salt

- S. aureus

extreme halophiles

requires salt

- Halobacterium spp.

(archaea ~~ extremophile)

compatible solutes

helps bacteria/cells to survive in high solute concentration (salt in environment)

- must equate or else water will go outside

osmotolerant

tolerate stuff outside but doesn't prefer to grow in it

higher solute → lower water activity (aw)

less water → aw decreases

- water availability necessary for microbial growth

all domains of life

Acidophiles

(0-5.5)

archaea

Neutrophiles

(5.5-8)

bacteria and protists

Alkaliphiles/alkalophiles

(8-11.5)

marine microorganisms

most microbes maintain an internal pH near

neutrality

- dies if internal pH is < 5.5-5.0 → denaturation

acid tolerance response

pumps protons out

- too many protons → pH drops → cell death

some synthesize acid and heat shock proteins that refold and protect proteins against denaturation of proteins

many microorganisms change what?

the pH of their habitat

microbes cannot regulate what?

their internal temperature

cardinal growth temperatures

minimum, optimum, maximum

30-40º C difference between minimum and maximum

- No bacteria grows at 0 and 100º C

optimum temperature

Optimum is generally closer to maximum

• Bc heat is energy, enzymatic activity increases as temp increases (which is at an optimum temp, which is the best speed)

  • Anything higher denatures the protein

Psychrophiles

cold lovers

Psychrotolerant

Tolerate cold

Mesophiles

moderate temperature loving (human, body temp)

majority of pathogens are ___?____ though one is psychrotolerant

mesophiles

psychrotolerant - Listeria monocytogenes

Thermophiles

heat loving

Hyperthermophiles

extreme thermophiles, grow in extreme heat

• fight against denaturation

psychrophiles

protein structure:

• more α-helix, less β-sheets

  • β-sheets can freeze and come together which is what they want to avoid 

• more polar, less hydrophobic amino acids

fatty acid chains:

• more unsaturated (more double bonds) and shorter

• Fatty acids found in bacteria and isoprenes (euk)

Compatible solutes to decrease freezing point

hyperthermophiles

protein structure:

• presence of chaperones & HSP

• more H bonds and proline

fatty acid chains:

• more saturated, branched and higher molecular weight

most are certain species of archaea

• monolayers and ether linkages

histone-like proteins stabilize DNA27

• Histones are in eukaryotes; histone-like found outside of eukaryotes

thiglycolate test tubes

as it solidifies, it inhibits oxygen from getting into the bottom → makes a gradient/mostly found at top

what color is the oxic zone when oxygen is present

pink

obligate aerobe

growth only at top

+SOD

+catalase

+peroxidase