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riddhisnewworld@yahoo.co.in
 Ôacterial Cell Division
‡ Cell growth is defined as an increase in
the number of cells, requires continued
growth to maintain species
‡ ~2000 chemical reactions with a wide
variety of types
± main rxn is polymerization reaction
‡ monomer to polymer
Ôinary Fission
‡ Cell growth continues until
divides into 2 new cells
‡ Cells create a —

between new cells
± starts as cytoplasmic
membrane and eventually
becomes cell wall
‡ Each batch of new cells is a
  

‡ Cellular components increase
proportionally so each cell gets
enough of everything to the
new cell
‡ Time to generate new cells is
dependent on nutritional and
genetic factors
± division is tied to chromosomal
replication
 Fts Proteins
‡ Filamentous temperature sensitive
proteins ± mutation in genes that encode
the Fts proteins
± bacteria without FtsZ have difficulty dividing
± FtsZ is universally distributed in all
prokaryotes
± see FtsZ-like proteins in mitochondria and
chloroplasts, also similar to tubulin in
eukarotes
 ‡ Fts interact to form the
division apparatus called
the divisome
Fts Complexes = ‡ FtsZ attach in a ring to
the cell at the membrane
Division Apparatus and then attracts FtsA
and Zip A
± FtsA ± ATP hydrolyzing
enzymes for proteins in
divisome
± ZipA ± anchor attachment
of FtsZ to membrane
‡ Also contain Fts proteins
involved in peptidoglycan
synthesis ± FtsI is a
penicillin-binding protein
(activity site for penicillin)
‡ Divisome makes new
cytoplasmic membrane
and cell wall in both
directions until large
enough to divide
 DNA Replication
‡ Occurs prior to FtsZ ring formation and
when done get the ring formation between
the 2 nucleoid regions using  

 —
‡   inhibits cell division until exact
center of the cell is found
‡   inhibits min C activity and attached
at center of cell, recruits the FtsZ and ring
formation
 Cell Shape
‡ Morphology = cell shape
‡ Peptidoglycans thought to dictate shape but now
know only a minor role
‡ Protein for shape is homologous to actin
‡ Major protein ± MreÔ ± forms an actin-like
cytoskeleton
± filamentous, spiral-shaped bands in cell under
cytoplasm membrane
± cocci lack MreÔ and its gene, default shape ± sphere
‡ Ôacteria make FtsZ and MreÔ ± tubulin- and
actin-like proteins
± evolutionary similarities between eukaryotes and
prokaryotes
Peptidoglycan Snthesis
‡ Must make the cell wall
before cell division ± add
new cell wall to existing
cell wall
‡ At FtsZ ± autolysins make
openings in wall
± enzyme similar to lysozyme
± present in the divisome
‡ Cell wall material added
thru the holes
‡ Ôetween new and old cell
wall, a ridge forms ± like
a scar
 Cell Wall Formation
‡ Precursors to the cell wall are spliced into
existing peptidoglycan
‡ If the precursors aren¶t coordinated with
the old, the cell goes through spontaneous
autolysis ± cell ruptures
 Ôiosynthesis of Peptidoglycan

‡ Cut pre-existing peptidoglycans by autolysins with

simultaneous insertion of precursors ± Ä

 
 ±
lipid carrier molecule, hydrophobic C55 alcohol
‡ Ôactoprenol makes the peptidoglycan precursors
hydrophobic so they can cross membrane to be
inserted, spend time in the periplasm to build cell wall
and make glycosidic bonds
 Transpeptidation and Penicillin
‡ Final step ± need to insert the peptide
components of the cell wall between the
muramic acid (refer to cell wall structure from
before)
‡ This reaction is inhibited by penicillins ± prevent
cell wall formation by binding to FtsI, autolysins
continue to weaken the cell wall and leads to
lysis
± used in humans
‡ since we do not have cell walls, can use drug at high levels
‡ virtually all bacterial pathogens have peptidoglycan so works on
most bugs
Final Interactions
‡ Interaction with several
amino acids based on the
organism
± E coli ± between
diaminopimelic acids and D-
Ala on adjacent peptides
‡ Removal of the 2nd D-Ala
drives the rxn as there is not
ATP (outside the cell)
‡ In gram +, glycine
interbridge is usaully
present, cross-link accur
across the interbridge on L-
Lys and D-Ala
urowth of Ôacterial Populations
‡ Increase in the number of organisms in a
population
‡ Terminology
± 1 cell to 2 cells is a   

± time for the new cell to form is the   
   ,
mass also doubles so also called 
Ä  
‡ These vary between organisms and are based
on growth medium, growth conditions
± usually differ out in nature vs. the test tube
Exponential urowth
‡ Where number of cells
double during a regular
tine interval
‡ uraph on linear scale,
see a dramatic increase
in the numbers over time
‡ uraph on semi-log
paper, you get a straight
line, meaning
exponentially growing
± use to estimate growing
time
Estimating urowth Rate
 urowth
‡ In exponential growth ± rate increase is
slow initially but increases in cell number
± in non-sterile, nutrient rich environments, such
as milk ± slow growth is good, leave milk out
an hour, not to many bacteria, but if leave out
several hours, the level of bacteria will be
much higher
 urowth Cycle

‡ Exponential growth cannot continue forever

‡ Cycle has 4 distinct areas ± lag, exponential,
stationary and death phases
 Lag Phase
‡ Delay in growth of bacteria
‡ Interval may be different
± based on organism and growth conditions
‡ See when using old or stationary phase
cultures to start your growth curve
‡ Lag is caused by cells being depleted in
essential constituents, must also repair if
damaged by heat or radiation, etc
± also see if moving from a rich medium to a
poorer medium
 Exponential Phase
‡ Cells divide for a brief time ± based on
resources and other factors
‡ Rate of growth vary greatly
± influenced by environmental conditions and
genetic characteristics of the organism
 Stationary Phase
‡ Limitation on growth caused by 2 factors
± essential nutrients of culture medium is used up
± some waste products of the organism build up in
medium and inhibit growth
± can be a combination of both
‡ Exponential growth stops and there is no net
increase or decrease in the cell number ± may
be slow growth
‡ Cellular functions continue ± energy metabolism
and biosynthetic processes
± some divide and some die ± 
 

 Death Phase
‡ Cells will die eventually
‡ Death accompanied by cell lysis
‡ ³Exponential death´ ± but slower than
growth

‡ Figure 6.8 is of a POPULATION and not a

single cell, this process does NOT apply to
them
 Measuring Microbial urowth
‡ 2 methods of direct measurement
± total counts
± viable count
‡ Important to know the number of bacteria
for some tests
 Total Counts
‡ 2

 using a microscope and
hemocytometer ± a special counting chamber
with a square on surface of glass with a
known volume under a cover slip
± count the number of cells on the grid and then
calculate the number of cells based on the
volume on the chamber
± also count dead cells, miss small cells, precision
is hard to achieve, requires phase contrast
microscopy when not stained, not good a low
density and motile cells must be immobilized
 ›iable Counts
‡ ›iable cells can divide and make offspring
‡ Determine whether capable of forming colonies on
suitable agar
± plate count or colony count, assume each cell can
yield a colony
‡ 2 methods ± spread plate and pour plate
± —
 
 ± use small volume of diluted cells and
spread over surface of agar, count colonies and
calculate number using dilution
±


 ± add volume of culture into Petri dish,
add melted agar, mix by swirling ± colonies form
throughout the agar, not just on top like above
method, examine carefully
 Dilutions
‡ Use dilutions to determine the number of
colonies in a countable range ± count/plate
should be between 30 and 300 colonies
‡ Must determine optimum conditions to grow
the bacteria ± temp, medium, time, etc
‡ Perform serial dilutions to get into the
³countable´ range
‡ Sources of error ± not using correct growing
conditions, errors also in technique ±
pipeting, mixing, etc.
Serial Dilutions
‡ Usually do serial 10-fold
dilutions by mixing 0.5 ml of
sample with 4.5 ml of fresh
medium (1 part in 9 parts =
10)
‡ Do consecutive dilutions in
the same manner and plate
a volume on the agar
‡ After growth, count the
colony forming units (cfu)
and calculate the number of
bacteria using dilution and
volume placed on plates
 Colony Forming Units
‡ Use colony forming units because occasionally
you may have 2 bacteria in the same area that
make a single colony you can¶t tell apart
‡ Can use selective media to count only a particular
organism
‡ ureat plate anomaly ± may be unreliable to
assess total number of cells in natural samples ±
soil, water plate count is usually lower than direct
count
± organisms may have really different nutrient needs
± may need selective media to get a better count
 Indirect Measurements

‡ Use turbidity as an indirect measurement ± more cells

mean it is more cloudy
‡ Use a photometer or spectrophotometer
± similar in use ± light scattered by cells and all light that passes
thru will be collected
± differences is with the light source ± photometer is a broad pass
filter and spec is prism or diffraction grating
± both measure only unscattered light but report in Klett units or
optical density
 ueneration of a
Standard Curve
‡ Can substitute turbidity for
direct counting methods but
need to make a standard curve
‡ Relate direct count to indirect
method
± may use cell number or cell mass
‡ Must use within limits ± really
³dense´ samples may deflect
light and then another cell re-
deflects them to the detector
± makes things non-linear
± can check sample repeatedly
without altering test outcome
Continuous Culture - Chemistat
‡ Previous growth was based on Ä — ±
fixed volume of medium ± altered by metabolism
in culture ± 
— —— 
‡ Constant environment needed over long periods
of time to generate a continuous supply of
exponential phase bacteria ± 
 
—
 ±

—— 
± add fresh medium and remove the ³old´ medium in a
chemistat
± volume, cell number and nutrient state are constant ±
— —
Chemistat
‡ Constant growth rate
and population
density
‡ 2 important factors
± dilution rate
± concentration of
limiting nutrient,
usually N or C
Affect of Nutrient on urowth and
Yield
‡ In batch cultures [nutrient]
can affect growth rate
and growth yield
‡ At low concentrations
only the rate is reduced
± cannot meet the needs of
organisms
‡ [Moderate] to [high] may
not change the rate but
the yield will increase
Chemistat ± Control of Rate and
Yield
‡ Ôoth rate and yield can
be controlled
independently by altering
the dilution rate which
effects the [] of nutrients
present
‡ Dilution rate ± at high and
low rates the steady state
breaks down, at [high] ±
bacteria aren¶t growing
fast enough and at [low] ±
not feeding fast enough
so cells are dying
‡ Cell density (cells/ml) ±
controlled by level of
limiting nutrient
 Environmental Effects -
Temperature
‡ Most important as alter the temperature to
drastically the bacteria will die
‡ Raising the temperature may speed up growth
rates but over a limited range ± may be
detrimental if too high ± maximum temperature
or too low ± minimum temperature
‡ Optimum temperature ± temperature that growth
occurs most rapidly ± usually nearer to the
maximum than the minimum
Cardinal Temperatures
‡ Maximum, optimum and
minimum are the cardinal
temperatures
‡ Cardinal temps are not fixed
and may fluctuate depending
on growth medium
‡ Maximal temperatures reflects
denaturation of 1 or more
proteins
‡ Not sure what causes minimal
temperature but may be the
composition of the cytoplasmic
membrane
± alter composition resulted in a
change in the maximum and
minimum temperatures
Temperature Classes of Organisms

‡ ߗ

  ± very low temperatures
‡  —

  — ± moderate temperatures
‡ 2 

  — ± high temperatures
‡ 
 

  — ± very high temperatures
‡ All but mesophiles can also be classified as
 

  —
 Important Thermophile
‡ Thermus aquaticus ± DNA polymerase
that works in ³artificial´ or á
á DNA
replication
‡ Enzyme is 
polymerase and is used in
PCR
Effect of pH
‡ pH scale ± logarithmic
scale that measure
the [H+] in a solution
± 10-fold difference
between numbers
‡ Ôacteria grow in
media with various
pH¶s
± 0-6.9 are acidophiles
± 7.1-14 are alkaliphiles
 pH
‡ Each organism has a range that it can grow in
(external pH) ± usually 2-3 pH units and
between pH 5-9
‡ Acidophiles usually live at < pH 2, fungi are
more tolerant of low pH, some obligate
acidophiles as they need a large amount of H+ to
maintain membrane structure
‡ Alkaliphiles usually > pH 10, some are also
halophilic (love salt) ± use the Na+ to
± proteases and lipases from alkaliphile bacteria seen
in household cleaners
‡ Neutrophiles live between pH 6-8
‡ Internal pH must remain close to neutral
 Ôuffers
‡ We add buffering chemicals to the media
to insure to proper pH for the organisms
‡ Metabolic reactions will increase or
decrease the pH depending on what is
happening in the cell
‡ Potassium phosphate is used quite
frequently, use others depending on the
pH range needed for the bacteria
 Osmotic Effects
‡ Water availability is expressed as water
activity
‡ Water diffuses from [high] to [low] thru a
membrane ± osmosis
‡ [Solute] usually higher outside the
organism so water moves into the cell
± cell in a positive water balance, in an area of
low water activity, then water leaves the cells
‡ causes many problems
 Halophile
s

‡ Osmotic effects seen in habitats with high [salt]

‡ Mild halophile ± 1-6%, moderate halophile ± 7-15% NaCl
‡ Halotolerant ± can adjust to increase in solute by
decreasing water in the cell
‡ Extreme halophiles ± 15-30% NaCl
 Other Types of Organisms
‡ Osmophiles ± grow in environments with a
high [sugar]
‡ Xerophiles ± grow in very dry
environments
 Compatible Solutes
‡ Organisms grown in an area of low water activity
need to adjust to this
‡ uain water by increasing the concentration of
internal solutes
‡ Accomplish this by
± pumping inorganic ions into cell from environment
± synthesizing or concentrating and organic solute
‡ Solute must not inhibit the biochemical
processes in the organism
Solutes
 Where do the Solutes Come
From?
‡ Synthesize or take up solute ± genetically
determined by the organism
‡ Staphylococcus species are salt tolerant ±
use to select over salt intolerant organism
and use proline as a compatible solute
‡ See glycine betaine in halophilic bacteria
and cyanobacteria
‡ Extreme halophiles produce ectoine (cyclic
derivative of aspartate)
 Oxygen and Microbial urowth
‡ Anoxic organisms ± grow without oxygen
‡ Classes of microorganisms ± vary in use of oxygen and
tolerance
‡ Aerobe ± grow in 21% O2 ± and respire O2 in metabolism
‡ Microphiles require less than 21% O2 ± may contain an
O2 labile protein, limited capacity to respire
‡ Facultative aerobe ± under appropriate nutrient and
culture conditions either grow anoxic or oxic condition
‡ Anaerobes cannot respire in O2
± 2 kinds ± aerotolerant anaerobes ±can tolerate O2 and grow in
the presence of O2 but do not use it and obligate or strict
anaerobes ± inhibited or killed by O2
3 Types of Obligate Anaerobes
‡ Prokayotes ± important one is clostridium
family that is gram positive spore forming
rob that causes food poisoning
‡ Some fungi
‡ Few protozoans
‡ Sensitivity to O2 varies in all these groups
 Culture ‡ Anaerobes need the O2
removed form the culture
Techniques ± use a reducing agent
such as thioglycolate in
broth to determine
oxygen requirements
‡ urowth at the top is
obligate aerobes,
facultative organisms
grow throughout the
medium and anaerobes
grow only at the bottom of
the tubes
‡ Also use resazurin in the
medium to indicate if O2
is present ± should see
only near the top
Anoxic Jar or Anaerobic Hood
‡ Use a tightly sealed
jar or bag that you
use a chemical
reaction to remove all
the O2 from it to grow
anaerobes
‡ Hood uses a series of
vacuum pumps to
remove O2 and
replace usually with
N2
Toxic Forms of O2 and Enzymes
 ‡ Catalase is the most
Enzymes common enzyme to
remove H2O2
‡ Used in conjunction with
superoxide dismutase
which generates H2O2
when combining 2
superoxide ions, also
makes O2
‡ Peroxidase removes
H2O2 but requires NADH
to make water
‡ Superoxide reductase in
Archaea ± reduce
superoxide to H2O2
without the production of
O2, remove H2O2 with
peroxidase-like enzyme