Chapter 4 - Prokaryotic Cell Structure & Function I Flashcards
The prokaryotic cell is ______ and structurally ____________.
Small
Structurally simple
You must be familiar with all of the structures listed in the following image.
Identify the labeled parts of the basic prokaryotic cell.
A. Pilus
B. Ribosome
BB. Storage granule
C. Flagellum
E. Cytoplasm
F. Cell membrane
G. Murein layer
GG. Outer membrane
T. Nucleoid
Prokaryotic organisms are in many ways the dominant life on earth. In terms of total protoplasmic volume, they make up about ___%.
90%
Why are prokaryotic organisms so successful from an evolutionary perspective?
They have very simple cell structures
What is the typical volume of a prokaryotic cell?
1 µm3
Structural simplicity does not mean ____________ simplicity.
Molecular simplicity
There is a range of sizes for prokaryotic cells. What are the ranges?
0.25 µm to 120 µm in diameter
Most prokaryotes are unicellular. True or false?
True
What are the three common cell shapes of prokaryotes?
- Cylindrical rods (bacillus)
- Spheres (cocci)
- Helical (spirilla)
Some prokaryotes are multicellular and are commonly _________________, made of a chain of identical cells attached end to end. Some are __________, meaning they are branching tubes of cytoplasm contained within walls.
Filamentous
Mycelial
Multicellular prokaryotes generally lack cell differentation. If they do, they usually are ____________, which merely have a different function, or in the case of cyanobacteria, _______________, which fix nitrogen and have a different metabolism.
Spores
Heterocysts
True of false: almost all prokaryotic cells have a cell envelope.
True
True or false: cell envelope structure and chemistry are the same among different groups of prokaryotes.
False - cell envelope strucutre and chemistry can differ among different groups of prokaryotes
True or false: the cell envelope is composed of a single layer.
False. The cell envelope can be a single homogeneous layer or a complex, multilayered structure.
What is the difference between a cell envelope and a cell wall?
The cell envelope comprizes the cell membrane, the cell wall and an outer membrane if present
How do prokaryotic cells protect themselves against osmotic lysis?
Via a cell envelope
Why do prokaryotic cells require protection against osmotic lysis?
Most of them live in environments in which solute concentration is less than their highly concentrated cytoplasm
Why strategy do prokaryotic cells use to protect against osmotic lysis?
The strategy is to contain the cell’s turgor pressure with a layer external to the cell membrane that resists expansion of the cell volume
Who developed the Gram stain process?
Hans Christian Gram in 1884
What is the Gram stain useful for?
Distinguishing some prokaryotic envelopes from each other
Outline the general steps of the Gram stain process.
- Stain with crystal violet (purple)
- Add iodine
- Add decolorizer, either ethanol or acetone
- Stain with safranin (pink)
Gram-positive cells stain _______________.
Purple
Gram-negative cells stain ___________.
Pink
What is the conventional theory regarding the mechanism of the Gram-stain process?
- Crystal violet and mordant freely crossed the outer membrane (OM), the peptidoglycan mesh (PM), and cell membrane (CM), binding to anions within the periplasm and cytosol
- Alcohol dissolves lipid membranes, attacking the structural integrity of the OM and CM. Because the PM of Gram-negative cells is covalently bound to the OM, it is significantly disrupted with the OM is torn away. Thus the crystal violet precipitate is washed away.
- In Gram-positive cells, there is no OM and only a thick PM coating the CM. Thus, the PM remains largely undisturbed when subjected to the decolorization process, retaining the crystal violet stain.
What does recent research suggest about the mechanism of the Gram-stain process?
Recent research,suggests that crystal violet slowly diffuses across the peptidoglycan mesh and does not penetrate the cell membrane; that crystal violet becomes “stuck” within the peptidoglycan layer; and that the crystal violet and mordant precipitate forms within the peptidoglycan layer
What method did researchers use to show that crystal violet does not, in fact, cross the cell membrane?
Second harmonic light scattering
What is second harmonic light scattering?
It is a type of nonlinear optical spectroscopy. A nonlinear vibrational spectroscopy in which two colors of light, one in the visible and one in the infrared, are mixed at an interface to create the sum frequency. The generated sum frequency light carries structural information about the molecules and chemical functionalities present at the interface.
What are the seven steps in the gram staining process?
- Crystal violet
- Iodine
- 95% ethanol
- Safranin
- Blot semi-dry, air dry, and/or heat fix
- Coverslip
- Oil
After each step in the gram staining process, what do you do?
Rinse with distilled water
How long do you saturate the sample with crystal violet?
60 seconds
How long do you saturate the specimen with iodine?
60 seconds
How long do you saturate with 95% ethanol?
Until the specimen becomes clear or 15 seconds
How long do you saturate the specimen with safranin?
30 seconds
What are three ways that a gram stain can go wrong?
- Excessive heat during fixation
- Low concentration of crystal violet
- Excessive washing between steps
Why should you avoid excessive heating during the gram staining process?
Heat fixing the cells, when done to excess, alters the cell morphology and makes the cells more easily decolorized
Why do low concentrations of crystal violet disrupt the gram stain process?
Concentrations of crystal violet up to 2% can be used successfully; however, low concentrations result in stained cells that are easily decolorized
Why does excessive washing disrupt the gram stain process?
The crystal violet stain is susceptible to wash out with water (but not the crystal violet- iodine complex.
How long should you rinse with water during each rinse in the gram stain process?
No longer than 5 seconds
What three results can occur from a gram stain?
- Gram positive - stain purple
- Gram negative - stain pink
- Gram variable - PITB (pilus backbone protein)
What seven microbe species can stain pink even though they are gram positive?
- Actinomyces
- Arthrobacter
- Corynebacterium
- Propionibacterium
- Bacillius
- Butyrivibrio
- Clostridium
Why do Actinomyces, Arthrobacter, Corynebacterium, and Propionibacterium stain pink even though they are gram positive?
Because they are part of a genera with cell walls that break during division
Why do Bacillus, Butyrivibrio, and Clostridium stain pink even though they are gram positive?
Because growing and elongating cells decrease their cell wall thickness
How old are most cultures that undergo gram staining?
Less than 24 hours
What bacterium is diplococci?
Neisseria gonorrhoeae
What does “diplococci” mean?
Refers to a bacterium that occurs as a pair of cocci
There are different types of cell envelopes among bacteria. Which is the most common?
The gram-negative envelope
Most bacteria have a _____ _____________ that consists of a thin rigid layer (the cell wall), overlain with a membrane, termed the outer membrane.
Cell envelope
In gram-______________ bacteria, the cell wall is between one to three layers thick.
Gram negative
What makes up the cell wall in bacteria?
Peptidoglycan
In what type of bacteria is the cell wall between 10 and 20 layers thick?
Gram-positive
One group of bacteria, the _______________________, has an envelope that appears to be a combination of gram-negative and -positive structures: a thick murein layer, overlain with an outer membrane; yet they stain gram positive.
Deinococci
The ________________________ group of bacteria has an envelope that consists of a layer of protein. These bacteria generally stain gram-negative because the thin layer of protein is unable to prevent the gram stain from rinsing out.
Planctomyces
Although planctomyces stain Gram negative - due to the thin layer of protein that surrounds their cell membrane - recent studies have suggested what about the structure of their cell envelopes?
Evidence of a cell wall with peptidoglycan
How many types of archael cell walls are there?
Three
What is the most common type of archael cell wall?
A layer of protein
Which archaea have a rigid wall made of a murein-like compound termed pseudomurein?
The methanogens
Some archae have walls that consist of what types of polysaccharides? Simple or complex?
Complex polysaccharides
True or false?
No archaea have been shown to contain true murein
True
What are the three types of archaeal cell envelope?
- A layer of protein
- Rigid wall of pseudomurein
- Wall of complex polysaccharide
Proteins without a defined cell envelope layer strengthen their membranes by what type of molecules?
Glycolipids
What sub-group of gram-positive bacteria has lost the ability to make a murein wall, instead using glycolipids?
The mycoplasma group
Mycoplasmas are obligate intracellular ____________.
Parasites
Two bacteria were discussed in glass that lack clearly defined cell walls. What were they?
- The mycoplasma group
- Mycobacterium
Mycobacterium have a cell membrane, peptidoglycan, and a unique outer layer that is covered with a waxy coating of ____________ ________, 60 to 90 carbon long fatty acids.
Mycolic acids
Are mycolic acids saturated or unsaturated hydrocarbon chains?
Saturated
What makes Mycobacterium difficult to treat?
The presence of a waxy outer covering of mycolic acids
Why do Mycobacterium not stain via the gram staining process?
Stains cannot penetrate the waxy layer of mycolic acids
Because Mycobacterium do not pick up the Gram stain, what type of staining process is used to identify them?
Acid-fast stain - they retain the dye carbolfuchsin despite decolorization with acid-alcohol
What color do Mycobacterium and Nocardia stain via the acid-fast stain?
Red/pink
What color do nonacid-fast bacteria stain?
Blue/purple
What is the counterstain in acid-fast stain?
Methylene blue
With the exception of ______________ and ________________, nearly all other genera of bacteria are nonacid-fast bacteria.
- Mycobacterium*
- Nocardia*
______________________ is a form of peptidoglycan in which individual glycan strands are cross-linked to each other by peptides
Murein
What is the wall’s principle function in bacteria?
Containing turgor pressure
Murein consists of two alternating sugar derivates. What are they?
N-acetylglucosamine
N-acetylmuramic acid
What is the structure of N-acetylglucosamine?
What is the structure of N-acetylmuramic acid?
Which component of the disaccharide of murein has a short peptide chain linked by its amino terminus to the carboxyl group of muramic acid? N-acetylglucosamine or N-acetylmuramic acid?
N-acetylmuramic acid
The repeating unit of murein is a ________________ of NAM and NAG with a peptide attached to the _____.
Disaccharide
NAM (N-acetylmuramic acid)
Murein contains a polysaccharide backbone and peptide side chains, which makes it a part of a class of heteropolymers termed ________________ and includes the archael pseudomurein
Peptidoglycans
How are N-acetylglucosamine and N-acetylmuramic acid linked in murein?
Beta 1–>4 linkages
The amino acid chain connected to the N-acetylmuramic acid molecule in murein contains peptides that alternate between which two conformations?
L and D
What is the usual sequence of the peptide chain in N-acetylmuramic acid?
L-Ala, D-Glu, L-Lys, D-Ala, D-Ala
The third amino group of the peptide chain of NAM is variable, but usually they all have a free ________ group on their R side chains.
Amino
What is the structure of murein?
What is the current belief regarding the reason that murein contains both L and D amino acids?
The alternation makes the murein more resistant to degradation by enzymes in the enviornment as most proteases have active sites that are specific for peptide bonds between two L amino acids
One murein chain contains up to 30 repeats, meaning that it is ___ carbohydrate monomers in length.
60
What type of “secondary” structure does murein adopt?
A helix
What is the spacing of sugars in the murein helix?
Each peptide is oriented 90 degrees to the previous and following chains
How many monomers are there per turn of a murein helix?
4 monomers
What is the name of the cross-linking reaction that links together NAG and NAM in murein?
Transpeptidation
What catalyzes a transpeptidation reaction?
Peptidase
How are the peptide chains cross-linked in murein?
Usually between the third amino acid with a free amino group and the second to last alanin amino acid
What gives murein its characteristic strength?
Cross linking between peptide chains
True or false?
All murein molecules have the same peptide chain composition.
Flase; both the peptide chain composition and the cross-linking between them can be different for different species
Murein consists of a single giant molecule: a single layer thick in the ____-_______ bacteria, and many layers thick in the ____-_______ bacteria
Gram-negative
Gram-positive
True or false?
No archaea have peptidoglycan although some have pseudopeptidoglycan.
True, no archae have been found with peptidoglycan
What are the three major differences bewteen murein and pseudomurein?
- Sugar composition
- N-acetylalosaminuronic acid instead of N-acetylmuramic acid
- Linkage
- Linkages are beta 1–>3 instead of beta 1–>4
- Amino acid conformation
- All amino acids in L conformation as opposed to mixed
What type of bacteria have cell walls that contain teichoic acid?
Gram-positive bacteria
There are two types of teichoic acids. What are they?
- Lipoteichoic acids that are linked to membrane phospholipids
- Wall teichoic acids that are covalently linked to murein
___________________ ____________ are polymers of sugar alcohol phosphates, usually glycerol phosphate or ribitol phosphate present in gram-positive bacteria.
Teichoic acids
What type of teichoic acids are covalently bound to a lipid at one of their ends?
Lipotechoic acids
What type of teichoic acids are covalent attached to murein strands?
Wall teichoic acids
How do we know that teichoic acids are important in gram-positive bacteria?
Mutants that lack both types of teichoic acids are unviable. Mutants that lack one of the two have decreased ranges of livable conditions and problems with cation binding.
What are autolysins?
Chemicals that break down the cell wall created by the cell
What do you believe are the roles of teichoic acids?
- Scaffolding
- Elongation and cell division
- Biofilm and host-tissue adhesion
What protects murein in gram-negative bacteria?
The outer membrane
Why are gram-positive bacteria able to tolerate some loss of their outer surfaces?
Because they have such a thick layer of murein
Why are gram-negative bacteria vulnerable to lysis by enzymes?
Because they have only a single or few layers of murein and lysozymes secreted by other bacteria and fungi can attack murein by cleaving the beta 1–>4 linkages between NAG and NAM
In gram-negative bacteria, the outer membrane is higly symmetric. True or false?
False, the outer membrane is highly asymmetric
How does penicillin work?
It binds the active site of peptidase preventing the bacteria’s ability to cross-link the murein cell wall, leading to lysis
What type of lipids constitute the inner leaf of the gram-negative outer membrane?
Phospholipids
What molecule characterizes the outer leaf of the outer membrane of gram-negative bacteria?
Lipopolysaccharides
What lipoprotein connects the outer membrane to murein in gram-negative bacteria?
The Braun lipoprotein
What does LPP stand for?
Outer membrane lipoprotein or murein lipoprotein
The outer leave of gram-negative bacteria is composed mostly of ________________ abbreviated LPS.
Lipopolysaccharide
Lipopolysaccharide or LPS is a known _____________, exclusive in bacteriology, and is toxic to humans.
Endotoxin
What confers the toxicity of lipopolysaccharide to humans?
Lipid A, a dimer of glucosamine phosphate
Lipopolysaccharide has two primary components. What are they?
A lipid core
A long polysaccharide chain
The lipid portion forms the outer leaf of the membrane, and the polysaccharide forms a dense mesh on the outer surface of the cell
What is the lipid portion of lipopolysaccharide known as?
Lipid A
What is lipid A?
The lipid portion of lipopolysaccharide that is composed of a phosphorylated glucosamine dimer to which are attached six to eight fatty acids
What two components make up the polysaccharide chain of lipopolysaccharide?
A core region of various sugars and sugar derivates
An O-side chain in which a sequence of five to six sugars is repeated many times to make a long chain
Which part of lipopolysaccharide is genus specific?
The R-core polysaccharide
What part of lipopolysaccharide is serotype specific?
The O-side chain
What does immunogenicity mean?
Immunogenicity is the ability of a particular substance, such as an antigen or epitope, to provoke an immune response in the body of a human and other animal. In other words, immunogenicity is the ability to induce a humoral and/or cell-mediated immune responses
What part of lipopolysaccharide is immunogenic?
The O-side chain
How does lipopolysaccharide initiate an immune response in humans?
Lipopolysaccharide binds to lipid binding protein on human CD14 macrophages and epithelial cells, which stimulates inflammatory cytokines and nitric oxide production, leading to cell death
What types of human cells does lipopolysaccharide bind?
CD14 macrophages
Epithelial cells
What does lipopolysaccharide binding to lipid binding protein in humans lead to?
The stimulation of inflammatory cytokines and nitric oxide, ultimately causing cell death
What animal’s blood is very sensitive to lipid A?
Horshoe crab blood, which is used to screen products, including insulin, for lipid A presence
The Braun lipoprotein is __________________ attached to the outer membrane and ______________ attached to murein
Noncovalently
Covalently
What do some antimicrobial peptides target?
The Braun lipoprotein
Why is the outer membrane of gram-negative bacteria impermeable to both hydrophobic and hydrophilic compounds?
- The outer membrane has a high density of charges at the outer surface due to the phosphate groups of lipid A and charged groups on the R core polysaccharide chain, forming hydrogen bonds to water (barrier)
- The hydrocarbon side chains of lipid A are saturated, which allows them to pack very tightly and leave little room for hydrophobic compounds to slip in between molecules
How do dissolved compounds enter the gram-negative periplasm?
Via porins
What is the periplasm?
A compartment between the cell membrane and outer membrane
Porins allow what sized molecules to enter the periplasmic space of gram-negative bacteria?
1 nm in diameter
Less than 1,000 Daltons
What does OmpF stand for?
Outer membrane porin F
What is OmpF?
A porin in E. coli
OmpF is composed of ___ identical subunits. Each monomer is a hollow __-____ with a diameter of approximately 20 angstroms.
Three (homotrimer)
Hollow beta-barrel
What is the constriction zone of OmpF?
The area in which OmpF narrows so that only ~600 Dalton molecules can enter; the constriction zone is between seven and 14 angstrons
What types of amino acid residues make up the constriction zone of OmpF?
Basic and acidic
Why does the constriction zone of OmpF contain basic and acidic amino acid residues?
To help block large molecules and solvents
What is the periplasm?
A dense solution of protein
The Braun lipoprotin physically links the outer membrane to what component of the cell wall in gram-negative bacteria?
Murein
What is the Braun lipoprotein also known as?
LPP = outer membrane lipoprotein = murein lipoprotein
What is the function of the Braun lipoprotein?
to anchor the cell wall and the outer membrane covalently via peptide bond at the carboxy terminus to peptidoglycan and noncovalently via the amino terminus to outer membrane (lipid moiety)
The Braun lipoprotein is small, approximately ___ kDa, and acts as a trimer.
5.8 kDa
What is the function of the periplasm?
Export/import
Peptidoglycan synthesis
