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Bacterial Physiology HSCI 5607

Chapter: 12 Inorganic Metabolism

Introduction: Most of the prokaryotes can utilize various derivatives of sulfur, nitrogen and iron as nutrient as well as energy source. These nutrients are metabolized using varieties of metabolic pathways:

1. Through assimilatory pathways microorganisms incorporate inorganic nitrogen and sulfur into organic cellular components. Some bacteria can also utilize chemically inert nitrogen gas as a nitrogen source through a process called ‘Nitrogen fixation’.
2. In dissimilatory pathways the inorganic compounds are used as electron acceptor instead of molecular oxygen through a process called ‘Anaerobic respiration’.  p is created in the same way in which it is created during aerobic respiration. There are many organisms which can use nitrate or sulfate as final electron acceptor but there are also bacteria which can use Fe 3+ or Mn 3+ as final electron acceptor.
3. There are oxidative pathway which oxidize inorganic compounds like H 2 , NH 3 , NO 2

, S, H 2 S, and Fe 2+ instead of organic compounds to obtain electrons and energy. These organisms are known as chemolithotrophs. If they use CO2 as carbon source then they are called Chemolithoautotrophs.

Sulfate Assimilation: Many bacteria can utilize inorganic sulfate as their sole source of sulfur. The sulfate (SO 4 2- ) is first reduced to sulfide (H 2

S

and HS-) and then incorporated into cysteine. In the first step there is a formation of adenosine-5’-phospho- sulfate (APS), a reaction catalyzed by the enzyme ATP sulfurylase. In the second step, APS is phosphorylated by APS kinase to form adenosine-3’-phosphate –5’-phophosulfate (PAPS). The PAPS is then reduced by thioredoxin, a sulfahydryl reductant to release AMP-3’-Phosphate and sulfite (SO3). Sulfite is then reduced by NADPH to hydrogen sulfide. H 2

S

is very toxic and doesn’t accumulate in cell but is immediately incorporated into cysteine. The sulfide enzymatically displaces acetate in O-acetylserine to form cysteine.

Dissimilation of Nitrate and Sulfate: Dissimilatory pathways use nitrate and sulfate as final electron acceptor but do not incorporate these elements in cellular materials rather excrete them out. Many facultative anaerobes utilize nitrate reduction pathway in the absence of oxygen while obligately anaerobic sulfate reducers use sulfate as final electron acceptors. Nitrate Dissimilation: Generally this takes place in facultative anaerobic bacteria under low oxygen level. It takes place in the membranes and the products can be nitrite, ammonia or nitrogen gas. When nitrate or nitrite is reduced to nitrogen gas or nitric or nitrous oxide, the process is known as ‘Denitrification’. Denitrification occurs in soil under anaerobic conditions, for example in water- logged soil. Examples of denitrifying bacteria are Alcaligenes and Pseudomonas species.

Dissimilatory Sulfate Reduction: Organisms like Desulfovibrio carry out anaerobic respiration, during which electrons flow in the cell membrane to sulfate as a terminal electron acceptor, reducing it to H 2

S.

The process also generates  p , which is used for ATP synthesis via respiratory phosphorylation. The flow of electron in this process is linked with utilization of lactate, which is converted to pyruvate generating 4e- that are used finally to reduce sulfate to sulfide. Several components are involved and the process spans across the membrane to the periplasm and back to the cytoplasm. It also generates  p which is used for ATP synthesis.

Nitrogen Fixation: Nitrogen fixation is ecologically one of the most important process carried out by prokaryotes. So far it has not been reported in eukaryotes. The ammonia produced via nitrogen fixation is incorporated into cellular components using glutamine synthetase and glutamate synthase system. Nitrogen fixation takes place when N 2 is the only source of nitrogen since the genes required for nitrogen fixation are repressed by exogenous nitrogen supply other than nitrogen gas. Industrial reduction of nitrogen is carried out by Haber process which requires 200 atm pressure and 800 °C while prokaryotes carry out same process at atmospheric pressure and temperatures.

Nitrogen Fixing Systems: Nitrogen fixing prokaryotes can be divided into three distinct groups:

1. Symbiotic legumes: These includes organisms belonging to Rhizobium , Bradyrhizobium , and Azorhizobium which form symbiotic relationship with leguminous plants like soybeans, clover, alfalfa, string beans and peas. These bacteria infect the roots of the plants and stimulate nodule formation, within which they fix nitrogen. Plants provide organic nutrients to the organism.
2. Symbiotic nonlegumes: There are nitrogen fixing bacteria in symbiotic relationship with non legume plants. The best example is that of water fern ‘Azolla’ and nitrogen fixing Cyanobacterium, Anaebaena azollae.
3. Non symbiotic nitrogen fixation: There are many free living soil and aquatic prokaryotes that can fix atmospheric nitrogen. The examples are Azotobacter , Clostridium , and certain species of Desulfovibrio , the photosynthetic bacteria and cyanobacteria. Certain archaea are also reported to fix nitrogen.

The Nitrogenase reaction: The nitrogenase reaction is a series of reductions during which 0.5 mole of N 2 and 4 moles of H

are reduced to 1 mole of NH 3 and 0.5 mole of H 2

. The overall reaction is, 4e

• 0.5N 2

+ 4H

• 8ATP NH 3

+ 0.5H

2

• 8ADP + 8P i Since the oxidation state of N2 is 0 and the oxidation state of NH 3 is -3, there is a need for three electrons per nitrogen atom. A fourth electron is transferred to a proton to produce hydrogen gas. The electrons are transferred one at a time in ATP-dependent reaction from the Fe4S4 cluster in Fe-protein to MoFe-protein to N 2

The details of the electron pathway and the role of ATP is not clearly understood but it is known that two molecules of ATP are required per electron transferred. Thus 16 moles of ATP are needed to convert one mole of nitrogen to two moles of ammonia. During the reduction of nitrogen, protons are used to make hydrogen gas, and thus electrons and ATP are apparently wasted but in few organisms like Azotobacter, the hydrogen gas is used to generate electrons for the nitrgenase.

The Nitrogen Fixation

Ammonia oxidizers: The bacteria that oxidize ammonia are called nitrifiers. They include, Nitrosomonas, Nitrococcus, Nitrosospira, Nitrosolobus , and Nitrosovibrio. They are all chemolithoautotrophs that acquire carbon by assimilating CO2 via calvin cycle. Nitrosomonas oxidizes ammonia to nitrite while Nitrobacter oxidize nitrite to nitrate. Both together are responsible for major portion of conversion of ammonia to nitrate, a process known as ‘Nitrification’. Sulfur oxidizers: The sulfur oxidizers are of two types:the photo-synthetic sulfur oxidizers and the nonphotosynthetic sulfur oxidizers. The acidophilic sulfur oxidizing bacteria can be isolated from sulfur and coal mines that produce sulfuric acid. The example is Thiobacillus thiooxidans which can grow at pH of 1.0 with optimum of 2 0r 3. The sulfur compound commonly used by these bacteria include hydrogen sulfide, elemental sulfur, and thiosulfate (S 2

O

3 2- ). All of them are oxidized to sulfate.

Iron oxidizing bacteria: Few bacteria derive energy from oxidizing ferrous ion to ferric ion. Most of these are acidophilic sulfur oxidizers which oxidize sulfide to sulfuric acid. The example is Thiobacillus ferroxidans which can obtain energy either from ferrous ion or from sulfur compounds.