City College of San Francisco Microbiology 12

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Prokaryotic Cells

Introduction

The earth is estimated to be about 4.6 billion years old and prokaryotes represent the oldest forms of life. Stromatolites, which are domed rocks formed from microbial sediments, have been found which are up to 3.8 billion years old. These ancient rocks provide important evidence that bacterial life arose or arrived and began to diversify shortly after the origin of the earth. It is not surprising that there is such great diversity among bacteria, since they have a history of at least 3.8 billion years versus about 1.5 billion years for eukaryotes.

Prokaryotes are distinct from all other living organisms as their cells lack nuclei and other membrane-bound organelles. Because of this fundamental difference, prokaryotes have traditionally been grouped together in the Kingdom Monera. However, it is evident using molecular criteria (primarily comparative ribosomal RNA sequencing data) that there are two distinct groups of prokaryotes, referred to as the Domains Bacteria and Archaea. A domain is a taxonomic category placed above the kingdom level. As molecular data are relatively recent and the information available is accumulating at a rapid pace, the taxonomy of the prokaryotes is frequently revised. Among the factors preventing new molecular (therefore known to be heritable) data from being a sound basis for an unambiguous classification system is the frequent exchange of genetic information between even the most distantly related bacteria. This type of genetic exchange is known as lateral gene transfer.

Although the Domains Bacteria and Archaea, as well as the Eukarya, are defined on the basis of ribosomal RNA sequence analysis, there are phenotypic traits (outward manifestations of a gene or genes) that are useful in delineating the phylogeny of the three domains. They include:

• Cell Walls

The cell wall is a very important structure in the identification of major phylogenetic groups of prokaryotes. The majority of bacteria have cell walls consisting of peptidoglycan while this substance is lacking in the Archaea (see table 1).

• Lipids

The chemical bonding of cell membrane lipids is among the most useful phenotypic criteria for distinguishing Archaea from Bacteria. In the Archaea, branched cell membrane lipids are linked to glycerol by an ether linkage. This type of chemical bond forms a strong cell membrane and allows cells to survive in harsh environments.

Ribosomes are the site of protein synthesis in cells and are typically comprised of two subunits, the size of which varies between prokaryotes and eukaryotes. The 30S (small) ribosomal subunits of the Archaea are more similar in ultrastructure to the eukarya than they are to those of the bacteria. Most antibiotics that interfere with bacterial protein synthesis do not affect archaeal or eukaryotic protein synthesis.

Table 1. Key differences between Prokaryotes and Eukaryotes

Feature	Prokaryotic cell	Eukaryotic cell
Nucleus	Absent. No nuclear envelope	Present with nuclear envelope and nucleolus
Membrane-bound organelles	Absent	Present. Includes mitochondria, chloroplasts (plants), lysosomes
Chromosome (DNA)	Single coiled chromosome in cytoplasm 'nucleoid' region in association with 'histone-like' proteins	Multiple linear chromosomes with histone proteins
Cell division (asexual)	No true mitotic apparatus. Divide by binary fission or fragmentation	Mitosis
Cell wall	Eubacteria have a cell wall of peptidoglycan Archaea have cell walls of pseudomurein	No cell wall in animal cells Plant cell walls = cellulose Fungal cell walls = chitin
Ribosomes	70S. Free in cytoplasm	80S. Both free in cytoplasm and attached to rough E.R. 70S in mitochondria and chloroplasts
Cytoskeleton	Detected in Bacillus subtilis; likely present in other bacteria (see notes under cytoplasm)	Present consisting of microtubules and filaments
Flagella	when present consist of protein flagellin	consist of 9+2 arrangement of microtubules
Cytoplasmic membrane lipids	Eubacteria= Fatty acids joined to glycerol by ester linkage Archaea= Hydrocarbons joined to glycerol by ether linkage	Fatty acids joined to glycerol by ester linkage

Cell sizes

There is a great range in size between prokaryotic and eukaryotic cells.

• Mycoplasma are some of the smallest bacteria known to date and range between 0.1 to 1.0 micrometers or microns (1x 10^-6m)
• Most bacteria range between 1-10 um in length
• Eukaryotic cells typically range between 10-100 um in diameter

 Bacterial cell morphology

• Coccus (plural = cocci, meaning berries): spherical cells
• Bacillus (plural = bacilli, meaning small staffs); rod-shaped cells. Cells have greater surface area-to-volume and absorption is more effective. Cells are often motile.
• Coccobacillus: cells that are not perfectly round, as are true cocci, but appear to have blunted ends
• Spirillum (plural spirilla): cells with spiral or curved bodies with one or more twists. Cells tend to be rigid and fairly inflexible. Spirilla are often motile by means of flagella.
• Spirochetes are also spiral shaped but are more flexible than spirilla and move by an internal flagellum known as an axial filament.
• Vibrio: comma shaped cells, typically motile by flagella.

Arrangement of bacterial cells

A full description of a microbe should include the arrangement and grouping of the cells. As certain bacteria divide the cells can remain attached to each other. Cocci tend to display more variations in grouping than bacilli as cocci can divide along more than one axis. Bacilli only divide along their short axis.

Groupings of cocci include:

• Diplococci: pairs of cocci
• Streptococci: chains of cocci.
• Staphylococci: clusters of geometrically arranged cocci.
• Tetrads: packets of 4 cells
• Sarcinus: packet of 8 cocci

Groupings of bacilli include:

• Diplobacilli
• Streptobacilli

General structure of bacterial cells

Note that not all of the following structures will occur in every bacterium. Besides this outline you can find more information (with good images) on bacterial cells by clicking here

Structures external to the cell wall

Glycocalyx

A gelatinous polysaccharide and/or polypeptide outer covering. The glycocalyx can be identified by negative staining techniques. The glycocalyx is referred to as a capsule if it is firmly attached to the cell wall, or as a slime layer if loosely attached. Capsules can serve numerous functions including:

• Virulence factors, protecting bacteria from phagocytosis by immune cells. Pathogens such as Streptococcus pneumoniae can cause pneumonia if protected by a capsule.
• Permit bacteria to adhere to cell surfaces and structures such as medical implants, catheters and so on. This is an important first step in colonization and sometimes leads to disease.
• Capsules can be a source of nutrients and energy to microbes. Streptococcus mutans, which colonizes teeth, ferments the sugar in the capsule and acid byproducts contribute to tooth decay.
• Prevent cell from drying out (desiccation)
• Polysaccharides from certain capsules can be the targets of protective immune responses and have therefore been included in 'conjugated' (linked to another immune-stimulating molecule) vaccines. Such a vaccine is used against Hemophilus influenzae (a leading cause of ear infections and meningitis in young children). Purified polysaccharide vaccines also exist for streptococcal pneumonia and meningococcal meningitis.

  Flagella

Bacterial flagella consist of a filament and a hook which pierces the cell wall and attaches to the base of ring-like structures. The flagella rotate and move the bacterium in a fashion similar to a propellor. Gram positive bacteria have one set of rings which anchor the flagellum into the cell while gram negative cells have two sets of rings. Bacterial flagella consist of intertwining chains of a protein called flagellin and are about 1/10 diameter of a eukaryotic flagellum. Flagella may also be identified by special staining techniques.

Functions of flagella

Primarily function in motility. Motile bacteria show 'taxis.' Positive taxis is movement toward a favorable environment whereas negative taxis is movement away from a repellent.

Flagella can help in identifying certain types of bacteria. For example, Proteus species show a rapid 'swarming' type of growth on solid media.

Flagellar antigens are used to distinguish different species and strains of bacteria known as serovars. Variations in the flagellar H antigen are used in such typing.

Basic arrangements of flagella

• monotrichous (trich = hair): a single flagellum at one pole of the cell
• lophotrichous: a tuft of flagella at one pole of the cell
• amphitrichous: one or more flagella at both ends of the cell
• peritrichous = flagella around the entire cell

   

Axial filaments

An internal flagellum (endoflagellum) occurring within the periplasmic space of a unique group of gram negative bacteria known as spirochetes. The filament consists of bundles of small fibrils existing between the cell wall and a multi-layer, flexible outer membrane sheath. When the filaments rotate this causes the cell to turn in a corkscrew-like manner. This movement is thought to aid the penetration of tissues by pathogenic spirochetes. By internalizing a flagellum, which is normally antigenic, this adaptation may help the spirochete from being effectively targeted by immune responses.

Fimbriae and Pili

• Fimbriae are short, hair-like structures present in many gram negative bacteria, which act as adhesins (analagous to velcro) and allow bacteria to colonize cells and sometimes cause disease. For example, Neisseria gonorrhoea uses its fimbriae to attach to the lining of the genital tract and initiate an STD.
• Fimbriae can also detect chemical signals and are important in bacterial cell communication and biofilm formation.

E. coli attaching to gut epithelium by fimbriae.

• Sex pili act to join bacterial cells for transfer of DNA from one cell to another (bacterial conjugation)
• Fimbriae also act as receptors for viruses that infect bacteria (bacteriophages)
• Fimbriae and cell walls of Streptococcus pyogenes are coated with M protein. This acts as an important virulence factor by adhering to host cells and resisting phagocytosis.

Bacterial cell wall

Functions of the cell wall

• maintains cell shape
• acts as a barrier, protects cell contents from external environment
• maintains cell integrity/osmotic pressure in a hypotonic environment
• determines reactivity to Gram stain
• attachment site for flagella
• contributes to sensitivity to certain antimicrobial agents and the immune system (antibodies, phagocytes)

  Cell wall composition

• Most eubacteria have a complex cell wall consisting of peptidoglycan (part protein and part sugar).
• The peptidoglycan backbone consists of consisting of alternating units of 2 sugars called NAG (N-acetylglucosamine) and NAM (N-acetylmuramic acid)
• The cell wall has an unusually high nitrogen content
• The sugar backbone is crosslinked by short chains of amino acids. There are also side chains of tetrapeptides attached to NAM.

  Gram positive cell walls

• Consist of a relatively thick layer of exposed peptidoglycan (60-90% of the cell wall) are are placed into the Division Firmicutes ('thick-skinned')
• cells stain purple due to retention of the crystal violet dye during the gram stain procedure
• Antigens called teichoic acids project out of the cell wall and aid in typing different gram positive bacteria
• If peptidoglycan is digested away from the cell, gram positive cells lose their cell walls and become protoplasts. Protoplasts must be maintained in isotonic solutions in order to survive.
• Mycobacteria also contain peptidoglycan in their cell walls and tend to stain weakly gram positive. However, the cell walls can contain as much as 60% mycolic acid, a thick waxy material. These type of bacteria are usually stained using the acid-fast procedure. The microbes grow slowly as lipids present a barrier to nutrients into cells.

   

  Gram negative cell walls

• Gram negative cells decolorize during the gram stain and appear pink due to retention of the counterstain safranin
• Consist of a relatively thin layer of peptidoglycan these bacteria are placed into the Division Gracilicutes ('thin-skinned')
• Contain a periplasmic space which contains digestive enzymes and other transport proteins
• An external layer of lipopolysaccharide (LPS) consists of polysaccharide O antigens used in typing gram negatives and a component known as lipid A or endotoxin. Endotoxin can trigger fever and septic shock in gram negative infections
• LPS also protects the cell from phagocytosis, penicillin's and the antibacterial enzyme lysozyme
• LPS acts as a permeability barrier interspersed with narrow, restrictive protein channels called porins. In the gram negative Enterics, porins exlude all hydrophobic molecules and only permit passage of low molecular weight hydrophilic molecules. This protects the bacteria from the action of bile salts and toxins of the gut. 

Structures internal to the cell wall

Cell membrane

• phospholipid bilayer with polar heads on either side of the membrane. Hydrophobic tails are oriented to the interior of the membrane
• a selectively permeable barrier: substances that pass through the membrane are limited by pore sizes and the hydrophobic nature of the membrane
• integral (transmembrane) proteins form channels and act as carriers
• peripheral proteins can act as receptors and as enzymes for metabolic reactions such as ATP production and photosynthesis

  Cytoplasm

• In prokaryotes the cytoplasm contains DNA, which is typically a single, closed circle (no ends) concentrated in a nucleoid region and not membrane bound as in eukaryotes.
• There are no membrane-bound organelles but membrane stacks occur in some species of cyanobacteria and many prokaryotes possess various inclusion bodies within their cytoplasm. Most inclusion bodies store some form of nutrient and energy reserves, such as glycogen and polyphosphate. Other inclusions occur in aquatic cyanobacteria that are capable of photosynthesis. For example, gas vacuoles allow the bacteria to adjust their position in the water so photosynthesis can be maximized. Other cyanobacteria house chlorosomes, which contain pigments necessary for photosynthesis.
• The cytoskeleton of eukaryotes has long been noted and consists of a network of filaments for support and movement. Many text books still state that bacterial cells lack cytoskeletons but this is no longer true. A study by Oxford University researchers (Cell, 3/23/01) clearly demonstrated the existence of a cytoskeleton in the bacterium, Bacillus subtilis. The study used fluorescent antibodies to detect two proteins, termed MreB and Mbl, involved in making a helical internal scaffold responsible for maintaining cell shape.
• Bacterial cells can contain thousands of ribosomes, the sites of protein synthesis. Bacterial ribosomes are termed 70 S ( for Svedberg units) and eukaryotic ribosomes are termed 80S. The difference between bacterial and eukaryotic ribosomes is often exploited during antibiotic therapy. The structural similarity of bacterial ribosomes to that of those found in eukaryotic mitochondria and chloroplasts has been interpreted as evidence of endosymbiotic theory.
• Plasmids are small, extrachromosomal DNA circles. Plasmids replicate independently of the chromosome carry genes that are not essential for cell survival but may give some advantage to an organism. For example, they can carry genes for antibiotic resistance and toxin production. Plasmids are also very useful vehicles for genetic engineering.

  Bacterial endospores: "AN ESCAPE POD FOR DNA."

• Spores are a survival mechanism: they are triggered to form during adverse environmental conditions. The ability to produce spores is a type of cell differentiation involving precise genetic regulation of proteins. This process has now become a model system for studying how cells of higher organisms divide and differentiate.
• endospores are NOT reproductive structures as only one cell gives rise to one spore.
• 10 genera of endospore-forming gram positive bacilli and cocci are known; many of them are pathogens
• endospores can be identified with special stains and differentiated from the vegetative cell
  • Spores are resistant to:
• heat: withstand boiling for over one hour
• desiccation: some viable spores recovered from bees trapped amber may be as old as 30 million years!
• UV radiation
• chemical disinfectants
• the resistance of these spores has serious consequences and some very pathogenic bacteria have the ability to produce such spores.
  • Sporulation and germination
• During spore formation the cell membrane invaginates and a copy of the DNA is pinched off into a separate part of the cell called the forespore
• A cross wall called a septum separates the spore from the rest of the cell
• The spore eventually becomes protected with a heat-resistant calcified spore coat which is surrounded by inner and outer spore membranes
• The spore contains DNA, a small amount of RNA and little free water. Most metabolic activity shuts down
• When conditions are favorable there is a rapid shift in metabolic activity and a return to the vegetative form of the cell

Classically, bacteria have been studied and considered as unicellular organisms, with few social and communication skills. Microbiologists are just beginning to realize that they have seriously underestimated the organizational capacity of prokaryotes and a big push is on to research the phenomenon known as a biofilm. Biofilms literally occur right under our noses and with great frequency in many environments. They also play a big role in disease causation and drug resistance. It is surprising then, that researchers are just waking up to what comes naturally to bugs! Microbes in biofilms find strength in numbers and can actually cooperate more like a multicellular organism. In this way, microbes are more likely to survive outside assaults, find food, and diversify when necessary.

Where do biofilms occur?

Literally on you and all around you. Some well known examples include:

• Sticky bacterial scum on teeth that promotes plaque formation
• Contact lenses (if not properly sterilized)
• Indwelling catheters contribute to large numbers of hospital-borne infections
• Your shower curtain!
• The lungs of people with Cystic Fibrosis disease
• Bacterial infection of heart valves
• Corrosion on metal pipes
• Thick, often colorful, biofilms called microbial mats live about the geysers of Yellowstone National Park and can be hundreds of years old.

How do biofilms form?

The major steps involved in biofilm formation have been summarized as follows:

• Free-swimming or floating bacteria land on and bind to a surface.
• The aggregated cells start synthesizing a sugary, slime layer.
• Cells communicate by signaling molecules, begin to multiply and form a microcolony.
• Within the slimy matrix cells can exist in complex communities, penetrated by water columns. The slime prevents bacteria from drying out, and may also improve nutrient access and waste elimination. The biofilm also protects bacteria from antibiotics and toxins. The center of the microcolony becomes anaerobic and cells may exist in a dormant state.
• Some cells break free from the biofilm and return to a free-swimming state. These free-swimming bacteria may then establish a biofilm at another site.