II. Biochemistry, Genetics, and Growth factors of Microorganisms.

In order to fully understand the role of microorganisms in the world and to better be able to utilize and/or control them, it is necessary to have a complete knowledge of how they function at both the molecular and cellular levels. In this unit we will consider the operation of microorganisms at the molecular layer, genetic processes, and the factors which affect growth and reproduction of microorganisms.

A. Basic chemistry - In order to understand the complex chemical changes that microorganisms carry out it is first necessary to have a basic knowledge of elementary chemistry, especially the principal kinds of chemical compounds which make up the organism. Students who have no background in chemistry should carefully read the material in chapter 2 of the textbook. The ensuing discussion will concentrate on those aspects of chemistry which are relevant to biological systems.

1. Elements, atoms, molecules, and covalent bonds



a. Element - These are the fundamental substances that make up the matter of the universe. There are 92 naturally occurring elements known. By definition, an element is any substance that cannot be subdivided further by chemical means. Another way of saying this is that an element is composed of only one kind of atom. Examples of elements include oxygen, nitrogen, sulfur, carbon, and calcium.



b. Atom - This is the smallest part of an element that still retains the chemical and physical properties of that element. Atoms in turn are composed of three types of subatomic particles.



(1) Protons - These are positively charged particles found in the center or nucleus of the atom.



(2) Neutrons - These are also found in the nucleus but bear no electrical charge (neutral).



(3) Electrons - These particles move around the nucleus in orbits. They have a negative electrical charge. Electrons are arranged in shells around the nucleus. Each shell can contain only a finite number of electrons before a new shell must be formed. The first shell is filled when it has two electrons. The second, and each succeeding shell (for our purposes) is filled with eight electrons.



The chemical properties of an atom are largely a function of the arrangement of its electrons. An atom is in its most stable state when the outer shell is filled with eight electrons.

c. Molecule - This represents the chemical combination of two or more atoms. Atoms chemically combine by interacts with their respective electron shells. Such interactions are termed bonds and they are what hold atoms together. One of the most important chemical bonds is the covalent bond.



d. Covalent bond - This is where two more atoms share their electrons with one another. This can effectively fill all of the outer shells of the participant atoms and thereby make the extremely stable. A good example is water.



(1) Water is composed of two atoms of hydrogen and one of oxygen. Oxygen has six electrons in its outer shell and requires two more for maximum stability. Hydrogen has one electron. A second electron would fill its first shell (its outer) and thereby make it stable. To atoms of hydrogen share each of their electrons with the six from oxygen. This effectively gives oxygen 8 and each hydrogen 2 so all are stable. The interaction holds the three atoms together and gives us the molecule known as water.



(2) Most organic molecules are held together by covalent bonds.

2. Macromolecules, dehydration synthesis, and hydrolysis.

a. Macromolecules - Term refers to large molecule. Many organic molecules are characteristically large and called macro. These macromolecules are usually formed from smaller repeating subunits. These macromolecules are often termed polymers and the subunits which compose them are termed monomers.

b. Dehydration synthesis - Monomers are assembled into polymers by means of a special kind of covalent bond forming reaction termed dehydration synthesis or condensation. The name comes from the fact that water is always one of the products. The reaction is as follows.

R - OH + H0 - R = R - 0 - R + HOH

Note that two small molecules are joined to form a large molecule and in the process a molecule of water is formed. Monomers which have the appropriate structure can form long chains by this method and therefore yield macromolecules.

c. Hydrolysis - Just as polymers can be formed by dehydration synthesis, likewise the reverse is true. Monomers can be formed from polymers. This reaction is termed hydrolysis and in it a molecule of water is added across the chemical bond that holds two monomers together. The result is the breakup of the polymer into its monomeric units. The process is illustrated below.



R - 0 - R + H0H = R - OH + R - 0H

3. Hydrogen bonds - These are weak electrical attractions that occur between the hydrogen atoms in a molecule and the oxygen or nitrogen atoms in the same molecules or in separate molecules. Hydrogen bonds are important because they give macromolecules a three dimensional shape which is critical to their proper functioning. In addition, hydrogen bonding between water molecules is what is responsible for many of water's unique properties.

4. Basic organic compounds - Organic compounds are those which contain carbon. Based upon the other atoms found in the compound, and upon the arrangement of those atoms, we can recognize four major classes of organic compounds which are significant to organisms.

a. Carbohydrates - These molecules are composed of carbon, hydrogen, and oxygen. They include the sugars, starches, glycogen,and cellulose.

(l) Simple sugars - These can be of various sizes, 3, 4, 5, 6, and 7 carbons per molecule. The ones mostly commonly found in microorganisms are those of 3, 5, and 6 carbons. Common 5 carbon sugars include ribose and deoxyribose. Common 6 carbon sugars include glucose, fructose, and galactose. Simple sugars are termed monosaccharides.

(2) Double sugars (disaccharides) - These are formed by the dehydration synthesis of two monosaccharides. When glucose and fructose are so joined the result is sucrose which is common table sugar.

(3) Carbohydrate macromolecules (polysaccharides) - These include starch, glycogen, and cellulose. They are all formed from simple sugar monomers by dehydration synthesis, just like the disaccharides were except now a number of units are added in a chain forming a large molecule. Starch, glycogen, and cellulose are all formed from glucose units. The differences in these polysaccharides reflect the different ways in which

the glucose molecules are put together.

(4) Functions of carbohydrates.

(a) Fuel molecules - Carbohydrates are the principal fuel molecules for organisms. Simple sugars are broken down for energy which in turn is used to do the work of the cell.

(b) Storage of energy - Starch and glycogen are stored by different microbes as an energy reserve. When extra energy is needed they are hydrolyzed into simple sugars which can be used as fuel.

(c) Cell walls - Cellulose is the principal constituent of many microbial cell walls. Another common cell wall material is chitin which also has a sugar component. The principal component of the procaryotic cell wall, peptidoglycan, also contains a carbohydrate component.

(d) Structural parts of other organic molecules - Many large organic molecules have a carbohydrate component. Glycoproteins, peptidoglycan, and the nucleic acids are all examples.



b. Lipids - These molecules are also composed of carbon, hydrogen, and oxygen, but the ratio of the atoms is different from that of the carbohydrates. Lipids have more hydrogens per carbon than do the carbohydrates.

(l) Lipids includes the fats, oils, waxes, and sterols.

(2) Structurally they are heterogeneous and are held together as a group by their solubility characteristics. Lipids are soluble in non-polar solvents such as acetone and benzene, but are insoluble in polar solvents such as water.

(3) Fats - It is not possible to generalize lipid structure as it was for carbohydrates, but by way of example, fats will be used as an example.

(a) Fats are composed of a molecules of glycerol which has been joined to three fatty acids by dehydration synthesis.

(b) Glycerol is a three carbon molecule while fatty acids are chains of carbons and hydrogens with an organic acid group (carboxyl group) at one end. They range in size from 2 to 26 carbons.

(c) A glycerol molecules combined with three fatty acids is termed a triglyceride.

(d) Triglycerides can be hydrolyzed back into glycerol and free fatty acids.

(4) Functions of lipids

(a) Long term energy storage - Fats contain about twice the energy per gram as do carbohydrates. Therefore, many microorganisms utilize fats and/or oils as energy storage products.

(b) Membranes - All cell membranes have a lipid component.

c. Proteins - These molecules are composed of carbon, hydrogen, oxygen, nitrogen, and usually sulfur. They are exceedingly large and complex polymers made up of monomers termed amino acids.

(l) Amino acids - There are 20 different amino acids but all share a common structure. All begin with a carbon atom designated the alpha carbon. To this is attached an amino group, an acid (carboxyl) group, a hydrogen atom, and an R group. It is the R that gives us the 20 different kinds. The simplest R is a single hydrogen atom, while other R groups consists of carbons and hydrogens, additional amino groups, acid, groups etc.

(2) Peptide bond - Amino acids are joined together by special dehydration synthesis bonds termed peptide bonds. This bond forms between the alpha amino group of one amino acid and the alpha carboxyl group of an adjacent amino acid. Two amino acids bonded together form a dipeptide, three bonded together form a tripeptide. When a dozen or more become bonded together it is termed a polypeptide, and when a

polypeptide reaches a certain size it is termed a protein.

(3) Levels of structure in proteins - Proteins have four

recognized levels of structural complexity.

(a) Primary - This is the sequence of amino acids in the protein. It ultimately determines the other three levels.

(b) Secondary - This is a twisting of the chain to form a helix or pleated sheet structure. This level is brought about by hydrogen bonding between the alpha carboxyl and alpha amino groups of adjacent amino acids.

(c) Tertiary - This is the complex folding and twisting which the chain undergoes due to hydrogen bonding atoms found in the R groups of the amino acids in the chain.

(d) Quaternary - This is the association of several polypeptide chains together to form a single protein.

It is the secondary, tertiary, and quaternary level of structure that determine the biological activity (function) of a protein.

(4) Denaturization - This is the process by which the tertiary level of a protein is altered. As the higher structural levels (especially the tertiary) determines the biological activity, loss of this structure will render a protein inactive. The denaturization can be reversible in which it returns to its normal shape, or irreversible, in which it stays altered and therefore inactive. As tertiary structure is maintained by very weak hydrogen bonds, relatively mild environmental changes can result in denaturization. Such changes can include temperature, acidity (pH), salt concentrations, and other chemical or physical factors.

(5) Diversity of proteins - The number of different kinds of proteins is virtually infinite. A protein can have as many as 500 amino acids and they can be in any order and any combination. With an alphabet of 20 letters and word lengths of 500 or more, the total possible protein molecules is almost without end.

(6) Functions of proteins

(a) They are the chief structural molecule of living things. Much of the living substance is formed from protein.

(b) They serve as major defense molecules against microorganisms that invade our bodies.

(c) They function as enzymes, biological catalysts which make possible all of the necessary chemical reactions that go on in a cell.

d. Nucleic acids - There are two of these, DNA and RNA. They are composed of carbon, oxygen, hydrogen, nitrogen, and phosphorous. DNA is the largest molecule known.

(l) Structure - The nucleic acids are macromolecules made up of repeating subunits held together by dehydration bonds. The subunit is termed a nucleotide and each nucleotide consists of a 5 carbon sugar, a nitrogen containing base group, and a phosphate group. Their structure will be considered in more detail later.

(2) Function - The nucleic acids transmit the genetic blueprint from generation to generation and are also responsible for implementing the blueprint by regulating protein synthesis.

5. Enzymes - All biochemical reactions occur at low temperatures. At biological temperatures most reactions proceed very slowly, too slowly for life to exist. Slow reactions may be sped up by the use of catalysts.

a. Catalysts - These by definition are substances that speed up reactions but are not altered by those reactions. Biological catalysts are termed enzymes.

b. Enzyme structure - A complete functioning enzyme is termed a holoenzyme. It consists of a protein part, the apoenzyme, and a non-protein part, the coenzyme. Coenzymes are small organic molecules, frequently vitamins. The enormous diversity of proteins insures that there will be adequate kinds of enzymes for each and every reaction.

c. Enzyme specificity - Each enzyme is specific for one particular reaction. This means that each of the hundreds of reactions that occur within the cell must have its own enzyme. It therefore follows that the types of reactions a cell can execute are dependent upon the kinds of enzymes present. As the types of reactions executed are what determine the nature of the cell then is obviously the types of enzymes that determine cell type.

d. Nomenclature of enzymes - Most enzymes are named by placing the suffix "ase" at the end of the substrate (molecule on which the enzyme acts) or reaction type. A enzyme which catalyzes a hydrolysis reaction would be termed a hydrolase. Based upon the major class of reactions in which they participate, six classes

of enzymes are recognized.

(l) Oxireductases - Catalyze oxidation-reduction reactions.

(2) Transferases - Catalyze movement of functional groups between molecules.

(3) Hydrolases - Catalyze breakdown of polymers by addition of water.

(4) Lyases - Catalyze breakdown of molecules but not by the addition of water.

(5) Isomerases - Catalyze internal rearrangements of molecules.

(6) Ligases, synthetases) - Catalyze the linkage of molecules together.

e. Enzyme function

(l) All chemical reactions are energy transformations. Some give off energy (exergonic reactions) and others absorb energy from their environment (endergonic reactions).

(2) Regardless of the reaction type, all reactions have a small energy "hill" which they must overcome before the reaction can occur. This is termed the energy of activation.

(3) Enzymes function by lowering this energy of activation so that the reaction can proceed.

(4) The energy of activation can be thought of as a problem in three dimensions. For two molecules to react, they must be brought together in the proper position. This positioning that must occur can be thought of as the energy of activation.

(5) Enzymes have a special "pocket" termed the active site which just fits the reactants and holds them in the proper reacting position. Thus the energy of activation or positioning is overcome.

(6) After the reaction has occurred, the resultant molecule has a new shape and will therefore not fit into the active site. It is then released by the enzyme which is now free to pick up new reactants.



6. Ribozymes - While biological catalysts are traditionally thought of as protein enzymes, recent discoveries have shown that the nucleic acid, RNA, also has limited catalytic activity. Specifically ribozymes specifically act on RNA molecules to cut out sections of the molecule and then splice the cut ends back together. The significance of this will be discussed later.

7 . Biological oxidations

a. Oxidation - This is a type of chemical reaction in which

electrons are removed from an atom or molecule.

b. Reduction - This is a type of chemical reaction in which

an atom or molecule gains electrons.

c. Oxidation and reduction always go together. As it is not

possible to have electrons floating freely about, whenever one molecule is oxidized another must be simultaneously

reduced, that is, the electron removed from one molecule

must be transferred to another molecule.

d. As a rule of thumb, atoms or molecules which are oxidized lose energy while those which are reduced gain energy.

e. In biological systems, oxidation-reduction almost always means the transfer of a hydrogen atom. Hydrogen consists of one proton and one electron. Therefore whenever hydrogen is removed from a molecule that molecule has effectively lost and electron and thus has been oxidized. Likewise an atom or molecule that gains a hydrogen has gained an electron and therefore been reduced.

B. Laws of Thermodynamics - It has been pointed out that chemical reactions are energy transformations. All energy transformations are governed by the two laws of thermodynamics.

l. First law - Energy cannot be created or destroyed, but can be

converted from one form to another.

a. This simply says that the energy content of the universe is finite. It does not increase or decrease but can be

transformed. For example, electrical energy can beconverted into heat energy. Nuclear energy can be converted into electrical energy.

2. Second law - In a closed system, energy tends to distribute itself in the most stable state which is total randomness or disorganization.

a. Note that the second law (and first law as well) applies only to a closed system. This is a system that receives nothing from the outside. It is totally isolated from its environment. As such systems are virtually non-existent in nature, the closed system is more of a theoretical ideal.

b. The second law essentially says that when anything is left to itself (no energy is put into it) it tends to run down and become disorganized. The degree of disorganization in a system is termed its entropy. A highly organized system has low entropy while a low organization system has a high degree of entropy.

c. A corollary to the second law is that there is no such thing as a one hundred percent efficient energy transformation. During any energy transformation a certain amount of energy is lost to the environment.

3. Significance of the laws of thermodynamics to living systems

a. Living systems are the most highly organized forms of matter known. Consequently, in accordance with the second law they are highly unstable and should become disorganized rapidly.

b. Living systems are not closed systems. They freely exchange matter and energy with their environment. As long as sufficient energy is put into the living system the high degree of organization can be maintained. In fact the second law tells us that this constant energy input is an absolute necessity. Whenever the energy input into a living system falls below the minimum required, death will result, followed by a rapid loss or organization (entropy gain).

c. The laws of thermodynamics thus predict that the price that must be paid for the high degree or organization (low entropy) that characterizes all living systems is a constant input of energy. It is important to note that as long as there sufficient energy input into a system any degree of organization is possible.

C. Cellular metabolism - Metabolism by definition is the sum total of all chemical reactions that occur within the cell or organism. It is broken down into two subsections.

l. Catabolism - These are breakdown reactions in which large complex molecules are reduced to smaller fragments. In accordance with the laws of thermodynamics these types of reactions release energy.

2. Anabolism - These are synthesis reactions whereby smaller molecules are joined to form larger or more energetic ones. In accordance with the laws of thermodynamics, these types of reactions require energy.

3. Catabolic reactions are exergonic (energy yielding) while anabolic reactions are endergonic (energy consuming).

4. In the cell, catabolic reactions provide the energy necessary to drive the anabolic reactions. Cells couple energy producing reactions to energy consuming reactions. Note that due to the mandatory loss of energy to the environment as predicated by the second law, the energy produced by the catabolic reaction must always exceed the energy required by the anabolic reaction.

D. Energy yielding mechanisms of the cell - The various reactions that provide the cell with the energy that it requires to maintain its organization are the subject of this section. As will be seen, during the history of life on earth, three principal patterns of energy production have evolved. They are the oxidation of organic compounds, the oxidation of inorganic compounds, and finally, photosynthesis.

l. Oxidation of organic compounds - Most organic compounds are rich in hydrogen and therefore highly reduced. This means that they have a lot of available energy. The great majority of organisms tap this energy source by oxidizing and releasing this stored energy. Even photosynthetic organisms which trap the radiant energy of the sun utilize this trapped energy to synthesize organic compounds which they then oxidize for their routine energy needs.

a. The basic pattern of this type of catabolism is that the carbon atoms of the organic molecule are sequentially oxidized (there hydrogens removed and transferred to hydrogen acceptors). The energy released during this process is then partially trapped in special high energy compounds which can then couple directly to endergonic reactions which require an energy boost.

b. The principal high energy compound is ATP (adenosine

triphosphate). This molecule contains two high energy phosphate groups which can be transferred to molecules which need to have their energy levels raised in order to react. For example, when two amino acids are joined by a peptide bond, two ATP molecules are converted to ADP (adenosine diphosphate) and the high energy phosphate groups which are lost transfer their energy to the amino acids making possible the dehydration synthesis.

c. Virtually every energy requiring reaction of the cell utilizes ATP as its energy source. Consequently ATP is frequently referred to as the energy currency of the cell.



d. Organic molecules may be completely oxidized in which case the end products are carbon dioxide and some hydrogen containing compound, usually water but in some cases a nitrogen or sulfur containing compound.

(l) Aerobic respiration - The complete oxidation of an organic compound in which the final hydrogen acceptor is oxygen and therefore carbon dioxide and water are the end products.

(2) Anaerobic respiration - Similar to aerobic accept that the final hydrogen acceptor is nitrate (N03) or perhaps sulfate (S04) instead of oxygen. Consequently water is not an end product. Certain bacteria have the ability to utilize nitrate when oxygen is not present.

e. Incomplete oxidation of organic molecules occurs when one of the end products is some other organic molecule. These molecules may include alcohol, acetic acid, lactic acid, butyric acid, propionic acid, and many others. Another name for incomplete oxidation is fermentation. Many microorganisms which are commercially valuable are fermentors. As you will see, fermentation is not as efficient at producing ATP as are the complete oxidations of respiration.

f. Energy generation by the oxidation of organic compounds is broken down into three major phases in most cells. These are glycolysis, Krebs cycle, and the electron transport system. Glycolysis the starting point for both fermentation and respiration in most cells. This is because most cells preferentially utilize carbohydrates for fuel molecules. The details of each phase are discussed below.



(l) Glycolysis - The oxidation of glucose - The most common fuel molecule is the 6 carbon sugar glucose. It undergoes a series of reactions in which it is split into two 3 carbon molecules known as pyruvic acid. These series of reactions are referred to a glycolysis or as the Embden-Myerhof pathway.

(a) During glycolysis two molecules of ATP are generated.

(b) Four hydrogen atoms are removed and transferred to two molecules of NAD. NAD is the first hydrogen acceptor and each NAD picks up two hydrogen atoms.

(2) Once pyruvic acid has been formed several possibilities can occur.

(a) Fermentation - In the absence of oxygen pyruvic acid may be converted to alcohol, lactic acid, or some other organic end product. If this is the case then the energy yielding reactions stop here and the net production of ATP is two molecules for every glucose processed.

(b) Oxidation of pyruvic acid into acetyl CoA - Organisms that utilize respiration covert pyruvic acid into a two carbon compound termed acetyl CoA. In eucaryotic cells this occurs inside of the mitochondria as do the Krebs cycle and the ETS. During the conversion of pyruvic acid into acetyl CoA the following events occur.

/1/ Each pyruvic acid molecules has one carbon

atom completely oxidized into carbon dioxide.

/2/ Four hydrogens are removed (two from each pyruvic acid) and transferred to NAD.



(3) Alternatives to the Embden-Myerhoff pathway - Many microorganisms have an alternative for the oxidation of glucose. There are two such pathways.



(a) Pentose phosphate pathway (hexose monophospate shunt). This can operate at the same time as the Embden-Myerhoff pathway and allows for the breakdown of both glucose and 5 carbon sugars (pentoses). This pathway results in the following.



/1/ Production of one ATP per glucose.



/2/ Production of pentose sugars used in the synthesis of other molecules (nucleic acids, amino acids, and glucose from carbon dioxide in photosyntesis).



/3/ Production of the reduced coenzyme, NADPH, which is used in many synthetic (anabolic) pathways.



(b) Entner-Doudoroff pathway - This is an alternative to the Embden-Myerhoff pathway. It produces pyruvic acid, ATP, and NADPH which can be used in synthetic pathways. This is found in some Gram negative bacteria, but not Gram positive.



Fungi have been found to utilize all three pathways for the oxidation of glucose

(4) Krebs's cycle - This is the central metabolic hub for those organisms which utilize respiration for energy production. Acetyl CoA enters into the Krebs's cycle where it is bonded to a four carbon acid known as oxaloacetic acid. This union forms a 6 carbon compound known as citric acid. This cycle is also referred to as the citric acid cycle. The citric acid so formed now undergoes a series of oxidation reactions in which the carbons brought in by acetyl CoA are sequentially oxidized into carbon dioxide. During the process a number of compounds are formed. One very key compound is the 5 carbon compound known as alpha glutaric acid. It is oxidized into a four carbon compound which then undergoes another series of reactions that eventually results in the regeneration of oxaloacetic acid which was the starting point for the cycle.

(a) The cycle must spin twice for each glucose molecule as two acetyl CoA's are produced from one glucose molecule. All of the accounting that follows is based on a per glucose molecule or two spins of the cycle.

(b) All of the carbons in the two acetyl CoAs are

completely oxidized which results in the production of four carbon dioxide molecules.

(c) Two molecules of ATP are produced, one for each spin of the cycle.

(d) Sixteen hydrogen atoms are removed and transferred to hydrogen acceptors. As there are only l2 hydrogens in the entire glucose molecule it is obvious that there must be another hydrogen source. This source is water which is an essential reactant during this cycle. The Distribution of the hydrogens to the acceptors are as follows.

/l/ Six pair are transferred to NAD.

/2/ Two pair are transferred to FAD. FAD is another hydrogen acceptor.

(4) Electron (hydrogen) Transport System - The large numbers of hydrogens which have been removed during the previous reactions undergo a series of oxidation-reduction reactions known as the electron transport system. This consists of passing the hydrogen from one set of acceptors to another.

The set that loses the hydrogen is oxidized while the set which gains them is reduced. Actually it is the electrons of the hydrogen that is transferred. The nucleus of the hydrogen atom (a proton) is pumped across a membrane and plays a key role in the actual generation of ATP.



(a) The first hydrogen acceptor is NAD and the second in sequence is FMN (flavine mononucleotide). FMN passes the electrons to coenzyme Q. There are then a series of acceptors known as the cytochromes.

(b) In aerobic respiration the final acceptor is oxygen and thus the final product is water.



(c) During the process energy is given off and much of it trapped in the form of ATP. Production of ATP through a respiratory chain is termed oxidative

phosphorylation.

(d) For every pair of hydrogens (electrons) that are

transported from NAD to oxygen, three ATP molecules are produced. One ATP is generated between NADH2 and Coenzyme Q, a second between coenzyme Q and the cytochromes, and a third between the cytochromes and oxygen. FADH2 from the Krebs cycle passes electrons directly to coenzyme Q. These electrons miss the first ATP generating zone and therefore, only two ATP are generated per FADH2.

/1/ 10 NADH2 are processed for a total of 30 ATP.

/2/ 2 FADH2 are processed for a total of 4 ATP.

/3/ The total ATP produced by the ETS per glucose is therefore 34 ATP.

(e) The actual generation of ATP from ADP during the ETS is best explained by the Chemiosmotic theory. According to this theory, during the ETS, the transport of the hydrogen electrons releases energy which is used to pump the hydrogen protons across a membrane (mitochondrial membrane in eucaryotes, plasma membrane in procaryotes). The accumulation of these protons creates a concentration gradient. Eventually, the protons are allowed to flow back across the membrane, down the concentration gradient. This is analogous to a rock rolling down hill. The energy released from this "downhill" movement is absorbed by ADP and inorganic phosphate, resulting in the synthesis of ATP



(5) Energy summary

(a) From glycolysis - 2 ATP.

(b) From Krebs's cycle - 2 ATP.

(c) From ETS - 34 ATP.

(d) Total per glucose is 38 ATP.

(e) Compared to the two ATP produced by fermentation per glucose molecule, it may be seen that respiration is a much more efficient process. It is not surprising that respiration is by far the most widespread form of energy generation.

(6) Oxidation of fats - Fats are first broken down into glycerol and fatty acids. Each fatty acid then undergoes a process known as Beta oxidation in which it is broken down two carbon atoms at a time into acetyl CoA. The acetyl CoA so formed then enters directly into the Krebs's cycle. It is now possible to understand why fats contain about twice the available energy of other organic molecules: they contain about twice as much hydrogen and that equates to ATP.

(7) Oxidation of proteins - Proteins are first broken down into amino acids. These are then deaminated (they have their amino group removed) which converts them into a form of carbohydrate. They then enter the metabolic process usually as an intermediate of the Krebs's cycle or the glycolytic pathway.

2. Oxidation of inorganic molecules - Certain bacterial species have the ability to oxidize inorganic compounds and convert that energy into ATP. They can then utilize that energy to "fix" carbon dioxide, a process by which carbon dioxide is combined with hydrogen to form organic compounds. Consequently, these organisms require no organic nutrients. This method of energy production is rare and found only in a few bacterial species. Inorganic compounds oxidized include molecular hydrogen, hydrogen sulfide, iron, ammonia, and others.

a. Hydrogen bacteria

b. Sulfur bacteria

c. Iron bacteria

d. Nitrifying bacteria



e. With the exception of the nitrifying bacteria, which play a role in maintaining soil fertility, the impact of these organisms on the environment is minimal. One exception is the deep sea smoking vents, regions of volcanic activity on the sea floor where superheated, rich in hydrogen sulfide and minerals, escapes from the earth. Bacteria use these compounds to generate organic compounds that support an entire community of organisms, including multicellular animals. In these communities, these bacteria function the same as the photosynthetic organisms do in the sunlit zones, they are the source of energy and organic molecules for other organisms.

3. Photosynthesis - Photosynthetic organisms have evolved the ability to convert radiant energy from the sun into chemical energy in the form of ATP. They then use this chemical energy to fix carbon dioxide into organic molecules. Photosynthesis is the ultimate source of molecules on the earth. Two patterns of photosynthesis have evolved.

a. Eucaryotic and cyanobacterial pattern - In these organisms chlorophyll traps the sun's energy and water is used as a source of hydrogen to fix carbon dioxide into carbohydrates. As a result, oxygen is released as a byproduct. This type of photosynthesis is the major source of oxygen in our atmosphere.

b. Bacterial pattern - Photosynthetic bacteria differ from the previous organisms in the following ways.

(l) Bacterial chlorophyll is chemically different.

(2) Bacteria do not use water has a hydrogen source and therefore do not liberate oxygen. The hydrogen source is frequently hydrogen sulfide.

c. A general equation for photosynthesis can be written for all photosynthetic organisms as follows.

E. Nutritional types - This is a classification based upon the way in which organisms obtain their essential energy.

1. Phototrophs - These organisms utilize radiant energy from the sun to convert carbon dioxide or rarely another organic molecule, and a hydrogen source into the organic compounds which they require. There are two patterns.



a. Photoautotrophs - These use light for energy and carbon dioxide as the carbon source.



(1) Oxygenic - These use water as their hydrogen source and liberate oxygen as a byproduct. These include the cyanobacteria, eucaryotic algae, and green plants.



(2) Anoxygenic - These use hydrogen sulfide or some other non water hydrogen source. Consquently the byproduct is not oxygen. This includes the green and purple sufur bacteria and the hydrogen bacteria.



b. Photoheterotrophs - Use light for energy but do not fix carbon dioxide. Carbon source is some other organic molecue. Include the freen and purple nonsulfur bacteria.

2. Chemotrophs - These organisms rely on the oxidation of compounds for their source of energy.

a. Chemoautrophs - Oxidize inorganic materials as previously discussed.

b. Chemoheterotrophs - Oxidize organic compounds (which must be supplied either directly or indirectly by the phototrophs and/or chemoautotrophs) for energy. These are the great bulk of non-photosynthetic organisms. They may be divided into three major groups based upon how they obtain their organic molecules.

(l) Ingestors - Engulf bulk organic matter. Of the microorganisms only the protozoa utilize this mode.

(2) Saprophytes - Absorb organic molecules from dead organisms. Most of the bacteria and fungi are found here.

(3) Parasites - Obtain their organic molecules from living organisms. Disease causing parasites are termed pathogens. Every kingdom has parasitic members.

F. Energy consuming processes of the cell - synthesis - The average cell is constantly involved in the production of all four classes of organic molecules. In accordance with the second law of thermodynamics, creation of larger more complex molecules requires an input of energy. Two classes of molecules will be considered here, the nucleic acids and the proteins.

l. Nucleic acids - A previously pointed out, there are two types of nucleic acid, DNA and RNA.

a. DNA - This is the largest molecule known. It has two essential functions.

(l) It is the hereditary molecule. It contains the genetic blueprint which it transmits from generation to generation.

(2) It regulates the activity of the cell by controlling protein synthesis.

b. DNA structure - The molecule is composed of two chains of

nucleotides spiraled around one another to form a double

helix.

(l) Each nucleotide consists of a 5 carbon sugar (deoxyribose), a nitrogen containing base group, and a phosphate group.

(2) The phosphate group of one nucleotide forms a dehydration bond with the sugar group of the adjacent nucleotide. In this manner a chain is built up that has a backbone composed of repeating sugar - phosphate units with the base groups projecting at more or less right angles to the long axis of the chain.

(3) The two chains are held together by hydrogen bonds that form between the base groups of the two chains.

(a) Base groups - These are ring shaped structures that contain nitrogen. They belong to two classes, the purines and pyrimidines. There are two in each class.

/l/ Purines include adenine and guanine.

/2/ Pyrimidines include thymine and cytosine.

(b) Purines can only form hydrogen bonds with pyrimidines, and specifically:

/1/ Adenine bonds with thymine.

/2/ Guanine bonds with cytosine.

(4) Each strand has two ends, one bearing a free phosphate group, (5'end) and the other with a free hydroxyl (OH) group (3' end) off of the sugar. The strands run in opposite directions or antiparallel.

c. DNA replication (synthesis) - DNA is unique in that it has the ability to make exact copies of itself. This process is as follows.

(l) DNA unspirals and the hydrogen bonds that hold the chains together break. Both of these events are enzyme catalyzed.

(2) The unwinding and breaking of the hydrogen bonds create replication forks, which are the actual sites of synthesis.

(3) At the replication forks free nucleotides lock into position on the two chains by means of pairing of complementary bases. This means that where a thymine is exposed in one of the chains an adenine will lock in because only adenine can hydrogen bond with thymine.

(4) Enzymes add new nucleotides only at the free hydroxyl end (3'). This means that synthesis must in the 3' to 5' direction. As the two strands run in opposite directions the replication of each chain actually proceeds in the opposite direction. Copying proceeds "up" one chain and "down" the other.

(5) Because of its antiparallel structure, DNA replication for each of the complementary strands differs. Because the nucleotides can only be added to the hydroxyl end, the growth of each chain can only move in a phosphate - hydroxyl direction. At the replication fork the chain that moves with the direction of the fork (leading strand) is synthesized in a continuous fashion with the aid of the enzyme DNA polymerase. The other strand (lagging strand), which is being synthesized in the direction opposite that of the moving fork is synthesized in a discontinuous fashion. This results in a series of fragments (Okazaki) that are eventually joined together to form a complete chain by the enzyme DNA ligase.

d. RNA - RNA differs from DNA is several respects.

(l) It is single stranded.

(2) The molecules are much smaller than DNA.

(3) The 5 carbon sugar ribose replaces deoxyribose.

(4) The base uracil replaces thymine, but it hydrogen bonds just like thymine, that is, only with adenine.

(5) There are three distinct kinds of RNA, messenger RNA

(mRNA), ribosomal RNA (rRNA), and transfer RNA (tRNA).

(6) All RNA is synthesized off of the DNA molecule. Part of the DNA unwinds and ribose containing nucleotides are locked into position in place of the deoxyribose nucleotides. Thus all RNA represents a copy of some of the information contained in the DNA molecule.

(7) All three types of RNA function in the regulation of protein synthesis.

2. Protein synthesis - The significance of protein was pointed out earlier. All enzymes, and thus all chemical reactions in the cell are protein dependent. The genetic code contained in the DNA is a code for all of the protein molecules that a cell can make. It is these kinds of proteins encoded in the DNA which will determine the nature of the cell.

a. The genetic code - The genetic code is a triplet code. It consists of three nucleotide bases in a row. These three bases code for one amino acid. Therefore a protein that consisted of 400 amino acids would require a segment of DNA l200 nucleotides long to code for it.

b. Implementation of the code - The DNA molecule is the blueprint which contains the code. It is the RNA that actually converts the code into a finished protein. There are two basic processes, transcription and translation.

(l) Transcription - This involves the copying of the code from the DNA into the species of RNA known as mRNA. Each triplet code word copied into the RNA is known as a codon. After the proper section of the DNA has been copied, the mRNA leaves the DNA and travels out into the cytoplasm where it attaches to the ribosomes and the process of translation begins.

(2) Translation - This is the conversion of the codons of the mRNA into the proper sequence of amino acids. All three types of RNA play a role in this process. The mRNA contains the code sequence to be translated, the rRNA is a part of the ribosome and plays a role in attaching the ribosome to the mRNA strand, and finally the tRNA transports the amino acids to their appropriate places. The details of the process are

as follows.

(a) Every mRNA has a special start codon, AUG, which binds to the ribosome. The first tRNA attaches at this point. The amino acid that is coded for by AUG is formylmethionine and therefore this is always the first amino acid coded for in every protein. It may be deleted later in the process so that it does not necessarily appear in the final product.

(b) Every ribosome has two binding sites for tRNA. At the beginning of the process the start tRNA binds to the first site on the ribosome while the next coded for tRNA binds to the second site. A peptide bond forms between the two amino acids. The start tRNA is now released and the ribosome moves down one codon so that the second tRNA is now at the first binding site on the ribosome and the third tRNA is on the second binding site. Another peptide bond forms and the process is

repeated. The process continues until one of the stop codons (UAG, UAA, or UGA) is reached and the process is terminated.

(3) Information coding - The flow of information from the DNA to the mRNA to the tRNA always involves pairing of complementary base groups. The codon of the mRNA is a complementary of the original triplet code word in the DNA. During the translation process, each tRNA transports one specific amino acid. It "knows" where to deposit that amino acid because each tRNA has a unique three base sequence termed the anticodon which binds with the codons of the messenger strand. The flow of information is therefore triplet code word (DNA) to codon (mRNA) to anticodon (tRNA).



c. RNA processing in eucaryotes - The description of protein synthesis so far applies only to procaryotes. Eucaryotes have their DNA organized in a different fashion. Each protein coding sequence (gene) contains sequences of bases that are nonj-amino acid coding. These are termed introns. The amino acid coding sequences are termed exons. When the mRNA transcript is synthesized, it contains both introns and exons. Before translation can occur this introns must be removed. This is the function of ribozymes. They excise the introns and splice the exons together. Translation then may proceed.



d. Energy requirements - In order to form a peptide bond requires the expenditure of two ATP molecules. The same is true for a bond between two nucleotides.

(l) A mRNA of l200 nucleotides would require 2400 ATP for the synthesis of one molecule. It would code for a 400 amino acid protein. It would require 800 ATP for one molecule of this protein. Thus to synthesize one molecule of this would initially require 3200 ATP. Each additional molecule would require another 800 ATP.

(2) The duplication of a single DNA molecules that consisted of l0,000 nucleotides would require l0,000 X 2 or 20,000 ATP for each strand. Being the molecule is double stranded the total energy cost to make one copy would be 40,000 ATP. It should now be obvious why a continuous input of energy into the cell is absolutely essential.

G. Microbial genetics - Genetics is the science that deals with the

inheritance of traits from generation to generation, and with the

expression of those traits within a given generation. All genetics, from microbe to man, is based upon the holy trinity of molecular biology, DNA, RNA, and protein. The DNA contains the code for all of the cells potential traits, the RNA implements the various traits as required, and the protein represents the actual expression of the traits.



l. Genes - The gene by definition is a segment of the DNA molecule which codes for a specific protein. A protein can be thought of as a trait of the cell or as the substance responsible for a trait.

a. By way of example a particular bacterium produces a red pigment. This pigment is a trait. This pigment can only be produced if a specific enzyme (protein) is present. The presence of this enzyme is dependent upon the appropriate gene being present in the bacterium's DNA.

b. The sequence of genetic expression is always as follows.

DNA RNA PROTEIN TRAIT

Gene Synthesis of protein Enzyme Reaction

c. It is the gene contained in the DNA that is passed from generation to generation. An organisms is capable of no more than it inherited in its genes.

2. Traits of microorganisms - Microorganisms as all organisms do possess many readily identifiable traits that are genetically controlled. Some of the more important ones include:

a. size.

b. shape.

c. virulence.

d. colony color and shape.

e. motility.

f. spore formation.

g. enzyme production.

h. drug resistance.

i. antigenic characteristics.

j. nutritional requirements.

3. Mechanisms of genetic recombination in microorganisms - In sexually reproducing organisms, the processes of meiosis and fertilization provide tremendous opportunities for new combinations of genes to be produced in the offspring. These differing combinations mean that a number of different traits can be found in combination in different individuals. Some of these combinations may prove to be more efficient than the combinations of the parents. Thus in a changing environment, it is advantageous to produce offspring with as many different combinations as possible. This is the great advantage of sex, genetic recombination.

a. Procaryotic organisms lack any true sex. There are several ways in which they can recombine their genes to provide for new genetic combinations. These mechanisms are sometimes referred to as "semi-sex." They include the following mechanisms.

(l) Transduction - This involves the movement of genetic material in procaryotes which is mediated by a virus. There are two forms.



(a) Generalized - This the movement of bacterial DNA from one bacterium to another by means of a viral vector (carrier). Often when a phage invades a bacterial cell and incorporates itself into the bacterial chromosome, it will remove a section of the bacterial DNA when it again becomes lytic and escapes. The bacterium which it next invade receives these bacterial genes and thus possesses new traits. Lytic viruses can also do this. When the lytic virus goes through its reproductive cycle it often produces enzymes which cleave the bacterial chromosome into pieces. The virus may mistakenly include the bacterial DNA in its capsid which will then release it into the next bacterial cell which is infected.

(b) Specialized (viral conversion) - In this case it is the viral nucleic acid per se which conveys new traits to an infected microorganism. Certain bacterial toxins can only be produced by converted bacteria. The diphtheria and botulism causing organisms are cases in point.

(2) Transformation - This is where a bacterium absorbs a part of a naked DNA molecule from its environment. The recipient cell then acquires some of the genetic traits from the cell which lost the DNA. It was transformation experiments performed in the laboratory back in the forties that first established DNA as the genetic material.

(3) Conjugation - This is more similar to traditional sex than the previous methods. Here two compatible cells come together and part of the genetic material from one is transported into the other. The genetic material transferred is termed a plasmid.

(a) Plasmids - These are small circular strands of DNA that are independent of the bacterial chromosome. Plasmids contain genes that govern traits that are not essential to the bacterium under normal conditions. Thus, most plasmids can be lost with no harm to the bacterial cell.

(b) Certain plasmids contain genes that make it possible for them to be transferred to appropriate recipient cells. Such cells contain a special conjugation pilus termed the F pilus. Cells having this plasmid are termed F+ while those which lack it are termed F-. The plasmid can be transferred from an F+ cell to an F- cell. These transferable plasmids not only contain genes that make conjugation possible, but they also frequently contain other genes, among them genes which convey drug resistance. Consequently a non-pathogenic organism that possesses drug resistance on a plasmid may pass plasmid to a pathogen which will then acquire the drug resistance.

(4) Hfr cells - It sometimes happens that the plasmid in a bacterium becomes incorporated into the bacterial chromosome. When that plasmid breaks out of the chromosome to be transferred it may take with it some of the bacterial DNA. The recipient cell then not only receives the genetic trait carried on the plasmid, but the traits from the section of bacterial chromosome which it received. Because such cells show many more new traits than is normal, they are termed high frequency recombination cells or Hfr for short.

4. Mutation - Not all genetic variation is due to the processes

mentioned. Sometime there is an alteration in the genetic code. Such an alteration leads to a change in the code and possibly a new gene is born. Such a change is termed a mutation and is ultimately responsible for all genetic variation. If the mutation results in a trait that is favorable or at least not harmful, it may spread throughout the population by reproduction and the recombination mechanisms that have been mentioned.



5. Transposons - These are small segments of a DNA molecule that are mobile. They can move from one in the DNA molecule to another, and can move from on DNA molecule to another molecule.



a. All transposons carry genes that allow them to be inserted into the new positions, the insertion sequences.



b. Complex transposons may also carry genes for antibiotic resistance or enterotoxin production.



c. Plasmids with resistance factors are often made up of groups of trasnposons.



d. Transposons may move within the same chromosome, between chromosomes, or into plasmids. Because of this they may be transferred to other bacteria, providing new genetic traits.

6. Regulation of genes - Although each cell produced by division has a complete copy of the entire genetic code, not all of the genes are expressed at all times. Some genes are turned on at appropriate times and then turned off when their product is no longer needed. Such genes are said to be inducible. Other genes seem to operate all of the time and are turned off only under certain special conditions. These genes are said to be repressible. The turning on and off of genes (genetic regulation) is one of the most intensely studied areas of biology. While our knowledge of genetic regulation in eucaryotes is still very limited, the regulation of genes in procaryotes is much better understood.

a. Operons - The genes of procaryotes are clustered into functional groups termed operons. Operons are either inducible which are usually off and turn on in the presence of a specific substance, or repressible which are usually turned on, unless specifically repressed by a substance.

(l) Inducible Operons - These operons consist of a series of genes which are as follows.

(a) Structural genes - A series of genes that code for enzymes that regulate a particular metabolic pathway. They are located adjacent to one another.

(b) Operator - A series of nucleotides next to the structural genes which serve as a switch, turning the structural genes on.

(c) Promotor - A series of nucleotides next to the operator. This is the attachment point for the enzyme RNA polymerase which catalyzes the assembly of mRNA.

(d) The structural genes, operator, and promotor are all located adjacent to one another and constitute the operon.

(e) Regulatory gene (RG) - This is a gene located a short distance from the operon which produces a repressor protein which binds to the operator and prevents the attachment of RNA polymerase to the promotor.

(f) The inducing substance is the substance that turns this system on. Frequently it is a nutrient like the sugar lactose. What happens is that when the inducing substance is present it combines with the repressor protein preventing it form binding with the operator. Now RNA polymerase can attach to the promotor and transcription takes place, resulting in the production of enzymes that will metabolize the inducing substance. A diagram of a typical operon is presented below.











(2) Repressible systems - These systems are always turned on unless specifically repressed. They are structured as follows.

(a) The system is essentially an operon but the repressor protein cannot attach to the operator by itself and therefore the operon is continuously transcribed.

(b) A substance which serves as a corepressor combines with the repressor protein and together they can attach to the operator and prevent the attachment of RNA polymerase to the operator, thus preventing transcription.

(c) Repressible systems usually the control the production of substances which are in constant supply by the cell such as ATP.

H. Genetic engineering - During the seventies techniques were discovered that permitted the removal of genes from the DNA of one individual or species and their insertion into the DNA of another individual or species. This technology is termed genetic engineering, gene splicing, or recombinant DNA. This permits scientists to insert new traits into individuals. Although it has been done with multicellular organisms, the technique is still most widely utilized with microorganisms. We now have microorganisms that produce human insulin and other valuable products.



1. Overview - The principal techniques of genetic engineering in microorganisms is outlined below.

a. Donor DNA is purified chemically and treated with a special class of enzyme known as a restriction endonuclease. There are many such enzymes and each one breaks the DNA chain in one specific place where a particular base sequence such as AATTAA is recognized.

b. The restriction enzyme leaves one DNA chain a few nucleotides longer that the other. This provides the so called "sticky ends" so named because of their ability to base pair.

c. Purified plasmids are now treated with the same endonuclease. This breaks open the plasmid at the identical recognition site of the donor, and provides sticky ends that are identical to those of the donor.

d. The plasmid fragments are mixed with the donor fragments and a ligase enzyme is added which causes the sticky ends to seal up. As the sticky ends of the plasmid and donor are identical, a certain percentage of the plasmids will incorporated the donor DNA as the ligase seals them up.

e. The plasmids are then mixed with microbial cells that can absorb them. Those bacteria that have absorbed plasmids containing the donor DNA are then cloned so that colonies are formed which contain the recombinant DNA.

f. The process is illustrated below.















































2. Restriction enzymes - These are the key to recombinant DNA technology. Most provide staggered cuts in the two strands, cuts that are not directly across from one another. This provides for the sticky ends that permit resealing of the cuts by complementary base pairing.



3. Vectors - These are the agents which carry the DNA fragment to be inserted into the host cell. Current vectors include the following.



a. Plasmids - These are the most widely used mechanisms to insert genes into organisms. Frequently plasmids are engineered so that they contain antibiotic resistance genes. These genes permit rapid identification of recombinant types by growth on selective media containing the antibiotics.



b. Viruses - Foreign genes are inserted into a virus, usually a retrovirus, that will incorporate itself into the chromosome of a cell.



4. Alternative methods - Besides vectors there are other methods to get foreign DNA into a cell. These include the following.



a. Protoplast fusion - Here the cell walls are removed from two cells and the protoplasms allowed to fuse. This will provide for recombination of the genetic material.



b. Electric current pores - Electric currents applied to protoplasts which develop pores in the cell membrane as a result. The DNA to be inserted can then enter directly into the cell.



c. DNA "gun" - This has proved especially useful in plant cells. Microscopic particles of metal are coated with the DNA to be inserted. These DNA pellets are then fired through the cell wall into the cell. One technique uses compressed helium as the propellant.



d. Micropipettes - These are glass pipettes that are so small they can be inserted through a cell membrane and directly inject DNA.



e. Liposomes - These consist of membrane envelopes into which the desired gene is inserted. The liposome will then fuse with the target cell membrane taking the gene inside. Liposomes may prove expecially useful for gene insertion into humans and other animals.



5. Sources of Donor DNA



a. Gene libraries - These are of two types, Genomic DNA and DNA made from RNA.



(1) Genomic (natural) DNA libraries - These are prepared by cutting up the entire genome with restriction enzymes. The fragments obtained are then cloned. Every attempt is made to assure that there will be a least one clone for every gene.



(2) cDNA libraries - These are libraries composed of complementary DNA (cDNA) which is synthesized from a mRNA template. These are prepared by obtaining mRNA transcripts of the genes and then preparing a cDNA compy by using the enzyme reverse transcriptase. Complementary DNA libraries have several advantages over genomic libraries.



(a) They are less complex because they contain only the protein - coding sequences and none of the regulatory sequences.



(b) In eucaryotic organisms there is an additional advantage in that the eucaryotic gene is split into protein coding sequences (exons) and non-protein coding sequences (introns). During transcription, all of the sequences are copied, but then the introns are sniped out of the transcript leaving mRNA which codes only for the gene product. Using cDNA avoids the difficulties of dealing with the non-coding introns found in genomic libraries.



(c) By using specific cell types, the chances of finding the clone of interest are greatly increased. cDNA molecules coding for liver enzymes will be found in abundance in liver cells but not in cDNA from a muscle library.



b. Synthetic DNA - When the sequence of a desired protein is known, it is possible to construct a synthetic DNA molecule from the genetic code. A computerized machine is used for this purpose. This is usually only practical for relatively small proteins whose amino acid sequence is known. The human insulin gene which was clone into bacteria was prepared by this method.



6. Identification of cloned genes - Finding the desired gene in a gene library is much the same as finding a book in a traditional library. The problem is finding the one desired among thousands of possibilities. Just as traditional libraries have systems for retrieving the desired volume, so do gene libraries. The gene library utilizes a probe to detect the desired gene.



a. Probes are short segments of single stranded DNA which are complementary to some unique sequence of the desired gene. It is therefore necessary to know something about the gene which is desired. Probes can be thought of as representing the book call number in a traditional library. Probes are tagged with radioactive isotopes and the clones of the library are allowed to react with the probe. Any clone that bases pairs with the probe contains the desired gene. The radioactive clones can be detected by use of photographic film.



7. Gene products - Once the desired gene has been found and inserted into the appropriate host cells the gene product will be produced. The product may be something of value in itself such as human insulin, growth hormone, or colony stimulating factor. It may also be a trait inserted into a complex organism such as pest resistance in food plants or increased size in food animals.



8. Polymerase Chain Reaction (PCR) - This is a method by which DNA may be amplified. A single molecule of DNA can be made to make copies of itself over and over again until clones consisting of millions or even billions of molecules exist. This can be done in a few hours. The methodology of the PCR are as follows.



a. A solution containing the DNA molecule is heated to cause the two strands to separate.



b. To this solution is added the four nucleotides, DNA polymerase, and short segments of DNA termed primers which start the reaction.



c. Each new strand synthesized serves as a template for another strand and therefore the reaction doubles the number of molecules during each suceeding cyle, an exponential increase.



d. At the end of each cycle the solution must be heated again to separate all of the new double strands into single strands.



e. The secret to the sucess of this reaction is the DNA polymerase enzyme which is used. The heat which is necessary to separate the strands will denature normal DNA polymerase, thereby stoping the reaction. However, the DNA polymerase used in the PCR is obtained from a thermophilic bacteria and functions at the high temperatures necessary.

f. The PCR has proved invaluable in the follwing areas.



(1) Forensic medicine - The DNA from a few cells from an individual can be amplified and used for identification. The DNA which is amplified can be treated with restriction enzymes, the fragments electrophoresed, and the bands which result can be compared with suspects. The result is an almost foolproof identification.



(2) Amplification of rare genes such as those causing hemophelia, muscular dystrophy, and cystic fibrosis. The large amount of material obtained via the PCR can then be researched by other methods.



(3) Amplification of disease causing viruses that otherwise are present in such small quantities as to be almost undetectable. The AIDS virus is a case in point.



(4) Systematic and evolutionary biology. Using the PCR to amplify DNA until adequate quantities have been obtained to either sequence or finger print is being used to compare both living and fossil organisms to determine their relationships. DNA found preserved in fossil plants has been amplified and compared with modern species. It is also known that fragments of bone from fossil human beings is currently be examined for the possibility of fossil DNA that might be amplified with the PCR and compared to that of modern human beings.



9. Recombinant DNA and agriculture - Agricultural scientists have been extremely interested in biotechnology techniques. The goal here is to incorporate into important crop plants various desirable genetic traits such as resistance to insect pests, herbicides, freeze resistance, and desirable metabolic traits such as increased protein content, ability to fix atmospheric nitrogen, and others. Plants have an advantage over animials in that virtually any single plant cell can grow into a complete individual. Therefore it is necessary only to insert a desirable gene(s) into one cell to have that gene incorporated into every cell produced in the adult plant.



a. DNA insertion into plant cells freqently relies upon the Ti plasmid. This plasmid occurs naturally in the bacterium Agrobacterium tumefaciens. When the bacterium inserts this plasmid into a plant cell where it results in a tumor like growth termed the "crown gall." Part of the plasmid inserts itself into the host cell DNA.



b. Genetic engineers can insert desired genes into this plasmid and have the bacterium insert it into the plant cell. Resistance to a major herbicide has been the most sucessful use of this technique, but a major goal is to insert genes that permit plants to fix nitrogen, i.e., convert atmospheric nitrogen into usable form by plants. Nitrogen is usually the limiting nutrient for most plants.



10. Applications of genetic engineering.



a. Product production - Everything from human growth hormone to enzymes for the production of stone washed jeans are currently being produced by recombinant DNA technology.



b. Gene therapy in humans - The techniques of recombinant DNA offer great potential for the treatment of human genetic defects. It is hoped that eventually all defective genes may be replaced with good ones. The first gene insertions have now taken place.



(1) Treatment of immune deficiency - Some children are born with genetic defects that prohibit the development of a functioning immune system. A certain number of them have this disease due to a lack of the enzyme adenosine deaminase (ADA). Recently the gene for ADA has been cloned and inserted into T lymphocytes. These lymphocytes were then injected into several deficient children. All of them start producing ADA and showed good improvement.



c. Recombinant Vaccines - It is possible to genetically engineer surface antigens from pathogenic organisms and incorporate them into a vaccine that will stimulate immunity. Such vaccines are far safer than tradition ones which are made from the whole organism. They also offer hope for more specific vaccines against organisms that have been difficult to prepare vaccines for by traditional methods. The current vaccine for hepatitis B is a case in point.



d. Agricultural improvement - Increased protein content in food crops, insect resistance, cold resistance, and other agriculturally significant traits are already in place. In the future genetic engineering may revolutionize food production and greatly alleviate world hunger.



e. Genomics - This is the determination of the entire DNA sequence (genome) for an organism. The entire genetic blueprint is then known. The genomes have been determined for nearly one hundred different microorganisms. In addition, the genome has been determined for several multicellular organisms. These include the mouse, fruit fly, several fish, and humans. This type of work is staggering in its complexity. For example, the human genome contains some three billion base pairs that had to be sequenced. This has only been possible due to revolutions in techniques for sequencing DNA.



(1) Random shotgun sequencing - Here the genome DNA is cut into small pieces and each piece is sequenced. The sequences are assembled into chromosomes using a computer program.



(2) Bioinformatics - Because of the enormous amount of data that DNA sequencing generates, new techniques of statistical analysis have to be developed. This has become the branch of mathematics/genetics known as bioinformatics. This discipline attempts to understand the functioning of sequenced genes by examining and comparing sequences from different organisms that are contained in vast online databases called gene banks.



(3) Applications of genomics



(a) Comparison of genomes among organisms - This has several practical applications.



/1/ Determination of evolutionary and taxonomic relationship. The closer the sequences between two species, the more closely related they are.



/2/ Determining gene functions - By comparing the genomes of several different organisms it is possible to determine the significance of various genes. When a number of organisms have a particular gene in common, that indicates that it must code for an important cellular function common to all species. Bioinformatics plays a major role here.



/3/ Identification of mutant forms - Once a gene sequence and function have been characterized, it is then possible to develop probes to identify mutant forms which may cause disease.



(b) Complete DNA sequencing is difficult, expensive and time consuming. Another technique which relies on similarities in DNA fragment lengths is far faster and can provide much of the same information as a complete genome sequence. The technique is as follows.



/1/ A genome is cut into a number of fragments with a series of restriction enzymes.



/2/ These fragments are place on a gel to which an electric current is applied (electrophoresis). The fragments are pulled by the electric field down the gel depending upon their size. They form definite bands. Each band represents fragments which are identical in size and charge. Such fragments are referred to as RFLPs (restriction fragment polymorpisms).



/3/ This procedure can be used to determine different kinds of information.



/a/ DNA Fingerprinting - Because the restriction enzymes always cut in exactly the same sites, the relatedness of two genomes can be compared. If the bands all match up exactly then the two genomes being compared are probably identical, i.e., the same individual. When not identical, the more similar the banding pattern, the more closely related the individuals are, as in paternity testing. Besides the legal aspects, fingerprinting is utilized in a wide variety of medical and biological

situations to identify individual organisms.



/b/ Genetic screening - Southern blotting - By further processing the gels, i.e. applying a nitrocellulose filter to the gel which picks up the bands, it is possible to identify mutant genes. Once the transfer has been made, the bands are treated with a radioactive tagged probe for the sought after gene. If the gene is present the probe will react with it and then can be detected by exposing the blot to X-ray film which will be exposed by the radioactivity. Several hundred genetic diseases can now be screen for. Potential parents may be screen for these abnormal genes as can fetal tissue.





f. Proteomics - This represents the identification of the protein products of genes. The goal is to have a complete proteomic map of the genome as we currently do for genes. This is more difficult than is genomic analysis, but new techniques are making rapid progess.































I. Population growth in microorganisms - A population of bacteria shows a distinct growth pattern. Under optimal conditions bacteria can divide about every 20 minutes. Other microorganisms can also reproduce very rapidly. Such conditions only hold for a very short time. When a sample of bacteria is inoculated into a new growth medium the following growth curve for the population will be observed.









l. Lag phase - Upon inoculation the numbers of cells does not increase immediately. It is during this period that increase in cell size occurs and molecules necessary for cell division are assembled. This represents a "tooling up" period.

2. Log phase - During this period organisms divide at their maximum rate.

3. Stationary phase - This is reached when the total number of viable cells no longer increases. It is generally brought about by an exhaustion of some essential nutrient, accumulation of toxic waste products or a combination of both.

4. Death phase - Now there is a decrease in the total number of viable cells. It is a continuation of the same problems that brought about the stationary phase, nutrient exhaustion and/or toxic waste accumulation.

J. Environmental factors affecting growth and reproduction - Based upon the growth curve presented above it is obvious that certain conditions must exist if optimum growth and reproduction is to take place. Maintaining such conditions is very important when microbes are used in industrial applications. However, in many cases, the aim is to prevent the growth of microbes completely. Such cases would be in food preservation or in clinical situations. Whether the aim is to make them grow or to stop them from growing, a knowledge of the environmental factors which affect their growth is absolutely essential. The major factors are listed below.

l. Moisture - A minimal amount of moisture is necessary for the growth and reproduction of all organisms. It is not essential for spores and other dormant cells. The necessity for moisture underlies the practice of drying food in order to preserve it. This practice is very common in agriculture where hay and grains may be dried and stored for years without spoilage.

2. Nutrients - These are often limiting factors for growth. Each species of microorganism has definite nutritional requirements which must be met if growth and reproduction are to occur. Phototrophs, requiring only simple inorganic substances, have the simplest needs, while certain chemoorganotrophs may require a large number of complex organic compounds in order to grow and reproduce. Regardless of the number of required nutrients, the one which is in shortest supply will constitute the limiting factor for growth.



3. Toxic substances - There are two general classes.

a. End products of metabolism - These are substances such as carbon dioxide, methane, acids, etc. which are produced by microorganisms. When they accumulate they will stop growth.

b. Environmental toxins - These are substances that occur in the environment but are not produced by the growing microorganism itself. They may be produced by other microbes, be introduced as a result of incidental pollution, or they may be introduced by design with the goal of controlling microbial growth.

4. Temperature - This is a limiting factor for most organisms. Microorganisms exhibit a wide range of temperature tolerance. Based upon these tolerances they may be grouped into three major groups.

a. Meosphils - These organisms grow best at temperatures ranging from 20 to 37 degrees C. The great majority of microbes fall into this group, including all pathogens.

b. Psychrophils - These organisms grow best at low temperatures, 4 to l0 degrees C. These would include those that live at the bottom of the oceans. Few species do well at low temperature and consequently reduced temperature has been a major means for protecting food substances from decomposition.

c. Thermophils - These are organisms that grow best above 50 degrees C. Certain species can grow at 75 degrees and some even higher (extreme thermophils). These organisms usually live around hot springs associated with hot rocks. Some archaebacterial species have been found living in the smoking vents (volcanic activity in the ocean floor) where the temperature of the water was 110O C. Fortunately no pathogens are found here, and treatment with temperatures of 75 degrees or higher for 20 minutes will kill

all pathogens (but not their spores).

5. Oxygen - Microorganisms may be grouped into four major categories based upon their utilization and/or tolerance of oxygen.

a. Obligate aerobes - These are organisms that require oxygen for oxidation of food materials. They utilized the Krebs's cycle and ETS.

b. Facultative aerobes - These are organisms that can use free oxygen when present as their final hydrogen acceptor, but in its absence, can utilize other inorganic compounds such as nitrate. This is termed anaerobic respiration.



c. Aerotolerant anaerobes - Cannot utilize oxygen, but are not harmed by it.

d. Obligate anaerobes - These organisms cannot grow in the presence of free oxygen. They must have an oxygen free environment in to grow and reproduce. All of the Clostridium species fall into this group, including the botulism causing organism. This is why improperly sterilized canned food can be so dangerous. The canning process provides an anaerobic environment in which these organisms can grow and produce toxins.

e. Microaerophiles - These are organisms which grow best in a reduced oxygen environment. Brucella abortus which causes the disease Brucellosis in both cattle and man is an example.

6. Osmotic pressure - This refers to the number of dissolved particles in a solution. As most microorganisms come in contact with solutions of some type is a very important physiological concept. In order to appreciate its significance, we must first investigate the phenomena of osmosis.

a. Osmosis - This is the movement of water across a selectively permeable membrane from an area of greater water concentration (lesser osmotic pressure) to an area of lesser water concentration (higher osmotic pressure). A selectively permeable membrane is one that permits water to pass but not the substances dissolved in later. Most cell membranes are of this nature.

(l) When two solutions are separated by a selectively permeable membrane water will move from the one with the greater water concentration (lesser dissolved particles) to the one with lesser water concentration (greater dissolved particles.

(2) The greater the number of dissolved particles, the greater is the osmotic pressure of a solution.

b. Osmotic pressure and cells - Using the cell's concentration as a point of reference, three different types of osmotic solutions can be recognized.

(l) Hypotonic - These solutions have less osmotic pressure (more water that does the cell. A cell placed into such a solution (providing it does not have a protective wall) will swell and burst due to the inward movement of water.

(2) Hypertonic - Such a solution has a greater osmotic pressure than does the cell. A cell placed into this solution will become dehydrated due to the loss of water.

(3) Isotonic - This solution has the same osmotic pressure as does the cell. A cell placed into this solution will not shrink or swell.

c. Cell walls - The rigid cell wall exerts back pressure when the cell is in a hypotonic solution and therefore prevents the cell from swelling and bursting. In a hypertonic solution, the wall cannot prohibit the loss of water, and thus the cell may become dehydrated even though the cell wall maintains its original shape.

d. Osmotic pressure and microorganisms - Osmotic pressure is major environmental factor with which microbes must deal.

(l) Microorganisms without a cell wall must have some way of eliminating water that moves into them by osmosis when they live in a hypotonic environment. Protozoa often have a contractile vacuole that squirts out water.

(2) As cell walls cannot protect against hypertonic environments, these have been utilized for preserving food. Brine, sugar, and other such high osmotic pressure solutions are virtually microbe free. There are exceptions.

(a) Although most bacteria cannot invade jam or jelly due to the high osmotic pressure induced by the sugar, fungi can. This is because the fungus has an osmotic pressure that is higher than the jam's.

(b) Halophilic (salt loving) bacteria will grow in brine.



7. Acidity and alkalinity, pH - This is another chemical phenomena Which is critically important to living things.

a. pH - This is a number which represents a measure of the hydrogen ion concentration of a solution. By definition it is equal to



1/log (H+)

Every aqueous solution has a hydrogen ion concentration and therefore a pH value. pH is measured on a scale of 0 to l4. Values less than 7 are said to be acid, while those greater than 7 are alkaline or basic. Note that acid solutions have greater hydrogen ion concentrations that do basic solutions.

b. Significance of pH - All enzymes have a pH value at which they operate best. This is because they assume a particular tertiary structure at a given pH. If the pH decreases or increases from the optimum value, the protein begins to denature, and then the enzyme stops functioning and the reaction which it catalyzed ceases to occur. Consequently, pH is critically important for all of life's processes. Microorganisms have a pH value at which they thrive best. Deviation from this value results in death.

c. pH tolerance - Most bacteria thrive best at neutral to slightly alkaline pH values. Fungi on the other hand seem to prefer somewhat acid environments. For this reason, acid bogs are usually deficient in bacteria but rich in fungi. During World War I, sphagnum moss was used as a sterile dressing because it grows in highly acid bogs and is therefore usually free of most bacteria. This lack of tolerance for acidity which most bacteria exhibit is the basis behind the pickling of foods in vinegar for preservation. Vinegar is a dilute solution of acetic acid.

8. Radiation - Radiation is a form of electromagnetic energy which is emitted from the sun, outer space, and the earth itself. Microorganisms have varying degrees of tolerance for different kinds of radiation. Photosynthetic organisms require radiation in the form of visible light, but many non-photosynthetic organisms are killed by direct exposure to sunlight. It is the UV portion of sunlight which has the most devastating effect. Most bacteria which can live in direct sunlight possess pigments that absorb the UV component.

a. Ionizing radiation - This is especially high energy radiation such as X-rays, gamma rays, neutrons, etc. All ionizing radiations are damaging to microorganisms. Because of this, some are used as sterilizing agents.