Download Cell Growth and Binary Fission

Microbial Growth
Microbial growth implies an increase in cellular constituents
- leads to rise in cell number when microorganisms
reproduce by processes like budding or binary fission
- ability to reproduce is a major criterion to determine if
a microbe is alive or not
- results when cell become longer/larger
Population growth is used to analyze the growth curve of a
microbial culture
Cell Growth and Binary Fission
Fts proteins
Min E proteins assist
in the location of the
actual cell midpoint
forms in the
space between
the duplicated
nucleoids
The FtsZ ring
and cell division
forms a ring around
the cylinder in the
center of the cell
2 copies of chromosomes
are pulled apart to each
daughter cell
Peptidoglycan Features
1. 3-D polymeric macromolecule
2. Formed from subunits by two types of
covalent bonds
3. ß-1,4 glycosidic bonds between hexose
sugars, and peptide bonds between amino
acids
4. Determines cell shape and prevents osmolysis
5. Dynamic structure
a) must grow as cell grows
b) must be regulated to allow septation
Peptidoglycan
Glycan
chains
MA
-1,4-linkages
L-ala
GA
D-glu
MA
L-lys
L-ala
GA
D-glu
L-lys
GA
MA
GA
L-ala
D-ala
D-glu
MA
L-ala
D-ala
L-lys
D-ala
D-glu
L-lys
D-ala
Peptide cross-link
Tetrapeptide
Gram - negative
Gram - positive
Peptidoglycan-Targeting Antibiotics
• Destruction of peptidoglycan causes bacterial lysis
• This can be accomplished in the laboratory using the enzyme
lysozyme, which hydrolyzes the glycosidic linkages
• Antibiotics should target bacteria-specific processes, such as
peptidoglycan synthesis
• DO NOT use these antibiotics on bacteria with no cell wall
(Mycoplasma) or a cell wall that is not susceptible to them
(Mycobacteria)
Antibiotics that Target the
Peptidoglycan
• Phosphonomycin
• Cycloserine
• Vancomycin
• Bacitracin
• Penicillin
• Cephalosporins
Peptidoglycan Antibiotic Targets
Synthesis-Cytoplasm
Start
NAG converted to NAM
L-ala, D-glu and L-lys
added one at a time
D-ala-D-ala
added as a
dipeptide
Synthesis-Membrane
Addition of NAG
results in dipeptide
precursor
Start
Transfer to lipid
carrier
Sites of action of different antimicrobial agents. PABA, paraminobenzoic acid;
DHFA, dihydrofolic acid; THFA, tetrahydrofolic acid.
Outer wall of Gram-positive and Gram-negative species and detail of
porin channels of Gram-negative bacteria. Antimicrobial agents diffuse
easily through the loose outer wall of Gram-positive bacteria, but must go
through the narrow channels of the Gram-negative species.
Structure of metronidazole and its mechanism of action. Metronidazole
enters an aerobic bacterium where, via the electron transport protein ferrodoxin,
it is reduced. The drug then binds to DNA, and DNA breakage occurs.
Diagrammatic representation of inhibition sites of protein biosynthesis
by various antibiotics that bind to the 30S and 50S ribosomes.
Inhibition of protein biosynthesis by aminoglycosides.
Structure of sulfonamide and trimethoprim with sites of inhibition of
folic metabolism.
MULTIDRUG RESISTANCE AMONG PATHOGENIC BACTERIA
How do they do it?
• Acquire ability to degrade the antibiotic
• Change their outer structure to prevent
drug entry
• Change the drug target so that it is no
longer affected by the drug
• Acquire and/or turn on an efflux pump to
eliminate the drug from the cell
Where do they get it?
• Chromosomal mutation(s) under selective
pressure by the antimicrobial
• Conjugal transfer of resistance plasmids
• Conjugal transfer of chromosomal
resistance genes
• Infection by bacteriophage
• An old system that found a new use
(efflux pumps)
Example of how two antibiotics (A and B) may interact with synergy,
indifference, or antagonism.
Types of Antibiotics
Penicillins have a common chemical structure which they share with the
cephalopsorins. Penicillins are generally bactericidal, inhibiting
formation of the cell wall.
Types of penicillin
•The natural penicillins are based on the original penicillin-G structure.
Penicillin-G types are effective against gram-positive strains of streptococci,
staphylococci, and some gram-negative bacteria such as meningococcus.
•Penicillinase-resistant penicillins, notably methicillin and oxacillin, are active
even in the presence of the bacterial enzyme that inactivates most natural
penicillins.
•Aminopenicillins such as ampicillin and amoxicillin have an extended spectrum
of action compared with the natural penicillins. Extended spectrum penicillins
are effective against a wider range of bacteria.
Penicillin
• Penicillin binds to proteins known as Penicillin-Binding Proteins
(PBPs)
• Multiple PBPs are made by each species, with different molecular
weights and different enzymatic activities
• PBPs are involved in the cross-linking reactions, and typically have
transpeptidase activity
• Inhibition of peptidoglycan cross-linking destabilizes the cell wall
Cephalosporins
Cephalosporins have a mechanism of action identical to that of the penicillins.
However, the basic chemical structure of the penicillins and cephalosporins differs
in other respects, resulting in some difference in the spectrum of antibacterial
activity.
Like the penicillins, cephalosporins have a beta-lactam ring structure that
interferes with synthesis of the bacterial cell wall and so are bactericidal.
Cephalosporins are derived from cephalosporin C which is produced from
Cephalosporium acremonium.
Tetracycline
Tetracyclines got their name because they share a chemical structure that has
four rings. They are derived from a species of Streptomyces bacteria.
Tetracycline antibiotics are broad-spectrum bacteriostatic agents, that inhibit
bacterial protein synthesis. Tetracyclines may be effective against a wide
variety of microorganisms, including rickettsia and amebic parasites.
Structure of tetracycline showing the
area critical for activity and major and
minor points of modification.
Macrolides
The macrolide antibiotics are derived from Streptomyces bacteria, and got their
name because they all have a macrocyclic lactone chemical structure.
The macrolides are bacteriostatic, binding with bacterial ribosomes to inhibit
protein synthesis. Erythromycin, the prototype of this class, has a spectrum and
use similar to penicillin.
The most commonly prescribed macrolide antibiotics are:
erythromycin
clarithromycin
azithromycin
dirithromycin
roxithromycin
troleandomycin
Fluoroquinolones
Fluoroquinolones (fluoridated quinolones) are the newest class of antibiotics.
Their generic name often contains the root "floxacin". They are synthetic
antibiotics, and not derived from bacteria. Fluoroquinolones belong to the family
of antibiotics called quinolones.
The older quinolones are not well absorbed and are used to treat mostly urinary
tract infections. The newer fluroquinolones are broad-spectrum bacteriocidal drugs
that are chemically unrelated to the penicillins or the cephaloprosins. Because of
their excellent absorption fluroquinolones can be administered not only by
intravenous but orally as well.
Aminoglycosides
Aminoglycoside antibiotics are used to treat infections caused by gram-negative
bacteria. Aminoglycosides may be used along with penicillins or cephalosporins
to give a two-pronged attack on the bacteria.
The aminoglycosides are drugs which stop bacteria from making proteins.
This effect is bacteriocidal.
The most commonly-prescribed aminoglycosides:
amikacin
gentamicin
kanamycin
neomycin
streptomycin
tobramycin
MICROBIAL GROWTH KINETICS
- describe how the microbe grows in the fermenter
- important to determine optimal batch times
- growth of microbes can be broken down into 4 stages;
accelerated
death phase
LAG PHASE
LOG/EXPONENTIAL PHASE
STATIONARY PHASE
DEATH PHASE
decelerated
growth phase
Lag Phase
accelerated growth phase
Cells have just been introduced into a new environment
Cell growth is minimal
Cell is synthesizing new components – no cell division takes place
- cell is old and depleted of ATP
- medium may be different from the one the microorganism was growing
- microorganism have been injured and require time to recover
LOG/EXPONENTIAL PHASE
Cells have adjusted to their environment
Rapid growth takes place
Cell growth rate is highest in this phase
At some point, cells growth rate level off
and become constant
STATIONARY PHASE
Cell growth rate has leveled off and become constant
Number of cells multiplying equals the number of cells dying
- nutrient limitation
- aerobic organism are limited by oxygen availability
- population growth cease due to the accumulation of toxic
waste products
DEATH PHASE
Decline in the number of viable cells
Log/Exponential Phase
The rate of increase in biomass is correlated with the specific
Growth rate µ and the biomass concentration X (g/L), whereas the
Rate of sincrease in cell number is correlated with µ and cell density N (1/L)
dX = µ•X
dt
or
dN = µ•N
dt
specific growth rate, µ,
3 parameters : the concentration of limiting substrate S
the maximum growth rate µmv
the substrate-specific constant Ks
µ = µm
___S___
Ks + S
Monod equation
Ks substrate concentration at which half
the maximum specific growth rate is
obtained (µ = 0.5 µm )
- equivalent to the Michaelis constant in enzyme
kinetics
Maximal specific growth rates (µm) of some fungi on glucose
Organism
T ºC
Aspergillus niger
30
0.20
3.46
Aspergillus nidulans
20
0.090
7.72
Penicillium
25
0.123
5.65
Mucor hiemalis
25
0.17
4.1
Fusarium avanaceum
25
0.18
3.8
Fusrium graminearum
30
0.28
2.48
Verticillium agaricinum
25
0.24
2.9
Geotrichum candidum
25
0.41
1.7
Neurospora sitophila
30
0.40
1.73
Anderson et al, 1975
µm (h-1)
Doubling time (h)
Effect of glucose chain length on the maximal specific growth rate (µm) in
Fusarium graminearum at 30 ºC
# of glucose units
µm
Doubling time
(h)
Glucose
1
0.28
2.48
Maltose
2
0.22
3.15
Maltoriose
3
0.18
3.85
Substrate
Anderson et al, 1975
PARAMETERS THAT MUST BE PRECISELY REGULATED
-
temperature
-
pH
-
rate and nature of mixing
-
oxygenation
- sterility and containment
Types of Cultures
1.
2.
3.
4.
Batch
Fed-batch
Continuous
Synchronous