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Learning objectives
In-depth knowledge of the morphology and function of the various structures that make up the microbes and viruses Relate the structures to the interest of a molecular microbiologist Knowledge will form a basis for understanding the other topics offered in the Molecular Microbiology module
Reading List
Brock Biology of microorganisms by Madigan, M. T., Martiniko, J. M. and Parker J. Bacterial pathogenesis: A molecular approach by Salyers B. A. and Whitt, D. D. Molecular biology of the cell by Alberts, B., Johnson, A., Lewis, J., Raff, M., Roberts, K. and Walter, P. Genetics of bacteria by Scaife, J., Leach, D. and Galizzi, A. Diagnostic Virology Protocols by Stephenson, J. A., and Warnes, A.
DNA (the hereditary material) Ribosomes (translation of genetic material into proteins
that perform the metabolic functions of the cell)
Eubacteria
Fungi Algae Protozoa
Prokaryotic
Eukaryotic Eukaryotic Eukaryotic
Eukarya
Bacteria
Archaebacteria
Eukaryotic
Prokaryotic
Prokaryotic
Nuclear membrane
# of chromosomes Chromosome topology Murein in cell wall Cell membrane lipids
1 Circular +
1 Circular -
Cellular Domain
Eukarya + +
80S (60S & 40S)
Archaebacteria 70S
Cytoplasmic steraming
Meiosis & Mitosis
+ + -
Eukarya Methionine
Archaebacteria Methionine
Cell arrangement
The Nucleoid
The bacterial nucleoid, which contains the DNA fibrils, lacks a limiting membrane Under the light microscope, the nucleoid can be visualized with the aid of Feulgen staining, which stains DNA Can be isolated by gentle lysis The DNA is a single, continuous, "giant" circular molecule with a molecular weight of approximately 3 109 The unfolded nuclear DNA is 1 mm long (length of 1 to 2 m for bacterial cells)
The mechanism of segregation is not fully understood, but seems the chromosome is always attached to the cell membrane throughout the various stages of the cell cycle
Ribosomes
The cytoplasm is densely packed with ribosomes; diameter of 18 nm Are not arranged on a membranous rough endoplasmic reticulum as they are in eukaryotic cells Consist of 2 subunits with sedimentation coefficients of 50S and 30S, while whole unit is 70S
The larger subunit has 2 RNA molecules; 23S and 5S plus 31 different polypeptides The smaller subunit contain a single RNA molecule (16S) and 21 polypeptides
Ribosomes contd
The role of rRNA include:
perform a scaffolding role by attachment of various ribosomal proteins are involved in recognition of mRNA Involved in catalytic events leading to peptide bond formation
Inclusions
Found in the cytoplasm as distinct granules Usually reserve materials of some sort Some are actually membranous vesicles or intrusions into the cytoplasm which contain photosynthetic pigments or enzymes
Inclusions contd
Cytoplasmic inclusions Glycogen Where found many bacteria e.g. E. coli Composition polyglucose polymerized hydroxy butyrate Function reserve carbon and energy source reserve carbon and energy source reserve phosphate; possibly a reserve of high energy phosphate reserve of electrons (reducing source) in phototrophs; reserve energy source in lithotrophs buoyancy (floatation) in the vertical water column
Sulfur globules
phototrophic purple and green sulfur bacteria elemental sulfur and lithotrophic colorless sulfur bacteria aquatic bacteria especially cyanobacteria
Gas vesicles
Inclusions contd
Cytoplasmic inclusions Parasporal crystals Where found endospore-forming bacilli (genus Bacillus) certain aquatic bacteria many autotrophic bacteria cyanobacteria Composition Function unknown but toxic to certain insects orienting and migrating along geomagnetic field lines site of CO2 fixation light-harvesting pigments protein
Magnetosomes
magnetite (iron oxide) Fe3O4 enzymes for autotrophic CO2 fixation phycobiliproteins
Carboxysomes
Phycobilisomes
Chlorosomes
Green bacteria
Endospores
Sometimes observed as an inclusion Formed by a few groups of bacteria as intracellular structures, but ultimately they are released as free endospores Is actually a type of dormant cell Biologically, they exhibit no signs of life, being described as cryptobiotic
Endospores contd
Are highly resistant to environmental stresses such as high temperature, irradiation, strong acids, disinfectants, etc. Although cryptobiotic, they retain viability indefinitely Germinate back into vegetative cells at appropriate environmental conditions Sporulation is a mechanism of survival rather than a mechanism of reproduction
Surface coats
Microscopic Non-refractile appearance Calcium dipicolinic Absent acid Cytoplasmic water High activity Enzymatic activity Present
High
High Resistant Resistant
Endospores
Endospores are highly heat-resistant, dehydrated resting cells formed intracellularly Sporulation, the process of forming endospores, is an unusual property of certain bacteria
Members of the genera Bacillus and Clostridium
The series of biochemical and morphologic changes occur & represent differentiation within the cycle of the bacterial cell
Sporulation
Usually begins in the stationary phase of the vegetative cell cycle It is initiated by depletion of nutrients (usually readily utilizable sources of carbon or nitrogen, or both) The cell then undergoes a highly complex, welldefined sequence of morphologic and biochemical events that ultimately lead to the formation of mature endospores As many as seven distinct stages have been recognized by studying sporulating Bacillus species
Sporulation stages
Stage 0: vegetative cells with 2 chromosomes at the end of exponential growth Stage I: formation of axial chromatin filament and excretion of exoenzymes, including proteases Stage II, forespore septum formation and segregation of nuclear material into two compartments Stage III, spore protoplast formation and elevation of tricarboxylic acid and glyoxylate cycle enzyme levels;
Sporulation stages
Stage IV: cortex formation and refractile appearance of spore Stage V: spore coat protein formation Stage VI: spore maturation, modification of cortical peptidoglycan, uptake of dipicolinic acid (a unique endospore product) and calcium, and development of resistance to heat and organic solvents Stage VII: final maturation and liberation of endospores from mother cells (in some species)
Illustration of an Endospore
Endospores contd
Newly formed endospores appear as round, highly refractile cells within the vegetative cell wall, or sporangium Some strains produce autolysins that digest the walls and liberate free endospores The spore protoplast, or core, contains a complete nuclear material, ribosomes, and energy generating components that are enclosed within a modified cytoplasmic membrane
Endospore contd
The peptidoglycan spore wall surrounds the spore membrane; on germination, this wall becomes the vegetative cell wall Surrounding the spore wall is a thick cortex that contains an unusual type of peptidoglycan, which is rapidly released on germination A spore coat of keratin-like protein encases the spore contained within a membrane (the exosporium)
Endospore contd
During maturation, the spore protoplast dehydrates and the spore becomes refractile and resistant to heat, radiation, pressure, desiccation, and chemicals Resistance correlate with the cortical peptidoglycan and the presence of large amounts of calcium dipicolinate
Phase microscopy of sporulating bacteria (above) demonstrates the refractility of endospores, as well as characteristic spore shapes and locations within the mother cell
Capsule
Capsules may be up to 10 m thick Not all bacterial species produce capsules; however, the capsules are often important determinants of virulence Encapsulated species are found among both Gram-positive and Gram-negative bacteria. Most capsules are composed of high molecularweight viscous polysaccharides that are retained as a thick gel outside the cell wall or envelope
38
Capsule contd
A true capsule is a discrete detectable layer of polysaccharides deposited outside the cell wall A less discrete structure or matrix is a called a slime layer Glycocalyx is a thin layer of tangled polysaccharide fibers which is almost always observed on the surface of cells growing in nature Capsules are generally composed of polysaccharide; rarely they contain amino sugars or peptides, are antigenic: capsular or K antigens Bacterial capsules are demonstrated by
India ink viewed by light microscopy Capsular swelling test (Quellung reaction) a serological test
Functions of a capsule
Often mediate adherence of cells to surfaces Capsules also protect bacterial cells from engulfment by predatory protozoa or white blood cells (phagocytes), or from attack by antimicrobial agents of plant or animal origin Protect certain soil bacteria from perennial effects of drying or desiccation Capsular materials (e.g. dextrans) may be overproduced to become reserves of carbohydrate
Flagella
Filamentous protein structures attached to the cell surface, antigenic, flagellar or Hantigen Provide the swimming movement for most motile procaryotes Flagellar distribution is a genetically-distinct trait that is occasionally used to characterize or distinguish bacteria
43
Monotrichous: one at a pole Amphitrichous: one on either pole Peritrichous: all over or around the body Lophotrichous: several on one pole
Bacterial Structure: Lecture notes by JLN 2006 44
Detection of flagella
Since motility is a primary criterion for the diagnosis and identification of bacteria
flagellar stains outline flagella and show their pattern of distribution. motility test medium demonstrates if cells can swim in a semisolid medium. direct microscopic observation of living bacteria in a wet mount shows transient movement of swimming bacteria.
Prokaryotic flagellum
Prokaryotic flagella are much thinner than eukaryotic flagella Lack the typical 9 + 2 arrangement of microtubules The diameter is about 20 nm The flagellar filament is rotated by a motor apparatus in the plasma membrane allowing the cell to swim in fluid environments
Structure of flagellum
The innermost rings, the M and S rings, located in the plasma membrane, comprise the motor apparatus The outermost rings, the P and L rings, located in the periplasm and the outer membrane respectively, function as bushings to support the rod where it is joined to the hook of the filament on the cell surface As the M ring turns, the rotary motion is transferred to the filament which turns to propel the bacterium
Flagellum contd
Response to chemical stimuli involves a sensory system of receptors that are located in the cell surface and/or periplasm and that transmit information to methyl-accepting chemotaxis proteins that control the flagellar motor Genetic studies have revealed the existence of mutants with altered biochemical pathways for flagellar motility and chemotaxis
Flagellum contd
Chemically, flagella are constructed of a class of proteins called flagellins The hook and basal-body structures consist of numerous proteins Mutations affecting any of these gene products may result in loss or impairment of motility About 50 genes are required for flagellar synthesis and function
Pili
Fimbriae and Pili are interchangeable terms Short, hair-like structures on the surfaces of procaryotic cells Are shorter and stiffer than flagella, and slightly smaller in diameter Fimbriae are very common in Gram-negative bacteria, but occur in some archaea and Gram-positive bacteria as well
Pili
Fimbriae are most often involved in adherence of bacteria to surfaces, substrates and other cells in nature Common pili (almost always called fimbriae) are major determinants of bacterial virulence
allow pathogens to attach to (colonize) tissues to resist attack by phagocytic white blood cells
Pili contd
A specialized type of pilus, the F or sex pilus, mediates the transfer of DNA between mating bacteria during the process of conjugation
Neisseria gonorrhoeae adheres specifically to the human cervical or urethral epithelium by means of its fimbriae ETEC adhere to the mucosal epithelium of the intestine by means of specific fimbriae M-protein and associated fimbriae of Streptococcus pyogenes help the bacterium resist engulfment by phagocytes
Provide ligands for adherence and receptor sites for drugs or viruses Cause symptoms of disease in animals Provide for immunological distinction and immunological variation among bacterial strains
Outer membrane proteins usually traverse the membrane and in one case, anchor the outer membrane to the underlying PG sheet
Composition of LPS
A complex molecule consisting of:
Lipid A anchor, a disaccharide of glucosamine containing both O-linked & N-linked fatty acids Oligosaccharide core, of about 10 sugars attached to the lipid A via the 8th sugar KDO Polysaccharide chain, the O-antigen consisting of 3-; 4- or 5-sugar monomer repeated 15 -20 times
Types of LPS
More than one type of LPS may be produced by a single strain Multiple forms of LPS continuosly during balanced growth (S-form) Discontinuously due to genetic changes (phase variation) or in response to physiological signals e.g. T, culture density & nutrition S-forms of LPS have 3 parts while R-forms have 2 parts, no O-antigen Presence or absence of the O-chain determines the shape of the bacterial colony, appearing as smooth or rough, respectively
LPS isolation
Treatment of cells with phenol/water for extraction of S-form LPS Treatment with petroleum ether/chloroform/phenol for R-form LPS Proteinase K digestion of lysates & analysis by SDS-PAGE
Illustration of the outer membrane, cell wall & plasma membrane of a Gramnegative bacterium
Plasma Membrane
Composed primarily of protein & phospholipid (about 3:1) Sequesters the molecules of life in a unit, separating it from the environment Involved in the processes such as respiration or photosynthesis or secretion Consequently, it has a variety of functions in energy generation and biosynthesis
Membrane proteins
Some are located and function on one side or another of the membrane Most proteins are partly inserted into the membrane, or possibly even traverse the membrane as channels from the outside to the inside Proteins can move laterally along a surface of the membrane, but unlikely to be rotated within a membrane (discounts mechanism of transport systems)
Archaea Membrane
Are in form of bilayers functionally equivalent to bacterial membranes, but
archaeal lipids are saturated, branched, repeating isoprenoid subunits that are attached to glycerol via an ether linkage ( ester linkage found in glycerides of eukaryotic and bacterial membrane lipids)
Transport Processes
The proteins involved are referred to variously as transport systems, carrier proteins, porters, and permeases Transport systems operate by one of three transport processes
Uniport process Symport processes (co-transport) Antiport processes (exchange diffusion
Uniport process
Symport processes
Two solutes must be transported in the same direction at the same time
Antiport processes
One solute is transported in one direction simultaneously as a second solute is transported in the opposite direction
Is a carrier-mediated system Does not require energy Does not concentrate solutes against a gradient
Use energy Concentrate molecules against a gradient e.g. Ion-driven transport & Binding proteindependent transport
Use energy during transport Modify the solute during its passage across the membrane E.g. Phosphotransferase (pts) system in E. coli
Membrane invaginations
Membrane of procaryotes may invaginate into the cytoplasm or form stacks or vesicles attached to the inner membrane surface Membrane foldings and vesicles may appear in electron micrographs of procaryotic cells as artifacts Such may be analogous to the cristae of mitochondria or the thylakoids of chloroplasts
Membranes contd
Some antibiotics (e.g. polymyxin), hydrophobic agents (e.g. bile salts), and proteins (e.g. complement) damage bacterial membranes
Degradative enzymes
Phosphatases proteases
Cytoplasm
The cytoplasmic constituents invariably include the procaryotic chromosome and ribosomes The distinct granular appearance is due to the presence and distribution of ribosomes Often contained in the cytoplasm of procaryotic cells is one or another of some type of inclusion granule
Co++
Function Maintenance of ionic strength; cofactor for certain enzymes Principal form of inorganic N for assimilation Cofactor for certain enzymes Present in cytochromes and other metalloenzymes Cofactor for many enzymes; stabilization of outer membrane of Gram-negative bacteria Present in certain metalloenzymes Trace element constituent of vitamin B12 and its coenzyme derivatives and found in certain metalloenzymes
Function Trace element present in certain metalloenzymes Trace element present in certain metalloenzymes Trace element present in certain metalloenzymes Trace element present in certain metalloenzymes Principal form of inorganic S for assimilation Principal form of P for assimilation and a participant in many metabolic reactions