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1

Metabolism

All the of the chemical processes that take place in a cell.

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Anabolism

Is the process by which a cell is formed or grows by converting simple nutritionist from the environment

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Catabolism

Is the process by which various
chemicals are broken down and energy is released for use by the cell in other processes, perhaps to drive anabolism

The conservation of energy from chemical
reactions involves oxidation-reduction reactions

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Carbon that is totally
reduced is bonded to....

4 hydrogens

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Totally oxidized carbon CO2 is bonded to ..

2 oxygens

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Totally reduced oxygen H2O is bonded to...

2 hydrogens

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Anaerobic bacteria

fail to grow in the presence of air or
molecular oxygen (i.e., OR are able to grow in the absence of oxygen
• The growth failure is due to a sensitivity to thepresence of oxygen

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Aerobic bacteria

Have sufficient levels of the enzymes

superoxide dismutase (SOD) and catalase

to remove the toxic metabolites that result from the incomplete reduction of molecular oxygen, thus they can grow in the presence of air

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Obligate aerobes

Require O2 for their energy metabolism;
they cannot carry out fermentation

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Obligate anaerobes come in two varieties:

Strict anaerobes


Moderate anaerobes

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Strict anaerobes

will grow in environments containing nomore than 0.5% O2 and may be brief exposures to air

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Aerotolerant anaerobes

Can grow in room air or in a 5% incubator BUT GROW BEST IN ANAEROBIC ENVIRONMENT

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Facultative anaerobes

Can grow either in the presence or absence of oxygen

They have sufficient SOD and catalase or peroxidase but grow best in its presence

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Facultative anaerobes can cope with changes in
environmental oxygen levels by

sensing oxygen concentration
and shifting their energy metabolism accordingly

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In the presence of the electron acceptors O
or nitrate, E. coli represses some..

Two growth moods......

Some anaerobicaly induced genes



Anaerobic respiration

Fermentation

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In the absence of inorganic electron acceptors, E. coli carries out

Mixed acid fermentation


It upregulates some of the enzymes of glycolysis and also the enzyme used for fermentative (anaerobic) disposal

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Oxygen has toxic metabolites...

Superoxide radicals O2-
Formed during aerobic respiration and in anaerobes when grown in presence of O2

Peroxide anions O2-2 (formed by SOD)

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Strict Anaerobes lack the enzyme...

Superoxide Dismutase SOD

Die in the presence of O2

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What bacterial cells use to convert H2O2 to non toxic molecules

Catalase

and

Peroxidase

Obligate anaerobes either lack both or have such a small amount that they die

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Bacteria metabolism

What does bacteria require to grow ?

They must obtain premade molecules or synthesize molecules for new cells

They require;
Carbon
Nitrogen
Energy
Water
Ions-to synthesize protein
Nuclei acids
Lipids
Carbohydrates

21

Why iron is important to the functioning of many enzymes in bacterial metabolism?

Fe is used in ETC

Some bacteria secrete SIDEROPHORES
-special proteins that help them concentrate iron from diluted solutions
And to take iron away from host tissue

22

What happens to the Fe in mammalian tissues ?

In mammalian tissues Fe is not left freely , it it found tightly bounded in variety different proteins

23

Where is Iron found?

As Heme (hemoglobin,myoglobin)

As Ferritin within cells

As Transferrin-transport between different tissues

In milk as Lactoferrin,

Also in tears,mucosal secretion (part of defense system)

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What does cells require to survive and grow (bacterial metabolism)?

Constant supply of energy

Controlled breakdown of carbohydrate, lipids and proteins (this energy is used in the synthesis of
Cell wall, cell membrane ,cytoplasm nucleoid

Large molecules broken down into smaller subunits---active/passive transport

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How is energy provided for medically important bacteria?

Organic molecules

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What does amino acids, sugars and lipids provide in bacterial metabolism?

They provide reduced carbon that can be oxidized via many catabolic pathways

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What is a Key intermediate in several metabolic pathways ?

Pyruvic acid

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What can happen to carbon that comes from Pyruvic acid?

It can be catabolized for energy

It can be used in anabolic reactions ;
Synthesis of amino acids
Lipids
Carbohydrates
As well as precursors for nucleic acids

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What are the pathways that bacteria use to convert glucose to pyruvic acid?

Glycolytic
Pentode Phosphate
Entner-Doudoroff

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What happens during catabolism of glucose ?(bacterial metabolism)

Chemical energy is captured by the creation of high energy molecules (ATP) or reduced coenzymes

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How can energy from reduced coenzymes be used?

It can be used to form proton gradient across the cell via aerobic or anaerobic respiration

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What is respiration?

Metabolic process that involves electron transport along a series of membrane associated electron carriers to final electron acceptor

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How can bacterial energy metabolism be divided?

Aerobic respiration-molecular oxygen is the final electron acceptor

Anaerobic Respiration -inorganic compound other than O2 is a final electron acceptor NO3- etc

Fermentation- organic molecule is the terminal electron acceptor

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How can pyruvic ever be used ?

It can provide fermentation products

It cans serve as an intermediate that enters the TCA cycle

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What happens in TCA cycle?

Reduced carbon are completely oxidized to CO2 and the maximum amount of energy is captured in the form of reduced coenzymes

The reduced coenzymes from TCA cycle are re oxidized via the ETC using either Aerobic or Anaerobic respiration

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What bacteria use Enter-Doudorff Pathway?

PSEUDOMONAS

ENTEROCOCCUS

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Where does the Glycolitic Pathway occur? For both eukaryotes and bacteria ?

CYTOPLASMIC MATRIX

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What conditions are required for Glycolytic Pathway to occur?

Both Aerobic and Anaerobic conditions are sufficient.
investment of two moles of ATP for every mole of glucose is required

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What is the net product for each more of glucose during Glycolytic Pathway?

2. Moles of Pyruvate
2 Moles of ATP
2 Moles of NADH

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Pentose Phosphate Pathway

Also known as hexes-monophosphate shunt
Maybe used at the same time as the other two

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Why is Pentose Phosphate Pathway important for biosynthesis and catabolism?

It serves as a function of NADPH for biosynthesis

Functions as a source of 5 carbon sugar
(Ribose-5-Phosphate for nucleic acid synthesis

CAN Yield 1ATP net if used for energy

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What are the key points about
Entner -Doudoroff Pathway?

Alternate pathway to catabolize glucose to pyruvate (different set of enzymes)
ONLY DESCRIBED IN PROKARYOTES

Generates; NADH. NADPH PYruvate 1ATP for each mole of glucose

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Why in some cases bacteria would prefer Entner-Doudoroff Pathways over Glycolysis? Examples!!

Because they may lack certain enzymes needed for glycolysis.


examples: PSEUDOMONAS AERUGINOSA
Gram positive

ENTEROCOCUS FAECALIS
Gram negative


44

What can be done to regenerate NAD+ from NADH?

Either fermentation or respiration
Fermentation: pyruvate or a pyruvate derivative
serves as the terminal electron acceptor with various compounds as waste products; no further ATP is created as the electron transport chain is NOT used
• Respiration: uses a membrane-embedded
electron transport chain and usually an inorganic terminal electron acceptor

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Product of Bacterial Fermentation

Streptococcus and Lactobacillus
;lactic acid and acetic acid

Enteric bacteria(E. coli, Salmonella)
;formic acid, acetic acid, ethanol ,lactic acid

Clostridium acetone, ethanol, bugaboo

Propionibacterium propionic acid (cheese)

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What is used for complete oxidation of Pyruvate to CO2?

TCA Cycle

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Energy production from TCA cycle?

2ATP
6NADH
2FADH2

For each molecule of glucose

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What is Biosynthetic function of TCA cycle ?

TCA cycle provides CARBON SKELETON

Some organisms only use part of the reactions

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What Happens to the NADH (and FADH2) Created by Glycolysis and the TCA Cycle?

Re oxidation via the ETC

Aerobic Respiration:
uses O acceptor with formation of H
H+ across the cell membrane

Anaerobic Respiration: could be NO
or CO produces less proton gradient (and hence fewerATPs)-2 as terminal electron acceptor, but then3

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When does the ETC occur in Bacterium?

Cell Membrane

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Summary of ATP production

2ATP ---Fermentation
If aerobic respiration available --glycolysis produces 8ATP

Complete conversion of Pyruvate to CO2
(30 ATP with aerobic respiration )

Total--38ATP per glucose molecule


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Bacterial growth-Binary Fission

1. cell elongates & DNA is replicated; note attachment of daughter chromosomes to cell membrane
• 2. cell wall & plasma membrane begin to divide cell in half between the two DNAs
• 3. cross wall completely
separates two daughter DNAs
• 4. cells separate

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How do we if the cell will be spherical or rod?(bacterial growth,)

If they synthesize peptidoglycan at the same rate, then the cell is spherical

if the cross-septum enzymes are slower than those at the cell's side, then the cell is a rod

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Differences between
Lag Phase
Log Phase
Stationary Phase
Death Phase

.....

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Methods to Identify Microbial Agents of Disease

▫ Direct microscopic visualization of the organism
▫ Cultivation and identification of the organism
▫ Serological and cytological methods to:
Detect microbial antigens
Detect the host's immune response, either a response of the innate or specific immune system
▫ Detection of microbial DNA or RNA

56

Light Microscopy

Glass or quartz lenses refract (bend) light because the glass is optically more dense than the surrounding air
Convex lenses refract light and can focus light on a focal point
This permits magnification (enlargement) of an image at distances farther away from the focal point

note that the magnified image is reversed and
upside down

57

Darkfield Microscopy

only oblique,scattered light reaches the specimen
This causes the specimen to appear
bright against a dark background
and increases the resolving power
of the microscope to 20nm vs.200nm for the brightfield

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What is Darkfield Microscopy good for?

useful for visualization of the
very thin spirochetes that cause
syphilis (Treponema pallidum) or
leptospirosis (Leptospira spp.)

59

Phase contrast microscopy

An optical microscopy
illumination technique in which
small phase shifts in the light
passing through a transparent
specimen are amplified to create
a 3D image. It enables
visualization of the internal
structures of unstained cells

60

Differential interference
contrast microscopy (DIC),

also known as Nomarski
Interference Contrast (NIC) or
Nomarski microscopy, is an
optical microscopy illumination
technique used to enhance the
contrast in unstained,
transparent samples

61

Flurorescent Microscopy

Short wavelength Uv light

62

Direct immunofluorescence

Employs a fluorescent dye linked to an antibody .
The antibody directly binds the target antigens

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Indirect immunofluorescence

The tagged antibody is second antibody it binds another antibody that actually binds the target antigen

64

Electron Microscopy - Transmission (TEM)

Magnetic coils focus beams of
electrons from a tungsten
filament onto or through
specimens for electron
microscopy. The electrons create
an image on a detector screen.

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Electron Microscopy - Scanning (SEM)

In SEMs the surface of
the specimen is coated with a thin layer of a heavy metal and the electron beam scans across its surface rather than passing thorugh the specimen

66

Wet mounts,Bacterial motility

To detec flung in tissue use solution of 10'5 KOH----lactophenol cotton blue
The KOH dissolves mammalian cells and
non-cellular material leaving the resistant fungal cell walls to be visualized

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Negative staining: India Ink

consists of carbon particles suspended in a fluid; it can be used to visualize the thick capsule that is characteristic of Cryptococcus neoformins in freshly obtained cerebrospinal fluid (CSF)

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Bacterial morphology

Cell shape

coryne- is a prefix applied to a
rod-shaped cell that is wider at one end than the other (e.g., club-shaped bacilli in diptheroids)

69

Bacterial morphology

Cell arrangements

diplococci and chains for
streptococci, grape-like clusters for staphylococci, palisades & v-shapes for diptheroids (look similar to Corynebacterium diphtheriae)

70

Basic Dyes

have a positive charge and bind to
negatively charged molecules such as nucleic acids, many proteins, and the surfaces of bacterial cells

Methylene blue, crystal violet

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Acid Dyes

have negatively charged groups and
bind to positively charged cell structures
▫ eosin, rose bengal, acid fuchsin

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Simple stain

single agent is used so can
only tell cell shape, size and cell arrangements

73

Differential stain

divide bacteria into separate
groups based on staining properties


Gram stein

Acid fast stain

74

Gram stain

stain with crystal violet, use a mordant, decolorize with alcohol, counterstain with safranin ⇨.

purple Gram positive &
red Gram negative;

used for many bacteria

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Acid fast stain

heat with basic fuchsin and phenol,
decolorize with HCl-alcohol, counterstain with methylene blue ⇨

red acid-fast cells with all others
blue;

important for a restricted group of bacteria

76

Acid fast stain is used for...

Used to stain cells of genus Mycobacterium and
Nocardia and oocysts of certain protozoa

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Negative staining

stains the background and
can show true cell size; also used to demonstrate capsules

78

Spore stains

drive malachite green into
vegetative cells and endospores with heat,
rinsewith water and counterstain with safranin
⇨green endospore and red vegetative cell

79

Metachromasia

staining in a color that is different
than that of the dye used to
create the stain
Albert's stain can be used
to detect volutin (black granules) within green Corynebacterium cells

80

Staining Bacterial Flagella

flagella are too slender to
visualize with the LM, thus flagellar stains coat them to make them apparent ⇨ provides important info for taxonomy but is not routinely done