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Four most common elements utilized by all microorganisms are; (1) Carbon (2) Hydrogen (3) Oxygen (4) Nitrogen Note: organic compounds usually supply the hydrogen and the oxygen to organism as well as carbon. Nitrogen Microorganims require large quantities of nitrogen for biosynthesis Inorganic sources of nitrogen can be utilized to produce organic compounds Nitrogen can be assimilated (incorporated) into organic molecules by different routes Nitrogen Assimilation – during anaerobic respiration, molecules other than oxygen are used as final electron acceptor in the electron transport system. Assimilation is the process by which inorganic compounds are reduced to serve this purpose during anaerobic conditions. (inorganic nitrogen sources listed in decreasing abundance) 1. N2 atmospheric nitrogen (78%) (Nitrogen fixation – the process of reducing atmospheric nitrogen to ammonia) a. few microorganisms can use nitrogen gas directly b. Free-living bacteria (Azotobacter, Klebseilla, Clostridium) c. Symbiotic bacteria (Rhizobium in association with legumes) d. Cyanobacteria (Anabaea) e. Atmospheric nitrogen, or N2, is converted into two molecules of ammonia under the action of the enzyme nitrogenase f. This is highly exergonic; utilizes 16 ATPs to make 2 NH3 g. N2 + H+ + e- + ATP →(nitrogenase)→ NH3 2. NO3 – nitrate a. much more oxidized than the nitrogen in ammonia b. must first be reduced to ammonia before conversion into an organic form c. can be formed by processes of nitrification (Nitrification is the aerobic process by which ammonia is oxidized to NO2, and then further oxidized to NO3. d. Nitrosomonas and nitrosococcus are involved in the first oxidative step e. Nitrobacter (a chemolithoautotroph) is involved in the second step. f. This process can occur anaerobically in Nitrosomonas eutropha but the products are nitrite and nitric oxide. Note: the electron acceptor in this reaction is nitrogen dioxide. 3. NH3 ammonia (ammonia incorporation/assimilation) a. can be incorporated into organic material relatively easily and directly b. It is more reductive than other forms of inorganic nitrogen (reduction is the process by which hydrogen atoms or protons are added to a compound or molecule thus reducing it oxidative state) Phosphorus is also important for cell survival 1. Phosphorus is obtained from organic and inorganic sources a. Inorganic sources can be utilized directly for cellular material b. Organic phosphate must be hydrolyzed at ester linkages by phosphatases 2. Phosphorus important in formation of nucleic acids, proteins, and phospholipids 3. it is an ATP coenzymes The role of sulfur and sulfate Reduction 1. Inorganic sulfate (SO4) is utilized directly but first must be activated 2. it’s reduced to H2S by utilizing 2 molecules of ATP and sulfate reductase 3. this is a main constituent of amino acids, cysteine and methionine, tRNA, coenzymes, and organic cofactor [nonprotein substance needed by an enzyme for normal activity] for an enzyme 4. organic sulfur can be added to cell directly by addition of cysteine and/or methionine in growth media Other elements are required for growth and metabolism but in much smaller amounts (K+, Mg, Ca++, Fe++) some organisms also require sodium or silicon but these are a select few Growth Factors – These compounds are essential to cell components by cannot be synthesized by the organism requiring them. There are three main classes of growth factors; 1. amino acids 2. purines & pyrimidines 3. vitamins Amino Acids Synthesized by cell or provided by exogenous sources (exogenous = derived or developed outside of the body; originating externally) Needed for protein synthesis Purines & pyrimidines Used for nucleic acids or coenzymes Vitamins Small organic molecules used as enzyme cofactors Refer to table 5.3 3. Nitrogen source Nitrogen is needed for the synthesis of such molecules as amino acids, DNA, RNA and ATP. Depending on the organism, nitrogen, nitrates, ammonia, or organic nitrogen compounds may be used as a nitrogen source. 4. Minerals a. sulfur Sulfur is needed to synthesisize sulfur-containing amino acids and certain vitamins. Depending on the organism, sulfates, hydrogen sulfide, or sulfur-containing amino acids may be used as a sulfur sorce. b. phosphorus Phosphorus is needed to synthesize phospholipids, DNA, RNA, and ATP. Phosphate ions are the primary source of phosphorus. c. potassium, magnesium, and calcium These are required for certain enzymes to function as well as additional functions. d. iron Iron is a part of certain enzymes. e. trace elements Trace elements are elements required in very minute amounts, and like potassium, magnesium, calcium, and iron, they usually function as cofactors in enzyme reactions. They include sodium, zinc, copper,molybdenum, manganese, and cobalt ions. Cofactors usually function as electron donors or electron acceptors during enzyme reactions. 5. Water 6. Growth factors Growth factors are organic compounds such as amino acids, purines, pyrimidines, and vitamins that a cell must have for growth but cannot synthesize itself. Organisms having complex nutritional requirements and needing many growth factors are said to be fastidious. 7. Some Relationships Between Bacterial Growth and Pathogenicity a. Ability to compete for limited nutrients Often the ability to be pathogenic is directly related to the bacterium's ability to compete successfully with host tissue and normal flora for limited nutrients. One reason the generation time of bacteria growing in the body is substantially slower than in lab culture is because essential nutrients are limited. To be pathogenic, a bacterium must be able to multiply in host tissue. The more rapid the rate of replication, the more likely an infection may be established. Pathogens, therefore, are able to compete successfully for limited nutrients in the body. Generally bacteria compete for nutrients by synthesizing specific transport systems or cell wall components capable of binding limiting substrates and transporting them into the cell. For example, the body makes considerable metabolic adjustment during infection to deprive microorganisms of iron. Iron is essential for both bacterial growth and human cell growth. Bacteria synthesize iron chelators (compounds capable of binding iron) called siderophores. Many siderophores are excreted by the bacterium into the environment, bind iron, and then re-enter the cell. Others are found on the cell wall where they bind iron and transport it into the bacterium. The body has iron chelators of its own (transferrin and lactoferrin). The ability of bacterial iron chelators to compete successfully with body iron chelators may be essential to pathogenic bacteria. Some bacteria such as Neisseria gonorrhoeae, Neisseria meningitidis, and Hemophilus influenzaea, however, have receptors for human lactoferrin and transferrin and can utilize iron bound to these compounds. b. Anaerobic environment In some cases, a normal product of fermentation may allow bacteria to be more pathogenic. For example, Clostridium perfringens, which causes gas gangrene, is an obligate anaerobe. When endospores enter a deep wound with no oxygen, the endospores can germinate. The vegetative Clostridia, which get their energy from fermentation, can ferment tissue sugar to produce gases (carbon dioxide and hydrogen) as end products. The gases accumulate in the enclosed space and physically tear the tissue allowing the organism to spread. This spreading ability is further aided by alpha, kappa, and mu exotoxins mentioned in Unit 1.) c. Temperature Most pathogens have evolved to grow best at body temperature (37C). 3. Some Relationships Between Factors Influencing Growth and the Control of Bacteria a. Temperature 1. Cold temperature, temperature below the minimum for growth, generally has a static effect. It inhibits growth by slowing the rate of metabolism and possibly causing ice crystals to form. Examples include refrigeration, freezing, and extreme low cold. 2. High temperature, temperatures above the maximum for growth, generally has a cidal effect. It kills organisms by denaturing enzymes (moist heat) or causing desiccation and oxidation of proteins (dry heat). Examples include autoclaving, boiling, dry heat, incineration, and pasteurization. b. Oxygen 1. The lack of oxygen may inhibit aerobic bacteria and molds (canned foods for example), but it has no effect on obligate anaerobes and facultative anaerobes. 2. The presence of oxygen may kill obligate anaerobes. For example, a hyperbaric chamber employing saturated oxygen under pressure may sometimes be of value in controlling gas gangrene caused by anaerobic Clostridium species. c. pH Organic acids, such as calcium proprionate, potassium sorbate, methylparaben, and sodium benzoate, are frequently used as food preservatives. The low pH denatures microbial proteins. d. Osmotic pressure 1. In conjunction with antibiotics or enzymes that alter peptidoglycan (penicillins, cephalosporins, monobactems, glycopeptides,lysozyme, etc.), a normal hypotonic environment which causes water to flow into the bacterium may lead to bacterial lysis. Without these agents, however, the peptidoglycan prevents osmotic lysis. 2. Making the bacterium's environment hypertonic by adding salts or sugars may have a static effect on microorganisms by causing plasmolysis (dehydration and shrinkage of the cytoplasmic membrane due to an outflow of water). This is sometimes used in food preservation.