Bacterial Endospore

Endospores

Some bacteria produce endospores within their cell by a process called sporulation. Endospore-producing bacteria occur most commonly in the soil and the genera Bacillus and Clostridium are the best studied of endospore-producing bacteria. These spores are extraordinarily resistant to environmental stresses such as heat, ultraviolet radiation, gamma radiation, chemical disinfectants, and desiccation and can remain dormant for extremely long periods of time. Endospores are of great practical significance in food, industrial and medical microbiology due to their resistance and dangerous pathogenic nature of several species of endospore producing bacteria. This is because it is essential to develop adequate methods to sterilize solutions and solid objects.

(i) Structure

The endospore (so named because of its formation within the cell), which is readily seen under the light microscope as strongly refractile bodiesdue to being very impermeable to usual dyes (e.g., methylene blue), is structurally much more complex in that it possesses many layers that are absent in vegetative cells The outermost layer is exosporium, a thin delicate covering made of protein. Beneath the exosporium, there is a thick spore-coat consisting of several protein layers which are spore-specific. The spore-coat is impermeable and responsible for the spores resistance to chemicals.

Bacterial Endospores

A. Intracellular locations of endospores (a = termianl, b = subterminal, c = Central).

B. Diagrammatic Sketch of the sectiones endospore of Bacillus anthracis showing different parts.

1.	Exosporium	5.	Core Wall
2.	Outer Coat Layer	6.	Core Membrane
3.	Inner Coat Layer	7.	Core
4.	Coretx	8.	Spore-Coat

Below the spore-coat is the cortex which may occupy as much as half the spore volume. Cortex consists of loosely cross-linked peptidoglycan. Inside the cortex, there is the core-wall which surrounds the core membrane and the core or spore protoplast. The latter possesses cytoplasm, nucleoid, ribosomes etc. but is metabolically inactive.
The core or spore protoplast of a mature endospore contains abundant dipicolinic acid and calcium ions normally existing in the form of calcium-dipicolinate complex and is in a partially dehydrated state as it contains only 10-30% of the water content of the vegetative cell. Because of it, the consistency of the core cytoplasm is that of a thick gel.

In addition to low water content, the pH of the core cytoplasm is about one unit lower than that of the vegetative cell and contains high levels of core-specific proteins, namely, small acid-soluble spore proteins (SASPs). SASPs are considered to perform at least two important functions: (i) they bind tightly to DNA in the core and protect it from ultraviolet radiation, dessication and dry heat and (ii) they function as a carbon and energy source at the time of endospore-germination to give rise to new vegetative cell

A. Structure of sipicolinic acid (DPA)	B. Cross-linking of Ca++ to DPA form calcium-dipicolinate complex

(ii) Formation of Endospore

Endospore formation (called sporulation or sporogenesis) involves a very complex series of events in cellular differentiation. Endospore formation takes place only in such a vegetative cell which ceases growth due to lack of nutrients. The complex process of sporulation can be divided into seven stages (I to VII).

(iii) Endospore Resistance

Bacterial endospores can retain viability for many years. A few viable endospores of Bacillus subtilis and B. licheniformis were found in the soil attached to plants that had been stored under dry conditions at the Kew Gardens Herbarium for 200-300 years. Endospores can even retain viability for millennia, and viable endospores have been found in geological deposits where they must have been dormant for thousands of years. What factors are responsible for such prolonged viability of endospores? It has long been thought that dipicolinic acid was directly involved in heat-resistance of endospore, but heat-resistant mutants now have been isolated in which dipicolinic acid is absent.

(iv) The Endospore Core and SASPs

Although both contain a copy of the chromosome and other essential cellular components, the core of a mature endospore differs greatly from the vegetative cell from which it was formed. Besides the high levels of calcium dipicolinate, which help reduce the water content of the core, the core becomes greatly dehydrated during the sporulation process. The core of a mature endospore has only 10–25% of the water content of the vegetative cell, and thus the consistency of the core cytoplasm is that of a gel. Dehydration of the core greatly increases the heat resistance of macromolecules within the spore. Some bacterial endospores survive heating to temperatures as high as 1508C, although 1218C, the standard for microbiological sterilization (121°C is autoclave temperature), kills the endospores of most species. Boiling has essentially no effect on endospore viability. Dehydration has also been shown to confer resistance in the endospore to chemicals, such as hydrogen peroxide (H₂O₂), and causes enzymes remaining in the core to become inactive. In addition to the low water content of the endospore, the pH of the core is about one unit lower than that of the vegetative cell cytoplasm. The endospore core contains high levels of small acid-soluble proteins (SASPs). These proteins are made during the sporulation process and have at least two functions. SASPs bind tightly to DNA in the core and protect it from potential damage from ultraviolet radiation, desiccation, and dry heat. Ultraviolet resistance is conferred when SASPs change the molecular structure of DNA from the normal “B” form to the more compact “A” form. A-form DNA better resists pyrimidine dimer formation by UV radiation, a means of mutation, and resists the denaturing effects of dry heat. In addition, SASPs function as a carbon and energy source for the outgrowth of a new vegetative cell from the endospore during germination.

(iv) Germination

The conversion of endospore into active vegetative cell appears a complex process and involves three steps: activation, germination and outgrowth. Activation is the process that prepares endospore for germination. It is most easily accomplished by heating at sublethal but elevated temperature. An activated endospore undergoes germination which is characterized by swelling and rupture or absorption of spore-coat, loss of dipicolinic acid, degradation of small acid-soluble spore proteins (SASPs), loss of resistance to heat and other stresses, loss of refractility, and enhancement in metabolic activity.The final stage is the outgrowth which involves visible swelling due to water uptake and synthesis of new RNA, DNA and proteins. The spore protoplast emerges from the broken spore-coat, develops into an active bacterial cell and begins to divide.

(v) Differences between Endospore and Vegetative Cell

A mature endospore differs greatly from the vegetative cell from which it was formed. These differences are given in.

Differences between Endospore and the Vegetative Cell

Characteristic	Endospore	Vegetative Cell
(i) Structure	Thick spore-cortex, spore-coat, exosporium	Typical gram (+) cell; a few gram (-) cells
(ii) Microscopic appearance	Refractile	Non-refractile
(iii) Dipicolinic acid	Present	Absent
(iv) Calcium content	High	Low
(v) Water content	Low (10-30% in core)	High (80-90%)
(vi) Heat resistance	High	Low
(vii) Radiation resistance	High	Low
(viii) Chemical resistance	High	Low
(ix) Enzymatic activity	Low	High
(x) Synthesis of macromolecules	Absent	Present
(xi) Messenger-RNA	Low or absent	Present
(xii) Oxygen-uptake	Low	High
(xiii) Lysozyme effect	Resistant	Sensitive
(xiv) Small acid-soluble proteins (SASPs)	Present	Absent
(xv) pH of cytoplasm	About 5.5-6.0	About 7

Then, if not DPA, what factors make endospore so resistant to heat and other lethal agents. It is now being believed, however, that there are several factors probably involved in endospore resistance. These are: calcium-dipicolinate and acid-soluble protein stabilization of DNA, protoplast dehydration, the spore-coat, DNA-repair, and the greater stability of cell proteins in bacteria adopted to growth at high temperatures.

Bacterial Nucleoid

The Nucleoid (Bacterial Chromosome)

Eukaryotic cells possess two or more chromosomes contained within a membrane-bound organelle called nucleus which lacks in prokaryotes. Exceptionally, membrane-bound DNA-containing regions have been found in two bacterial genera, namely, Pirellula and Gemmata. Pirellula has a single membrane that surrounds a region called pirellulosome which contains a fibrillar nucleoid and ribosome-like particles. The nuclear body of Gemmata obscuriglobus is delimited by two membranes.

However, the prokaryotes possess a single chromosome located in an irregular discrete region, namely, nucleoid (nuclear body, chromatin body, nuclear region are the other names used). Usually the bacteria (prokaryotes) contain single, circular, ds-DNA chromosome, but some have a liner DNA chromosome. Recently it has been discovered that some bacteria (e.g., Vibro cholerae possess more than one bacterial chromosome.

Nucleoids have been isolated intact and free from membranes. When chemically analysed, they are found composed of about 60% DNA, 30% RNA and 10% protein (mostly RNA polymerase) by weight. The DNA is looped and coiled extensively with the aid of RNA and nucleoid proteins which are different from the histone proteins occurring in eukaryotic nuclei and are recognized by different names such as HU, NS and DNA-binding protein-II. However, following two features of bacterial DNA are particularly distinctive:

(i) Lack of histone proteins. Contrary to the eukaryotic organisms in which the DNA is packaged by wrapping if around special beads of protein called histones to form structure called nucleosome, the monerans (prokaryotes) have no histone proteins and no nucleosome. (Ingle, 1986).

(ii) Lack of introns. Eukaryotic DNA, which codes for protein is interrupted by noncoding    sequences (introns). The latter are absent in bacteria (Ingle, 1986).

The closed circular DNA of E. coli (a rod-shaped cell about 2-6 μm long) measures approximately 1400μm having molecular weight 3 x 10⁹ daltons. Obviously, it is extremely efficiently packaged to fit within the nucleoid. The DNA is looped and coiled extensively with the aid of RNA and nucleoid proteins, as stated earlier. Worcel and Bury (1972) have proposed the structure of the coiled DNA of E. coli and showed as seven loops, each twisting into a superhelix

Diagrammatic representation of coiling and supercoiling of bacterial (E.coli) DNA (after Worcel and Burgi, 1972)

1.	Unfolded circular DNA	6.	Super-coiled
2.	Coiled	7.	DNA
3.	DNA	8.	Super Coiling
4.	Loop       5. RNA	9.	Superhelix

Electron microscopic studies have often shown that the bacterial chromosome is in contact with either the mesosome or plasmamembrane. This shows that the membranes may be involved in the separation of DNA into daughter cells during division. The nucleoid is observed as a coral-like (coralline) structure the branches of which spread and occupy most of the cytoplasmic part of the cell.

Plasmids

In addition to the nucleoid, bacterial cytoplasm normally contains one or more circular molecules of ds-DNA called plasmids (linear plasmids are also known). Naturally occurring plasmids vary in size from approximately 1 to more than 1000 kilobase pairs. The typical ds-DNA circular plasmid is less than 1/20 the size of the bacterial chromosome. Plasmids, the extra-chromosomal genetic materials, existing independently of the bacterial chromosome and are present in many bacteria (they are also present in some yeasts and other fungi). They replicate autonomously as they possess their own replication origins.

Because of the small size of plasmid DNA relative to the bacterial chromosome, the whole replication process takes place very quickly, perhaps in 1/10 or less of the total time of cell division cycle. Plasmids have relatively few genes, generally less than 30 and their genetic information is not essential for the bacteria because the latter lacking them function normally. Those plasmids that can reversibly integrate into the bacterial chromosome are called episomes. The plasmids share many characteristics with viruses.

Types of Plasmids.
(a) F-Plasmids tor F-factors)

These are the first described plasmids that play major role in conjugation in bacteria. It is a circular ds-DNA molecule of 99,159 base pairs. The genetic map of the F-plasmid is shown in One region of the plasmid contains genes involved in regulation of the DNA replication (rep genes), the other region contains transposable elements (IS3 Tn 1000, IS3 and IS2 gene involved in its ability to function as an episome, and the third large region, the tra region, consist of tra genes and possess ability to promote transfer of plasmids during conjugation. Example F-­plasmid of E. coli.

(b) R-Plasmids

These are the most widespread and well studied group of plasmids conferring resistance (hence called resistant plasmids) to antibiotics and various other growth inhibitors. R-plasmids typicallyhave genes that code for enzymes able to destroy and modify antibiotics.

Genetic Map of the F (Fertility Plasmid of Escherichia Coli. tea region contains tra genes involves in conjugative transfer; Ori T sequences is the origin of transfer during conjugation; transposable element region responsible for functioning as episome, and the rep genes regulate DNA replication.

Genetic map of the resistance plasmid R100. Cat = Chloramphenicol resistance gene; str = Streptomycin resistance gene; sul = sulfonamide resistance gene, mer = mercury ion resistance gene, IS = insertion sequences

They are not usually integrated into the host chromosome. Some R-plasmids possess only a single resistant gene whereas others can have as many as eight. Plasmid R 100, for example, is a 94.3 kilobase-pair plasmid that carries resistant genes for sulfonamides streptomycin and spectinomycin, chloramphenicol, tetracyclin etc. It also carries genes conferring resistance to murcury. Many R­-plasmids are conjugative and possess drug-resistant genes as transposable elements, they play an important role in medical microbiology as their spread through natural populations can have profound consequences in the treatment of bacterial infections.

(c) Virulence-Plasmids

These confer pathogenesity on the host bacterium. They make the bacterium more pathogenic as the bacterium is better able to resist host defence or to produce toxins. For example, Ti-plasmids of Agrobacterium tumefaciens induce crown gall disease of angiospermic plants: enterotoxilgenic strains of E. coli cause travelers diarrhea because of a plasmid those codes for an enterotoxin which induces extensive secretion of water and salts into the bowel.

(d) Col-Plasmids

These plasmids carry genes that confer ability to the host bacterium to kill other bacteria by secreting bacteriocins, a type of proteins. Bacteriocins often kill cells by creating channels in the plasmamembrane thus increasing its permeability. They also may degrade DNA or RNA or attack peptidoglycan and weaken the cell-wall. Bacteriocins act only against closely related strains. Col E1 plasmid of E. coli code for the synthesis of bacteriocin called colicins which kill other susceptible strains of E. coli.

Col plasmids of some E. coli code for the synthesis of bacteriocin, namely cloacins that kill Enterobacter species. Lactic acid bacteria produce bacteriocin NisinA which strongly inhibits the growth of a wide variety of gram (+) bacteria and is used as a preservative in the food industry.

(e) Metabolic Plasmids

Metabolic plasmids (also called degradative plasmids) possess genes to code enzymes that degrade unusual substances such as toluene (aromatic compounds), pesticides (2, 4-dichloro­phenoxy acetic acid) and sugars (lactose). TOL (= pWWO) plasmid of Pseudomonas putida is an example. However, some metabolic plasmids occurring in certain strains of Rhizobium induce nodule formation in legumes and carry out fixation bf atmospheric nitrogen

Cell membrane and cytoplasmic inclusions-Re-positing

The Plasma Membrane

Plasma membrane (cytoplasmic membrane) is an absolute requirement for all living beings as it is the chief point of contact with the cells environment and thus is responsible for much of its relationships with the outside world. If the membrane is broken, the integrity of the cell is destroyed and the internal contents leak into the environment resulting in the death of the cell.

(i) Structure

The plasma membrane is approximately 7.5 nm (0.0075 μm) thick, forms the limiting boundary of the cell and is made up of phospholipids (about 20-30%) and proteins (about 60-70%). Several models have been proposed to explain the ultrastructure of the plasma membrane, the most widely accepted one is Fluid Mosaic Model introduced by Singer and Nicolson (1974). According to this model the membrane is a bi-layer of phospholipids and the two opposing layers of phospholipids overlap slightly; each phospholipid molecule consisting of a phosphate group and a lipid.

Each phospholipid is structurally asymmetric with polar and nonpolar ends and is called amphipathic. The polar ends interact with water and are hydrophilic; the nonpolar ends do not interact with water (i.e. insoluble in water) and are hydrophobic. The hydrophilic ends occur towards the outer surface of the membrane whereas the hydrophobic ends are burried in the interior away from the surrounding water.

Digrammatic representation of the fluid mosaic model of plasma membrane

1.	Outside Cell	7.	Hydrophobic Region of Integral Protein
2.	Inside Cell	8.	Polar (Phosphate)
3.	Extrainsic or Peripheral Protein	9.	Non Polar (Lipid)
4.	Intrinsic or Integral Protein	10.	Non Polar (Lipid)
5.	Hopanoids	11.	Polar (Phosphate)
6.	Hydrophilic Region of Integral Protein	12.	Phospholipid Bilayer

The structure of Phosphatidylethanolamine, an amphipathic phospholipid usually occuring in bacterial plasma membrane. R = long, nonpolar fatty acid chains

The bi-layer phospholipid is interrupted by proteins which are distributed in a mosaic-like pattern. Some of the proteins are confined to the outer surface of bilipid layer (extrinsic or peripheral proteins) and others are partially or totally buried within it (intrinsic or integral proteins). The integral proteins, like membrane lipids, are amphipathic. Their hydrophobic regions are burried in the lipid while the hydrophilic regions project out from the plasma membrane surface.

A common hopanoid (bacteriohopanetetrol) occuring in bacterial plasma membrane

Often carbohydrates are attached to the outer surface of plasma membrane proteins and seem to perform important functions. Both proteins and lipids move within the phospholipid matrix of the membrane. However, many bacterial plasma membranes do contain pentacyclic sterol-like molecules called hopanoids which are synthesized from the same precursors as steroids. Like steroids in eukaryotic cells, hopanoids are thought to provide stability to bacterial plasma membrane.

(ii) Differences with Eukaryotic Plasma membrane

Although the bacterial plasma membrane resembles its counterpart of eukaryotic cells, it differs from the latter in two distinctive features:

(a) Sterols (such as cholesterols) that occur in eukaryotic cell membranes are absent in bacteria (except in the mycoplasmas that do not have cell wall). These substances help stabilize the phospholipids in eukaryotic membrane and make it more rigid.

(b) The proportion of protein to phospholipids is high (typically 2: 1 in prokaryotes, and 1: 1 or less in eukaryotes).

Functions
The plasma membrane is of extreme importance to the cell and performs following important functions:
1. Being differentially permeable barrier, the plasma membrane regulates the flow of materials, in and out of the cell i.e. it selectively restricts movement of molecules in and out. Thus, the plasma membrane prevents the loss of essential components through leakage while allowing the movement of other molecules.
2. It contains enzymes that mediate in the synthesis of membrane lipids and various other macromolecules that compose the bacterial cell wall.
3. It is the site of enzymes and carriers of electron transport system that generates ATP from ADP.
4. It contains specific attachment sites of the chromosome and for plasmids, and that it plays an active role in their replication at the time of cell division.

Mesosomes
In most of the bacteria cells (particularly Gram-positive ones) the plasma membrane shows characteristic infoldings either superficially or significantly deep, invading the cytoplasm. These infoldings are called Mesosomes, the term coined by Fitzjames. The bacterial DNA (chromosome) is always attached to or closely associated with mesosomes. Mesosomes are considered to play in important role in the intiation of replication of bacterial DNA and the septa formation at the time of cell division (Higgins and Shockmann, 1971). They act as sites of respiratory activity as well.

Mesosomes Real Structures

Although many functions have been proposed for Mesosomes, it has been found during the recent past that the bacterial cells with no apparent Mesosomes were not defective for such functions. The promoted to reevaluate the evidence for the existence of Mesosomes were always observed attached to or closely associates with bacterial DNA in electron microscopic observations were frozen in liquid nitrogen and then exposed to X-rays to break up the DNA before the cells were dehydrates for electron microscopy.

When this procedure was followed, no Mesosomes were observed in such cells. This suggests that the observed Mesosomes were artifacts of preparations for electron microscopic observation, formed by DNA pulling on the plasma membrane when the cell was dehydrated. The current view, therefore, is that Mesosomes are artifacts rather than real structures of the bacterial cell with definite functions.

The Cytoplasmic Inclusions

The cytoplasm of most prokaryotes lacks chloroplasts, mitochondria, and all other membrane-­bound organelles of cytoplasmic origin such as endoplasmic reticulum and Golgi bodies. Therefore, it is a homogenous aqueous solution of soluble proteins, cell solutes, metabolites of smaller molecular weights, and inorganic ions. It contains many enzymes, tRNAs, amino acids and a large amount of RNA collected into ribosomes. Granules and cell inclusions of various types, e.g. polyphosphates (volutin granules or metachromatin granules), poly-β-hydroxybutyrate (DHB), glycogen, gas vacuoles, magnetosomes, sulphur inclusions, carboxysomes, etc., are sometimes observed in the cytoplasm.

Ribosomes
Ribosomes are small granular bodies of 10-20 nm in diameter freely lying in the cytoplasm and composed of ribosomal ribonucleic acid (rRNA) and proteins. Bacterial ribosomes are thought to contain about 80-85% of the bacterial RNA. Sometimes, they are found in small groups called polyribosomes or polysomes, which are formed when several ribosomes begin to translate a single mRNA molecule. Each ribosome has sedimentation coefficient of 70 S and a mass of 2.8 x 10⁶ daltons, and is made up of two subunits of 50 S and 30 S, each subunit consisting of roughly equal amounts of rRNA and protein. Ribosomes are functional only when the two subunits are combined together.
The association and dissociation of two subunits of ribosomes depend on the concentration of Mg⁺⁺ ions. Each 50 S subunit (mass of 1.8 x 10⁶ daltons) contains one molecule of 23 S rRNA (having approximately 3200 nucleotides), one molecule of 5 S rRNA (having only about 120 nucleotides) and 34 different proteins designated as L1 to L34; while the 30 S subunit (mass of 0.9 x 10⁶ daltons) contains one molecule of 16 rRNA (having approximately 1540 nucleotides) and 21 different proteins designated as S 1 to S21.

The Structure of the prkaryotic ribosome

As in eukaryotes, ribosomes are the sites of protein synthesis and, therefore, antibiotics such as streptomycin and chloramphenicol specifically inhibit protein synthesis by attacking ribosomes. Generally, the ribosomes are a few hundred in. number in each bacterial cell, but when the cell undertakes active protein synthesis, they increase in number to as many as 15,000-20,000 per cell, about 15% of the cell mass.

Polyphosphates (Volutin Granules or Metachromatin Granules)

Many bacteria and microalgae accumulate phosphates in the form of polyphosphates (Fig. 3.21). Because they were first described in Spirillum volutans and because they bring about characteristic changes in the pigmentation of certain dyes, they have been given the name volutin granules and metachromatin granules respectively. These granules are composed of polymetaphosphate and are common in diphtheria bacillus and in certain lactic acid bacteria.
These granules stained reddish with blue dyes (e.g., methylene blue), are highly refractive and hence are easily observable under light microscope. The volutine granules represent intracellular phosphate reserve when nucleic acid synthesis does not occur, and when the latter starts, this phosphate is incorporated into the nucleic acids.

Polyphosphate Structure

Poly-β-hydroxybutyrate (PHB)

Poly-β-hydroxybutyrate (PHB) is a lipid-like compound. It is formed from β-hydroxybutyrate units joined by ester-linkages resulting in long PHB polymer which aggregate into granules of around 0.2-0.7 μm in diameter. PHB is accumulated by aerobic and facultative bacteria when the cells are deprived of oxygen and must carry out fermentative metabolism. On return of aerobic conditions, PHB is used as an energy and carbon source and incorporated into the oxidative metabolism.

Structure of polu-β-hydroxy butyrate

Some bacteria produce co-polymers of PHB often referred to as poly-β-hydroxy-alkanoate (PHA). The latter can be thermoplastically moulded and used as new plastics that shows advantage over conventional plastics (polypropylene or polyethylene) of being biodegradable.

Glycogen

Glycogen like PHB, is another storage product formed by prokaryotes. It is a polymer of glucose units composed of long chains formed by α(l → 4) glycosidic bonds and branching chains connected to them by α (1 → 6) glycosidic bonds. Glycogen is dispersed more evenly throughout the cytoplasmic matrix as small (about 20 - 100 nm in diameter) and is a storage reservoir for carbon and energy. Glycogen is also known as animal starch and, besides prokaryotes, is found in fungi.

Glycogen Structure

Gas Vacuoles

These are single membrane vacuoles formed as a result of the aggregation of enormous number of small, hollow, cylindrical structures called gas vesicles. Each gas vacuole appears about 75 nm in diameter with conical ends and about 200-1,000 nm in length. They characteristically occur in many aquatic bacteria, especially purple and green photosynthetic ones. These bacteria float at or near the surface of water because gas vacuoles give them buoyancy. Bacteria possessing gas vacuoles can regulate their buoyancy to float at the depth necessary for proper light intensity, oxygen concentration, and nutrient levels. They descend by simply collapsing vesicles and further float upward when new one are formed.