Fundamentals of typical bacteria

Shape, size and aggregation

Bacteria occur in a variety of shapes and sizes, determined not only by the nature of the organisms themselves but also by the way in which they are grown (Fig. 13.1).

In general, bacterial dimensions
lie in the range from 0.75 µm to 5 µm.

The most common shapes are the sphere (coccus) and the rod (bacillus).

Some bacteria grow in the form of rods with a distinct curvature, e.g. vibrios are rod-shaped cells with a single curve resembling a comma, whereas a spirillum possesses a partial rigid spiral; spirochaetes are longer and thinner, exhibit a number of turns and are also more flexible.

Rod-shaped cells occasionally grow in the form of chains but this is dependent
on growth conditions rather than being a characteristic of the species.

Cocci, however, show considerable variation in aggregation, which is characteristic of the species.

The plane of cell division and the strength of adhesion of the cells determine the extent to which they
aggregate after division.

Cocci growing in pairs are called diplococci, those growing in groups of four are called tetrads and those growing in groups of eightbare called sarcina.

If a chain of cells is produced resembling a string of beads this is termed a streptococcus and demonstrates division in one plane only and adhesion between cells after division.

An irregular cluster similar in appearance to a bunch of grapes is called a staphylococcus and shows division
in a number of different directions, as well as adhesion between cells after division.

In many cases the aggregation of cells is sufficiently characteristic to give rise to the name of the bacterial genus, e.g.
Staphylococcus aureus or Streptococcus pneumonia


Anatomy

The image above shows a diagrammatic representation of a typical bacterial cell.

The various components are described in the following section.

Capsule

Many bacteria produce extracellular polysaccharides, which may take the form of either a discrete capsule, firmly adhered to the cell, or a more diffuse layer of slime.

Not all bacteria produce a capsule, and even those that can will only do so under certain circumstances.

For instance, many encapsulated pathogens, when first isolated, give rise to colonies on agar which are smooth (S) but subculturing leads to the formation
of rough colonies (R).

This S to R transition is due to loss of capsule production.

Reinoculation of the R cells into an animal results in the resumption of
capsule formation, indicating that the capacity has not been lost and that the cell can determine when production is required.

The function of the capsule is generally regarded as protective, as encapsulated cells are more resistant to disinfectants, desiccation and phagocytic attack.

In some organisms, however, it serves as an adhesive mechanism; for example, Streptococcus mutans is an inhabitant of the mouth that metabolizes sucrose to
produce a polysaccharide capsule enabling the cell to adhere firmly to the teeth.

This is the initial step in the formation of dental plaque, which is a complex
array of microorganisms and organic matrix that adheres to the teeth and ultimately leads to decay.

The substitution of sucrose by glucose prevents capsule formation and hence eliminates plaque.

A similar picture emerges with Staphylococcus epidermidis. This bacterium forms part of the normal
microflora of the skin and was originally thought of as nonpathogenic.

With the increased use of indwelling
medical devices, coagulase-negative staphylococci, in particular S. epidermidis, have emerged as the major
cause of device-related infections.

The normal microbial flora has developed the ability to produce
extracellular polysaccharide, which enables the cells to form resistant biofilms attached to the devices.

These biofilms are very difficult to eradicate and have profound resistance to antibiotics and disinfectants.

It is now apparent that the dominant mode of growth for aquatic bacteria is not planktonic (free swimming) but sessile, i.e. attached to surfaces and
covered with protective extracellular polysaccharide or glycocalyx.

Cell wall

Bacteria can be divided into two broad groups by the use of the Gram-staining procedure which reflects differences
in cell wall structure.

The classification is based on
the ability of the cells to retain the dye methyl violet after they have been washed with a decolourizing agent such as absolute alcohol.

Gram-positive cells retain the stain, whereas Gram-negative cells do not.

As a very rough guide, the majority of small rodshaped cells are Gram negative.

Most large rods, such as the Bacillaceae, lactobacilli and actinomycetes, are Gram positive.

Similarly, most cocci are Gram
positive, although there are notable exceptions, such as the Neisseriaceae.
Bacteria are unique in that they possess peptidoglycan in their cell walls.

This is a complex molecule with repeating units of N-acetylmuramic acid and N-acetylglucosamine (Fig. 13.3).

This extremely long molecule is wound around the cell and cross-linked
by polypeptide bridges to form a structure of great rigidity.

The degree and nature of cross-linking vary between bacterial species.

Cross-linking imparts to the cell its characteristic shape and has principally a protective function. Peptidoglycan (also called murein or mucopeptide) is the site of action of a number of antibiotics, such as penicillin, bacitracin, vancomycin and cycloserine.

The enzyme lysozyme is also capable of hydrolysing the β-1–4 linkages between N-acetylmuramic acid and N acetylglucosamine.

Fig. 13.4 shows simplified diagrams of a Grampositive and a Gram-negative cell wall.

The Gram-positive cell wall is much simpler in layout, containing peptidoglycan interspersed with teichoic acid polymers.

These latter compounds are highly
antigenic but do not provide structural support.

Functions attributed to teichoic acids include the regulation of enzyme activity in cell wall synthesis, sequestration of essential cations, cellular adhesion
and mediation of the inflammatory response in disease.

In general, proteins are not found in Gram-positive cell walls. Gram-negative cell walls are more complex,
comprising a much thinner layer of peptidoglycan surrounded by an outer bilayered membrane.

This outer membrane acts as a diffusional barrier and is the main reason why many Gram-negative cells are much less susceptible to antimicrobial agents than are Gram positive cells.

The lipopolysaccharide component of the outer membrane can be shed from the wall on cell death.

 

 

It is a highly heat-resistant molecule known as endotoxin, which has a number of toxic effects on the human body, including fever, shock and even
death.

For this reason, it is important that solutions for injection or infusion are not just sterile but are also free from endotoxins.

Cytoplasmic membrane

The cytoplasmic membranes of most bacteria are very similar and are composed of protein, lipids, phospholipids and a small amount of carbohydrate.

The components are arranged in a bilayer structure with a hydrophobic interior and a hydrophilic
exterior.

The cytoplasmic membrane has a variety
of functions:

  • It serves as an osmotic barrier.
  • It is selectively permeable and is the site of carrier-mediated transport.
  • It is the site of ATP generation and cytochrome activity.
  • It is the site of cell wall synthesis.
  • It provides a site for chromosome attachment.

The cytoplasmic membrane has very little tensile strength, and the internal hydrostatic pressure of up to 20 bar forces it firmly against the inside of the
cell wall.

Treatment of bacterial cells with lysozyme may remove the cell wall and, as long as the conditions are isotonic, the resulting cell will survive.

These cells are called protoplasts and, as the cytoplasmic membrane is now the limiting structure, the cell assumes a spherical shape.

Protoplasts of Gramnegative bacteria are difficult to obtain because the layer of lipopolysaccharide protects the peptidoglycan from attack. In these cases, mixtures of EDTA and lysozyme are used, and the resulting cells, which still retain fragments of the cell envelope, are termed spheroplasts.

Nuclear material

The genetic information necessary for the functioning of the cell is contained within a single circular molecule of double-stranded DNA.

When unfolded, this would be approximately 1000 times as long as
the cell itself and so exists within the cytoplasm in a considerably compacted state.

It is condensed into discrete areas called chromatin bodies that are not
surrounded by a nuclear membrane.

Rapidly dividing cells may contain more than one area of nuclear material but these are copies of the same chromosome, not different chromosomes, and arise because DNA
replication proceeds ahead of cell division

In addition to the main chromosome, cells may contain extra pieces of circular double-stranded DNA which are called plasmids.

These can encode a variety
of products which are not necessary for the normal functioning of the cell but confer some sort of selective advantage.

For example, the plasmids may contain
genes conferring antibiotic resistance or the ability to synthesize toxins or virulence factors.

Plasmids replicate autonomously (i.e. independent of the main chromosome) and in some cases are able to be transferred from one cell to another (maybe of a different species).

Mesosomes

These are irregular invaginations, or infoldings, of the cytoplasmic membrane which are quite prominent in Gram-positive bacteria but less so in Gram-negative bacteria.

It has been proposed that they have a variety of functions, including cross-wall synthesis during cell division and furnishing an attachment site for
nuclear material, facilitating the separation of segregating chromosomes during cell division.

They have also been implicated in enzyme secretions and may act as a site for cell respiration.

However, it has also been suggested that they are simply artefacts which arise as a result of preparing samples for electron
microscopy.

Ribosomes

The cytoplasm of bacteria is densely populated with ribosomes, which are complexes of RNA and protein
in discrete particles 20 nm in diameter.

They are the sites of protein synthesis within the cell, and the numbers present reflect the degree of metabolic
activity of the cell.

They are frequently found organized in clusters called polyribosomes or polysomes.

Prokaryotic ribosomes have a sedimentation coefficient of 70 svedberg units (1 S = 1 × 10−13 s), compared
with 80 S for ribosomes of eukaryotic cells.

This distinction aids the selective toxicity of a number of antibiotics. The 70S ribosome is made up of RNA and protein, and can dissociate into one 30S subunit
and one 50S subunit.

Inclusion granules

Certain bacteria tend to accumulate reserves of materials after active growth has ceased, and these become incorporated within the cytoplasm in the
form of granules.

The most common are glycogen
granules, volutin granules (containing polymetaphosphate) and lipid granules (containing poly(β- hydroxybutyric acid)).

Other granules, such as sulphur and iron, may also be found in the more primitive
bacteria.

Flagella

A flagellum is made up of protein called flagellin and it operates by forming a rigid helix that turns rapidly like a propeller.

This can propel a motile cell a distance
up to 200 times its own length in 1 second.

Under the microscope, bacteria can be seen to exhibit two kinds of motion: swimming and tumbling.

When tumbling, the cell stays in one position and spins on its own axis, but when swimming, it moves in a straight
line.

Movement towards or away from a chemical stimulus is referred to as chemotaxis.

The flagellum arises from the cytoplasmic membrane and is composed
of a basal body, hook and filament.

The number and arrangement of flagella depend on the organism and vary from a single flagellum (monotrichous) to a
complete covering (peritrichous).

Pili and fimbriae

These terms are often used interchangeably but in reality these structures are functionally distinct from
each other.

Fimbriae are smaller than flagella and are not involved in motility. They are found all over the surface of certain bacteria (mainly Gram-negative cells) and are believed to be associated with adhesiveness and pathogenicity.

They are also antigenic. Pili (of which there are different types) are larger and
of a different structure to fimbriae and can be involved in the transfer of genetic information from one cell to another.

This is of major importance in the transfer of drug resistance between cell populations.

Other types of pili have been shown to be involved in a form of movement known as twitching.

Pseudomonas aeruginosa, for example, exhibits three types of motility; swimming, swarming and twitching.

Swimming and swarming are interlinked and are brought about by the use of flagella.

Swimming is a characteristic of individual cells, whereas swarming is ac oordinated migration of groups of cells.

Twitching occurs on solid substrates when the cells are attaching to a surface during biofilm formation.

It results from the repeated extension and retraction of type IV pili allowing the cells to translocate across the surface and thus form discrete microcolonies.

Endospores

Under conditions of specific nutrient deprivation, some genera of bacteria, in particular Bacillus and Clostridium, undergo a differentiation process at the
end of logarithmic growth and change from an actively metabolizing vegetative form to a resting spore form.

The process of sporulation is not a reproductive mechanism, as found in certain actinomycetes and filamentous fungi, but serves to enable the organism
to survive periods of hardship.

A single vegetative cell differentiates into a single spore.

Subsequent encounter with favourable conditions results in germination of the spore and the resumption of vegetative activities.

Endospores are very much more resistant to heat, disinfectants, desiccation and radiation than are
vegetative cells, making them difficult to eradicate from foods and pharmaceutical products.

Heating at 80°C for 10 minutes would kill most vegetative bacteria, whereas some spores will resist boiling for several hours.

The sterilization procedures now routinely used for pharmaceutical products are thus designed specifically with reference to the destruction of the bacterial spore.

The mechanism of this extreme heat resistance was a perplexing issue for many years.

At one time it was thought to be due to the presence of a unique spore component, dipicolinic acid (DPA).

This compound is found only in bacterial spores, where it is associated in a complex with calcium ions.

The isolation of heat-resistant DPA-less mutants, however, led to the demise of this theory.

Spores do not have a water content appreciably different from that of
vegetative cells, but the distribution within the different compartments is unequal, and this is thought to generate the heat resistance.

The central core of the spore houses the genetic information necessary
for growth after germination, and this becomes dehydrated by expansion of the cortex against the rigid outer protein coats.

Water is thus squeezed out of the central core. Osmotic pressure differences
also help to maintain this water imbalance.

Endospores are also highly unusual because of their ability to remain dormant and ametabolic for prolonged periods of time.

Bacterial spores have been isolated from
lake sediments where they were deposited 1000 years previously, and there have even been claims of spores
revived from geological specimens up to 40 million years old.

The sequence of events involved in sporulation is illustrated in Fig. 13.5. It is a continuous process, although for convenience it may be divided into six
stages.

The complete process takes approximately 8 hours, although this may vary depending on the species
and the conditions used.

Occurring simultaneously with the morphological changes are a number of
biochemical events that have been shown to be associated with specific stages and occur in an exact
sequence.

One important biochemical event is the
production of antibiotics. Peptides possessing antimicrobial activity have been isolated from the majority
of Bacillus species and many of these have found pharmaceutical applications.

Examples of antibiotics
include bacitracin, polymyxin and gramicidin.

Similarly, the proteases produced by Bacillus species during sporulation are used extensively in a wide variety of
industries.

 

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