BACTERIAL STRUCTURE, FUNCTION AND GROWTH

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1 Dr. Ronald E. Gill Wednesday, February 15, :00 11:50 AM BACTERIAL STRUCTURE, FUNCTION AND GROWTH I. Introduction Bacteria are unicellular organisms with a relatively simple structure. Unlike the cells of higher organisms (eukaryotes), bacteria do not have a nuclear membrane and therefore their genetic material lies within their cytoplasm. Bacteria have 70S instead of 80S ribosomes, and they do not have an endoplasmic reticulum. In addition, bacteria do not have a mitotic spindle, mitochondria or chloroplasts. All cells, including bacteria, consist of the same chemical elements and share many basic biochemical units, such as amino acids, nucleotides, fatty acids, vitamins, etc, and they also share a number of closely related biochemical pathways for energy metabolism and biosynthesis. Nevertheless, bacteria display great diversity with respect to their minimal nutritional requirements for growth, the range of nutrient sources they can use, and the ways that they derive energy from the nutrients they consume. Bacterial growth is a highly regulated process in which numerous controls are imposed on metabolism by the nutritional environment as well as by intracellular regulatory circuits. An understanding of bacterial structure, function and growth is clinically important because it provides the basis of appropriate methods for collecting, transporting, cultivating, isolating, and identifying bacterial pathogens and for developing and using antimicrobial agents to treat infectious diseases caused by pathogenic bacteria. Bacterial Cell Eukaryotic Cell

2 II. Structure and Function of Bacterial Components A. The bacterial cell wall and cell surface structures. 1. Most bacteria have a rigid cell wall external to the cytoplasmic membrane that contains peptidoglycan. Gram-positive bacteria and gram-negative bacteria have different cell wall structures and react differently to the Gram staining procedure that you will perform in the laboratory. The rigidity of the cell wall is essential for resisting osmotic lysis (the internal osmotic pressure is typically ~5 atmospheres for gram-negative species and ~20 atmospheres for gram-positive species) and for maintaining cell shape. 2. Each bacterial isolate typically has a characteristic, rigid shape. 3. Bacterial shape is determined both by intracellular cytoskeletal elements and by rigid components of the cell wall. a. Bacteria have cytoskeletal elements that play essential roles in establishing cell shape. The conformation of FtsZ resembles that of tubulin in eukaryotes; MreB and ParM resemble actin in eukaryotes; and CreS (crescentin) appears to function like intermediate filament proteins. The typical intracellular localization of each type of protein is illustrated:

3 b. The peptidoglycan layer forms a rigid mesh that surrounds the cytoplasmic membrane. Peptidoglycan consists of a polymer with repeating units of two hexose sugars, N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc). The MurNAc residues are linked to tetrapeptide chains that contain amino acids found only in bacterial cell walls (e.g., meso-diaminopimelic acid [DAP], D-glutamic acid and D-alanine). The tetrapeptides are cross-linked from one chain (via DAP in gram-negative bacteria or L-lys in gram-positive bacteria) to D-ala on another chain, and cross-linking in gram-positive bacteria occurs via an intervening peptide such as pentaglycine in Staphylococcus aureus. The extent of cross-linking of peptidoglycan chains is typically much greater in gram-positive bacteria than in gram-negative bacteria. The enzyme lysozyme, which is present in many body secretions and which contributes to innate host defenses against bacteria, hydrolyzes peptidoglycan by specific cleavage of the glycosidic bond between MurNAc and GlcNAc. 4. Gram-negative bacteria have a thin, sparsely cross-linked peptidoglycan layer and other major components that are located exterior to the peptidoglycan. Grampositive bacteria have a thick, extensively cross-linked peptidoglycan layer that also contains teichoic acids. Gram-positive cell surface Gram-negative cell surface

4 a. The outer membrane (OM) of gram-negative bacteria is a lipid bilayer that contains lipopolysaccharide (LPS), lipoproteins (which are linked covalently to the peptidoglycan), and porins (which form transmembrane channels permitting diffusion across the membrane of hydrophilic molecules <600 MW), other membrane proteins, and phospholipids. The OM is a barrier to entry of some antibiotics and also protects the cell against the action of detergents and other toxic compounds. b. The LPS of gram-negative bacteria is located exclusively in the outer leaflet of the outer membrane, and the inner leaflet consists of phospholipids. LPS contains Lipid A (the toxic component of endotoxin), core polysaccharide, and O side chain oligosaccharides that function as somatic antigens (O antigen), c. Teichoic acids of gram-positive bacteria have a repeating polyglycerol-p or polyribitol-p backbone substituted with other molecules (sugars, aminosugars, D-alanine), and they are covalently attached to the peptidoglycan layer. Lipoteichoic acids are attached to the underlying cytoplasmic membrane and help to anchor the cell wall to the membrane. 5. Other cell surface structures that are found on many bacteria include: a. Capsules are firmly attached, gelatinous outer surface layers that usually consist of complex polysaccharides (although the capsule of Bacillus anthracis is a polymer of D-glutamic acid), and coats the surface of individual cells. Capsules often enhance virulence by enabling the encapsulated bacteria to resist phagocytosis. Most capsular polysaccharies are antigenic, and some are used as components of vaccines to prevent specific bacterial infections (e.g., in the protein-polysaccharice conjugate vaccines used to immunize against Streptococcus pneumoniae or Hemophilus influenzae type b). b. An extracellular glycocalyx, responsible for the formation of microbial biofilms is relatively common feature of pathogenic and environmental bacteria. Many (perhaps most) bacteria exist in their natural ecosystems (e.g., soil, intestinal, urinary tract) in communities called biofilms. Within the biofilm, the community bacterial cells are embedded within an adherent, irregular, diffuse polysaccharide/glycoprotein matrix or slime layer. Biofilms allows the bacteria to adhere to inert surfaces (e.g., rocks, teeth, indwelling catheters), to host cells (e.g. Streptococcus pneumoniae attachment to lung cells) or to other bacteria (e.g., within complex, multi-species communities). It also protects cells from phagocytosis, host defensive proteins (complement, antibody, defensins), and serves to limit access of many antimicrobial agents to the embedded cells. b. Flagella are appendages originating in the cytoplasmic membrane that function as organs of motility. Some bacteria have flagella distributed over their surface (peritrichous); others may have one or several flagella at one end of the cell (polar). Bacterial

5 chemotaxis (movement toward attractive nutrients or away from toxic substances) involves the control of flagellar rotation (counterclockwise results in swimming; clockwise results in tumbling). Motile bacteria that exhibit chemotaxis spend more time swimming and less time tumbling when attractants or repellents are present, resulting in directed motion. Most flagella are antigenic, and the H antigens used for classification of enteric bacteria are flagellar antigens. c. Pili (also known as fimbriae) are long, slender, proteinaceous, antigenic, hair-like structures on the surface of many bacteria. Pili often play a role in bacterial adherence to surfaces and tissues, and antibodies against pili may block adherence and confer resistance to infection. Sex pili that play a role in bacterial conjugation are found in small numbers on some bacterial cells. d. Specialized secretion systems are used to deliver proteins from the cytoplasm of the bacterium directly to a target cell. Particularly notable are the Type III, IV and VI secretory systems of certain (but not all) Gram-negative bacteria. These specialized secretory systems allow the microbe to inject effector proteins directly into a host cell, which then alters the cells function. These proteins have a wide range of effects inside the cell including altering the tubules, which determines function; altering the membranes and the signaling from the membranes; altering the tagging systems that allow the cell to destroy microbes; and direct alteration of DNA function to change proteins inside the cell, and create the microbe s own machinery. When they are present, these secretion systems may provide functions for the bacterium that play an essential role in its pathogenesis. B. The Cytoplasmic membrane (also called the inner membrane in gram-negative bacteria) is the anatomical and physiological barrier between the inside and outside of the bacterial cell. It is a lipid bilayer made up primarily of phospholipids and proteins, but unlike plasma membranes of animal cells it usually contains no sterols and has a much higher content (60-70%) of proteins. 1. The cytoplasmic membrane exhibits selective permeability. It is essentially impermeable to all charged substances, even H +. Only hydrophobic molecules or uncharged molecules no larger than glycerol can diffuse through it. Thus, essential metabolites are not readily lost from the cytoplasm. 2. The electron transport system, which is the principal source for generating the proton motive force during respiration in bacteria, is located in the cytoplasmic membrane. Other functions of the cytoplasmic membrane include transport of metabolites into the cytoplasm, biosynthesis of lipids and other cell envelope components, certain aspects of DNA replication, and flagellar rotation. C. The cytoplasm consists of an aqueous solution of proteins and metabolites and is the site where many metabolic processes occur. Principal intra-cytoplasmic structures include: 1. Ribosomes. Bacterial 70S ribosomes are closely related to the 70S ribosomes of mitochondria from eukaryotes, but they are less closely related to the 80S cytoplasmic ribosomes from eukaryotes. Protein synthesis occurs on the ribosomes. Polyribosomes are formed by the interaction of several ribosomes with a single messenger RNA. Bacterial mrnas may by polycistronic (e.g., encode more than one protein product). 2. The Nucleoid. The DNA of bacteria is located within a distinct region of the cytoplasm known as the nucleoid or nuclear body. The DNA is tightly packed and supercoiled, and there is no nuclear membrane surrounding the nucleoid. The older name prokaryote referred to this primitive nuclear structure. The name prokaryote is outdated as a taxonomic term because members of the bacteria and the archea, which constitute different biological kingdoms, both lack nuclear membranes. Because there is no nuclear membrane, transcription and translation can occur as coupled processes in bacteria. Several different genetic elements can contribute to the bacterial genome, including:

6 a. The chromosome often consists of a single, double-stranded, circular DNA molecule with a contour length hundreds to thousands of times greater than the longest dimension of the bacterium. Some bacterial chromosomes are linear, and some bacteria have more than one chromosome. Cytoskeletal components appear to function as a primitive mitotic apparatus during bacterial cell division. b. Plasmids are extra-chromosomal, self-replicating DNA molecules, much smaller than bacterial chromosomes, and they are usually not essential for bacterial viability. Plasmids in pathogenic bacteria often encode virulence factors. Plasmids called R factors carry genes that determine resistance to antibiotics in many pathogenic bacteria. c. Bacteriophages (phages) are viruses that infect bacteria. The DNA genomes of temperate bacteriophages can integrate into bacterial chromosomes and replicate as part of those chromosome. Temperate bacteriophages often carry genes that encode bacterial toxins, other bacterial virulence factors or resistance to antibiotics. Phage conversion is defined as a change in the phenotype of a host bacterium as a consequence of expression of a gene that is encoded by a bacteriophage within the host bacterium (e.g., production of diphtheria toxin by isolates of Corynebacterium diphtheriae harboring a prophage that carries a gene encoding the toxin). III. Bacterial Growth and Nutrition A. In a nutritionally complete medium, a bacterial cell grows larger and eventually divides by binary fission to form two cells of nearly equal size. Each of these grows to about twice its original size or mass, and divides again. The products of the division are physiologically the same each is equally young. Unlike the cells of higher life forms, there is no aging process in bacteria that leads inevitably to senescence and cell death. Therefore, the concept of age for a bacterium applies only to its progression through the cycle of cell division. If the environment remains favorable, bacterial cells are capable of unlimited growth and division. B. The bacterial growth curve typically reveals at least three and sometimes four distinct phases. 1. An initial lag phase is a period of physiologic adjustment for the starting cells, or inoculum, involving the induction of new enzymes and the establishment of a proper intracellular environment for optimal growth in the new medium.

7 2. During the exponential (logarithmic) phase of growth, the rate of increase in cell number/cell mass is proportional to the cell number/cell mass already present. A constant interval of time (ranging from about 20 minutes up to about 1 day) is required for doubling of cell number/cell mass, and this interval is termed the generation time (doubling time). During exponential growth, the rate of cell division is maximal for the available nutritional conditions. 3. The stationary phase occurs as essential nutrients are consumed and toxic products of metabolism accumulate. Cell growth may slow dramatically or cease, and growth that occurs is balanced by cell death. Such non-growing or slow-growing cells may exhibit markedly increased resistance to antibiotics such as penicillin or other β-lactam antibiotics that act on growing cells. In nature, bacteria probably spend most of their time in stationary phase. 4. Some bacterial species remain viable for long periods of time in stationary phase, but others are less hardy. If a death phase occurs, the number of viable bacteria will decrease over time. If spontaneous cell lysis (autolysis) occurs, the mass of intact bacteria in the culture will also decrease. C. Bacterial Nutrition. Nutrition may be broadly defined as the provision of proper environmental conditions for promoting the growth of bacteria. This includes factors such as nutrients, ph, temperature, aeration (O 2 tension), salt concentration, and osmotic pressure. 1. Minimal requirements for growth. Most bacteria require a nutrient medium that contains several inorganic ions (NH 4 +, PO4 =, SO4 =, K +, Mg ++, Fe ++, etc.) plus sources of carbon and energy. Bacteria that require an organic carbon source (including most bacterial pathogens) are heterotrophic; bacteria that obtain their carbon exclusively from CO 2 are autotrophic. Many bacterial pathogens are deficient in one or more biosynthetic pathways. Such bacteria (often called fastidious bacteria) require, in addition to sources of carbon and energy, a number of essential growth factors such as amino acids, vitamins, purines, pyrimidines and inorganic ions. They are typically grown in rich, complex growth media. Some bacterial pathogens are obligate intracellular bacteria that can grow within eukaryotic cells but cannot be cultivated on artificial media. 2. Bacterial responses to oxygen. Growth Response Type of Bacteria Aerobic Anaerobic Comment Example Aerobe (strict aerobe) + - Anaerobe (strict anaerobe) * - + Requires oxygen; cannot ferment Killed by oxygen; fermentative metabolism Mycobacterim tuberculosis Clostridium sp Bacteroides sp Indifferent (aerotolerant anaerobe) + + Ferments in presence or absence of O 2 Streptococcus pyogenes Facultative (facultative anaerobe) + + Respires with O 2 ; ferments in absence of O 2 Escherichia coli Staphylococcus aureus Microaerophilic ( + ) + ( + ) indicates small amount of growth Grows best at low O 2 concentrations; can grow without O 2 Campylobacter jejuni Organisms that grow in the presence of oxygen produce toxic oxygen metabolites, such as hydrogen peroxide and superoxide. Professional human phagocytes such as neutrophils and

8 macrophages use reactive oxygen species as defense mechanisms against ingested bacterial pathogens. Bacteria that can grow in the presence of oxygen usually produce catalase (or peroxidase) and superoxide dismutase (SOD) that protect them against toxic reactive oxygen species. Anaerobes that are frequently associated with disease tend to be more aeroterant than most strict anaerobes, and they may possess small amounts of catalase or SOD. 3. Bacterial Energy Metabolism. a. There are two forms of energy currency in bacteria and higher cells: ATP and electrochemical gradients (the proton motive force). ATP drives many biosynthetic reactions, and electrochemical gradients drive other functions like flagellar rotation and certain substrate transport systems. These two types of potential energy are interconvertible by the membrane ATPase. Bacteria also require reducing power in the form of NADH and NADPH to drive various metabolic interconversions. Heterotrophic bacteria obtain both energy and reducing power by subjecting nutrients to fermentation or respiration. b. In fermentation, organic compounds serve as both electron donors and electron acceptors, and no net oxidation of substrates occurs. Both anaerobic and facultative or indifferent bacteria grown under anaerobic conditions obtain energy by fermenting organic substrates. Indifferent organisms (aerotolerant anaerobes, see table above), obtain energy by fermentation under either anaerobic or aerobic conditions, because they are incapable of respiration. c. In respiration, many bacterial species, like the mitochondria of higher organisms, generate ATP through electron transport and use molecular oxygen as the final electron acceptor. In anaerobic respiration, certain bacteria may use inorganic substrates such as nitrate or nitrite as terminal electron acceptors instead of O Sporulation is a response to adverse nutritional conditions. Spores are specialized cells that are produced by certain bacteria, such as Clostridium sp. and Bacillus sp., when the nutritional supply of carbon, nitrogen or phosphorus is limited. During sporulation, these bacteria differentiate to form highly resistant, dehydrated forms (spores) that have no metabolic activity. Spores are adapted for prolonged survival under adverse conditions such as heat, drying, freezing, the presence of toxic chemicals, and radiation. When spores find themselves once again in a nutritionally satisfactory environment, they may convert back into vegetative cells through the process of germination. IV. Principles of Antibiotic Action A. Antimicrobial agents. Antimicrobials work on the principle of selective toxicity, namely the selective inhibition of microbial growth at drug concentrations tolerated by the host. Many aspects of microbial metabolism are very similar to those of eukaryotic organisms (including humans). However, there are some components of bacteria that are not present in eukaryotes or are sufficiently different from their counterparts in eukaryotes be effective as targets for antimicrobial agents. Some representative antimicrobials are arranged here according to their mode of action and basis of selective toxicity. 1. Cell wall-active antimicrobials. Selective toxicity is due to the lack of peptidoglycan in mammalian cells. a. β-lactams (penicillins, cepalosporins, etc) inhibit the final transpeptidation reaction in cross-linking of peptidoglycan. b. Vancomycin inhibits utilization of lipid-linked intermediate at an intermediate step in peptidoglycan synsthesis, e.g., elongation of the peptidoglycan chain. c. Cycloserine inhibits alanine racemase, preventing formation of muramyl pentapeptide, an early intermediate in peptidoglycan synthesis.

9 2. Outer and cytoplasmic membrane-active antimicrobials: Polymyxins are cationic surfactants that disrupt bacterial outer and cytoplasmic membranes. They are less active on mammalian cell membranes. 3. Inhibitors of protein synthesis at the ribosomal level. Selective toxicity is due to differences between bacterial and mammalian ribosomes. a. Aminoglycosides (including streptomycin, kanamycin, gentamicin, neomycin, tobramycin, amikacin, etc) bind to specific target proteins in the 30S ribosomal subunit and inhibit protein synthesis. b. Tetracyclines reversibly bind to the 30S ribosomal subunit and inhibit binding of aminoacyl trna. c. Chloramphenicol binds reversibly to the 50S ribosomal subunit and inhibits peptidyl transferase and peptide bond formation. d. Macrolides (such as erythromycin) and lincomycins (such as lincomycin and clindamycin) bind to the 23S ribosomal RNA of the 50S subunit and inhibit peptidyl transferase. 4. Inhibitors of nucleic acid synthesis. a. Quinolones inhibit DNA gyrase and topoisomerase and therefore interfere with DNA replication. b. Rifampicin inhibits RNA polymerase and interferes with the initiation of transcription. 5. Metabolic inhibitory antimicrobials. a. Sulfonamides are structural analogs of p-aminobenzoic acid (PABA), which is a component of folic acid. Enzymes that use folic acid derivatives as coenzymes are needed for one-carbon transfer reactions in the synthesis of many compounds, including thymidine and purines. Sulfonamides inhibit the formation of folic acid by competing with PABA, and this in turn prevents nucleic acid synthesis. The inhibition is selective because only bacteria, and not the host, possess enzymes for making folic acid (we get ours from the bacteria), whereas bacteria, in contrast to human cells, cannot utilize pre-formed folic acid. b. Trimethoprim also interferes with folate metabolism by inhibiting the enzyme dihydrofolate reductase. Since both bacterial and host cells both possess this enzyme, the basis of selective toxicity lies in the 50,000-fold greater sensitivity of the bacterial enzyme to this drug. c. Isoniazid inhibits lipid synthesis (probably mycolic acid synthesis) in susceptible Mycobacteria. metabolism. d. Metronidazole appears to specifically interfere with anaerobic

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