The following points highlight the six main components of cytoplasm of bacteria. The components are: 1. Ribosomes 2. Molecular Chaperones 3. Nucleoids 4. Plasmids 5. Cytoplasmic Inclusions 6. Spore and Cysts.
Contents
Component # 1. Ribosomes:
All living cells contain ribosomes which act as a site of protein synthesis. High number of ribosomes represents high rate of protein synthesis and vice versa. Cytoplasm of a prokaryotic cell contains about 10,000 ribosomes which account upto 30% of total dry weight of the cell. Presence of ribosomes in high number gives the cytoplasm a granular appearance.
The eukaryotic ribosomes are found attached to cell membrane, whereas the prokaryotic mesosomes are free in cytoplasm. Prokaryotic ribosomes are smaller and less dense than eukaryotic ribosomes.
Ribosomes of prokaryotes are often called 70S ribosomes and that of eukaryotes as SOS ribosomes. The letter ‘S’ refers to Svedberg unit which indicates the relative rate of sedimentation during ultracentrifugation. Sedimentation rate depends on size, shape and weight of particles.
(a) Subunits:
In general the ultrastructure of ribosomes reveals that these are composed of two subunits, a larger 508 subunit and a smaller 30S subunit. Each subunit is composed of protein and ribosomal RNA (rRNA).
Their association and dissociation depend on the concentration of Mg++ ions. The structure of a ribosome is very complex. The proteins and RNA are inter-wined. James A. Lake (1981) presented the structure and function of ribosome.
According to him the smaller subunit of ribosome consists of a head, a base and a platform. With the help of a cleft the platform and head are separated from the base. The larger subunit comprises of a ridge, a central protuberance and a stalk; the former two are separated by a valley (Fig. 4.16).
Fig 4.16 : Three dimensional model of E.coli ribosome shown in two different orientation (A and B)
(b) Chemical Composition:
The ribosomes of E. coli consist of three types of RNAs, 5S, 16S and 23S, and 53 proteins. The SOS subunit consists of 5S and 23S RNA, and 34 proteins; 30S subunit consists of 16S RNA and 21 proteins. The 5S RNA is 120 nucleotides long, 16S RNA is about 1,600 nucleotides long and 23S RNA is about 3,200 nucleotides long.
Base sequence of 5S RNA has been strongly conserved throughout the evolution and that of 16S form double stranded hairpin loops. Only 30-35% bases of 16S form single stranded loops. Interactions between rRNA and cellular RNA (mRNA and tRNA) occur (Fig.4.17).
From 30S RNA of E. coli, 21 proteins have been isolated that are designated as SI to S21. Similarly from larger subunit (50S), 31 proteins (LI to L34) have been isolated. Protein map of ribosome showing their sites on two subunits is presented in Fig.4.18.
The function of ribosome in protein synthesis is a well established fact. However, they are not specific in nature. Ribosome of one species can be used for protein synthesis in other species.
There are several antibiotics such as streptomycin, neomycin and tetracycline that inhibit protein synthesis on the ribosomes. Even antibodies can kill the prokaryotic microorganisms but not the eukaryotic microorganisms. This is due to differences in prokaryotic and eukaryotic ribosomes.
Component # 2. Molecular Chaperones:
It was thought for many years that polypeptides after synthesis fold into native stage and this folding is not determined by its amino acids. It is now clear that there are certain helper proteins called molecular chaperones or chaperones which recognise the newly formed polypeptides and fold to its proper shape.
Proteins fold rapidly into the secondary structure. This unusually open and flexible conformation is called as molten globule. It is the starting points for slow process which results in correct tertiary structure.
There are several chaperones involved in proper protein folding in bacteria. Chaperones were first identified in E. coli mutant that did not allow to replicate, phage lambda. In E. coli at least four chaperones viz., DnaK, DnaJ, GroEL and GroES, and stress protein GrpE are involved in folding process.
They play an important role because after protein synthesis the cytoplasmic matrix is filled with nascent polypeptides and proteins. It is possible that these polypeptides become folded and form a nonfunctional complex. The chaperones check wrong in-folding and promote correct folding. The chaperones are found both in the cells of prokaryotes and eukaryotes.
Fig 4.19 : Mechanism of polypeptide folding by chaperones.
After synthesis of a sufficient length of polypeptide from the ribosome, DnaJ binds to unfolded chain (Fig.4.19). DnaK complexed with ATP attaches to polypeptide. These two chaperones check the polypeptide from folding. After binding of DnaK to polypeptide ATP is hydrolysed to ADP which increases the ability of DnaK to bind with unfolded peptide.
When polypeptide synthesis is completed, GrpE protein binds to DnaK-polypeptide complex and causes the DnaK to release ADP. Thereafter, ATP binds to DnaK, and DnaK and DnaJ are released from the polypeptide. During these events, polypeptide is folded and reaches to its final native conformation. At this stage if polypeptide is partially folded it binds to DnaJ and DnaK, and repeats the same process again.
Mostly DnaK and DnaJ transfer the polypeptide to GroEL and GroES where final foldings occurs. GroEL is a long hollow barrel shaped complex of 14 subunits which are stacked in two rings, whereas GroES contains four subunits arranged in one ring and can combine to both ends of GroEL. ATP binds to GroEL and changes the ability of the later for polypeptide binding. GroES binds to GroEL and helps in binding and release of refolding peptide.
Heat Shock Proteins:
When E. coli cells are exposed to high temperature, metabolic poisons and other stressful conditions, the concentrations of chaperones increase. In E. coli cultures, at temperature between 30 and 40°C, 20 different chaperones often called heat shock proteins are produced within 5 minutes.
These protect the cell from thermal damage and stress, and promote the proper folding of polypeptides. In hypo-thermophiles (e.g. Pyrodictum occultum) that grow at about 110°C, a large amount of chaperones are present.
Mutant E. coli resistant to phage X produces slightly two changed chaperones like heat shock proteins 60 and 70 (hsp60 and hsp70). The eukaryotic cells have families of hsp60 and hsp70 proteins, and different family members function in different organelles.
The mitochondria contain their own hsp60 and hsp70 molecules which are different from those functioning in the cytosol. A special hsp70 helps to fold proteins in the endoplasmic reticulum. The other function of chaperones is the transport of proteins across the membrane.
Component # 3. Nucleoids (Bacterial Chromosome):
As in eukaryotes, in prokaryotes too the basic dye stains the nuclear material and reveals as dense and centrally located bodies of irregular outline. Upon observation with electron microscope it was found that this central region is not separated from the cytoplasm by a membrane and consists of nuclear structure besides the DNA fibrils. The eukaryotes contain a well organised nucleus in which the genetic material is enclosed by a nuclear membrane.
Therefore, the DNA material is not enclosed by any covering. Hence the bacterial chromosome is known as chromatin bodies or nucleoids (Fig. 4.20). The nucleoid is a single long circular double stranded DNA molecule devoid of highly conserved histone protein. The histone is present in eukaryotes, therefore, results the eukaryotic DNA into the beaded structures i.e. nucleosomes.
By the turn of 1960s, the nuclear material was studied in detail. In 1963, J. Cairns succeeded in extracting E. coli DNA under conditions that minimize its shearing. Auto-radiographic studies showed that the DNA was extremely long threads measuring about 1mm in length.
A few threads were circular. Hobot (1985) found that the fully extended E. coli DNA was 1 mm long (4 × 106 × 3.4 Å) having molecular weight 3 × 109 Dalton. Through electron microscope they also observed the compaction of chromosome into irregular shaped nucleoids.
The nucleoid is observed as a coralline (coral like) shaped structure the branches of which spread far into the cytoplasm and over the entire area of the cell. By using serial sections it has been possible to reconstruct the ribosome free area of the nucleoid.
Two types of nuclear bodies can be observed, an envelope associated nucleoid and an envelope free nucleoid. Associated with the first type a large amount of RNA, proteins, lipids and peptidoglycan are found, whereas the second type contains less amount of it.
Generally, the number of nucleoid per bacterial cell is one, but in some bacteria the number may go even to four or more. The DNA molecule appears to be present in 10 to 80 super coils. Worcel and Burg (1972) proposed the structure of the folded chromosome of E. coli and showed as seven loops, each twisting into a super helix. These loops are held together by a core of DNA (Fig.4.21).
Fig. 4.21 : A model representing the process of folding and super coiling of bacterial chromosome.
Super coiling may be induced enzymatically. Possibly it may be a factor for the formation of nucleoids. The folded structure was found to be attached to a fragment of cell membrane. This shows that the bacterial chromosome remains associated to a point of cell membrane. This helps in separation of newly replicated DNA molecules.
Component # 4. Plasmids:
During 1950s, working on conjugation process it was found that maleness in bacteria is determined by a transmissible genetic element. When male and female bacteria conjugate, every female is converted into a male. This inherited property of male is called the F (fertility) factor which is transmitted by cell to cell contact. Therefore, F is a separate genetic element.
In 1952, J. Lederberg coined the term plasmid as a genetic name for this element. Hence the plasmids may be defined as a small circular, self replicating and double stranded DNA molecule present in bacterial cell, in addition to its chromosome. It replicates independently during cell division and inherited by both of daughter cells. Therefore, its function is not governed by the bacterial chromosome.
In 1960, Jacob Schaeffer and Wollman for the first time used the term episome to denote the extra-chromosomal genetic element that integrated the bacterial chromosome during replication. The number of plasmids ranges from one to hundreds or more per bacterial cell.
A plasmid contains 5-100 genes that determine several biological functions. Under certain circumstances they provide special characteristics to the bacterial cell and help them in survivability. They may even lose without harming the bacterial cell.
Plasmids are the circular DNA molecule but in resting stage helix twists in right hand direction at every 400-600 base pairs and forms supercoils. The twisted form is called covalently closed circular- DNA. After cleaving the twists this form is converted into an open circular form of double stranded DNA molecule (Fig.4.22).
Component # 5. Cytoplasmic Inclusions:
The cytoplasm of prokaryotic and eukaryotic cells contains several reserve deposits which are called inclusions. Some inclusions are common to most of bacteria and some are restricted to certain species only. These inclusions serve as the basis for identification of bacteria. Shiveley (1974) has given an excellent account of inclusion bodies of prokaryotes.
The inclusion bodies are of two types (a) free inclusion bodies (e.g. polyphosphate granules and cyanophycean granules), and (b) single-layered non-unit membrane enclosed inclusion bodies (such as poly P-hydroxybutyrate granules, glycogen granules, sulphur granules, carboxysomes and gas vaculoes). The membrane of inclusion bodies is made up of proteins or lipids. Allen (1984) has reviewed the cyanobacterial cell inclusions.
Some of the inclusions are discussed here:
(a) Volutin Granules:
The volutin granules are also known as polyphosphate granules or metachromatic granules because after staining the bacteria with blue dye (e.g. methylene blue) these granules take stain and appear reddish purple in colour. Polyphosphate is a linear polymer of orthophosphates joined by ester bonds.
These granules are found in algae, fungi, protozoa and bacteria. These are present in high amount in Corynebacterium diphtheriae, hence it can easily be diagnosed. The volutin granules are composed of polyphosphates i.e. inorganic phosphates which are used in synthesis of ATP.
The phosphate is incorporated into nucleic acid. These are generally formed when the cells grow in phosphate rich environment. Growing cells of Aerobacter aero-genes contain traces of inorganic phosphate when there is nutrient deficiency; a large amount of phosphate is accumulated when synthesis of nucleic acid gets ceased.
(b) Polysaccharide Granules:
The polysaccharide granules are found in protozoa, yeasts, fungi and algae. These can be identified by using iodine solution. After reacting with iodine, glycogen turns into reddish brown and starch into blue colour.
(c) Lipid Inclusions:
Lipids are found in high amount in several species of Bacillus, Azotobacter, Mycobacterium, Spirillum, etc. These are present as storage material and are polymer of poly β-hydroxybutyric acid. It is formed by the condensation of acetyl CoA.
In Bacillus megaterium poly β-hydroxybutyrate (PHB) accounts for 60% of total dry weight when the bacterium has grown on acetate or butyrate (PHB) The monomers of poly β-hydroxybutyrate are linked by ester linkage forming the long poly β-hydroxybutyrate polymer.
The chemical structure of poly β-hydroxybutyrate is given in Fig. 4.25. The poly p- hydroxybutyrate polymer accumulates as granules of 0.2-0.7 Hm diameter.
The collective term poly β-hydroxybutyrate represents the all classes of carbon storage reservoir polymer acting as a source of energy and biosynthesis. These are found naturally both in Archaea but not in Eukarya. The presence of lipid inclusions can be demonstrated by using fat soluble dyes, for example sudan dye.
(d) Glycogen:
Glycogen is another storage product of prokaryotes. It is polymer of glucose sub-units looking like starch. The glucose units are linked by α(1→4) glycosidic bonds. The branching chains are connected by α(1→6) glycosidic bonds. Glycogen is also a storage reservoir for carbon and energy.
(e) Carboxysomes (Polyhedral Bodies):
Carboxysomes are found in photosynthetic bacteria, nitrifying bacteria, cyanobacteria and thermo bacilli. These are polyhedral or hexagonal inclusions containing ribulose-1, 5-bi-phosphate carboxylase. This enzyme is required in carbon dioxide fixation during photosynthesis.
(f) Sulphur Granules:
Sulphur granules are also temporarily stored by some bacteria which are also called a second type of inorganic inclusion body. It is exemplified by photosynthetic purple sulphur bacteria (e.g. Thiobacillus, Thiospirillum, Thiocapsa, and Chromatium) which are found in anaerobic, sulphide-rich zones of lake.
They oxidize H2O to S and internally deposit as sulphur granules within invaginated structures of plasma membrane. Sulphur is oxidised into sulphate as the reduced sulphur source becomes limiting. Fig. 4.26 shows the intracellular sulphur granules found in Chromatium vinosum.
(g) Maghetosomes:
Magnetosomes are the intracellular chains of 40-50 magnetite (Fe3O4) particles of about 40-100 nm diameter found in magneto tactic bacteria (Fig. 4.27). These bacteria are motile, highly microaerophilic spirilla isolated from fresh water habitats such as Aquaspirillum magnetotacticum. In addition, some bacteria isolated from sulphide habitats have also been found to possess magnetosomes containing greigite (Fc3S4) and pyrite (FeS2).
Magnetosomes are surrounded by membrane containing phospholipids, proteins and glycoproteins. The membrane proteins of magneto-some precipitate Fe3+ as Fe3O43 in the magneto-some. The shape of magnetosomes varies from square to rectangular or spike-shaped.
Each iron particle is a tiny magnet. Hence, the bacteria employ their magnetosomes to determine northward and downward directions. With the help of magneto-some they swim to nutrient- rich sediments or locate the optimum depth in fresh water and marine habitats. Besides, magnetosomes are also found in some eukaryotic algae, heads of birds, dolphins, green turtles and other animals to aid navigation.
(h) Gas Vesicles:
There are many prokaryotic microorganisms found in floating forms in lakes and sea that possess gas vesicles. Gas vesicles are gas-filled structures which contain the same gas in which the organisms are suspended. Gas vesicles provide buoyancy and keep the cells in floating form.
The most interesting ‘floating and sinking’ phenomenon is found in those cyanobacteria which cause water blooms in lakes such as Microcystis aquaticus and Anabaena flosaquae. Besides, gas vesicles are also found in certain purple and green phototrophic bacteria and some archaebacteria such as halo bacteria e.g. Thiopedia and Amoebobacter.
Gas vesicles look like spindles of varying dimension (300-700×60-110 nm) and numbers (a few to hundreds per cell). They contain a rigid membrane and internally a hollow structure (Fig. 4.28). The membrane consists of only proteins called GvpA and GvpC. They are 2 nm thick and impermeable to water and solutes; but they are permeable to most of gases.
Gas vesicles cannot resists high hydrostatic pressure and can be collapsed resulting in loss of buoyancy. The collapsed vesicles cannot resume the normal shape. Gas vesicles attain a density of about 5-20% of the cell. Presence of gas vesicles in photoautotrophic microorganisms provides a mechanism to adjust in a water column to move in a region to rescue from high or low light intensity.
(i) Chlorobium Vesicles (Chlorosomes):
In Heliobacteria (strict anaerobic, green coloured phototrophs) bacteriochlorophylls are associated with the cytoplasmic membrane. But in green bacteria photosynthetic apparatus is different. These bacteria consist of a series of cylindrical, flat sheets (lamellae) or ellipsoidal vesicles called chlorosomes or chlorobium vesicles (Fig. 4.29). Structure of chlorosomes varies in different bacteria.
Chlorosomes are attached to cytoplasmic membrane. They lack bilayered thin membrane called non-unit membrane. Depending upon bacterial species bacteriochlorophylls a, c, d or e is present in chlorosomes. But most of the bacteriochlorophyll a (and component of electron transport chain) are found in the cytoplasmic membrane. Chlorosomes are the site of photosynthesis.
Component # 6. Spores and Cysts:
Certain species of bacteria produce metabolically dormant structures known as spores. The spores may be endospores (i.e. produced inside the cell) or exospores (i.e. produced external to the cell). After return of suitable conditions spores undergo germination and produce vegetative cells.
In addition, some bacteria such as Azotobacter produce thick-walled dormant, and resistant spores which are known as cysts. Cysts develop after differentiation of a vegetative cell. To some extent cysts resemble endospores that differ in structure and chemical composition.
(a) Endospores:
The endospore formation is restricted to six bacterial genera including two Gram-positive bacteria such as Bacillus (e.g. B. megaterium, B. spharericus) and Clostridium (C. tetani). They form endospores when there is lack of water or depletion of essential nutrients in the environment.
Endospores, therefore, are highly durable and dehydrated resting bodies produced inside plasma membrane of the cells. These have additional layers of thick walls which are made up of peptidoglycan (Fig. 4.30).
Frankein and Bradlay (1957) reported the spores in a majority of species of Bacillus and Clostridium which differed by surface layer. The surface may be smooth or ribbed. Van den Hooff and Aninga (1956) found that the spore coat consists of an outer and an inner layer separated by a space. The outer layer is called exine and the inner layer intine. The central core is separated from the intine by a regular non- osmophilic space or cortex.
Some of the endospores can remain viable over 100 years. Several species of endospore forming bacteria are the dangerous pathogens. The endospores are of much practical importance in food, industrial and medical microbiology. Several physicochemical processes are adopted to kill the vegetative cells.
If the endospores are produced the process of killing by chemicals is useless. In the processed food the endospores are adapted and produce toxins when suitable growth conditions prevail. In such conditions special methods are adopted. Generally the endospores are difficult to stain for deletion. Therefore, specially prepared stain i.e. the Schaeffer-Fulton endospore stain is commonly used for detection purpose.
Sporulation:
The process of endospore formation is called sporulation or sporogenesis. It occurs normally when growth of bacterium ceases due to lack of nutrients. Sporulation is a complex process and occurs in several stages (Fig.4.31). Generally sporulation process requires 10 hours to get accomplished. Formation of endospores begins with the development of ingrowth of plasma membrane known as spore septum which becomes double layered.
The spore septum surrounds the cytoplasm and chromosome. A structure entirely enclosed within cell is formed which is known as fore-spore. Between the two membranes a thick layer of peptidoglycan is laid down.
A thick coat of protein is formed around this structure that provides resistance to endospores. All the endospores contain DNA, small amount of RNA and large amount of organic acid (dipicolinic acid). These cellular components are important for resuming metabolism in later stage.
Spore Germination:
The endospores may be produced terminally, sub-terminally or centrally depending upon species. After maturity wall layer dissolved and endospores are set free (Fig. 4.31). This process is called spore germination. Germination occurs only in favourable environmental conditions which are accomplished in three stages: activation, germination and outgrowth.
(b) Exospores:
Cells of Methylosinus (methane oxidizing bacterium) produce exospores through budding at one end of the cell. The exospores do not contain dipicolinic acid. They can resist desiccation and heat.
Conditions promoting spore germination:
Following are some of the conditions that stimulate spore germination:
(a) Heat shock at sub-lethal temperature for varying period of time from 1 minute to 1 hour or more (time of heating varies with species, age of spores and conditions of storage).
(b) Incubation of spores at 30°C for 5 minutes in water-ethanol mixtures.
(c) Addition of yeast extract in active components viz., glucose, a few amino acids with or without nucleotides.
(d) Presence of various metal ions such as Ca++, Mg++, etc.
(e) Presence of dipicolinic acid.
(f) Addition of L-alanine.