FUNDAMENTALS OF MICROBIOLOGY

Lesson 11. CELL WALL, CYTOPLASMIC MEMBRANE, MEMBRANE TRANSPORT SYSTEMS

Module 4. Structure and functions of prokaryotic cells

Lesson 11

CELL WALL, CYTOPLASMIC MEMBRANE, MEMBRANE TRANSPORT SYSTEMS

11.1 Bacterial Cell Wall

The bacterial cell wall is a unique structure which surrounds the cell membrane. Although not present in every bacterial species, the cell wall is very important as a cellular component. Structurally, the wall is necessary for:

• Maintaining the cell's characteristic shape- the rigid wall compensates for the flexibility of the phospholipid membrane and keeps the cell from assuming a spherical shape.

• Countering the effects of osmotic pressure- the strength of the wall is responsible for keeping the cell from bursting when the intracellular osmolarity is much greater than the extracellular one.

• Providing attachment sites for bacteriophages - teichoic acids attached to the outer surface of the wall are like landing pads for viruses that infect bacteria.

• Providing a rigid platform for surface appendages - flagella, fimbriae, and pili all emanate from the wall and extend beyond it

All the members of domain Bacteria, with the exception of the genera Mycoplasma, Ureaplasma, Spiroplasma, and Anaeroplasma contain cell walls.

11.2 Chemical Composition of Bacterial Cell wall

Cell walls are chemically peptidoglycans i.e. peptides (short amino acids chains) and glycans (sugars); peptidoglycans are also known as mureins, mucopeptide (Fig. 11.1).

11.2.1 Gram positive bacterial cell wall

The cell wall of Gram positive bacteria is as thick as 20-80 nm and chemically contains mostly peptidoglycan (>50%). Proteins and other molecules diffuse freely into and rough the peptidoglycan (Fig. 11.2)

11.2.1.1 Glycans

These are modified sugars viz. N-acetyl muramic acid (NAM or M) and N-acetly glucose amine (NAG or G). M and G are linked to each other by a beta 1, 4 glycosidic bond and alternate to form the wall backbone. Lysozyme (an enzyme produced by organisms that consume bacteria, and normal body secretions such as tears, saliva, and egg white = protect against would-be pathogenic bacteria) digests beta 1, 4 glycosidic bonds. It growing or non growing cells but cell wall-less microbes are not affected high osmotic pressure in high solute concentrations prevents lysis of Gram positive and Gram negative cells when treated with lysozyme. As such, it has mild antibacterial action and indeed was one of the first antibiotics studied by Sir Fleming, the discoverer of penicillin.

11.2.1.2 Peptides

Short peptides (4 amino acids, tetrapeptides) are attached to M. Some of the amino acids are only found in cell walls and not in other cellular proteins (D- amino acids, eg D-alanine and diaminopimelic acid, DAP). Tetrapeptides chains are crosslinked (interlinked) by a peptide bridge (the carboxyl group of one tetrapeptide with an amino group of an adjacent (direct interbridge) or a different tetrapeptide chain (indirect interbridge). Transpeptidase enzyme builds peptide bridges in actively dividing cells; penicillin binds to it, stopping cell wall synthesis. Autolysins restructure and reshape cell walls by breaking specific bonds in the peptidoglycan in actively growing cells. Cell wall synthesis stops but cell degrading enzymes still function resulting in weakened cell walls and ultimately death. Glycans and peptides therefore form a single, large and strong cross- linked molecule in a form of a multilayered sheet, (sacculus, Latin = little sac) that surrounds the entire bacterial cell.

11.2.1.3 Teichoic acid

It consists of glycerol, phosphates and sugar alcohol, ribitol; occurs in polymers of 30 units long; extends beyond the cell wall and its functions include:

• Attachment sites for phages
• Binds protons and thus maintain cell wall at low pH which prevents autolysis by autolysins
• Teichuronic acid: formed when phosphate concentration low; help conserve phosphate essential for ATP, DNA and other cellular components
• Are phosphate chains of uronic acid and NAG

11.2.1.4 Synthesis of peptidoglycan

The peptidoglycan monomers are synthesized in the cytosol of the bacterium where they attach to a membrane carrier molecule called bactoprenol (Fig. 11.3). Bacterial enzymes called autolysins break both the glycosidic bonds at the point of growth along the existing peptidoglycan, as well as the peptide cross-bridges that link the rows of sugars together. Bactoprenol and transglycosidase enzymes then insert the new peptidoglycan monomers into the breaks in the peptidoglycan. Transglycosidase enzymes catalyze the formation of glycosidic bonds between the NAM and NAG of the peptidoglycan momomers and the NAG and NAM of the existing peptidoglycan. Finally, transpeptidase enzymes reform the peptide cross-links between the rows and layers of peptidoglycan to make the wall strong. During normal bacterial growth, bacterial enzymes called autolysins put breaks in the peptidoglycan in order to allow for insertion of peptidoglycan building blocks (monomers of NAG-NAM-peptide). These monomers are then attached to the growing end of the bacterial cell wall with transglycosidase enzymes. Finally, transpeptidase enzymes join the peptide of one monomer with that of another in order to provide strength to the cell wall. Penicillins and cephalosporins bind to the transpeptidase enzyme and block the formation of the peptide cross-links. This results in a weak cell wall and osmotic lysis of the bacterium.

11.2.2 Gram negative bacterial cell wall

The cell wall of Gram negative bacteria contains relatively thin (~10 nm) layer of peptidoglycan comprising only10 – 20 % of wall (Fig. 11.4).
Outside the peptidoglycan layer exists bilayered outer membrane which contains Phospholipids, protein,lipoprotein and Lipoplysaccharides. Its inner layer consists of phospholipid while outer layer is made of lipopolysaccharide (LPS or endotoxin). Functionally, outer membrane is a coarse molecular sieve and permeability to nutrients is partly due to Omp, called porins, which form cross-membrane channels through which some molecules can diffuse. Molecules with molecular weight 800 kDa, for E. coli and higher (3000-10000 kDa) for Pseudomonas neisseria can pass through outer membrane. Pores in porin don’t allow molecules as large as proteins to cross. The outer membrane excludes external protein from the periplasm and keeps proteins secreted by cell to periplasm . It also protects peptidoglycan from Lysozyme and antibiotic by keeping them out. Bacterial lipopolysaccharides are toxic to animals. When injected in small amounts endotoxins activate macrophages to produce pyrogens, activate the complement cascade causing inflammation, and activate blood factors resulting in intravascular coagulation and hemorrhage. Endotoxins may play a role in infection by any Gram negative bacterium. The toxic component of endotoxin is lipid A. The O-specific polysaccharide may provide ligands for bacterial attachment and confer some resistance to phagocytosis. Variations in the exact sugar content of the O polysaccharide (also referred to as the O antigen) accounts for multiple antigenic types (serotypes) among Gram negative bacterial pathogens. Therefore, even though lipid A is the toxic component in lipopolysaccharide, it contributes to virulence of Gram negative bacteria.

A bacterial surface component plays an indispensable role in the pathogenesis of infectious disease. Bacterial surface structures may act as:

• Permeability barriers that allow selective passage of nutrients and exclusion of harmful substances (e.g. antimicrobial agents);
• Adhesins used to attach or adhere to specific surfaces or tissues;
• Enzymes to mediate specific reactions on the cell surface important in the survival of the organism;
• Protective structures against phagocytic engulfment or killing;
• Antigenic disguises;
• Sensing proteins that can respond to temperature, osmolarity, salinity, light, oxygen, nutrients, etc., resulting in a molecular signal to the genome of the cell that will cause expression of some determinant of virulence (e.g. an exotoxin).

In medical situations, the surface components of bacterial cells are major determinants of virulence for many pathogens. Pathogens can colonize tissues, resist phagocytosis and the immune response, and induce inflammation, complement activation and immune responses in animals by means of various structural components.

Table 11.1 Functions of the outer membrane components of escherichia coli

11.2.3 Archaeal cell walls

Archaeal cells have more variations in their cell wall chemistries, and some do not contain cell walls (e.g. Thermoplasma). Methanobacterium sp. contains glycans (sugars) and peptides in their cell walls. Glycans are modified sugars viz. N-acetyl talosaminouronic acid (NAT or T) and N-acetly glucose amine (NAG or G). T and G are linked to each other by a beta 1, 3 glycosidic bond and alternate to form the cell wall backbone. Lysozyme (an enzyme produced by organisms that consume bacteria, and normal body secretions such as tears, saliva, and egg white; protect against would-be pathogenic bacteria) cannot digest beta 1, 3 glycosidic bonds. Peptides are short peptides attached to T. The amino acids are only of the L-type. Penicillin is ineffective in inhibiting the cell wall peptide bridge formation. Some examples of unique cell wall composition are given below:

• Methanosarcina sp. cell walls contain non-sulfated polysaccharides
• Halococcus sp. contain sulfated polysaccharides similar to Methanosarcina sp.
• Halobacterium sp. contain negatively charged acidic amino acids in their cell walls which counteract the positive charges of the high Na+ environment. Therefore, cells lyse in NACl concentrations below 15%
• Methanomicrobium sp. and Methanococcus sp. cell walls are exclusively made up of proteins subunits.

11.3 Periplasmic Space

It is the region between cell membrane and outer membrane/peptidoglycan, an area where diverse reaction occurs - osmoregulation, solute transport, protein secretion and hydrolytic activities. The term gel implies that peptidoglycan may actually fill the region between cell membrane and outer membrane. Several proteins are found e.g. binding proteins (transport), chemoreceptors (chemotaxis), hydrolytic enzymes (transportation of small products). It also stores enzymes like alkaline phosphatase which degrade incoming DNA from other bacteria. It contains oligosaccharides that help in adjustment with change in osmolarity of medium. The periplasmic space is 20 to 40% of total volume of cells grown under typical conditions.

Entry of sugars into periplasmic space: The outer membrane is penetrated by water-filled pores. Each pore consists of a trimer of porin and has a diameter of 1.5-2.0 nm. Substances of molecular weight less than 650 may diffuse rapidly through the pore (if hydrophilic). Substances of molecular weight greater than 900-1000 are excluded. The porins are arranged on the outer surface of the mucopeptide in a regular array and are mostly beta-sheet. They are tightly, but non-covalently, bound to the mucopeptide. Vesicles, reconstituted from lipid, LPS and porins show the same diffusion properties as an intact cell. E. coli K-12 has two porins, coded by genes ompC and ompF. (omp = Outer Membrane Protein). A regulatory gene ompB controls the expression of the ompC and F proteins, for example, when osmotic pressure increases, ompF decreases and ompC increases. Somehow, ompB detects changes in osmotic pressure. Both ompC and ompF act as receptors for certain bacteriophages.

11.4 Plasma Membrane

Plasma membrane (cytoplasmic membrane) is a biological membrane that separates the interior of all cells from the outside environment. The cell membrane is selectively-permeable to ions and organic molecules and controls the movement of substances in and out of cells.

11.4.1 Functions of plasma membrane

The plasma membrane retains the cytoplasm, particularly in cells without cell walls, and separates (the cell) from the surroundings. The plasma membrane of prokaryotes and eukaryotes are functionally equivalent, though the prokaryote plasma membrane additionally serves in roles that eukaryotes reserve for internal membranes. Various functions of the prokaryotic plasma membrane are as follows:

• Osmotic or permeability barrier
• Location of transport systems for specific solutes (nutrients and ions)
• Energy generating functions, involving respiratory and photosynthetic electron transport systems, establishment of proton motive force, and transmembranous, ATP-synthesizing ATPase
• Synthesis of membrane lipids (including lipopolysaccharide in Gram-negative cells)
• Synthesis of murein (cell wall peptidoglycan)
• Assembly and secretion of extracytoplasmic proteins
• Coordination of DNA replication and segregation with septum formation and cell division
• Chemotaxis (both motility per se and sensing functions)
• Location of specialized enzyme system

11.4.2 Structure of plasma membrane

The plasma membrane is approximately 7.5 nm (0.0075 μm) thick, forms the limiting boundary of the cell and is made up of phospholipids (about 20-30%) and proteins (about 60-70%) as illustrated in Fig. 11.5. Several models have been proposed to explain the ultra-structure of the plasma membrane; the most widely accepted one is Fluid Mosaic Model introduced by Singer and Nicolson (1974) as depicted in Fig.11.6. According to this model the membrane is a bi-layer of phospholipids and the two opposing layers of phospholipids overlap slightly; each phospholipid molecule consisting of a phosphate group and a lipid. Each phospholipid is structurally asymmetric with polar and nonpolar ends and is called amphipathic (Fig. 11.7). The polar ends interact with water and are hydrophilic; the nonpolar ends do not interact with water (i.e. insoluble in water) and are hydrophobic. The hydrophilic ends occur towards the outer surface of the membrane whereas the hydrophobic ends are burried in the interior away from the surrounding water. The arrangement of hydrophilic heads and hydrophobic tails of the lipid bilayer prevent polar solutes (e.g. amino acids, nucleic acids, carbohydrates, proteins, and ions) from diffusing across the membrane, but generally allows for the passive diffusion of hydrophobic molecules. This affords the cell the ability to control the movement of these substances via transmembrane protein complexes such as pores and gates.

Fig. 11.7 Structure of lipid bilayer and phospholipids

Unsaturated fatty acids remain liquid at low temperatures and become denatured as the temperatures increase, however saturated fatty acids are more stable than unsaturated fatty acids at high temperatures, e.g. butter is solid at room temperature. The membranes of psychrophilic (cold-loving) bacteria have high content of polyunsaturated fatty acids. Thermophilic bacteria use mainly saturated fatty acids, otherwise their membrane would be too soft to maintain cell structure and function at high temperature.

The bi-layer phospholipid is interrupted by proteins which are distributed in a mosaic-like pattern (Fig. 11.4). Some of the proteins are confined to the outer surface of bilipid layer (extrinsic or peripheral proteins) and others are partially or totally buried within it (intrinsic or integral proteins). The integral proteins, like membrane lipids, are amphipathic. Their hydrophobic regions are buried in the lipid while the hydrophilic regions project out from the plasma membrane surface.

The cell membrane plays host to a large amount of protein that is responsible for its various activities. The amount of protein differs between species and according to function, however the typical amount in a cell membrane is 50%. Proteins are in dynamic state and distribution is according to the fluid mosaic model. On the basis of their location and interaction in plasma membrane these proteins can be broadly termed as integral, lipid anchored and peripheral proteins and can be categorized in following six groups on the basis of their functions:

• Transport proteins
• Receptor proteins
• Enzymatic proteins
• Cell recognition proteins
• Attachment proteins
• Intercellular junction proteins

Sterols are not present in bacteria but are present in cell wall less bacteria (Mycoplasma, Ureaplasma, Spiroplasma, Anaeroplasma) - required for growth, provide stability e.g. sterols. Poylene antibiotics (e.g. nystatin, candicidin) inhibit growth by interacting with sterols and destabilizing eukaryotic and cell wall-less bacterial membranes (but do not inhibit growth of cell wall containing bacteria). Often carbohydrates are attached to the outer surface of plasma membrane proteins and seem to perform important functions. Both proteins and lipids move within the phospholipid matrix of the membrane. However, many bacterial plasma membranes do contain pentacyclic sterol-like molecules called hopanoids which are synthesized from the same precursors as steroids. Like steroids in eukaryotic cells, hopanoids are thought to provide stability to bacterial plasma membrane.

11.5 Differences with Eukaryotic Plasma Membrane

Although the bacterial plasma membrane resembles its counterpart of eukaryotic cells, it differs from the latter in two distinctive features:

1. Sterols (such as cholesterols) that occur in eukaryotic cell membranes are absent in bacteria (except in the mycoplasmas that do not have cell wall). These substances help stabilize the phospholipids in eukaryotic membrane and make it more rigid.
2. The proportion of protein to phospholipid is high (typically 2:1 in prokaryotes, and 1:1 or less in eukaryotes).

11.6 Membrane Transport Systems

There are four basic types of transport systems (Fig.11.8).

11.6.1 Passive diffusion

Passive diffusion is the net movement of gases or small uncharge polar molecules across a phospholipid bilayer membrane from an area of higher concentration to an area of lower concentration. Examples of gases that cross membranes by passive diffusion include N2, O2, and CO2; examples of small polar molecules include ethanol, H2O, and urea.

11.6.2 Facilitated diffusion

The rate of diffusion across selectively permeable membranes is greatly increased by the use of carrier proteins, sometimes called permeases, which are embedded in the plasma membrane. Since the diffusion process is aided by a carrier, it is called facilitated diffusion. The rate of facilitated diffusion increases with the concentration gradient much more rapidly and at lower concentrations of the diffusing molecule than that of passive diffusion.

11.6.3 Active transport

Active transport is the transport of solute molecules to higher concentrations, or against a concentration gradient, with the use of metabolic energy input. In active transport the target is not altered and a significant accumulation occurs in the cytoplasm with the inside concentration reaching many times its external concentration. Active transport proteins are molecular pumps that pump their substrates against a concentration gradient. As in all pumps, fuel is necessary and in the case of cells, this fuel comes in two forms, ATP or the proton motive force (PMF). Ion driven transport systems (IDT) and binding-protein dependent transport systems (BPDT) are active transport systems that are used for transport of most solutes by bacterial cells. The former is driven by PMF while ATP is used to drive later system. IDT is used for accumulation of many ions and amino acids; BPDT is frequently used for sugars and amino acids.

11.7 Group Translocation

In this system, a protein specifically binds the target molecule and during transport a chemical modification takes place. No actual concentration of the transported substance takes place, because as it enters the cell, it becomes chemically different. The best known group translocation system is the phosphoenolpyruvate: sugar phosphotransferase system (PTS), which transports a variety of sugars into prokaryotic cells, while simultaneously phosphorylating them using phosphoenolpyruvate (PEP) as the phosphate donor. Table 11.2 enlists the distinct features of various prokaryotic transport systems.

Table 11.2 Distinguishing characteristics of bacterial transport systems