Metabolic Diversity Overall, the chemical reactions that take place within microbial cells are highly diverse There are 2 key “ingredients” in this diverse collection of recipes Energy-to make things work Carbon-for precursors and building blocks

Part Two-Carbon An emphasis on the flow of carbon and other elements in fundamental metabolic pathways 3.8, 3.9 Glycolysis and Fermentative Diversity 3.12 Respiration: Citric Acid and Glyoxylate Cycle 13.5 (390-391) Autotrophic Pathways

Broad Overview of Metabolism Energy to run metabolism comes from ATP generated by catabolism Microbes have different ways to acquire or make precursor molecules

3.8 Glycolysis (Stage I and II of Figure 3.14) Glycolysis (Embden–Meyerhof-Parnas or EMP pathway): a common and central pathway for catabolism of glucose End product of glycolysis is pyruvate (pyruvic acid) Stage III shows one type of fermentation

Diagram of reactions of Glycolysis Start = Glucose End = Pyruvate

3.9 Fermentative Diversity Pentose Phosphate Pathway (Shunt) “Alternate” pathway Runs “parallel” to glycolysis Different reactions thus different intermediates Generates different precursor metabolites Generates reducing power but no ATP Allows metabolism of “unusual” or “weird” sugars

3.9 Fermentative Diversity In food science fermentation can refer to the production of foods such as yogurt In chemical engineering it can refer to the production of ethanol as an additive for gasoline In microbiology it refers to basic and important process in which there is breakdown of carbon compounds (eg glucose) to smaller compounds with no oxygen required Final steps different in different species But…microbiologists use the term in different ways

3.9 Fermentative Diversity Fermentations may be classified by products formed (See Sec.13.12) in final stages Ethanol Lactic acid (homolactic vs heterolactic) Propionic acid “Mixed acids” Butyric acid (extra ATP generated) Butanol

3.9 Fermentative Diversity Some authors consider glycolysis to be a part of fermentation (Fig. 3.14) while others do not Fermentations may be classified by substrate fermented (See Sec.13.12) Amino acids Purines and pyrimidines Aromatic compounds Usually glucose NOT specifically named

Figure 3.14 Reactions of Fermentation (lactic acid produced) Figure 3.14 Embden–Meyerhof–Parnas pathway (glycolysis). Figure 3.14

3.9 Fermentative Diversity Fermentation Helps detoxify and eliminate waste products Generates metabolites for other microbes in the environment May help to recover additional ATP Maintains redox balance (page 87 and Fig. 3.14) of NAD and NADH.

3.12 Respiration: Citric Acid and Glyoxylate Cycle Citric acid cycle (CAC): pathway through which pyruvate is completely oxidized to CO2 (Figure 3.22a) (aka Krebs or TCA cycle) Follows after glycolysis Pyruvate oxidized Subsequently 6 CO2 molecules released and NADH and FADH generated Plays a key role in both catabolism AND anabolism

3.12 Respiration: Citric Acid and Glyoxylate Cycle The citric acid cycle generates many compounds available for biosynthetic purposes - α-Ketoglutarate and oxaloacetate (OAA): precursors of several amino acids; OAA also converted to phosphoenolpyruvate, a precursor of glucose in gluconeogenesis Succinyl-CoA: required for synthesis of cytochromes, chlorophyll, and other tetrapyrrole compounds Acetyl-CoA: necessary for fatty acid biosynthesis

Figure 3.22a The citric acid cycle.

3.12 Respiration: Citric Acid and Glyoxylate Cycle The Citric Acid Cycle is also a key collection point and entry point for metabolites C4-C6 citric acid cycle intermediates (e.g., citrate, malate, fumarate, and succinate) are common natural plant and fermentation products and can be readily catabolized through the citric acid cycle alone Fatty acids metabolized via Acetyl-CoA

3.12 Respiration: Citric Acid and Glyoxylate Cycle A variation of the citric acid cycle with glyoxylate as a key intermediate Shares enzymes with citric acid cycle Anabolic-allows Ac-CoA from fatty acids to be used for carbohydrate synthesis-not energy harvest Ac-CoA converted directly to succinate with glyoxylate as key intermediate Also allows utilization of C2-C3 organic acids if larger molecules not available (Figure 3.23)

In other words-the citric acid cycle generates key precursor metabolites As well as harvesting energy

13.5 Autotrophic Pathways (pp 390-391) Carbon fixation is reduction of CO2 to carbohydrate-a key feature of autotrophy At least 6 pathways exist in various Archaea and Bacteria But the Calvin cycle (Figure 13.16, 13.17) is the most important

13.5 Autotrophic Pathways (pp 390-391) Named for its discoverer, Melvin Calvin Fixes CO2 into cellular material for autotrophic growth Requires NADPH, ATP, CO2 and special enzymes e.g. ribulose bisphophate carboxylase (RubisCO), 6 molecules of CO2 are required to make 1 molecule of glucose (Figure 13.17)

Figure 13.17 The Calvin cycle.

13.5 Autotrophic Pathways: RUBISCO Responsible for most carbon fixation on planet Unusual enzyme-”weak”- when CO2 is low easily inhibited by oxygen Often found sequestered in carboxysomes to increase CO2 and lower O2

Reductive Ac-CoA Pathway for Carbon Fixation Found in some Bacteria and Archaea Electrons from hydrogen reduce CO2 to Ac-CoA “acetogens” Non-cyclic Most ancient form of carbon fixation?

3.13 Catabolic Diversity Chemolithotrophy Uses inorganic chemicals as electron donors Examples include hydrogen sulfide (H2S), hydrogen gas (H2), ferrous iron (Fe2+), ammonia (NH3) Begins with oxidation of inorganic electron donor Uses an electron transport chain and transmembrane ion gradient

Dissimilative Iron Oxidizers are chemolithotrophs (13.9 and 14.15) Oxidize Fe2+ to Fe3+ Very widely distributed in many environments where Fe2+ is available Autotrophic or heterotrophic Aerobic or anaerobic Archaea or Bacteria

Acidithiobacillus ferrooxidans is a representative iron oxidizer Acidophile at pH 2-3 Acid environments with Fe2+ Fe2+ -> Fe3+ -> FeOH3

Figure 13.24 (pH 2) (pH 6) + ATP ATP e– e– Out Outer membrane cyt c Electron transport generates proton motive force. Rusticyanin e– Periplasm Reverse e– flow e– NAD+ Q cyt bc1 cyt c cyt aa3 Figure 13.24 Electron flow during Fe2+ oxidation by the acidophile Acidithiobacillus ferrooxidans. In (pH 6) + ATP NADH Cell material ADP ATP Figure 13.24

Figure 13.23 Iron-oxidizing bacteria.

3.17 Nitrogen Fixation (Sec 3.17 also pp 438-439) Living systems require nitrogen in the form of NH3 or R-NH2 “Fixed” or “reduced” nitrogen, not N2 Only some prokaryotes can fix atmospheric nitrogen: diazotrophs

3.17 Nitrogen Fixation Some nitrogen fixers are free-living, and others are symbiotic Cyanobacteria are free-living nitrogen fixers Soybean root nodules contain endosymbiotic Bradyrhizobium japonicum

3.17 Nitrogen Fixation Energetically expensive (8 ATP per N atom) Requires electron donor, often pyruvate Reaction is catalyzed by nitrogenase Sensitive to the presence of oxygen Fe plus various metal cofactors Can catalyze a variety of reactions