Microbiology 2
Escherichia coli: a Gram-negative bacterium of the gut microbiome, Part 1
FIGURE 3.1 ■ Escherichia coli: a Gram-negative bacterium of the gut microbiome. The envelope includes the outer membrane; the cell wall and periplasm; and the inner (cell) membrane. Embedded in the membranes is the motor of a flagellum. The cytoplasm includes enzymes, messenger RNA extending out of the nucleoid, and ribosomes. Ribosomes translate the mRNA to make proteins, which are folded by chaperones. The nucleoid contains the chromosomal DNA wrapped around binding proteins. (PDB codes: ribosome, 1GIX, 1GIY; DNA-binding protein, 1P78; RNA polymerase, 1MSW)
*
Bacterial Cell Structure: What is seen in Gram-negative & Gram-positive bacteria
- Bacteria can be placed in 2 groups based on the thickness and placement of the cell wall
- Gram-negative
- Gram-positive
- Plasma membrane
- Absence of a nucleus
- DNA is located in the nucleiod region
- No histone proteins, but DNA-binding proteins present to keep genomic DNA compact
- Plasmids: DNA that is independent of the genome.
- Flagellum
Biochemical composition of bacteria
- Water
- Essential Ions
- Needed for enzymatic reactions
- Small organic molecules: lipids and sugars
- Lipids are almost as abundant as RNA molecules
- Found in the cell wall
- peptidoglycan
- Macromolecules: nucleic acids, proteins, fats, & sugars
Goal: Isolate proteins
- Purpose of cell fractionation is to isolate components of choice from a bacterial cell
- The first step is cell lysis
- EDTA
- Sucrose
- Lyzozymes
- Ultracentrifugation
FIGURE 3.2 ■ Fractionation of Gram-negative cells.
Cell periplasm fills with sucrose, and lysozyme breaks down the cell wall. Dilution in water causes osmotic shock to the outer membrane, and periplasmic proteins leak out. Subsequent centrifugation steps separate the proteins of the periplasm, cytoplasm, and inner and outer membranes. Photo
Source: Lars D. Rennera and Douglas B. Weibel. PNAS 108(15):6264.
*
Goal 2:Protein Analysis
FIGURE 3.3 ■ Protein analysis.
A. Gel electrophoresis of total cell proteins compared to outer membrane proteins from cell fractionation. B. Outer membrane proteins are identified by tryptic digest and mass spectrum analysis. The resulting peptide sequence is compared with those predicted from genome data.
*
FIGURE 3.3a ■ Protein analysis.
A. Gel electrophoresis of total cell proteins compared to outer membrane proteins from cell fractionation.
*
FIGURE 3.3b ■ Protein analysis.
B. Outer membrane proteins are identified by tryptic digest and mass spectrum analysis. The resulting peptide sequence is compared with those predicted from genome data.
*
Understanding the role of a protein
FIGURE 3.4 ■ Genetic analysis of FtsZ.
A. E. coli with aspartate (D) at position 45 replaced by alanine (A) (D45A) elongate abnormally, forming blebs from the side, with no Z-rings. Cells with aspartate replaced by alanine at position 212 (D212A) elongate to form extended nondividing cells that contain spiral FtsZ complexes. FtsZ was visualized by immunofluorescence. B. Model of FtsZ protein monomer based on X-ray crystallography shows the position of the mutant residues, D212A and D45A.
*
FIGURE 3.5 ■ Bacterial cell membrane.
The cell membrane consists of a phospholipid bilayer, with hydrophobic fatty acid chains directed inward, away from water. The bilayer contains stiffening agents such as hopanoids. Half the membrane volume consists of proteins.
*
LeuT sodium/leucine cotransporter
- Homology to human neurotransmitter sodium sympoters
- Has been used as a blueprint to understand structure and function, and pharmacology of NSS human transporters.
FIGURE 3.7 (part 1) ■ A cell membrane–embedded transport protein: the LeuT sodium/leucine cotransporter of Aquifex bacteria.
The protein complex carries leucine across the cell membrane into the cytoplasm, coupled to sodium ion influx. (PDB code: 3F3E)
*
Transport across bacterial membranes
- Passive diffusion
- Membrane proteins
- Aquaporins
- Permease (lac operon)
- Osmosis
- Greater osmotic pressure can lead to bacterial cell lysis (seen with certain antibiotics)
- Membrane-permeant weak acids and bases: can cross the plasma membrane
- Transmembrane ion gradients
FIGURE 3.8 ■ Common drugs are membrane-permeant weak acids and bases.
In its charged form (A– or BH+), each drug is soluble in the bloodstream. The uncharged form (HA or B) is hydrophobic and penetrates the cell membrane.
*
FIGURE 3.8a ■ Common drugs are membrane-permeant weak acids and bases.
In its charged form (A– or BH+), each drug is soluble in the bloodstream. The uncharged form (HA or B) is hydrophobic and penetrates the cell membrane.
*
FIGURE 3.8b ■ Common drugs are membrane-permeant weak acids and bases.
In its charged form (A– or BH+), each drug is soluble in the bloodstream. The uncharged form (HA or B) is hydrophobic and penetrates the cell membrane.
*
- NAM and NAG are linked together by a β-(1,4)-glycosidic bond
- Lysozymes target this bond
- The peptidoglycan monomer will have 5 peptides
- Once this monomer becomes incorporated into the existing polymer, 4 peptides are seen.
FIGURE 3.14b ■ The peptidoglycan sacculus and peptidoglycan cross-bridge formation.
B. A disaccharide unit of glycan has an attached peptide of four to six amino acids.
*
FIGURE 3.16 ■ Cell envelope: Gram-positive (Firmicutes) and Gram-negative (Proteobacteria).
A. Firmicutes (Gram-positive) cells have a thick cell wall with multiple layers of peptidoglycan, threaded by teichoic acids. A inset: Gram-positive envelope of Bacillus subtilis (TEM). B. Proteobacteria (Gram-negative) cells have a single layer of peptidoglycan covered by an outer membrane; the cell membrane is called the inner membrane. B inset: Gram-negative envelope of Pseudomonas aeruginosa (TEM).
*
Gram +
FIGURE 3.19a ■ Gram-negative cell envelope.
A. Murein lipoprotein has an N-terminal cysteine triglyceride inserted in the inward-facing leaflet of the outer membrane. The C-terminal lysine forms a peptide bond with the m-diaminopimelic acid of the peptidoglycan (murein) cell wall.
*
FIGURE 3.20 ■ Lipopolysaccharide (LPS).
A. Lipopolysaccharide (LPS) consists of core polysaccharide and O antigen linked to a lipid A. Lipid A consists of a dimer of phosphoglucosamine esterified or amidated to six fatty acids. B. Repeating polysaccharide units of O antigen extend from lipid A.
*
FIGURE 3.20a ■ Lipopolysaccharide (LPS).
A. Lipopolysaccharide (LPS) consists of core polysaccharide and O antigen linked to a lipid A. Lipid A consists of a dimer of phosphoglucosamine esterified or amidated to six fatty acids.
*
FIGURE 3.20b ■ Lipopolysaccharide (LPS).
B. Repeating polysaccharide units of O antigen extend from lipid A.
*