Cell Biology - Chapters 3 & 4

Today we want to begin to exam some of the structure and function relationships in prokaryotic cells. Remember that cells are essentially well organized assemblages of macromolecules with many common features shared among diverse types of cells.
Begin the discussion with the microscope since it has been instrumental in microbiological studies.

Microscopes

Two features:
Magnification - pretty much increase the magnification of an image indefinitely. Magnification is the product of the eye piece magnification times the objectives magnification.
Resolution - the ability to separate or resolve to close objects. There is a limit which is a function of the wavelength of the light source. Resolution equals 0.5 times [lambda] divided by the numerical aperture. Blue light gives the greatest resolution.
Two types of microscopes:
Compound light microscope - resolution of about 0.2 micron.
Electron microscope which we will come back to later.
Compound light microscopes
Bright field microscope - Microscope you used in class the other day. Usually visualize specimens by contrasting the cells against the light background. We can use dyes to stain the cells.
Types of dyes used are cationic, meaning that they are positively charged since the cell and much of the macromolecules in a cell are negatively charged. Methylene blue, saffarin, and crystal violet are common dyes used in the laboratory.
Phase contrast microscope - view living cells and don't need to stain the cells. How? Light passing through a cell is retarded relative to the light passing through the liquid medium. The changes in the phase of the light waves is translated into changes in the intensity of light seen through the ocular lens.
Dark field microscope - A dark field ring placed in the condenser ring causes the light to be focused into a hollow cone with the specimen at the apex of the cone. The light is moving in a direction such that it would not enter the objective lens. Therefore without anything on the stage the field of observation would be dark. A specimen on the stage will cause the light to bend and enter the objective lens causing a bright image on a dark background when viewed through the ocular lens. Very high resolution and good to view flagella.
Fluorescence microscope - a specialized microscope which illuminates the specimen with light of one wavelength, e.g., ultraviolet, and the specimen emits light at a higher wavelength which is observed. Detect fluorochromes that may be attached to antibodies. The fluorochromes have resonating double bonds that when hit by high energy light such as ultraviolet causes the electrons to become excited. The excited electrons return to ground state and emit light rather than simply heat.
Electron microscope - very high resolution microscope which used electrons rather than light. Can see proteins and nucleic acids using the electron microscope.
Transmission electron microscope - Thin sections of cells impregnated in a matrix are made. The thin sections are then viewed using the transmission electron microscope. To enhance contrast, the sample is coated with an electron dense stains such as osmic acid, uranium, or lead. These stains cause the electrons to be scattered thus increasing the contrast between sub-cellular constituents.
Scanning electron microscope - Used to observe intact cells. The cells are coated with something like gold. Electrons are scattered by the gold plated cells which allows them to be viewed. Magnify up to 100,000X.

Specimen preparation -

in most situations the specimen is heat fixed to the slide before all staining procedures. This is to bind the cells to the slide so that they will not be subsequently washed off in the staining procedure.

Dyes are ionic chromophores meaning that they carry an electronic charge and are colored. basic dyes are cations with positive charges. Most bacteria have a slightly negative charge at neutral pH. Therefore basic dyes will be attracted and bind to the cells. acidic dyes are anions with a negative charge. They are not attracted to cells and usually are used to provide a dark background with the cells highlighted.
Simple stains are used to simply provide contrast to see the cells. Cells are mostly water and therefore would be very difficult to see with a light microscope. A simple stain such as methylene blue binds to the cell and highlights the whole cell.
Differential stains are used to differentiate between microorganisms. The most obvious differentiation based on staining is the Gram positive versus Gram negative species of bacteria. Gram staining was devised by Hans Christian Gram. Cells are first stained with crystal violet, followed by a mordant such as iodine, followed by a alcohol wash and subsequently stained with a counter stain such as safranin.
Special stains include the negative stain, capsule stains and flagella stain.

Common features of a prokaryotic cell

Cell wall - rigid structure that provides strength and shape to cell.
Cytoplasmic membrane - semi-permeable barrier between outside and inside cell.
Ribosomes - small particles in cell which are responsible for the translation of mRNA into a polypeptide.
Inclusion bodies - storage "organelles" found in prokaryotic cells.
Nucleoid - nuclear region of the prokaryotic cell. Remember there is no true nucleus like in a eukaryotic cell.
Size of prokaryotes
Average 1 x 3 microns but there is lots of variation.
Smallness affects their biological activity: Small cocci have a greater surface to volume ratio than bigger cocci. This effect exchange of nutrients and waste products in and out of cell. This in turn allows small prokaryotes to have more rapid growth rates and larger population sizes than most eukaryotes in a microbial habitat.
Basic shapes include coccus (spheres), bacillus (rod), and spiral.

Cell structure outside in Flagella -

used for motility. Not found in all bacteria, i.e., there are nonmotile bacteria. Arranged differently on different species of bacteria - polar flagella are attached to one (monotrichous) or both ends of the cell (amphitrichous). Lophotrichous flagella are tufts of flagella attached to one end of the cell. Peritrichous flagella are attached at many places around the cell.

Structure
Flagella composed of subunits of a protein called flagellin.
Base of the flagellum is the hook which is composed of a single protein and serves to attach the flagellum to the motor apparatus.
The motor, called the basal body, is anchored in the cytoplasmic membrane and the cell wall. Consists of a rod inserted through two rings in gram positive cells and three rings in gram negative cells. The inner two rings of all prokaryotes are anchored in the cytoplasmic membrane and peptidoglycan layer of the cell wall. The third ring of the gram negative cells is anchored in the outer membrane.
Mot proteins - proteins that surround the inner ring in the cytoplasmic membrane. These are the actual motor proteins. They cause the flagella to rotate.
Fli proteins - proteins associated with the inner ring. They are responsible for switching the direction in which the flagella rotates.
Overall there are some 40 genes required to make an operational flagella.
Growth
Flagella grow at the tip not the base. Flagellin is passed up through the hollow core of the flagella. They than self assemble at the tip of the flagella.
Function
Used for motility. Flagella is a semirigid structure with a helical shape- like a cork screw - that actually rotates. Proton motive force drives the motor as the protons move through the mot proteins. Cells can move about 60 cell lengths per second - faster than a cheetah which moves at 25 body lengths per second.

Cells are always in gradients and need to be able to respond to gradients either positively or negatively by moving into or away from signal molecules respectively.
chemotaxis - bacterial response to a chemical gradient. Bacteria sense the gradient temporally not spatially since they are so small. In the absence of a gradient, the bacteria move randomly about in a series of runs and tumbles and moves nowhere. Runs are due to the flagellar motor moving counterclockwise and tumbles occur when the motor is moving clockwise. As they move into a gradient of an attractant, movement becomes biased with longer runs and fewer episodes of tumbling. Similarly as the cell moves away from a repellent, there are longer runs and fewer tumbles.
The actual sensing mechanism is complex and will be discussed when we talk about gene regulation. Suffice it to say that the cell has chemoreceptors for different molecules and these proteins interact with cytoplasmic proteins that affect the motor direction.
phototaxis - analogous to chemotaxis but the cells are responding to light gradients. This type of taxis is found in phototrophic bacteria which may want to orient themselves in the best position to carry out photosynthesis. Photoreceptors rather than chemoreceptors sense the light gradients.
other taxes - response to other types of gradients such as oxygen, magnetic fields, or ionic strength. Similar processes as discussed above.

Fimbriae and pili -

Fimbriae are similar to flagella in that they consist of protein. There are many more fimbriae than flagella. Their function is not always known, but they are used for attachment in the case of some pathogens or forming pellicles or scums on surfaces.
Pili are structurally like fimbriae but there are fewer per cell. Function in gene transfer = sex pili and attachment to host tissues.

Glycocalyx

A general term referring to the slime layer or capsule of a cell. Made up of polysaccharide and proteins which is highly variable depending on the bacterial species.
Function to aid in attachment to surfaces, resist phagocytosis -a virulence factor for pathogens, and resist desiccation since the glycocalyx binds lots of water.

Outer membrane of gram negative cells
Structure
Based on a differential stain, called the Gram stain, prokaryotes can be divided into gram positive and gram negative microbes. The outer membrane is found only in gram negative prokaryotes. Referred to as lipopolysaccharide layer or simple LPS.

Composed of phospholipids, polysaccharides and protein.
Polysaccharides consists of core polysaccharides and O-polysaccharides.
Core polysaccharides contains ketodeoxyoctonate (KDO) and various sugars.
O-polysaccharides consist of repeating units of sugars and are linked to the core polysaccharides. These are linked to Lipid A.
Lipid A is not a glycerol lipid but rather fatty acids are linked to a disaccharide of N-acetyl-glucosamine phosphate via an ester amine linkage. The O-Polysaccharide is linked to lipid A via KDO.
Lipoproteins are found in the inner half of the outer membrane. These anchor the outer membrane to the peptidoglycan of the cell wall.
Other proteins are found in the outer half of the outer membrane.
Notice that the lipids of the outer surface of the outer membrane is made up primarily of LPS and the inner surface is made up of phospholipids.
Function
The outer membrane of many gram negative bacteria is toxic to animals. Referred to as endotoxin - lipid A is the toxic portion. Includes Salmonella, Shigella, and Escherichia coli.
Provides resistance to some toxins and antibiotics.
Acts as a second lipid bilayer. Contains porin, protein channels, that allow hydrophilic, low molecular weight molecules to move through the bilayer. Porins are responsible for some antibiotic resistance properties of gram negative bacteria.

Periplasm

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space between the outer and inner (cytoplasmic) membranes - gram positive lack a well defined periplasm. Contains numerous proteins of hydrolytic enzymes, which break down food, binding proteins, which bind molecules and transport them to the inner membrane carrier proteins, and chemoreceptors.

Cell wall

Cell wall found in both gram positive and gram negative prokaryotes.
Function
Resist turgor pressure due to dissolved solutes inside the cell - pressure about that of a car tire.
Provide shape and rigidity of cell.
Cocci - spherical shape
Rod or bacilli - rod shaped
Spirilla - twisted rod about an axis
Spirochetes - tightly coiled
Appendaged bacteria - possess extensions such as stalks or long tubes
Filamentous bacteria - form long chains of cells
The basis of gram positive vs gram negative prokaryotes. The cell wall of these two are distinctly different.
Structure
Peptidoglycan composed of two sugars, N-acetyl-glucosamine (NAG) and N-acetyl-muramic acid (NAM) and a small group of amino acids including L-alanine, D-alanine, D-glutamic acid, and either lysine or diaminopimelic acid (DAP). Rare instance where you find D forms of amino acids used in biological systems.
Thin sheet of alternating NAG and NAM linked by glycosidic linkages. NAM has a short peptide of 4 amino acids linked to it.
In gram negative bacteria, there is a direct linkage between the amino group of DAP of one NAM and the carboxyl group of the terminal D-alanine of a second NAM.
In gram positive prokaryotes, there is a oligopeptide linker between the amino acid side chains of adjacent NAMs. There is lots of variation on this theme
90% of the gram positive cell wall consists of peptidoglycan. There may be up to 25 layers of it in the cell wall. In gram negative cell only about 10% of the cell wall is peptidoglycan. The majority is the complex outer membrane layer.
Diversity of peptidoglycans due to differences in the tetrapeptide - though these are small differences, and more differences in the crosslinking interbridge between the tetrapeptides of adjacent NAMs.
Summary - what is constant is the two sugars are always NAG and NAM linked by beta 1,4 linkages and NAM is always the sugar that is crossed linked.
Differences
Gram positive cell wall contains teichoic acids. Not found in gram negative prokaryotes.

Protoplast formation
Protoplasts lack or have their cell wall integrity disrupted by a treatment.
Lysozyme breaks the beta 1,4 glycosidic linkage between NAM and NAG. Water may enter into the cell and cause it to burst in a process called lysis. Lysozyme found in tears, saliva and other bodily fluids presumably as a defense against bacteria.
Protoplasts are cells where the cell wall is degraded with lysozyme but placed in an isotonic solution so water does not enter the cell and cause it to burst.
There are some bacteria that lack cell walls and peptidoglycan layers - these are the mycoplasmas. Essentially free-living protoplasts that are able to survive under specific conditions.

Inner membrane or Cytoplasmic membrane - thin structure that surrounds the cell's cytoplasm. Highly selective barrier allowing concentrating of nutrients inside the cell and excretion of wastes.

Composition
Phospholipids forming a phospholipid bilayer with the hydrophilic phosphate groups facing outwards and the hydrophobic hydrocarbon tails pointing inwards to form a hydrophobic core. Referred to as unit membrane.

Proteins associated with the membrane:
Integral proteins that span the membrane. They contain a hydrophobic core region which is embedded in the hydrophobic core of the membrane and hydrophilic regions that are stick out of the membrane.
Peripheral proteins are associated with either the internal or external surface of the membrane. Frequently associated with integral proteins. Some of them associated with lipids and are referred to as lipoproteins. The lipids function to anchor the protein in a certain position on the membrane.
Fluid mosaic - though the membrane looks rigid it is fluid - viscosity like light motor oil. And the proteins move about the membrane like ships on water - hence the fluid mosaic model for the cytoplasmic membrane.

Function
Selectively permeable - allows specific ions, and molecules to move across.

Small, non polar and fat-soluble substances can move across the membrane freely. Water moves across the membrane freely.

Polar solutes, charged molecules, e.g., amino acids, organic acids, and inorganic salts, do not move across the membrane. Hydrogen anions do not since they hydrate to H3O+ which is charged.

Membrane transport proteins
3 classes
Uniports
Symports
Antiports

Facilitated diffusion
Protein allows or moves molecules across the membrane down the molecules concentration gradient - a source of potential energy. Still requires a protein. Some proteins are non specific and move related molecules and others are specific as to what they let cross the membrane.

Active transporters
General features
1. Requires a carrier protein
2. Requires energy to move the molecule. The hydrolysis of ATP or the dissipation of a proton gradient generated during energy releasing reactions in the cell.
3. Can move the molecule up its concentration gradient (i.e., against a concentration gradient) - which is why it requires energy.
Group translocation - the transported molecule is modified as it is transported across the membrane. The phosphotransferase system or PTS is the most intensely studied group translocation system. A complex system of many cytoplasmic and membrane bound proteins. Phosphoenol pyruvate is the intermediate which donates the phosphate group.
Active transport - the molecule is not modified as it is transported across the membrane. Energy derived from hydrolysis of ATP or the dissipation of a proton gradient -a so called proton motive force.

DNA
A chromosome with few associated proteins. No nuclear membrane. In addition, there are extrachromosomal elements called plasmids. Chromosome normally circular molecule of a couple of thousand kilobase pairs (1000 bps). Tends to aggregate and form a nucleoid. Linearized chromsomes of 4000 kilobase pairs would be about 1 mm in length while the bacteria is only 3 microns. To compact the chromsome, the DNA is supercoiled with 50 or more domains maintained by proteins that interact with each domain.
Because a cell can divide faster than the chromsome can replicate there are usually more than one copy or partial copy of the chromosome in a cell. We call bacteria haploids since they contain only on chromosome.
Genetic exchange - conjugation, transduction, and transformation

.Endospores
Differentiated cells that are extremely resistant to a number of harsh treatments and remain dormant yet viable for long periods of time. Very important in how we preserve food since several food borne pathogens form spores including Clostridium botulinum which causes botulism - potentially lethal.