Premed · Premed · Microbiology
Lecture 8: Microbial Metabolism II -- Anabolism and Metabolic Diversity
Microbiology
Learning Objectives
By the end of this lecture, students will be able to:
- Describe the major anabolic pathways used by microorganisms for biosynthesis of amino acids, nucleotides, lipids, and carbohydrates
- Explain how catabolic and anabolic pathways are interconnected (amphibolic pathways)
- Describe the mechanisms of carbon fixation, including the Calvin-Benson-Bassham cycle
- Compare and contrast oxygenic and anoxygenic photosynthesis in microorganisms
- Explain chemolithotrophy and its ecological significance
- Describe nitrogen fixation, nitrification, and denitrification
Lecture Content
I. Principles of Anabolism
Anabolic reactions are endergonic, requiring energy input from ATP or GTP and reducing power from NADPH or NADH. These biosynthetic pathways draw their starting materials -- precursor metabolites -- from catabolic pathways. From glycolysis come glucose-6-phosphate, fructose-6-phosphate, glyceraldehyde-3-phosphate, phosphoenolpyruvate, and pyruvate. The TCA cycle contributes acetyl-CoA, alpha-ketoglutarate, succinyl-CoA, and oxaloacetate. The pentose phosphate pathway supplies erythrose-4-phosphate and ribose-5-phosphate.
Amphibolic pathways serve both catabolic and anabolic functions simultaneously; the TCA cycle is the prime example. When intermediates are siphoned off for biosynthesis, they must be replenished through anaplerotic reactions. PEP carboxylase converts phosphoenolpyruvate plus CO2 to oxaloacetate, and pyruvate carboxylase converts pyruvate plus CO2 to oxaloacetate. In organisms growing on acetate or fatty acids, the glyoxylate cycle allows net synthesis of oxaloacetate from acetyl-CoA.
II. Biosynthesis of Major Macromolecules
A. Amino Acid Biosynthesis
Amino acids are assembled from carbon skeletons derived from central metabolic intermediates. They are organized into families based on their precursor: the alpha-ketoglutarate family includes glutamate, glutamine, proline, and arginine; the oxaloacetate family includes aspartate, asparagine, methionine, threonine, isoleucine, and lysine; the pyruvate family includes alanine, valine, and leucine; the 3-phosphoglycerate family includes serine, glycine, and cysteine; the aromatic amino acids (phenylalanine, tyrosine, tryptophan) are synthesized from phosphoenolpyruvate and erythrose-4-phosphate via the shikimate pathway; and histidine derives from ribose-5-phosphate. The amino group is typically introduced by transamination from glutamate or by direct amination, with glutamate dehydrogenase and glutamine synthetase serving as key enzymes for nitrogen assimilation.
B. Nucleotide Biosynthesis
Purine nucleotides (adenine and guanine) are assembled directly on ribose-5-phosphate, with the purine ring being built one atom at a time from amino acids, CO2, and formyl-tetrahydrofolate. Pyrimidine nucleotides (cytosine, uracil, thymine) take the opposite approach: the ring is synthesized first from aspartate and carbamoyl phosphate and then attached to ribose-5-phosphate. Deoxyribonucleotides are produced by reduction of ribonucleotides, catalyzed by ribonucleotide reductase. Salvage pathways recycle free bases and nucleosides, conserving the substantial energy investment of de novo synthesis.
C. Lipid Biosynthesis
Fatty acids are built from acetyl-CoA units by fatty acid synthase, using NADPH as the reducing agent and malonyl-CoA as the two-carbon donor at each elongation step. Bacterial fatty acids are typically 16--18 carbons long and may be saturated or unsaturated. Phospholipids are formed when glycerol-3-phosphate is acylated with two fatty acids and a polar head group is added. In bacteria, membrane phospholipids feature ester linkages, while archaea use ether-linked isoprenoid chains with opposite glycerol stereochemistry.
D. Polysaccharide and Cell Wall Biosynthesis
Peptidoglycan synthesis is a multistep process spanning three cellular compartments. In the cytoplasm, UDP-NAG and UDP-NAM are synthesized and a pentapeptide is added to NAM. At the membrane, the NAM-pentapeptide is transferred to undecaprenyl phosphate (bactoprenol), NAG is added to form the disaccharide-pentapeptide unit, and the unit is translocated across the membrane by a flippase. Outside the cell, transglycosylation polymerizes the disaccharide units into glycan chains, and transpeptidation -- catalyzed by penicillin-binding proteins (PBPs) -- cross-links the peptide side chains, which is the step targeted by beta-lactam antibiotics.
LPS biosynthesis in Gram-negative bacteria begins with Lipid A synthesis at the inner membrane, followed by addition of the core polysaccharide and O-antigen and transport to the outer leaflet of the outer membrane. Capsule synthesis typically proceeds through Wzy-dependent or ABC transporter-dependent pathways.
III. Autotrophic CO2 Fixation
The Calvin-Benson-Bassham (Calvin) cycle is the most widespread carbon fixation pathway. Its key enzyme, RuBisCO (ribulose-1,5-bisphosphate carboxylase/oxygenase), catalyzes the addition of CO2 to ribulose-1,5-bisphosphate. Three turns of the cycle consume 3 CO2, 9 ATP, and 6 NADPH to produce one molecule of glyceraldehyde-3-phosphate (G3P). The Calvin cycle is used by cyanobacteria, purple sulfur bacteria, nitrifying bacteria, and all plants and algae. In some bacteria, RuBisCO is concentrated within carboxysomes, polyhedral protein microcompartments that increase the local CO2 concentration around the enzyme.
Several alternative autotrophic pathways exist. The reverse (reductive) TCA cycle is used by some anaerobic and microaerobic bacteria such as Chlorobium and Helicobacter. The Wood-Ljungdahl (acetyl-CoA) pathway fixes two CO2 molecules to form acetyl-CoA and is used by acetogens and methanogens. Additional pathways include the 3-hydroxypropionate pathway in some photosynthetic bacteria and archaea, and the dicarboxylate/4-hydroxybutyrate cycle found in certain archaea.
IV. Photosynthesis in Microorganisms
A. Oxygenic Photosynthesis
Oxygenic photosynthesis is performed by cyanobacteria, algae, and plants and employs two photosystems operating in series. Photosystem II (PSII) absorbs light at 680 nm and splits water molecules, releasing O2, protons, and electrons. Photosystem I (PSI) absorbs light at 700 nm and reduces NADP+ to NADPH via ferredoxin. Electrons flow from water through PSII, plastoquinone, the cytochrome b6f complex, plastocyanin, PSI, ferredoxin, and finally NADP+ reductase to produce NADPH. The proton gradient generated during electron flow drives ATP synthase in a process known as photophosphorylation. The ATP and NADPH produced are then used in the Calvin cycle to fix CO2. Cyanobacteria are the ancestral organisms from which chloroplasts evolved through endosymbiosis.
B. Anoxygenic Photosynthesis
Anoxygenic photosynthesis is carried out by purple bacteria and green bacteria, which possess only a single photosystem and therefore do not produce O2. Instead of water, these organisms use alternative electron donors such as H2S, elemental sulfur, H2, or organic compounds. Purple sulfur bacteria (such as Chromatium) contain bacteriochlorophyll a or b and oxidize H2S through elemental sulfur to sulfate. Green sulfur bacteria (such as Chlorobium) contain bacteriochlorophyll c, d, or e and use the reverse TCA cycle for CO2 fixation. Purple non-sulfur bacteria (such as Rhodospirillum) are photoheterotrophs that use organic compounds as electron donors. Cyclic photophosphorylation generates ATP by returning electrons to the reaction center, while reverse electron flow is used to generate NADPH when needed.
<image>A side-by-side comparison of oxygenic and anoxygenic photosynthesis. Panel A (Oxygenic -- Cyanobacteria): Diagram showing PSII and PSI connected by an electron transport chain (plastoquinone, cytochrome b6f, plastocyanin). Water is oxidized at PSII releasing O2. NADPH is produced at the end of PSI. Proton gradient drives ATP synthase. Panel B (Anoxygenic -- Purple Sulfur Bacteria): Single photosystem with cyclic electron flow generating ATP via proton gradient. H2S shown as the electron donor producing S0. Reverse electron flow to generate NADPH indicated by a dashed arrow. Key differences highlighted: O2 production (yes vs. no), electron donor (H2O vs. H2S), and number of photosystems (2 vs. 1).</image>
V. Chemolithotrophy
Chemolithotrophic organisms obtain energy by oxidizing inorganic compounds without light. The electrons harvested from inorganic donors are passed through an electron transport chain to generate a proton motive force and ATP, while carbon is typically fixed via the Calvin cycle, making these organisms chemolithoautotrophs.
Major types include hydrogen-oxidizing bacteria (such as Ralstonia), which oxidize H2 to protons and electrons; nitrifying bacteria, comprising ammonia oxidizers like Nitrosomonas (converting NH4+ to NO2-) and nitrite oxidizers like Nitrobacter (converting NO2- to NO3-), both crucial for the nitrogen cycle and wastewater treatment; sulfur-oxidizing bacteria like Thiobacillus, which oxidize H2S or elemental sulfur to sulfate (and can produce sulfuric acid responsible for acid mine drainage); iron-oxidizing bacteria such as Acidithiobacillus ferrooxidans, which oxidize Fe2+ to Fe3+ and are used in biomining; and methanogens (archaea), which reduce CO2 with H2 to produce methane via the Wood-Ljungdahl pathway.
VI. Nitrogen Metabolism
Nitrogen fixation converts atmospheric N2 to ammonia (NH3), a reaction catalyzed by the nitrogenase complex, which is extremely oxygen-sensitive and requires 16 ATP per N2 molecule fixed. Free-living nitrogen fixers include Azotobacter, Clostridium, and cyanobacteria, which protect their nitrogenase in specialized heterocysts that exclude oxygen. Symbiotic nitrogen fixers, most notably Rhizobium in legume root nodules, provide biologically available nitrogen to ecosystems.
Assimilatory nitrate reduction converts NO3- through NO2- to NH4+, which is then incorporated into amino acids. Dissimilatory nitrate reduction (denitrification) is a form of anaerobic respiration in which NO3- is sequentially reduced to NO2-, NO, N2O, and finally N2, returning molecular nitrogen to the atmosphere. This process, carried out by organisms such as Pseudomonas and Paracoccus, is important in soil and aquatic systems. Nitrification is the aerobic oxidation of NH4+ to NO2- and then to NO3- by nitrifying bacteria. Anammox (anaerobic ammonium oxidation) is a more recently recognized process in which NH4+ and NO2- are combined to produce N2 and water, performed by Planctomycetes such as Candidatus Kuenenia. Anammox is significant in marine nitrogen cycling and has been harnessed for wastewater treatment.
<image>A diagram of the biogeochemical nitrogen cycle. Central elements: N2 gas in the atmosphere, NH4+ (ammonium) in soil/water, NO2- (nitrite), and NO3- (nitrate). Arrows labeled with processes and key organisms: nitrogen fixation (Rhizobium, Azotobacter, cyanobacteria) converting N2 to NH4+; nitrification (Nitrosomonas: NH4+ to NO2-; Nitrobacter: NO2- to NO3-); assimilatory nitrate reduction (plants and microbes: NO3- to NH4+ to organic N); denitrification (Pseudomonas: NO3- to N2 returning to atmosphere); ammonification (decomposers: organic N to NH4+); and anammox (NH4+ + NO2- to N2). Each process color-coded and annotated with whether it is aerobic or anaerobic.</image>
VII. Sulfur and Carbon Cycling (Brief Overview)
The sulfur cycle involves sulfate reduction under anaerobic conditions (SO4 2- to H2S, carried out by Desulfovibrio), sulfur oxidation under aerobic conditions (H2S or elemental sulfur to SO4 2-, by Thiobacillus), and assimilatory sulfate reduction, which incorporates sulfate into the amino acids cysteine and methionine.
The carbon cycle is driven by CO2 fixation by autotrophs through photosynthesis and chemolithotrophy, mineralization of organic carbon back to CO2 by heterotrophic decomposition, methane production by methanogens, and methane consumption by methanotrophs. Microbes are indispensable drivers of all of these global biogeochemical cycles.
