Biosynthesis

Bio 314 Microbiology Lecture Notes

Bacterial Metabolism- Biosynthesis

Instructor

• Mark Kainz, PhD

Semester

• Fall 2025

Lecture Outline

• Shared Intermediates & Pathways

• Nitrogen Assimilation

• Macromolecule Synthesis

• DNA Replication

• Gene Structure

• Transcription

• Translation

• Coupled Transcription & Translation

Biosynthesis (Anabolism)

• Definition:

  • The process of creating large and complex molecules from smaller precursor molecules.

• Characteristics:

  • Energy-requiring process.

  • May utilize some pathways and/or enzymes that also function in catabolism.

Gluconeogenesis

• Involves intermediates and enzymes from glycolysis.

• Certain reactions are unidirectional.

• Energetic Cost:

  • Requires 6 ATP to convert 2 pyruvates to 1 glucose.

Intermediates in Catabolic and Anabolic Pathways

• Many catabolic pathways serve as sources of intermediates for anabolic pathways.

• Examples of metabolites and their origins:

  • Glucose

  • Nucleosides

  • Glucose-6-P

  • Ribose

  • Lipids (e.g. Glycerol P)

  • Amino Acids (e.g. Serine, Glycine, Cysteine, etc.)

  • Purines (e.g. Histidine)

  • Pyruvate

  • Acetyl-CoA

  • Several Metabolic Precursors (e.g. 3-phosphoglycerate, oxaloacetate, etc.)

Levels of Biosynthetic Activity

• Inorganic Molecules:

  • CO₂, NH₃, H₂O, PO₄

• Monomers:

  • NTPs, Amino Acids, Sugars, Fatty Acids

• Macromolecules:

  • Nucleic Acids, Proteins, Polysaccharides, Lipids

• Supermolecular Structures:

  • Membranes, Enzyme Complexes, Ribosomes, Flagella

• Cells:

  • The culmination of all the above materials.

Nitrogen Assimilation

• Uses:

  • Amino Acids

  • Nitrogenous Bases

• Sources:

  • Ammonia (preferred source, although not all bacteria can utilize it)

  • Nitrate

  • Molecular Nitrogen (N₂)

Mechanisms of Nitrogen Assimilation

• Bacteria assimilate nitrogen primarily in the form of NH₃ by synthesizing glutamine in ATP-requiring reactions.

• Glutamine can then be converted into other nitrogenous compounds via other energy-requiring reactions.

Nitrate Assimilation

• Some bacteria can assimilate nitrogen in the form of nitrate.

• Key enzyme: Nitrate Reductase

• Energetics:

  • Conversion of NO₃ to NH₃ is energetically expensive.

  • After conversion, NH₃ must be incorporated into glutamine, also requiring energy.

Nitrogen Fixation

• Certain bacteria can fix molecular nitrogen (N₂) from air.

• Energetics of the Process:

  • 1 N₂ yields 2 NH₃

  • This multi-step process requires the hydrolysis of 16 ATP.

  • NH₃ serves as a substrate for Glutamine Synthetase, which requires additional energy input.

Macromolecular Synthesis

Self-Assembly

• Most proteins contain all the information needed for assembly within their primary structure.

• Similar situations apply for many supermolecular structures including flagella and ribosomes.

• Role of Chaperonins:

  • Assist in the correct folding and assembly of proteins.

Types of Macromolecular Synthesis

• Proteins:

  • Composed of amino acids linked via peptide bonds.

• Nucleic Acids:

  • Formed from nucleotides linked by phosphodiester bonds.

• Polysaccharides:

  • Built from monosaccharides linked by glycosidic bonds.

The Central Dogma

• Representation of the flow of information in biological systems:

  • DNA → RNA → Protein

Processes:

• DNA Replication

• Transcription

• Translation

Bacterial Chromosomes

• Most bacteria possess circular chromosomes.

• Origin of Replication:

  • DNA replication initiates at specific locations termed origins of replication.

• DNA replication occurs in a bidirectional manner, resulting in the formation of a second chromosome.

Rolling Circle Mechanism

• Some bacterial chromosomes, certain plasmids, and some bacteriophages utilize a different mechanism for DNA replication.

• Initiation and synthesis differ from classical models, even though replication starts at an origin.

Gene Organization in Bacteria

• Many bacterial genes are organized into operons.

  • Operons consist of genes that encode proteins involved in a specific process.

  • Genes are usually physically grouped and expressed under the control of the same promoter, producing a shared mRNA.

General Operon Structure

• The layout of a typical operon consists of:

  • 5’ to 3’ orientation with genes interspersed:

  • +1 (start of transcription)

  • -1 (first nucleotide on the left of the promoter)

  • 0 (promoter)

  • -3 (indicates upstream genes)

  • Terminator sequence

Sigma Factors in Bacterial Gene Expression

• Function:

  • Sigma subunits confer promoter recognition on RNA Polymerase (RNAP).

• Types of Sigma Factors:

  • σ70: Primary sigma factor, recognizes most promoters.

  • σ32: Heat shock sigma factor, active during stress conditions.

  • σN: Recognizes promoters involved in nitrogen metabolism.

  • σF: Associated with flagella synthesis promoters.

  • σS: Recognizes promoters of genes expressed in the stationary phase.

Characteristics of Promoter Recognition

• Different sigma factors recognize various promoter features which are characterized by specific nucleotide sequences.

Operon Terminators

• Operons encompass terminators located at the 3’ ends of genes:

  • Intrinsic Terminators:

  • Characterized by G+C rich stem-loops followed by U rich sequences; they function as structures in the transcript.

  • Rho-dependent Terminators:

  • These require the rho protein and transcription pausing, involving non-translated RNA.

Requirements for Translation

• Conditions necessary for effective translation include:

  • mRNA: Proper ribosomal binding sites (RBS) and translation start sites.

  • Ribosomes: Essential for protein synthesis.

  • Charged tRNAs: Achieved through the activity of aminoacyl-tRNA synthetases.

  • Initiation Factors: Important for starting translation.

  • Elongation Factors: Assist in the elongation phase of translation.

  • Energy: Significant amounts of energy are required.

Features of Prokaryotic Gene Expression

• Key aspects include:

  • Coupled nature of transcription and translation.

  • Minimal to no mRNA processing.

  • Internal initiation of translation.

  • mRNAs are typically very short-lived, undergoing rapid turnover.