1. Active transport
2. Group translocation
Ø Active
transport systems facilitate the movement of a compound to a higher
concentration or against a concentration gradient with the use of metabolic
energy.
§
ENEGRY DEPENDENT PROCESS
Ø Involves
Protein Carrier (premeases)
§
Premeases has a great specificity for binding the
transport molecule.
§
Similar molecules can compete for the same carrier
protein in both AT and FD.
§
AT – carrier saturation effect at high
concentrations.
Active Transport differ from Facilitated
Diffusion:
§ AT requires metabolic energy input
§ Has ability to concentrate substances
§ Metabolic inhibitors that block energy
production will inhibit AT but will not effect facilitated diffusion
ABC Transporters
§ Binding
protein transport system
(Active
in Bacteria, Archea, and Eucaryote
§ Consist
of two (2) hydrophobic membrane-spanning domains associated on their
cytoplasmic surfaces with two nucleotide-binding domains. (figure 5.3)
n forming
a pore in the membrane
n nucleotide
binding domains bind and hydrolyze ATP
to drive uptake
§ ABC
transporters employ special substrate binding
proteins
q
In gram negative bacteria: in perplasmic space
q
In gram positive bacteria: attached to membrane lipids on the external face of the plasma
membrane
q
These binding molecules – bind the molecule to be
transported and then interact with the membrane transport proteins to move the
solute molecules inside the cell. (E.coli: sugars & amino acids).
q
Many transport
proteins use the energy of proton and sodium gradients to move molecules across
the membrane (coupled transport).
§ Symport: transport of two substances in the same direction
§
Antiport:
Transported substances move in the
opposite direction.
Group Translocation
Ø Molecules
are transported into the cell while being chemically altered and metabolic
energy is used.
n Phosphenolpyruvate: sugar
phosphotranseferase system (PTS)
Transports
a variety of sugars into procaryotic cells while
phosphorylating
them using phosphoenolpyruvate (PEP)- a
high energy phosohate as the
phosphate donor.
PEP
+ sugar (outside the cell) à
pyruvate + sugar---P (inside the cell)
Ø PTS are widely distributed in prokaryotes
Ø Aerobic bacteria lack PTS – except for
species like Bacillus that have glycolysis and PTS
Ø Escherchia, Salmonella, Staphylococcus and
other Facultatively Anaerobic have PTS.
Ø Clostridium: obligately anaerobic have PTS.
Iron Uptake
Siderophores.
Ø
Under aerobic
conditions iron assumes the Fe3+ form and is highly insoluble.
Ø
In order to
facilitate its uptake many bacteria synthesize compounds called siderophores (iron bearers) which are
secreted from the cells and chelate the iron ion.
Ø
This process
solublizes the iron. The
iron-siderophore complex is transported into the cell by a mechanism that
remains unclear.
Ø
In E. coli the
siderophore is called enterochelin
Culture Media
o
Solid or liquid
preparation used to grow, transport, and store MO.
o
Medium must
contain all necessary nutrients the MO requires for growth.
o Medium is used to select and grow specific
Mo to help ID a particular species.
Ø Synthetic
or Defined Media
Medium in which all of the components are
known
Ø Complex
Media
Media that contain some ingredients of
unknown chemical components
q
Rich and complete
to meet the nutritional requirements of many MO
q
Used b/c
nutritional requirements are unknown (used for fastidious MO)
q
Contain
components like peptones, meat extracts
and yeast extract:
Peptones: protein
hydrolysates prepared by partial digestion of meat, casein, soya, meal, gelatin, and other protein sources, serving
as C, energy
and N sources.
n Beef
extract contain AA, peptides,
nucleotides, organic acids, vitamins, and minerals
n Yeast
Extract: source of B vitamins, nitrogen, and carbon compounds.
n Three commonly complex media: Nutrient
broth/agar
Tryptic broth/agar, MacConkey
agar
Ø Broth
can be solidified with the addition of agar (1.5%)
o Agar is a polysaccharide (long chain of
monosaccharides linked by glycosidic bonds.
o Agar: extracted from red algae
o Solidifying agent b/c most bacteria cannot degrade it.
o Melt at temp above 80º Cand solidify at temp below 42º C.
Types of Media:
Example: Tryptic soy broth ot
Tryptic soy agar
Example: Blood agar
Selective Media:
o A medium that is specific and favors the growth or isolation of a particular MO.
Examples:
Bile salts or dyes like basic fuchsin and crystal violet favor
the growth of gram – by inhibiting
the growth of gram +.
Endo agar, eosin methylene blue agar, and MacConkey agar used for the detection of E.coli
Differential Media:
-- media
that incorporates different ingredients that cause certain organisms to develop
a different appearance from other microbes growing on the same medium.
Example: Blood agar is both
a differential medium and enriched.
It distinguished between
hemolytic and nonhemolytic bacteria.
It allows you to determine
if a bacterial colony has produced the
enzyme hemolysin and whether the
resulting hemolysis is partial (alpha), complete (beta), or gamma (no hemolysis).
Isolation
of Pure Cultures
In nature, MO usually grow in a
complex mixed populations containing several species, therefore it is necessary
to isolate the bacteria into single species for study.
Pure Culture:
n a population of cells arising from a single
culture cell characterize an individual species.
n A culture containing a single type of MO
Spread Plate:
Spread Plate: a
technique where a mixture of cells is spread out on an agar surface so that
every cell grows into a completely separate colony.
Colony: a
macroscopically visible growth or cluster of MO on a solid medium, where each
colony represents a pure colony.
§
A small volume no
more than 0.1 ml is transferred to the center of an agar plate and spread
evenly over the surface with a sterile bent-rod glass.
§
The dispersed
cells develop into an isolated colonies
§
The number of
colonies should equal the number of viable organisms in the sample
§
Used to count the
microbial population
Viable Plate Count contains 30-300 bacterial colonies
TMTC - more than 300 colonies
on the plate
TFTC – less than 30.
Streak Plate:
Streak Plate: Technique used to isolate pure colonies.
Using an inoculating loop or swab, obtain bacteria, transfer to the
edge of the agar plate and then streaked out over the surface in one or several
patterns. Single cells drop of the loop
at it is rubbed along the surface of the agar and develop into separate
colonies
§
Successful
isolation depends on spatial separation of single cells inboth streak and
spread plate..
The Pour Plate:
Pour Plate: original
sample is diluted several tined to reduce microbial population to obtain
separate colonies when plating.
§
Small volume of
several diluted samples are mixed with liquid agar (cooled to 45º C)
§
Mixtures are
poured immediately in sterile culture Petri dishes
§
After agar is
solidified, each cell is fixed in place and forms an individual colony.
Colony Morphology and Growth:
§
Form of the
colony and shape of the edge or margin can be determined by looking down at the
top of the colony.
§
Elevation of the
colony when viewed from the side as the plate is held at eye level.
The most rapid cell
growth occurs at the colony edge:
§
At colony edge,
oxygen and nutrients are plentiful.
Slower growth takes
place at the colony center:
§
Much thicker
growth than at the edge.
§
Cells autolysis
takes place in the older central portions of some colonies.
§
Oxygen does not
diffuse readily into the center, toxic metabolic products cannot be eliminated
quickly, and growth in the colony center is slowed or stopped.
(Differences in growth are due to gradients of oxygen, nutrients, and
toxic products within the colony).