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In lecture 5, we will briefly discuss one of the most primitive forms of bacteria reproduction which is known as binary fission or transverse fission. Because the method is so characteristic bacteria are referred to as fungi fission. The contents of the cell undergo reorganization to distribute the material for two cells resulting from the initial process. Special screening techniques and microscopic procedures, particularly electron-microscopic examinations have revealed some of the cytological changes that occur in this process. Bacteria of certain groups may reproduce by methods other than binary fission. This type of reproduction which is considered to be sexual and involves methods of fusion or conjugation, and other transformations will be discussed in a proceeding lecture. These genetic changes involve gene substance. Chemically speaking genes are nucleic acids, and more specifically deoxyribonucleic acid (DNA). Evidence presented from bacteria and viruses has identified DNA as the chemical basis of heredity, and it is assumed that this same substance is the carrier of genetic information in all forms of life. In the foregoing discussion in this chapter emphasis will be placed on the isolation of genetic material of the bacterial cells and on some hypothetical models of how DNA replicates. Methods of preservation of the bacterial cell by endospore formation as well as the biochemistry and biologic properties is discussed. Chemical composition of the cell wall and its biological function will also be discussed.
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The most important process of the usual growth cycle of bacterial is known as binary fission or transverse fission. The method of reproduction is so characteristic that bacteria are often referred to as fission fungi (Schizomycetes). The terminal events of this reproductive process find the single cell dividing into two, after the development of a transverse cell wall to separate the intracellular contents, Figure 9 of (Handout). Bacterial reproduction by binary fission is an asexual reproductive process. The exact morphological transition leading to binary fission is not clearly understood.
However, starting with a single viable bacterium we can postulate the following developments: Nutrients from the medium are taken into the cell by a selective process. The enzyme system of the bacterium then converts the chemical (nutrient) which has been assimilated into protoplasmic material characteristic of the particular organism. When increased amounts of nuclear materials are produced, cell elongation may follow (this is more evident in bacilli than cocci). The contents of the cell undergo reorganization to distribute the material for two cells, which are formed by a transverse wall or septum, that subsequently develops by an invagination of the cytoplasmic membrane.
With organisms as small as bacteria, there are numerous technical obstacles to observing and interpreting the detailed morphological changes that take place during the process of reproduction. However, special staining techniques and microscopic procedures, particularly electron microscope examination of ultra-thin sections, have revealed some of the cytological changes that occur in this process.
When bacteria are treated with a chemical fixative and stained with basic dye, the dye is taken up evenly throughout the cell. This behavior is due to the presence of ribosomes in the prokaryotic cell more so than in the eukaryote. The uniform staining of the cell by basic dyes was interpreted to mean that bacteria have dispersed genetic material rather than discrete nuclei before it was known that the cytoplasm of bacteria is rich in RNA.
If, however, fixed bacteria cells are first treated with an agent that destroys RNA (such as HCl or ribonuclease), staining with a basic dye reveals discrete basophilic bodies, or nucei. The presence of genetic material in these bodies is confirmed by their ability to stain with the Feulgen reagent, which is specific for DNA.
The bacterial nuclei may also be observed in living cells by phase contrast in a medium of appropriate contrast. The division of the nuclei can be followed in such preparations.
At the time that this cytological picture of the bacterial nucleus had emerged, genetic analysis had revealed that E. coli and related enteric bacteria contained only one genetic linkage. Thus, each nucleus should contain only one element of structure, or chromosome. In addition, the genetic data yielded a closed (circular) genetic map. Although a circular genetic map does not necessarily mean that the physical chromosome is a closed circular structure, it does seem possible that this is the case in bacteria.
In 1963, evidence was presented by J. Cairn that the bacterial nucleus might represent one continuous, circular structure, folded
and packed into a compact mass. Cairn succeeded in extracting bacterial DNA under conditions that minimized its shearing or degradation. Upon examination of the final preparation of the material, the DNA was found to be present as extremely long threads, the longest of which were slightly more than 1 mm in length. Further, a few of the threads were circular. These threads were contained within cells which had an average length of approximately 2 millimicrons.
The length of 1 mm for the DNA thread agreed well with the amount of DNA per bacterial nucleus as determined chemically, assuming that the radioactive DNA in Cairn's pictures is an extended double helix. This amount of DNA represented approximately 5 x 106 base pairs, with a molecular with of about 3 x 109. Since an intact structure was obtained after treatments that degrade lipid, proteins, and RNA, it was suggested that the bacterial chromosome consists of a single circular molecule of DNA, about 5 x 106 base pairs in length,(see figure 10 of handout).
The bacterial nucleus may be defined as the region of the bacterial cell into which is packed the single molecules of DNA representing the bacterial chromosome. The nucleus and chromosome can be considered to be equivalent since there is no nuclear membrane and no visible internal structure other than fibrillar DNA.
Replication of DNA may be described as occurring at a replication fork in the molecule. The replication fork represents a "growing point"; it moves along the growing point of the growing point of the molecule with the two branches of the fork acting as templates for the replication enzyme system, Figure 11 (Handout). For this to take place, the double helix must unwind. If the two branches of the fork are thought of as remaining stationary, unwinding requires that the original structure rotate along its axis, Figure 12 (Handout).
The Watson-Crick structure of DNA suggests a model for its reproduction called semiconservative, whereby each strand (i.e., half molecule) serves as a template for, and combines with, a new strand, Figure 13 (Handout), thereby being conserved in the new duplex. By taking advantage of the symmetry built into the DNA structure, this model accounts for the transmission of genetic information from parent to progeny.
The semiconservative model was verified by Meselson and Stahl in a classic experiment, presented in Figure 14 (Handout). The most notable feature is the total conversion of the DNA, after one generation, into a form with hybrid density, i.e., containing half old and half new DNA. Since the density remained unchanged after the DNA was fragmented into short pieces, the old and new strands were complementary rather than joined end to end.
Replication of a circular double helix may be considered to begin by the separation of the two strands at a fixed point on the circle; this point, the replicator, may be defined as the site of initiation of DNA synthesis. The replicating enzyme system attaches to the DNA at this site and begins to move along the circle as the double helix rotates and unwinds. Such unwinding would be impossible, however, if both strands of the duplex molecules formed intact, covalent circles. It must therefore be presumed that one strand of the double helix is broken at the replicator site, the unbroken phosphodiester bond of the other strand serving as a swivel to permit rotation and unwinding. Replication of the circular structure can then proceed as shown in Figure 15 (Handout).
Photomicrographs (phase contrast) of the dividing nuclei have shown that the newly replicated sister chromosomes gradually separate from each other, so that the nuclei become more or less equally spaced apart in the cell. It may be recalled that in a eukaryotic cell such movement is brought about by the formation and action of a mitotic spindle. In bacteria, however, no mitotic apparatus exists.
To what, then is the mechanism of chromosomal separation in bacteria attributed?
To answer this question, Jacob and Brenner proposed a model for chromosomal replication. This model, in somewhat modified form, can be summarized by presenting a sequential chain of hypothetical events, Figure 16 (Handout).
1. At the beginning of the replication cycle, the chromosomal replicator is attached to the cell membrane. At the site of chromosomal attachment, the replicating enzyme is localized in the membrane. One strand of DNA of the double helix, which has served as a template during the preceding cycle of replication, is a closed circle; the newly synthesized strand, is a broken circle.2. Immediately following completion of the previous replication cycle, a new attachment site forms in the cell membrane, adjacent to the old attachment site. One free end of the recently synthesized strand of DNA attaches to this new membrane site.
3. Replication of the chromosome begins. As it progresses, the replication enzyme system remains fixed in the membrane, and the chromosomes move past it. The replicating fork, hence, remains at the membrane attachment site at all times. Its movement along the chromosome results from the movement of the chromosome past the attachment site.
4. During replication, the synthesis of new cell membrane takes place in an annular region between the two membrane attachment sites. The two daughter chromosomes are thus separated from each other as the attachment sites are spread farther and farther apart. The cell wall also elongates during the process; the synthesis of new cell wall material may or may not take place adjacent to the region of membrane growth.
5. When replication is complete, one daughter chromosome again contains one circular and one broken strand. The other daughter chromosome, which is completely broken, must undergo ring closure of one strand to complete the cycle. The strand that becomes closed is the one which acted as a template during replication.
Some bacteria have the capacity to transform themselves into small spheres or ovals which are highly resistant cells known as spores or endospores, since they are produced intracellularly. All of the organisms in the genus Bacillus as well as Clostridium are characterized in part by their ability to produce spores. Some other genera of true bacteria produce spores but only in isolated cases. Endospores can be readily recognized microscopically by their characteristic intracellular location, by their extreme refractivity, and by their resistance to staining by basic aniline dye that readily stains the vegetative cells. They also possess remarkable physiological properties. Endospores have been shown to remain dormant for long periods (in some cases as much as half a century) and are highly resistant to heat, shortwave radiation and toxic chemicals.
The endospore-forming true bacteria share certain general properties in addition to spore formation, which suggests that they constitute a large natural group. With one exception, the vegetative cells are rod shaped. The Gram reaction is typically positive. However, it must be determined on exponentially growing cells since many sporeformers tend to become Gram negative very rapidly after populations enter the stationary phase of growth. Most sporeformers are motile, and the mode of attachment of the flagella is always peritrichous. All sporeformers (with the exception of one strict anaerobe) are chemoheterotrophs; the mode of energy-yielding metabolism found in the group include aerobic and anaerobic respiration and fermentation. The G + C content of the DNA ranges from 25 - 50 mole percent.
5.8.1 Formation of Endospores: Cytological Events
Development of an endospore involves the formation, within a vegetative cell, of a new kind of cell, with a completely different fine structure, enzyme constitution, and chemical composition.
Upon germination, an endospore gives rise once more to a typical vegetative cell. Sporulation normally begins some hours after a population of vegetative cells enters the stationary phase and is first manifested by the appearance in these cells of a relatively transparent forespore, which then gradually changes into the characteristically refractile, thickwalled mature spore. The development of the forespore is proceeded by unusual nuclear changes: the pair of nuclear bodies in the vegetative cell fuse into a rod shaped structure, which then divides. Part of the DNA enters the spore and part remains in the sporangium. Electron microscopy of thin sections of sporulating bacilli have added much further information. Division of the rod shaped nucleus is accompanied by unequal cell division: a new septum grows across the cell near one pole, segregating in the smaller daughter cell the DNA destined to enter the spore. The formation of the septum is not followed, as in normal cell division, by the development of a transverse cell wall which separates the daughter cells. In contrast, the newly formed septum grows back around the pole of the small cell, which thus becomes cut off from, and finally completely enclosed by, the large cell. The forespore is only visible with the light microscope at this stage. Once completely enclosed by the large cell, or sporangium, the forespore rapidly develops into a mature endospore. The maturation process involves the elaboration around the forespore of additional outer layers. These include a layer known as the cortex, which enclosed by a thick multilayer known as the spore coat. In certain bacilli, the spore coat is enclosed by a loose outer investment, the exosporium. The spore development process is shown in Figure 17 (Handout).
5.8.2Physiological and Biochemical Events in the Formation of the Endospore
The cytological transformation in endospore formation is accompanied by profound changes in both the chemical and enzymatic composition of the cell. Many enzyme systems characteristic of the vegetative cell are reduced to a very low level, or completely absent, in endospore. Some enzymes are synthesized preferentially during the maturation of spores; for example, alanine racemase is present at a far higher level in spores than in vegetative cells. The most remarkable chemical change associated with spore formation is the synthesis in large amounts of a substance of low molecular weight, dipicolinic acid. In endospores, dipicolinic acid is principally present as the calcium salt.
5.9.1 Resistance of the Endospore to Heat
Dipicolinic acid, present in the mature spore as the calcium salt, accounts for some 10-15% of its dry weight but is undetectable in vegetative cells. The calcium dipicolinate of the spore appears to be located in the cortex, between the spore cell and the outer spore coat. The calcium and dipicolinate in spores can be decreased by lowering the calcium content of the medium. Heat resistance is then seen to parallel calcium dipicolinate content. The mechanism is not clear. In contrast to the effect of calcium dipicolinate content on heat resistance, it does not influence resistance to killing by drying or membran-lysing agents (phenol, acetyl alcohol, or to staining). These properties evidently are dependent on the imperviousness of the spore integument and not on the state of the cell content.
5.9.2 Endospore Germination and Outgrowth
Endospore germination is manifested by simultaneous structural and physiological changes. The spore loses refractivity and becomes readily stainable; at the same time heat stability is lost. These changes occur rather rapidly. They are accompanied by the disappearance of the cortex and the liberation of soluble organic material. The materials released consist of calcium dipicolinate, protein, peptides and peptidoglycan material of low molecular weight. If the nutrients necessary for growth are present, germination is followed by the conversion of the spore cell into a vegetative cell: after 30-60 minutes, the outer spore coat ruptures, and the vegetative cell emerges.
The factors required to trigger germination vary from specie to species. In some spore formers, rapid and massive germination can be elicited by heat activation. In other species specific chemicals are required for germination. In the B. cereus group, adenosine triggers the process; other species require L-alanine. Spore germination can also be induced by mechanical treatment, such as rapidly shaking the spore suspension for a few minutes with fine glass beads.
5.9.3 Protoplasts, Spheroplasts and L forms
The normal bacterial cell has a very rigid cell wall. The structure is responsible for the characteristic shape of an organism, and it also protects the cell from osmotic damage. If the cell wall is removed from a bacterium, the remaining body enclosed by the fragile cytoplasmic membrane normally will burst because of "osmotic shock". Recently, experimental procedures have been developed whereby the bacterial cell wall can be removed without destroying the viability of the remaining portion of the cell. The viable structure derived from a vegetative cell after the removal of the entire cell wall is called a protoplast. Protoplasts may be prepared from many species of bacteria. In general this is accomplished in the following manner:
1. Removal of the cell wall by treating the cells with an enzyme (lysozyme) which selectively dissolves the cell wall material; or2. Growing the organisms in an environment (e.g., the presence of penicillin in the medium) which prevents the synthesis of cell wall substance but otherwise does not interfere with growth and reproduction.
In the second instance, a portion of the cell wall material is synthesized, and these cells are called sphereplasts. However, the osmotic pressure of the medium must be adjusted to maintain the structural integrity of the cell now devoid of its rigidity in either of the above cases.
L forms
are regarded as nonrigid bacterial forms, or "soft protoplasmic elements", without defined morphology, which can be propagated indefinitely on solid medium independent of the bacterial species from which the growth was derived. Hence, there is a relationship between the specimens designated protoplasts, speroplasts and L forms in that they are all bacterial forms devoid of the rigid cell wall.
Protoplasts
are round bodies; they assume a spherical shape since they posses no rigid outer wall. They have been shown to posses many of the physiological characteristics of their cell counterpart under experimental conditions provided to maintain their viability.
Another means of reproduction observed in some bacteria (e.g., Actinomycetales) is the formation of a filamentous growth followed by fragmentation into small units which can develop into cells of normal size. Some bacteria (e.g., Hyphomicrobiales) are capable of reproducing by budding. An outgrowth, or bud, develops from the parent as a new cell.
Some bacteria also have a sexual mode of reproduction. Two types of 'parent' cells of the same species, possessing stable but slightly different characteristics, are grown in the same tube of medium. After a suitable period of incubation the individual cells are isolated and examined for the identifying characteristic of each parent type. Most of the new cells resemble one or the other type, but few posses characteristics of both parents. The occurrence of new cells possessing some properties of both bacterial types is explained on the basis of fusion or conjugation of cells (sexual reproduction). Evidence for conjugation in bacteria is discussed in a section which follows.
5.10.1 Chemical Composition of the Cell Wall
The cell wall represents a major fraction of the total material in the cell and accounts for approximately 20-35% of the dry weight. Substances unique to the bacterial cell and closely related organisms consist of diaminopimelic acid, muramic acid and teichoic acid. Other major constituents of the bacterial cell wall are amino acids, sugars and lipids. These substances are joined together to form a complex polymeric substance (protein, carbohydrates, lipids) that make up the cell wall. It is thought that the common monomeric building blocks of these complex polymers consist of D-alanine, D-glutamic acid, glucosamine and diaminopimelic acid.
There are significant differences between constituents of the cell wall of Gram negative and Gram positive organisms. For example, Gram positive bacteria contains fewer amino acids than do the Gram negative. The lipid content of Gram negative organisms is significantly higher than that of Gram positive. This fact has been used to explain the Gram reaction. It has been suggested that a species might be identified on the basis of knowledge of the cell wall constituents, Fig. 18 (Handout).
The only macro-molecular compound that is universally present in the wall of all bacteria is the murein sacculus. The rigid, covalently linked framework surrounding the bacterial cell is actually one large saclike molecule (referred to as a sacculus). It is a complex polysaccharide peptide called peptidoglycan or murein. The murein sacculus of some bacterial cell walls may be isolated after the accessory molecules have been stripped off. The glycan component invariably contains two amino sugars, glucosamine and muramic acid. These occur in chains of varying links as alternating beta-1, 4 linked n-acetyl-d glucosamine n-acetylmuramic acid linked through its lactic acid carboxyl group to the amino acid terminus of a tetrapeptide, the muropeptide.
As a general rule, the wall of Gram-positive bacteria contain a much larger amount of murein sacculus than do the walls of Gram-negative bacteria. Moreover, in some Gram-positive bacteria the murein sacculus is the only protoplasm; it is accompanied by a simple polysaccharide and a class of polymers, the teichoic acid. The teichoic acids consist mainly of the phosphate ester of a sugar alcohol either ribotol or glycerol which is built into a polymer together with glucose and one amino acid (alanine).
5.10.1.1 Function of the Cell Wall
The murein sacculus can be hydrolyzed to subunits of relatively low molecular weight by a class of enzymes known as lysozymes. The function of the cell wall of bacteria is thought to be purely mechanical since it appears to restrict the protoplast it encloses to a fixed maximal volume. This prevents the uptake of water, with consequent swelling and rupture, when the cell exists in a strong hypotonic media (as is commonly the case for most bacteria). The wall does not seem to play any role in regulating permeability or in the metabolic activities of the cell. The murein sacculus has been shown to be of considerable importance in the maintenance of the normal cell wall structure in another way. The antibiotic penicillin is an effective (as well as non-toxic for animals) therapeutic agent since it prevents the incorporation of subunits of the murein sacculus into the bacterial cell wall. Therefore, penicillin is toxic for growing bacteria and not for resting cells. When cells are grown in the presence of the antibiotic, the newly synthesized regions of the cell are deprived of their tensile strength as a result of the absence of the murein sacculus and bizarre, distorted cells are produced by bulging of the protoplast into these regions. Eventually osmotic lysis occurs. Lysis and consequent death of the cells can be prevented if penicillin treatment is conducted in hypertonic media. Under these conditions the cells are eventually converted to speroplasts and can usually revert again to normal cells if they are allowed to grow in the absence of penicillin.
5.10.1.2 Bacterial Movement (Motility)
Bacterial movements are of three types. The primary form of bacterial movement may be described as mechanical or by the use of flagella. Flagella are long, fine, whiplike filamentous appendages. The appendages apparently originate from a granular body just beneath the cell wall in the cytoplasm. The length of a flagellum is usually several times that of the cell, but its diameter is only a small fraction of the cell. Flagella are readily visualized by darkfield microscopy but are not seen in ordinary light microscopy unless they are heavily coated with stain. Not all bacteria posses flagella. For bacteria belonging to the order Eubacteriales it may be generalized by saying that many species of bacilli have flagella but only rarely do they occur in cocci. The flagella may be peritrichous (over the whole cell surface) or polar (flagella at one end or both poles). The pattern of attachment of the flagella is used to classify
bacteria into the orders Psedomonadales (all bacteria with polar flagella) and Eubacteriales (all bacteria with peritrichous flagella).
5.10.2 Structure and Chemical Composition of Flagella
While most of the flagellum appears uniform, it has a hook-like shape and a slight constriction near its attachment to a basal body just beneath the cytoplasmic membrane. It is composed of individual spherical subunits formed into three parallel chains wound in a triple helix. Chemical analysis of flagella suggest they are composed of a single homogeneous protein having a molecular weight of about 20,000. This protein has been named flagellin.
5.10.2.1 Movement
Because flagella is responsible for the motility of bacteria and since all bacteria are not flagellated, it follows that there are motile and non-motile species. The exact method by which the flagella move the bacterial cell is not known, but it is postulated that the macromolecular protein chain alternately contracts and relaxes, much as muscle fiber does, producing a wave motion that pulls or pushes the organism.
5.10.2.2 Detection of Motility
Motility can easily be observed by microscopic examination of a hanging-drop preparation. It can also be detected by observing a stab inoculation in a semisolid medium/motility test agar. With motile bacteria, growth proceeds away from the line of inoculation, whereas non-motile bacteria grow only along the line of inoculation.
5.10.2.3 Spirochetes
Spirochetes are mobile by another mechanism. The cell proper forms a helix around a relatively rigid axial filament. Contraction of the cell around this filament causes the cell to flex and bend, thus moving.
5.10.2.4 Gliding Motility
Some of the so-called higher bacterial types are capable of moving by what has been described as a "gliding motility". This is true of bacteria in the order Myxobacteria and some algae (Cyanophycae). They do not possess flagella or other organelles that might be interpreted as structures for locomotion. They move or "glide" on the surface of solid media by means of a sinuous, flexing motion.
Study Questions: 1. What was the evidence presented that the bacteria nucleus is packaged as a continuous circular in the bacteria cell?2. Explain the semiconservative replication of DNA.
3. What is the double helix?
4. Explain the difference between hydrogen and covalent bonds or bonding.
5. Explain the Jacob and Brenner model for chromosomal replication.
6. Define the following:
endospore, dipicolinic acid, protoplasts, spheroplasts, L- forms, lipozymes, teichoic acid.
7. Describe the murein sacculus and its function.
8. Describe bacterial motility.
9. What is the relationship between protoplasts, spheroplasts, and L forms of bacteria.
10. What structures of the bacterial cell are likely to contain significant amounts of diaminopymelic acid? calcium dipicolinate? DNA, Teicholic acid?
11. Why is the significance of a position and size of the endospore in a vegetative cell of value for the identification of a bacterium? Supplement your answers with specific examples.
12. Is endospore formation in bacteria a method of reproduction or a means of multiplication? Explain.
13. What generalizations can be made with respect to morphology and sporulation? Morphology and motility?
14. Compare the bacterial nucleus with the discrete nucleus in cells of higher organisms.
15. Describe the processes by which bacteria multiply.
16. Does an active culture of bacteria contain "old" cells? Explain.
17. How does the term growth, as used in bacteriology, differ from the same term applied to higher plants and animals?
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Microbiology-Davis, Dulbeco, Eisen, Ginsberg and Wood. 4th edition, 1990. |
