Chapter 5

Chapter 5 is difficult. Read Chapter. Alot of test material.

Chapter 5
Microbial Nutrition, Culture and Metabolism

Biochemical Reactions Within Cells.
Every cell acquires nutrients
Metabolism requires energy (light or nutrients)
Energy stored in adenosine triphosphate (ATP)
Cells catabolize nutrients to form precursors
Precursors +ATP +enzymes =anabolic reactions
Cells grow by assembling macromolecules
Growth Requirements
Organisms use a variety of nutrients for their energy needs and to build organic molecules and cellular structures

Most common nutrients – those containing necessary elements such as carbon, oxygen, nitrogen, and hydrogen

Microbes obtain nutrients from variety of sources
Sources of Carbon, Energy, Electrons
Organisms categorized into groups
Those using an inorganic carbon source (carbon dioxide) are autotrophs
Those catabolizing reduced organic molecules (proteins, carbohydrates, amino acids, and fatty acids) are heterotrophs
Those that acquire energy from redox reactions involving inorganic and organic chemicals are chemotrophs
Those that use light as their energy source are phototrophs

Other Chemical Requirements
Phosphorus: membranes, DNA, RNA, ATP, some proteins
Sulfur: sulfur-containing amino acids, disulfide bonds critical to proteins, and vitamins
Trace elements: tap water
Growth factors: organic chemicals not synthesized by certain organisms (vitamins, some amino acids, purines, pyrimidines, cholesterol, NADH, heme)
Anabolism can cease due to insufficient nitrogen needed for proteins and nucleotides
Nitrogen from organic and inorganic nutrients; cells recycle nitrogen from amino acids and nucleotides
The reduction of nitrogen gas to ammonia (nitrogen fixation) by certain bacteria is essential to life on Earth because nitrogen is made available in a usable form

Iron and other Growth Factor Requirements
Iron needed in cellular respiration. Under anoxic conditions, iron is soluble. Under oxic conditions, siderophore is needed for transport into cell.
Growth factors: Organic compounds needed only in trace amounts; include vitamins and amino acids.

Culture Media
Majority of prokaryotes have never been grown in culture medium
Six types of general culture media
Defined media
Complex media
Selective media
Differential media
Anaerobic media
Transport media
Culture Media:
Chemically defined (defined medium)
Undefined (complex medium).
Selective, differential, and enriched are terms that describe media used for the isolation of particular species or for comparative studies of microorganisms.

Culturing Microorganisms
Inoculum introduced into medium (broth or solid)
Environmental specimens
Clinical specimens
Stored specimens

Culture – refers to act of cultivating microorganisms or the microorganisms that are cultivated
Obtaining Pure Cultures
Cultures composed of cells arising from a single progenitor
CFU: colony forming unit
Aseptic technique: prevent contamination
Two common isolation techniques
Streak Plates
Pour Plates
Culture Media
Culture media: nutrient solutions that supply the nutritional needs of microbes

Streak Plate Method

ENZYMOLOGY   

Enzymes regulate metabolic reaction rates   
Control metabolism  
molecules (mostly protein) that accelerate or catalyze chemical reactions (A--->B) in cells by  breaking old covalent bonds & forming new covalent bonds                                                 
Biological catalyst

different from a chemical catalyst - have complex structure (sequence of aa’s)
act only upon a specific substrate
do not change direction (energetics) of reactions of catalysis* 

Enzymes Catalysts
Enzymes are organic catalysts –help reactions occur but are not permanently changed
Most are protein
Some are RNA-ribozymes
To catalyze a reaction the enzyme must bind the correct substrate and position the substrate relative to the catalytic active site

Enzyme Activity
How is energy produced? Electrons
Electrons transfer from “donor” molecule to “acceptor” molecule
These reactions are always coupled…Meaning what?
EVERY ELECTRON THAT IS GAINED MUST FIRST BE GIVEN
REDOX

Electron Carriers
Cells use electron carrier molecules to carry electrons (often in H atoms) from donors to acceptors. Each carrier transport a pair of electrons.
Three important electron carriers (derived from vitamins)
Nicotinamide adenine dinucleotide (NAD+) → NADH
Nicotinamide adenine dinucleotide phosphate (NADP+) → NADPH
Flavine adenine dinucleotide (FAD) → FADH2

Electron carriers
External carriers
NADH dehydrogenases: 2 e and 2 H+
Flavoproteins: 2 e and 2 H+
Accepts and donates e
Membrane Carriers
Cytochromes: iron containing prophyrin ring, quinone, flavins
NAD as a Redox Electron Carrier
NAD+ and FAD accept electrons and hydrogen during reactions
They become NADH and FADH2
They deliver electrons and hydrogen to the electron transfer chain to make ATP
(Electron Carriers)

NAD+ (Nicotinamide Adenine Dinucleotide)
NAD+ functions in cellular respiration by carrying two electrons. With two electrons, it becomes NADH.
NAD+ oxidizes its substrate by removing two hydrogen atoms. One of the hydrogen atoms bonds to the NAD+. The electron from the other hydrogen atom remains with the NADH molecule but the proton (H+) is released. 
NAD+ + 2H ® NADH + H+
NADH then donates the two electrons (one of them is a hydrogen atom) to another molecule. The carrier FAD works in a similar manner.

ATP Production/Energy Storage
Organisms release energy from nutrients

Phosphorylation – organic phosphate is added to substrate; i.e. carbohydrate

Cells phosphorylate ADP in 3 ways:
Substrate-level phosphorylation
Oxidative phosphorylation
Photophosphorylation


Adenosine TriPhosphate
Substrate Level Phosphorylation and Oxidative Phosphorylaltion
Substrate Phosphorylation: ATP is produced during the breakdown of specific substrates in the breakdown of sugar.
Oxidative Phosphorylation: ATP is produced as a result of the proton motive force.

Energy Conservation: Options
Two Options
Fermentation: Produces energy for ATP synthesis with no outside electron acceptors.
Respiration: A electron acceptor is present to act as a terminal electron acceptor.

Glucose
A simple sugar (C6H12O6)
Atoms held together by covalent bonds

Glycolysis-Fermentation
Glycolysis is an anoxic process that occurs in the cytoplasm and can be divided into three major stages:
Preparatory reactions
ATP and pyruvate production
Making fermentation products

Cellular Respiration
Glycolysis (breakdown of glucose)

Produces: PYRUVIC ACID

In Cellular Respiration, pyruvic acid used to produce ATP by a series of reactions
Three stages of cellular respiration
Synthesis of acetyl-CoA
Citric Acid Cycle
Final series reactions -electron transport chain

Anaerobic Pathways
Do not use oxygen
Produce less ATP than aerobic path
Use Fermentation pathways
Fermentation
Essential function – regeneration of NAD+ for glycolysis, so that ADP can be phosphorylated to ATP

Not as efficient as respiration – most of the potential energy remains in the bonds of fermentation products

Fermentation products are waste to bacteria. Many are useful to humans (ethanol, acetic acid, and lactic acid)

Summary: Glycolysis to Krebs Cycle
Starts with Glucose
Breakdown of Glucose to Pyruvic acid (Glycolysis)
Pyruvic Acid converted to Acetyl-CoA (Respiration)
Acetyl-CoA converted to ATP and e- Carriers (Krebs)
CO2 and H2O produced as by products
Purpose?
To produce energy to fuel reactions

Electron Transfer Phosphorylation
Occurs in the bacterial membrane
Coenzymes deliver electrons to electron transfer chains
Electron transfer sets up H+ ion gradients
Flow of H+ down gradients powers ATP formation

Chemiosmosis-Electron chain
H+ ions,
propelled by proton motive force
flow down electrochemical gradient
Through ATP synthases (protein channels) that phosphorylate ADP to ATP
Called oxidative phosphorylation because proton gradient created by oxidation of components of ETC
A total of ~34 molecules of ATP are formed from one molecule of glucose

Biosynthesis of Sugars
Polysaccharides are biosynthesized from activated forms of their monomers.
Monomers: hexoses or pentoses
If unavailable, gluconeogenesis occurs.
Breakdown of different substances to make sugars
Anabolic Pathways – Carbohydrate Biosynthesis

Anabolic Pathways – Amino Acid Biosynthesis
Nucleotides and Lipids
Nucleotides (purines and pyrimidines) are biosynthesized using carbon from several sources

Fatty acids are synthesized two carbons at a time and then attached to glycerol to form lipids
Anabolic Pathways – Lipid Biosynthesis
Anabolic Pathways – Nucleotide Biosynthesis

Know Enzyme Regulation by Feedback inhibition, Isoenzymes, Covalent Modification