Bacteria

Bacterial morphology

1. Bacteria By: Mrs. Mali Dhanashri R. Assistant professor, GES’s Sir Dr. M. S. Gosavi College of Pharmaceutical Education and Reseach

2. Bacteria Bacteria represent a large and diverse group of microorganisms that can exist as single cells or as cell clusters. Bacteria prokaryotic microorganisms a single-celled microscopic organisms that lack nuclei and other organized cell structures. "Bacteria" is the plural form of “bacterium”. The small size, simple design, and broad metabolic capabilities of bacteria allow them to grow and divide very rapidly and to inhabit and flourish in almost any environment.

3. Both deoxyribonucleic acid (DNA) and Ribonucleic acid (RNA). Prokaryotic microorganisms Do not contain chlorophyll: need a host to supply food and also a supportive environment Present in water, soil, air, food, and all natural environment. They can grow in a multiplicitynof environments ranging from hot sulphur springs (65 ° C) to deep freezers ( − 20 ° C), from high (pH 1) to low (pH 13) acidity and high (0.7 m) to low osmolarity (water). In addition, in both nutritionally rich (compost) and nutritionally poor (distilled water) situations.

4. Terms Used To Describe Bacteria

5. MORPHOLOGY Bacteria display a wide diversity of shapes and sizes called morphologie s Cannot be seen with human eyes (microscopic) Their presence was only first recognized in 1677, when the Dutch naturalist Antonie van Leeuwenhoek saw microscopic organisms in a variety of substances with the aid of primitive microscopes. Now bacteria are usually examined under light microscopes capable of more than 1,000-fold magnification Details of their internal structure can be observed only with the aid of much more powerful transmission electron microscopes. Unless special phase-contrast microscopes are used, bacteria have to be stained with a coloured dye so that they will stand out from their background.

6. Cell size The smallest free - living organisms Their size being measured in micrometres (microns) The size & shape vary between the dimensions of 0.75 to 4.0 μm . The cocci diameter near about 1 μm An average rod-shaped bacterium measures approximately 1 μm in diameter and 4 μm in length. They usually vary in size considerably from < 0.5 to 1.0 μm in diameter to 10–20 μm in length in some of the longer spiral forms.

7. smallest bacteria- Mycoplasma , which measure about 0.1 to 0.25 μm in diameter, as small as the largest viruses. E. coli , a normal inhabitant of the intestinal tract of humans and animals, is about 2 μm long and 0.5 μm in diameter Spherical cells of Staphylococcus aureus - up to 1 μm in diameter. Some bacteria are relatively large, such as Azotobacter , which has diameters of 2 to 5 μm or more

8. Cell Shape On basis of shape, bacteria are classified as follows…. Cocci Bacilli Vibrios Spirilla Spirochetes Actinomycetes Mycoplasams

9. Cocci Cocci are small, spherical or oval in shape In greek ‘ kokkos ’ means berry E.g. Micrococcus

10. Bacilli They are rod in shapes. It is derived from greek word ‘ Bacillum ’ meaning stick. Some of the bacilli the length of the cell may be equal to width those are called coccobacilli E.g. Bracella

11. Vibrios Vibrios are comma in shaped, curved rods E.g. Vibrio comma

12. Spirilla Those are longer rigid rods with several curves or coils. Those are helical in shape & rapid bodies. E.g. Spirillum ruprem .

13. Spirochetes They are slender & flexuous spiral forms.

14. Acetinomycetes These are branching filamentous bacteria. The characteristics shape due to the presence of rigid cell wall. E.g. Streptomyces species.

15. Mycoplasma Those are cell wall deficient bacteria So that, they do not have stable morphology. They occur as round or oval bodies with interlacing fillaments .

16. Arrangement of Bacteria Cocci appears as several characteristics arrangement or grouping. 1.Diplococci 2.Streptococci 3.Tetracocci 4.Staphylococci 5.Sarcinae

17. Cocci- Arrangement Diplococci They split in one plane and remains in pair. Eg : diplococcus pneumoniae. Streptococci These cells divide in one planes and remain attached, to form chains. Eg : streptococcus lactis.

18. Tetracocci They divide in two planes and live in groups of four. Eg : Gaffyka tetragena . Staphylococci Cocci cells divide in three planes in an irregular pattern. These cells produce bunches of cocci as in grapes . Eg : staphylococcus aureus, staphylococcus albus .

19. Sarcinae Sarcinae cells divide in three planes in a regular pattern. These cells produces a cuboidal arrangement of group of a eight cells. Eg : Micrococcus tetragena .

20. Bacilli- Arrangement Arrangement of grouping formed by bacilli species 1. Diplobacilli 2. Streptobacilli 3. Trichomes

21. Diplobacilli Arrangement of groupings formed by bacilli species are limited & those split across their short axis. They may appear as pair those called Diplobacilli e.g. klebisella pneumoniae

22. streptobacilli Some bacilli species are found in chain like structure those called streptobacilli e.g Bacillus subtilis.

23. trichomes Some bacilli species are found in chain like structure but have much large area of contact between the adjacent cells those are called trichomes e.g. Saprospira species

24. Bacterial Structures Flagella Pili Capsule Plasma Membrane Cytoplasm Cell Wall Ribosomes mesosomes Inclusions Spores

25. 1. Flagella Flagella are long, slender, thin hair-like cytoplasmic appendages, which are responsible for the motility of bacteria. These are the organs of locomotion. They are 0.01 to 0.02 μm in diameter, 3 to 20 μm in length. Flagella are made up of a protein- flagellin.

26. Flagella found in both Gram-positive & negative bacteria. Few coccal forms, most bacilli & almost all of the spirilla & vibrios are motile by flagella. They can be seen by compound microscope with special staining technique & can be seen easily under electron microscope & dark filled microscope.

27. Flagellum The flagellum has three basic parts , 1. Filament 2. Hook 3. Basal body

28. Filament is the thin, cylindrical, long outermost region with a constant diameter. The protein in filament is flagellin similar to keratin, Mol. Wt 20,000-40,000. The filament is attached to a slightly wider hook. It consist of diffent proteins. The basal body is composed of a small central rod inserted into a series of rings.

29. Gram negative bacteria contain four rings as L-ring, P-ring, S-ring, M-ring. Gram positive bacteria have only S and M rings in basal body. L- ring is embedded in lipolysaccharide layer of outer membrane P- ring in pepetidoglycan layer S- ring- Just above cytoplasmic membrane ( Semi position of membrane) M- ring- within cytoplasmic membrane

30. Flagella may be seen on bacterial body in following manner. 1. Monotrichous : These bacteria have single polar flagellum. Eg : vibrio cholera 2. Lophotrichous : These bacteria have two or more flagella only at one end of the cell. Eg : pseudomonas fluorescence. 3. Amphitrichous : These bacteria have single polar flagella or tuft of flagella at both poles. Eg : Aquaspirillum serpens . 4. Peritrichous: Several flagella present all over the surface of bacteria. Eg: Escherichia coli, Salmonella typhi.

31. Fimbriae Fimbriae are similar structure like flagella but not involved in motility. shorter than flagella (3 μm ). distributed over the entire surface of the cell. act primarily as adhesions & allow to microorganism to attach to surface. Responsible for haemagglutination & cell clumping in bacteria. Each bacteria possess 100 to 200 fimbriae

32. Pili Pili are hair-like microfibrils, 0.5 to 2 μm in length and 5 to 7 nm in diameter. thinner, shorter and more numerous than flagella. present only on gram negative cells. composed of protein known as pillin . unrelated to motility and are found on motile and non-motile cells.

33. Pili are usually longer than fimbriae and number only one or two per cell. Pili play an important role in attachment to surfaces. Hence pili is also called organ of adhesion. Specialized pili, the sex pili, allows the transfer of genetic material from one bacteria to another in a process called conjugation where they are called conjugation pili or "sex pili"

34. Capsule Glycocalyx : sticky coating produced by many bacteria covering the surface of cell. The glycocalyx is composed of polysaccharidesn (sugars) and proteins. Bacteria synthesize loose amorphous organic exopolymer which is deposited outside and tightly to cell wall The EPS( extracelluar polysaccharides) contains 2% carbohydrate & 98% water so, they produce gummy exterior to the cell.

35. Morphologically two extreme forms exist… i . Capsules and ii. Slime Capsules: which forms rigid, tightly & closely associated with cell Slimes: which are loosely associated with cell. Capsulated bacteria produces smooth colonies and non capsulated bacteria produces rough colonies on the surface of agar media.

36. Function of capsule & slime They protect from desiccation. They provide a protection barrier against the penetration of biocides. They protect against engulfment by phagocytes & protozoa. They may promote the stability of bacterial suspension by preventing the cells from aggregation & settling. They may promote attachments of bacteria to surface.

37. Cell wall Cell wall is rigid structure which gives definite shape to cell ( spherical, rod and spiral). situated between the capsule and cytoplasmic membrane. It is about 10 – 20 nm in thickness and constitutes 20-30 % of dry weight of cell. The cell wall cannot be seen by direct light microscopy and does not stain easily by different staining reagents. Composed of peptidoglycan (polysaccharides + protein) Mycoplasma are bacteria that have no cell wall and therefore have no definite shape.

38. The cell wall of bacteria contains diaminopimelic acid (DAP), muramic acid and teichoic acid. These substances are joined together to give rise to a complex polymeric structure known as peptidoglycan or murein or mucopeptide. Peptidoglycan is the major constituent of the cell wall of gram positive bacteria (50 to 90 %) where as in gram negative bacterial cell wall its presence is only 5 -10 %.

39. Peptidoglycan = (polysaccharides + protein), Peptidoglycan (murein) is a large macromolecules containing glycan (polysaccharide) chains that are cross-linked by short peptide bridge. peptidoglycan is made from polysaccharide chains cross-linked by peptides containing D-amino acids The glycan chain act as a backbone to peptidoglycan Those short peptide bridge composed of alternating residues of N-acetyl muramic acid (NAM) & N-acetyl glucosamine (NAG).

40. Each molecule of NAM attached a tetrapeptide. Tetrapeptide consisting of the amino acids L-alanine, Dalanine , D-glutamic acid & lycine or diaminopimelic acid (DAP). This glycan tetrapeptide repeat unit is cross -linked to adjacent glycan chain. This adjacent glycan chain occurs through a direct peptide linkage or a peptide interbridge . The type & number of cross linking amino acids vary from organism to organism.

41. Functions of cell wall Cell wall is involved in growth and cell division of bacteria. It gives shape to the cell. It gives protection to internal structure and acts as supporting layer. To prevent rupture of bacteria caused by osmotic pressure differences between intra cellular and extra cellular environment. To provide support for flagella. To regulate a certain degree of passage of molecules into and out of the cell. It serve as the sites of attachment for most bacterial viruses.

42. On the basis of structure of cell wall, bacteria can be classified as Gram-positive Gram- negative The staining technique used to distinguish this bacteria is called as Gram staining technique.

43. Gram Staining Developed in 1884 by Danish scientist Christian Gram. It is a differential stain. In this, bacteria are first stained with crystal violet, then treated with a mordant - a solution that fixes the stain inside the cell. Bacteria are then washed with a decolorizing agent, such as alcohol, and counterstained with safranin, a light red dye.

44. Gram-positive bacteria are those that are stained dark blue or violet by Gram staining. Gram-negative bacteria cannot retain the crystal violet stain, instead take up the counterstain and appearred or pink. The walls of gram-positive bacteria have more peptidoglycans than do gram-negative bacteria. Thus, gram-positive bacteria retain the original violet dye and cannot be counterstained.

45. Gram-positive Cells Gram positive bacterial cell wall consist of a single type of molecules. Cell wall thick near about 20 to 80 nm. In that present of 60 to 80 % peptidoglycan. Gram positive walls frequently contains acidic polysachrides are called teichoic acids.

46. Teichoic acid are either ribitol phosphate or glycerol phosphate molecules that are connected by phosphodiester bridge. In some gram positive bacteria glycerol-teichoic acids are bound to lipids membrane and termed as lipoteichoic acid. Those lipoteichoic acid create infection by killing bacteria & shows inflammation.

47. Gram-negative Gram negative cell wall are multilayered & complex type structure . Gram negative cell wall consist 10 to 20 % peptidoglycan. In that second layer found outside the peptidoglycan layer. This layer is asymmetrical & contains proteins, lipoproteins , phospholipids & lipopolysaccharide (LPS).

48. This outer layer is attached to peptidoglycan & the other end is fixed in the outer membrane. In the inner leaf of the outer layer conatins phospholipids & it’s outer layer composed with LPS ( lipopoysaccharide ), a polysaccharide-lipid molecule. In gram negative cell, the LPS is an important molecule because it determine the antigenicity & it is extremely toxic to animal cell. In the LPS molecules contains three regions i . lipid A ii. Core polysaccharide iii. O-specific polysaccharide

49. Lipid A linked to core polysaccharide by the molecule KDO ( ketodeoxyoctonate ) & other end of core polysaccharide is the O-polysaccharide. In the O-polysaccharide or O-antigen usually contains six-carbon sugars as well as one or more usually deoxy sugars such as abequose In the lipid A components are gives toxic & pathogenic properties to the gram-negative bacteria. Gram negative bacterial outer membrane is relatively permeable to small molecules but not for enzymes or large molecules. The region between the outer surface of the cytoplasmic membrane & the inner surface of the outer membrane is called the periplasm .

50. outer membrane • Similar to the plasma membrane, but is less permeable . This membrane has tiny holes or openings called porins . Porins block the entrance of harmful chemicals and antibiotics, making G- ve bacteria much more difficult to treat than G+ve cells. Composed of lipopolysaccharides (LPS). LPS is a harmful substance classified as an endotoxin. Lipopolysaccharides , which acts as an endotoxin , are composed of polysaccharides and lipid A (responsible for much of the toxicity of G- ve bacteria). These differences in structure can produce differences in antibiotic susceptibility Ex : vancomycin can kill only Gram + ve bacteria and is ineffective against Gram - ve pathogens, such as Haemophilus influenzae or Pseudomonas aeruginosa .

51. Cell wall If the bacterial cell wall is entirely removed, it is called a protoplast while if it's partially removed, it is called a spheroplast . Antibiotics such as penicillin inhibit the formation of peptidoglycan cross-links in the bacterial cell wall. The enzyme lysozyme , found in human tears, also digests the cell wall of bacteria and is the body‘s main defense against eye infections.

52. CytoplasmicMembrane The cytoplasmic (plasma) membrane is a thin ( 5 to 10 nm). It separates the cell wall and cytoplasm. It composed of phospholipids (20 to 30 %) and proteins ( 60 to 70 %). Prokaryotic plasma membranes are less rigid than eukaryotic membrane due to lack of sterols.

53. The phospholipids molecules are arranged in two parallel rows, called a phospholipid bilayer . Each phospholipid molecule contains a polar head & tail. Polar head composed of a phosphate group & glycerol. The non-polar tails are interior of the bilayer . numerous proteins moving within or upon this layer are primarily responsible for transport of ions, nutrients and waste across the membrane.

54. Functions of cytoplasmic membrane They including in transportation of nutrients. It provides mechanical strength to the bacterial cell. It helps in DNA replication. It contains the enzymes involved in the biosynthesis ofmembrane lipids & various macromolecules of the bacterial cell wall.

55. Periplasmic space Gram-negative bacteria : space between the cytoplasmic membrane and the cell wall and space found between cell wall and the outer membrane Periplasm may constitute up to 40% of the total cell volume in G- ve species. Gram-positive bacteria : space between the cytoplasmic membrane and the cell wall. The periplasm is filled with water and proteins and is reminiscent of the cytoplasm.

56. However periplasm contains proteins and other molecules distinct from those in the cytoplasm because the membrane prevents the free exchange between these two compartments. Periplasmic proteins have various functions in cellular processes including: transport, degradation, and motility. Periplasm controls molecular traffic entering and leaving the cell.

57. Cytoplasm Portion of the cell that lies within the PM substances within the plasma membrane, excluding the genetic material. Gel-like matrix composed of mostly water(4/5 th ), enzymes, nutrients, wastes, and gases The bacterial cytoplasm is a suspension of organic, inorganic solutes in a viscous water solution. The cytoplasm of bacteria differs from that of higher eukaryotic microorganisms in not containing endoplasmic reticulum, Golgi apparatus, mitochondria and lysosomes .

58. • It contains the ribosomes , proteins and other water soluble components and reserve material. • In most bacterial, extrachromosal DNA ( plasmid DNA ) is also present. It is relatively featureless by electron microscope - although small granules can be seen. • carries out very important functions for the cell - growth, metabolism, and replication .

59. Constituents Proteins including enzymes Vitamins Ions Nucleic acids and their precursors Amino acids and their precursors Sugars, carbohydrates and their derivatives Fatty acids and their derivatives

60. Mesosomes Mesosomes are respiratory sites of bacteria. The mesosomes are attached to the bacterial chromosomes and is involved in DNA segregation during cell division. They are predominant in Gram positive bacteria. In most of the bacteria, particularly in Gram-positive bacteria the growth condition depending upon the membrane appears to be infolded at more than one point. Such infoldings are called mesosomes . Mesosomes presents in two types… In central ( septal ) mesosomes & peripheral (lateral) mesosomes

61. Central mesosomes present deep into the cytoplasm & locate near the middle of the cell. These are involved in the DNA segregation & in the formation of cross walls during cell division. The peripheral mesosomes are not present at central location & are not associated with nuclear material. Mesosomes are also called as chondroids & are visible only under electron microscope. Larger numbers of mesosomes have a higher respiratory activity e.g. Azotobacter .

62. Nucleoid Unlike the eukaryotic (true) cells, bacteria do not have a membrane enclosed nucleus. The nucleoid is a region of cytoplasm where the chromosomal DNA is located. It is not a membrane bound nucleus, but simply an area of the cytoplasm where the strands of DNA are found. The genome consists of a single molecule of double stranded DNA arrangement in a circle. The bacterial chromosome is haploid & replicated by simple fission instead of mitosis as in an eukaryotic cell.

63. Plasmids small extra-chromosomal DNA contain genes for antibiotic resistance or virulence. Structure Similar to most bacterial chromosomes, but considerably smaller. plasmids are covalently closed circular DNA In a few species linear plasmids have been found. Size : Chromosomal DNA is typically about 4000 kb, plasmid DNA ranges from 1-200 kb. • Number of plasmids: 1-700 copies of plasmid in a cell.

64. Plasmid Function The function of plasmids is not always known, but they are not normally essential for survival of host, although their presence generally gives the host some advantage. Antibiotic resistance - Some plasmids code for proteins that degrade antibiotics-a big advantage for pathogens. Some encode for proteins which confer virulence factors on the host. For example- E. coli plasmid Ent P307 codes for an enterotoxin which makes E. coli pathogenic. Conjugative plasmids - These allow exchange of DNA between bacterial cells.

65. Plasmids and the associated traits can be transferred between bacteria, even from one bacterial species to another. Plasmids are not involved in reproduction. Plasmids replicate independently of the chromosome. Plasmids are passed to other bacteria by two means. For most plasmid types, copies in the cytoplasm are passed on to daughter cells during binary fission. Other types of plasmids ,form tube like structure at the surface called a pilus that passes copies of the plasmid to other bacteria during conjugation, a process by which bacteria exchange genetic information.

66. Plasmids have been shown to be instrumental in the transmission of special properties, such as antibiotic drug resistance, resistance to heavy metals, and virulence factors necessary for infection of animal or plant hosts. The ability to insert specific genes into plasmids have made them extremely useful tools in the area of genetic engineering/RDNA Technology

67. Ribosomes Ribosomes are most important structure in bacterial cytoplasm. They involved in protein synthesis. Ribosomes numbers varies with the rate of protein synthesis. If greater the number of ribosomes then the greater the protein synthesis. They have 200 Ao in diameter. They are characterised by their sedimentation properties.

68. These bacterial ribosomes are called as 70 S ribosomes . S= svedberg unit..unit of sedimentation.  After sedimentation carried in ultra-centrifuge & then placed in low concentration of magnesium that time 70 S ribosomes dissociated into 50 S & 30 S particles.  Each 50 S particles contain…one molecule of 23 S RNA, one molecule of 5 S RNA & 32 different proteins.  And, each 30 S particles contains…one molecule of 16 S RNA & 21 different proteins.  During protein synthesis these ribosomes are associated with the m-RNA & such association are called polysomes This 70s ribosomes are made up of two subunits namely a large subunits 50s and a small subunit 30s. During active protein synthesis the ribosomes are associated with mRNA and such associations are called polysomes .

69. They translate the genetic code from the molecular language of nucleic acid to that of amino acids—the building blocks of proteins. • Bacterial ribosomes are similar to those of eukaryotes, but are smaller and have a slightly different composition and molecular structure. • Bacterial ribosomes are never bound to other organelles as they sometimes are bound to the endoplasmic reticulum in eukaryotes, but are free-standing structures distributed throughout the cytoplasm. • There are sufficient differences between bacterial ribosomes and eukaryotic ribosomes that some antibiotics will inhibit the functioning of bacterial ribosomes , but not a eukaryote's, thus killing bacteria but not the eukaryotic organisms they are infecting. • Streptomycin binds 70S ribosome and stops protein synthesis but it can not bind 80S ribosome of eukaryotes and thereby eukaryotic cell remains unaffected.

70. Bacterial Chromosome - Genophore • The bacterial chromosome consists of a single, circle of deoxyribonucleic acid. • DNA is double stranded- two strands line up antiparrallel to each other and the bases are linked together with hydrogen bonds. • It includes most of the genetic material of the organism .

71. Inclusion bodies Inclusion bodies: Bacteria can have within their cytoplasm a variety of small bodies collectively referred to as inclusion bodies. Some are called granules and other are called vesicles. Inclusions are considered to be nonliving components of the cell that do not possess metabolic activity and are not bounded by membranes. The most common inclusions are glycogen, lipid droplets, crystals, and pigments.

72. Inclusion bodies - Granules Granules: Densely compacted substances without a membrane covering. Nutrients and reserves may be stored in the cytoplasm in the form of glycogen, lipids, polyphosphate, or in some cases, sulfur or nitrogen for later use. Each granule contains specific substances, such as glycogen (glucose polymer) and polyphosphate (phosphate polymer, supplies energy to metabolic processes). Sulfur bacteria contains reserve granules of sulfur. These granules are depleted in starvation

73. Inclusion bodies-vesicles Some aquatic photosynthetic bacteria and cyano bacteria have rigid gas-filled vacuoles and it helps in floating at a certain level - allowing them to move up or down into water layers with different light intensities and nutrient levels. Some magnetotactic bacterium, eg Aquaspirillium magnetotacticum , stores Magnetitite (Ferric oxide). The presence of such magnetic inclusions enables these bacteria to responds to magnetic fields.

74. Spores Many bacterial species produce spores inside the cell & outside the cell. endospores Inside the spores are called & outside the spores are called exospores. E.g Bacillus anthracis , Bacillus subtilis etc. Spores are extremely resistant to desiccation, staining, radiation, disinfecting chemicals & heat. Each bacterial spore on germination forms a single vegetative cell.

75. The process of endospore formation is known as sporulation and it may take 4 to 8 hours in a vegetative cell. Endospores are thick-walled, highly refractile bodies that are produced one per cell. They remain viable for long time & help bacteria to survive for long period under unfavourable condition. Endospore can remain dormant for thousand of years. Spores of all medically important bacteria are destroyed by moist heat sterilization ( autoclave ) at 121 °C for 20 minutes.

76. All the endospores contain large amount of DPA ( dipicolinic acid). It occurs in combination with large amount of calcium , which is present in central part of the spore (core ). Calcium & DPA complex play important role in the heat resistant of endospores . Endospores consists of a core or envelope or protoplast. In the core or protoplast consist of DNA & ribosomes , t-RNA & enzymes. The spore envelop consist of the inner membrane, outer membrane, cortex & spore coat. In some species have the outer layer called exosporium which bears ridges & fold.

77. SPORULATION The process of endospore formation is known as sporulation It may take 4 to 8 hrs in a vegetative cell. Sporulation process Firstly the bacterial chromosomes replicate A newly replicated bacterial chromosomes & small portion of cytoplasm are isolated by an in growth of the plasma membrane called a spore septum .

78. The septum derived from the cytoplasmic membrane It is then formed by a process of invagination which divides into a forespore & sporangium. The forespore is subsequently encircled by a dividing septum as a double layered membrane. Between the two layers is laid a spore cortex & outer layer is transformed into spore coat which consists of several layer . After formation of spore mother cell is lyses to release it