Structure Of Microbes .ppt 6w114y

Organisational Structure of Microbes and Viruses

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.

Reading List cont’d • Diagnostic molecular microbiology: Principles and Applications by Persing, D. H., Smith, T. F. S., Tenover, F. C. and White, T. J. • Introduction to Modern Virology by Dimmock, Easton and Leppard • Principles of Virology Molecular Biology, Pathogenesis and control by Flint, S. J., Enqinst, L. W., Krug, R. M., Racaniello, V. R. and Skalka, A. M. • Methods for General and Molecular Bacteriology by Gerhardt, Murray, Wood and Krieg

Underlying properties of cells • All cells contain common functional and structural properties – Cytoplasmic membrane (boundary between the living cell & the environment)

– Cytoplasm (substance where biological reactions take place)

– DNA (the hereditary material) – Ribosomes (translation of genetic material into proteins that perform the metabolic functions of the cell)

– ATP (the Universal energy currency)

Major groups of Microbes Microbial Group

Structure

Viruses

No cell or acellular

Arch(a)ebacteria

Prokaryotic

Eubacteria

Prokaryotic

Fungi

Eukaryotic

Algae

Eukaryotic

Protozoa

Eukaryotic

Prokaryotic vs Eukaryotic structure • Fundamental difference is presence or absence of a membrane-bound nucleus and membranous organelles • Basing on the small subunit ribosomal RNA (ssrRNA) analysis there are three cellular domains of life: Archaea, Bacteria, (both Procarya) and Eukarya • Procaryotic cells lack a nuclear membrane; are the simplest of cells & the first types of cells to evolve

Differences in the cell types Property Cell configuration

Nuclear membrane # of chromosomes Chromosome topology Murein in cell wall Cell membrane lipids

Cellular Domain

Eukarya

Bacteria

Archaebacteria

Eukaryotic

Prokaryotic

Prokaryotic

+ >1 Linear Ester-linked glycerides; unbranched; polyunsaturated

1 Circular +

1 Circular -

Ester-linked Ether-linked glycerides; branched; unbranched; saturated saturated or monounsaturat ed

Differences in the cell types Property Cell membrane sterols

Organelles (Mitochondria & Chloroplasts) Ribosome size

Cytoplasmic steraming Meiosis & Mitosis

Transcription & translation coupled

Cellular Domain

Eukarya + +

Bacteria -

Archaebacteria -

80S (60S & 40S)

70S (50S & 30S)

70S

+ + -

+

+

Differences in the cell types Cellular Domain

Property

Eukarya

Bacteria

Archaebacteria

Amino acid initiating protein synthesis

Methionine

N-fomyl methionine

Methionine

Protein synthesis inhibited by streptomycin & chloramphenicol

-

+

-

Protein synthesis inhibited by diphtheria toxin

+

-

+

Illustration of a Typical Prokaryotic Cell

Bacterial Cell: Gross Morphology • Determined by use of a light microscope on stained or unstained films of bacterial suspension • Bacteria have characteristic shapes (cocci, rods, spirals, etc.) • Often occur in characteristic aggregates (pairs, chains, tetrads, clusters, etc.) • These traits are usually typical for a genus and are diagnostically useful

Cell shape and arrangement

Cell arrangement

Bacterial Cell Structure • Bacteria have a very simple internal structure • Prokaryotes have a nucleoid (nuclear body) rather than an enveloped nucleus • Lack membrane-bound cytoplasmic organelles • The plasma membrane in prokaryotes performs many of the functions carried out by membranous organelles in eukaryotes • Multiplication is mainly by binary fission

Pictorial presentation of a Bacterial Cell Structure

Bacterial Structure: Components Cell envelope Cytoplasmic membrane* Cell wall** Capsule Slime Flagella Fimbriae/Pilli

Intracellular components Nuceloid*

Ribosomes* Inclusion granules Endospores Plasmids

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 µm for bacterial cells)

The bacterial Nucleoid • Bacterial chromatin does not contain basic histone proteins, but low-molecular-weight polyamines and magnesium ions may fulfill a function similar to that of eukaryotic histones. • Contain a single chromosome • The number of copies of the chromosome in a cell depends on the stage of the cell cycle (chromosome replication, cell enlargement, chromosome segregation, etc)

• 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 cont’d • 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 cont’d Cytoplasmic inclusions

Where found

Composition

Function

Glycogen

many bacteria e.g. E. coli

polyglucose

reserve carbon and energy source

Polybetahydroxyutyric many bacteria e.g. acid (PHB) Pseudomonas

polymerized hydroxy butyrate

reserve carbon and energy source

Polyphosphate (volutin many bacteria e.g. granules) Corynebacterium

linear or cyclical polymers of PO4

reserve phosphate; possibly a reserve of high energy phosphate

Sulfur globules

phototrophic purple and green sulfur bacteria elemental sulfur and lithotrophic colorless sulfur bacteria

reserve of electrons (reducing source) in phototrophs; reserve energy source in lithotrophs

Gas vesicles

aquatic bacteria especially cyanobacteria

buoyancy (floatation) in the vertical water column

protein hulls or shells inflated with gases

Inclusions cont’d Cytoplasmic inclusions

Where found

Composition

Function

Parasporal crystals

endospore-forming bacilli (genus Bacillus)

protein

unknown but toxic to certain insects

Magnetosomes

certain aquatic bacteria

magnetite (iron oxide) Fe3O4

orienting and migrating along geomagnetic field lines

Carboxysomes

many autotrophic bacteria

enzymes for autotrophic CO2 fixation

site of CO2 fixation

Phycobilisomes

cyanobacteria

phycobiliproteins

light-harvesting pigments

Green bacteria

light-harvesting lipid and protein and pigments and bacteriochlorophyll antennae

Chlorosomes

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 cont’d • 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

Endospores vs vegetative cells Property

Vegetative cells

Surface coats

Thick spore coat, Typical Gramcortex, and positive murein cell peptidoglycan core wall polymer wall

Microscopic Non-refractile appearance Calcium dipicolinic Absent acid Cytoplasmic water High activity Enzymatic activity Present

Endospores

Refractile Present in core Very low Absent

Endospores vs vegetative cells cont’d Property

Vegetative cells

Heat resistance Low Resistance to chemicals and Low acids Radiation Low resistance Sensitivity to Sensitive lysozyme Sensitivity to dyes Sensitive and staining

Endospores High

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 – 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 cont’d • 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 cont’d • 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 cont’d • 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

Shape; Size and location of endospores

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 µm 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

Bacterial Structure: Lecture notes by JLN 2006

38

Capsule as illustrated by India ink

Capsule cont’d • 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

Role of slime layer • Some bacteria produce slime materials to adhere and float themselves as colonial masses in their environments • Other bacteria produce slime materials to attach themselves to a surface or substrate • Bacteria may attach to surface, produce slime, divide and produce microcolonies within the slime layer, and construct a biofilm, which becomes an enriched and protected environment for themselves and other bacteria

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

Bacterial Structure: Lecture notes by JLN 2006

43

Different arrangements of bacterial flagella

• • • •

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

Prokaryotic Flagellum cont’d • Powered by proton motive force (chemiosmotic potential) established on the bacterial membrane, rather than ATP hydrolysis which powers eukaryotic flagella • ≈ 50% of the bacilli & all of the spiral and curved bacteria are motile by means of flagella • Very few cocci are motile (adapted to dry environments and their lack of hydrodynamic design)

Structures of the flagellum • The flagellar apparatus consists of several distinct proteins: – a system of rings embedded in the cell envelope (the basal body) – a hook-like structure near the cell surface – and the flagellar filament

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 the rod where it is ed 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

Ultrastructure of the flagellum

Flagellum cont’d • 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 cont’d • 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 • 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 cont’d • 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

The Cell Wall • Most prokaryotes have a rigid cell wall • Is an essential structure that protects the cell protoplast from mechanical damage and from osmotic rupture or lysis • The membrane is a delicate, plastic structure, it must be restrained by an outside wall made of porous, rigid material that has high tensile strength; that is murein, the ubiquitous component of bacterial cell walls

Uniqueness of bacterial cell wall • Essential structure for viability • Composed of unique components found nowhere else in nature • One of important sites for attack by antibiotics – Cross-linking transpeptidase enzymes are some of the targets for β-lactam antibiotics

• 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

Bacterial Cell wall composition • Contain a unique type of peptidoglycan called murein & many types of PG exist • PG is a polymer of disaccharides (a glycan) crosslinked by short chains of amino acids (peptides) • All Bacterial peptidoglycans contain Nacetylmuramic acid, which is the definitive component of murein • In Archaea, PG may be composed of protein, polysaccharides, or peptidoglycan-like molecules, but never do they contain murein

Gram positive cell wall

Gram-positive cell wall • Most Gram-positive bacteria have a relatively thick (about 20 to 80 nm), continuous cell wall (often called the sacculus) • Is composed largely of peptidoglycan (also known as mucopeptide or murein) • Other cell wall polymers (such as the teichoic acids, polysaccharides, and peptidoglycolipids) are covalently attached to the peptidoglycan

Profiles of the cell walls of Gram negative bacteria

Gram-negative cell wall • The peptidoglycan layer thin (about 5 to 10 nm thick • Outside the PG layer in the Gram-negative envelope is an outer membrane structure (about 7.5 to 10 nm thick) • The membrane structure is anchored non-covalently to lipoprotein molecules (Braun's lipoprotein), which, in turn, are covalently linked to the PG • The lipopolysaccharides of the Gram-negative cell envelope form part of the outer leaflet of the outer membrane structure

Chemical components in bacterial walls

Muramic acid subunit of the peptidoglycan of Escherichia coli

Peptidoglycan of Staphylococcus aureus

Comparison of G-ve and G+ve cell walls

Cell wall components: Teichoic acids/Teichuronic acids • Polyol phosphate polymers bearing a strong negative charge • Covalently linked to the peptidoglycan in some Gram-positive bacteria • Strongly antigenic, but are generally absent in Gram-negative bacteria • May be released from murein – by acidic or basic hydrolysis of sensitive phosphodiester linkages between teichoic acid and the C-6 of muramic acid; – lysozyme or neuraminidase digestion of murein

Cell wall components: Lipoteichoic Acids • Like membrane teichoic acids are polymers of amphiphitic glycophosphates with the lipophilic glycolipid and anchored in the cytoplasmic membrane • Are antigenic, cytotoxic and adhesins (e.g., Streptococcus pyogenes) • Isolated using same methods as for teichoic acid

Outer Membrane of Gramnegative Bacteria • The outer membrane is a discrete bilayered structure on the outside of the PG sheet • For the bacterium, the outer membrane is – a permeability barrier, – due to its LPS content, it possesses many interesting and important characteristics of Gramnegative bacteria

• Outer membrane proteins usually traverse the membrane and in one case, anchor the outer membrane to the underlying PG sheet

Outer membrane cont’d • It is a lipid bilayer intercalated with proteins, superficially resembling the plasma membrane – Inner face is composed of phospholipids similar to the phosphoglycerides that compose the plasma membrane – Outer face may contain some phospholipid, but mainly it is formed by a different type of amphiphilic molecule, which is composed of lipopolysaccharide (LPS)

Cell wall components: Lipopolysaccharides/Endotoxin • One of the major components of the outer membrane of Gram-negative bacteria • Sugars in the polysaccharide chains confer serologic specificity • The LPS molecule is composed of 2 or 3 biosynthetic entities: the lipid A; the core-; and the O-polysaccharide (O-antigen)

Positioning of LPS molecule • The Lipid A head of the molecule inserts into the interior of the membrane • The polysaccharide tail of the molecule faces the aqueous environment • Where the tail of the molecule inserts into the head there is an accumulation of negative charges such that a magnesium cation is chelated between adjacent LPS molecules • This provides the lateral stability for the outer membrane, and explains why treatment of Gramnegative bacteria with a powerful chelating agent,

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

• Composition of the O-chains depend on the bacterial environment – e.g. 6-deoxy sugars are prevalent in phytopathogenic bacteria – acidic monosaccharides are frequent in marine bacteria

Biological effects of LPS • Most are due to the lipid A part • There is an increasing evidence indicating that O-antigen plays an important role in – effective colonization of host tissues – resistance to complement-mediated killing – in the resistance to cationic antimicrobial peptides that are key elements of the innate immune system

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

Energy generation by the Plasma procaryotic membrane • Site for or location of the – electron transport system that couples aerobic respiration and ATP synthesis – photosynthetic chromophores that harvest light energy for conversion into chemical energy – oxidative phosphorylation and photophosphorylation; analogous to the functions of mitochondria and chloroplasts in eukaryotic cells

Functions of plasma membrane • Mainly acts as a selective permeability barrier • Allows age of water and uncharged molecules up to mw of ≈ 100 daltons • age of larger molecules or any charged substances by means of special membrane transport processes and transport systems

Bacterial Membrane composition • Phospholipids (40%) are amphoteric molecules with a polar hydrophilic glycerol "head" attached via an ester bond to two nonpolar hydrophobic fatty acid tails • They naturally form a bilayer in aqueous environments • Within the bilayer, are various structural and enzymatic proteins (60%), which carry out most membrane functions

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)

Membrane proteins cont’d • The arrangement of proteins and lipids to form a membrane is called the fluid mosaic model • Bacterial membranes vs eukaryotic membranes – Structurally similar but former – consist of saturated or monounsaturated fatty acids (rarely, polyunsaturated fatty acids) – do not normally contain sterols

Fluid mosaic model of a biological membrane

Types of Membrane Proteins • Transport proteins selectively mediate the age of substances into and out of the cell • Sensing proteins that measure concentrations of molecules in the environment or • Binding proteins that translocate signals to genetic and metabolic machinery in the cytoplasm • Enzymes for metabolic processes e.g. wall & membrane synthesis, septum formation, DNA replication, CO2 fixation & NH4 oxidation

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)

• Structure is an adaptation to survival in extreme environments

Bacterial membrane phospholipid

Archaeal membrane phospholipid

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

• One solute es through the membrane unidirectionally

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

Bacterial Transport systems • Presence of various transport processes and transport systems reflects the need to concentrate substances inside the cytoplasm against the concentration (gradient) of the environment • This requires the operation of an active transport system (2 in bacteria) – ion driven transport systems (IDT) – binding-protein dependent transport systems (BPDT)

Bacterial transport system cont’d • Accumulation of the solute in the cytoplasm at concentrations far in excess of the environment is active transport & requires energy • Are operated by transport proteins (also called carriers, porters or permeases) in the plasma membrane – Facilitated diffusion – Active transport systems e.g. IDT & BPDT – Group translocation systems e.g. phosphotransferase

Bacterial transport systems: Facilitated Diffusion

• Is a carrier-mediated system • Does not require energy • Does not concentrate solutes against a gradient

Facilitated diffusion systems (FD) • Least common type in bacteria • Glycerol uniporter in E. coli is the only well known FD system • Involves age of a specific solute through a carrier that forms a channel in the membrane • The solute can move in either direction to the point of equilibrium on both sides • Although it is carrier-mediated and specific, no energy is expended, thus • Solute cannot be accumulated against the concentration gradient

Bacterial transport systems: Active transport systems

• Use energy • Concentrate molecules against a gradient • e.g. Ion-driven transport & Binding proteindependent transport

Ion driven transport systems (IDT) • Together with BPDT are the systems that are used for transport of most solutes by bacterial cells • Is used for accumulation of many ions and amino acids • IDT is a symport or antiport process that uses a hydrogen ion (H+) i.e., proton motive force (pmf), or some other cation, i.e.,chemiosmotic potential, to drive the transport process – e.g. Lactose permease of E. coli is a single transmembranous polypeptide that spans the

Binding-protein dependent transport systems • BPDT is frequently used for sugars and amino acids • BPDT, such as the histadine transport system in E. coli, are composed of four proteins – Two proteins form a membrane channel that allows age of the histadine – A third protein resides in the periplasmic space where it is able to bind the amino acid and it to a forth protein which its the amino acid into the membrane channel – Going through the channel involves the expenditure of energy, provided by the hydrolysis of ATP

Bacterial transport systems: Group translocation systems

• Use energy during transport • Modify the solute during its age across the membrane • E.g. Phosphotransferase (pts) system in E. coli

Group translocation systems (GT) • More commonly known as the phosphotransferase system (PTS) in E. coli, are used primarily for the transport of sugars • Like BPDT systems, they are composed of several distinct components, but • GT systems specific for one sugar may share some of their components with other group transport systems

Group translocation systems (GT) cont’d • In E. coli, glucose may be transported by a group translocation process that involves the phosphotransferase system • The actual carrier in the membrane is a protein channel fairly specific for glucose • Glucose specifically enters the channel from the outside, but in order to exit into the cytoplasm, it must first be phosphorylated by the phosphotransferase system (PTS)

Group translocation systems (GT) cont’d • The PTS derives energy from the metabolic intermediate phosphoenol pyruvate (PEP) • PEP is hydrolyzed to pyruvate and glucose is phosphorylated to form glucose-phosphate during the process • Thus, by the expenditure of a single molecule of high energy phosphate, glucose is transported and changed to glucosephosphate

Types of carrier-mediated transport systems • A carrier is a protein (or group of proteins) that functions in the age of a small molecule from one side of a membrane to the other • A transport system may be: – a single transmembranous protein that forms a channel that its age of a specific solute – a coordinated system of proteins that binds and sequentially es a small molecule through the membrane

Specificity of transport system for the solute • Some transport a single solute with the same specificity and kinetics as an enzyme • Some transport (structurally) related molecules, although at reduced efficiency compared to their primary substrate • Most transport specific sugars, amino acids, anions or cations that are of nutritional value to the bacterium

Distinguishing characteristics of bacterial transport systems Property Carrier mediated Concentration against gradient Specificity Energy expended Solute modification

PD -

FD + -

IDT BPDT + + + +

GT + N/A

-

+ -

+ + pmf ATP -

+ PEP +

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

Membrane invaginations cont’d • Increase the surface area of membranes to which enzymes are bound for specific enzymatic functions • e.g. mesosomes, photosynthetic apparatus is located in such structures • Mesosomes may also represent specialized membrane regions involved in DNA replication and segregation, cell wall synthesis, or increased enzymatic activity.

Membranes cont’d • Some antibiotics (e.g. polymyxin), hydrophobic agents (e.g. bile salts), and proteins (e.g. complement) damage bacterial membranes

Representative periplasmic proteins • Detoxifying enzymes – Beta-lactamases (e.g. penicillinase) – Aminoglycoside-phosphorylating enzymes

Representative periplasmic proteins • Binding proteins – For amino acids (e.g. histadine, arginine) – For sugars (e.g. glucose, maltose) – For vitamins (e.g. thiamine, vitamin B12) – For ions (e.g. phosphate, sulfate)

Representative periplasmic proteins cont’d • Biosynthetic enzymes – For murein assembly (e.g. transglycosylases, carboxypeptidases, transpeptidases) – For fimbrial subunit secretion and assembly (e.g. chaperonins)

• Degradative enzymes – Phosphatases – proteases

Schematic view of the plasma membrane

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

Molecular composition of E. coli under conditions of balanced growth Molecule

% dry weight

Protein 55 Total RNA 20.5 DNA 3.1 Phospholipid 9.1 Lipopolysaccharide 3.4 Murein 2.5 Glycogen 2.5 Small molecules: precursors, metabolites, 2.9 vitamins, etc. Inorganic ions 1.0 Total dry weight 100.0

Small molecules present in a growing bacterial cell Approximate Molecule number of kinds •Amino acids, their precursors and 120 derivatives 100 •Nucleotides, their precursors and derivatives •Fatty acids and their precursors 50 •Sugars, carbohydrates and their 250 precursors or derivatives •quinones, porphyrins, vitamins, 300 coenzymes and prosthetic groups and their precursors

Inorganic ions present in a growing bacterial cell Ion K+ NH4+ Ca++ Fe++ Mg++ Mn++

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

Inorganic ions present in a growing bacterial cell cont’d Ion Cu++ Mo++ Ni++ Zn++ SO4-PO4---

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