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INTRODUCTION

In higher organisms there is a distinction between physiology and biochemistry. Physiology is the study of the relation between structure and function, e.g. how does the mammalian intestine fulfill its function of digesting food? Biochemistry is the study of life processes at the molecular level. Because bacteria are small, single-celled organisms with no distinct organs or tissues there is no valid distinction between anatomy, physiology, and biochemistry. For example, the cell wall of most bacteria consists of a single peptidoglycan molecule and bacterial nutrition involves uptake of discrete molecules, not large lumps of food.

PROKARYOTES VERSUS EUKARYOTES

Living cells are divided into two major types - PROKARYOTES and EUKARYOTES, on the basis of their genetic organization. Higher organisms together with microorganisms such as fungi, algae and protozoa are eukaryotes. Bacteria (including the blue green "algae") are prokaryotes and comprise the subject of this course.

In eukaryotes the genetic information is separated from the rest of the cell in a membrane bound compartment - the nucleus. In prokaryotes the DNA is free in the cytoplasm. The presence or absence of a separate nucleus defines the difference between the prokaryotic and eukaryotic cell. The presence of a separate nucleus allows eukaryotes to accumulate much more DNA per cell than prokaryotes. Consequently eukaryotic cells are usually much larger than prokaryotes. In addition the DNA of eukaryotes is generally partitioned among several chromosomes. The greater genetic complexity of eukaryotes facilitates the differentiation of cells into the complex tissues and organs seen in higher organisms.

The presence of a nucleus brings problems as well as advantages. The major problem is to divide the genome equally upon cell division. This is performed by the complex process of mitosis. Mitosis involves the temporary dissolution of the nuclear membrane and the creation of the mitotic spindle. The spindle is made up of microtubules projecting from structures known as centrioles. Between cell divisions, microtubules, with associated microfilaments, form an internal scaffolding system for the eukaryotic cell - the cytoskeleton. The centrioles also function as the basal bodies of eukaryotic flagella and cilia.

Eukaryotic cells contain several membrane-bound organelles and membrane systems. By increasing the surface to volume ratio, these allow eukaryotic cells to be larger. Note that growth of a cell is limited by the surface area available to take up oxygen, nutrients, etc. [Many bacteria have infoldings of the cell membrane known as mesosomes. These may be involved in cell division, though they are poorly characterized and have had almost every possible function attributed to them at some time.] The two most prominent organelles of eukaryotes are the mitochondria and chloroplasts. Both possess their own DNA, their own ribosomes, which are of the prokaryotic 70S type, and are in several respects semi-autonomous. New mitochondria and chloroplasts arise only by the division of pre-existing mitochondria or chloroplasts. Thus these organelles are in some ways equivalent to prokaryotic cells. The endosymbiont theory proposes that mitochondria and chloroplasts evolved from symbiotic bacteria which were trapped inside larger eukaryotic cells and lost their independence.

	PROKARYOTES	EUKARYOTES
a) Size	1-10 microns	10-100 microns
b) Complexity	unicellular, rarely small clusters or filaments	sometimes unicellular more often multicellular
c) Membrane bound organelles	none (mesosome is infolding of cytoplasmic membrane)	nuclei, mitochondria, chloroplasts, lysosomes, endoplasmic reticulum, golgi, & vacuoles
d) Nucleus	no	yes
e) Chromosomes	single & circular	usually several & linear
f) Introns & Exons	occasionally	frequent
g) Histones	no	yes
h) Ploidy	haploid	diploid
i) Mitosis & Meiosis	absent	present
j) Sexual reproduction	none, or unidirectional from donor to recipient	usually, involves fusion of haploid gametes
k) Ribosomes	70s (50s + 30s subunits)	80s (60s + 40s) in cytoplasm (mitochondria & chloroplasts have prokaryotic ribosomes)
l) Cytoskeleton	absent	microtubules and microfilaments
m) Cell wall	usually present, contains peptidoglycan	absent in animals present in fungi (chitin) & plants (cellulose)
n) Motility	simple, prokaryotic, flagella, gliding motion	complex "9+2" flagella or cilia with centrioles
o) Endocytosis & cytoplasmic streaming	absent	present
p) Differentiation	usually absent	cells differentiate to form tissues & organs
q) Energy metabolism	many diverse pathways in various bacteria	glycolysis in cytoplasm, Krebs Cycle and ETC in mitochondria
r) Oxygen	aerobic and/or anaerobic	usually aerobic
s) Sterols	usually absent	used as hormones and in plasma membrane

Differentiation is mostly confined to eukaryotes, however, there are a few prokaryotic examples. Some blue-green bacteria form filaments in which certain cells are specialized to fix nitrogen. These differentiated cells are called heterocysts. Differentiation is also seen in Caulobacter and Hyphomicrobium., both of which have cell cycles in which two cell types alternate: motile, with flagella, and non-motile, without flagella.

While eukaryotes show much greater cellular differentiation and structural diversity than prokaryotes, the converse is true of metabolism. Eukaryotes are very stereotyped metabolically. Prokaryotes are diverse biochemically and possess many pathways not found in eukaryotes, e.g. nitrogen fixation, methane production and anaerobic respiration.

STRUCTURE OF BACTERIA

Since bacteria contain no internal organelles except ribosomes and the bacterial chromosome, the structure of the bacterial cell is largely the structure of its surrounding layer - the cell envelope, together with the appendages (flagella and pili) which project outwards from this layer. There are two major forms of cell envelope structure, gram-positive and gram-negative. Originally these two classes of bacteria were recognized by a staining reaction. However, this difference in staining is due to fundamental differences in envelope structure and chemical composition.

Gram-positive envelope: Thick cell wall with multiple peptidoglycan layers. No outer membrane, periplasmic space or lipopolysaccharide. They possess long chain teichoic acids intertwined among the peptidoglycan.

Gram-negative envelope: Single thin peptidoglycan layer, but no teichoic acids. Possess an outer membrane containing lipopolysaccharide and a periplasmic space between the outer and inner membranes.

Peptidoglycan (Also called mucopeptide or murein): Long chains of a polysaccharide consisting of alternate residues of N-acetyl-glucosamine (NAG) and N-acetyl-muramic acid (NAM) are crosslinked by short chains of amino acids. The amino acids side chains are attached to the NAM residues and some of the amino acids are in the D-configuration (i.e. opposite to that found in proteins). In E. coli and most gram-negative bacteria there is a single molecular layer of peptidoglycan. In Staphylococcus aureus and most gram-positives there are many layers of peptidoglycan forming a thick wall. In gram-positives some of the teichoic acid is covalently attached to the peptidoglycan. In gram-negatives the peptidoglycan is covalently attached to lipoprotein molecules which project into the outer membrane.

Teichoic acids: Teichoic acids are only found in gram-positive bacteria. They are polymers in which glycerol or ribitol residues alternate with phosphate groups. The glycerol or ribitol residues may carry amino acid and/or sugar substituents. Some teichoic acids are attached to the peptidoglycan and extend throughout the cell wall layer. Other teichoic acids are covalently attached to lipids in the cytoplasmic membrane - so called "lipoteichoic acids". They are immunogenic but their function is unclear.

Outer Membrane (OM): Found in gram-negatives only. Provides an outer diffusion barrier to molecules greater than 700 to 1500 molecular weight (exact value depends on species). Protects cells against antibiotics, toxic metals, and other noxious chemicals. Consists of protein, lipid, and a unique molecule found only in the OM of gram-negative bacteria - the lipopolysaccharide (LPS).

Lipopolysaccharide is found only in the outer half of the OM. LPS contains a hydrophobic region, Lipid A buried in the membrane and then two polysaccharide regions which project outwards - the core and O-antigen. LPS is sometimes called endotoxin because of its toxic effects on mammalian cells. The toxicity is due to the Lipid A portion of the molecule which consists of two glucosamine residues with 6 long chain fatty acids covalently attached. The core region contains 7 and 8 carbon sugars as well as hexoses and hexosamines. It is often substituted with phosphate and phosphoethanolamine side chains. On the distal end of the core is attached the O-antigen, a chain made of many repeating oligosaccharide units each usually consisting of 3 or 4 sugars. There is great variety in the structure and composition of the O-antigen between different strains of bacteria. E. coli, for example, has over 150 possible types of O-antigen, many of which contain rare sugars, e.g. colitose, abequose. The O-antigen is highly immunogenic and is used in the antibody typing of Salmonella and other Enterobacteria.

Outer Membrane Proteins (OMP's) are of three major classes:

I. Structural Proteins. The lipoprotein is covalently bound to the peptidoglycan and serves to bind the OM to the rest of the cell wall. OmpA protein is a structural protein and is also the receptor for the sex pilus during conjugation.

II. The porins are the major proteins of the OM. There are two or three different porins which form pores through the OM. These pores admit molecules up to a size limit (700 to 1500 M.W.) which is characteristic of the bacterial species. Specialised porins exist eg for phosphate or maltose transport which are induced only under appropriate conditions.

The porins, lipoprotein and OmpA are the most numerous proteins in the bacterial cell along with ribosomal proteins, translation factors, and acyl carrier protein.

III. Specific receptor proteins are found in the OM for certain nutrients which are too large to get through the porins. Many of these are also used as receptors by bacteriophages. Other phages may use LPS or porins as their receptors.

Periplasmic Space: Between the OM and inner membrane (IM) of gram-negative cells. This space contains various degradative enzymes eg: 5'-nucleotidase, alkaline phosphatase. It also contains binding proteins involved in the uptake of amino acids, sugars, etc. When gram-negative cells contain plasmids specifying antibiotic resistance the enzymes which break down antibiotics are located in the periplasmic space, e.g. beta-lactamase. In E. coli about 20-40% of the cell volume is in the periplasmic space. The osmotic pressure of the periplasmic space cannot be maintained with simple sugars or inorganic ions as these are too small to be retained by the OM. Instead the periplasmic space contains the membrane-derived oligosaccharides (MDO).

Inner (Cytoplasmic) Membrane (IM): A typical biological membrane comprising about one third lipid and two thirds protein. The lipid bilayer is penetrated by hydrophobic proteins - "intrinsic" proteins. In addition the surface of the lipid layer is covered by 2 to 3 layers of "extrinsic" protein molecules. The lipid bilayer behaves as a 2-dimensional liquid allowing membrane proteins to drift laterally. The IM controls entry into the cytoplasm and acts as an electrical insulator for the electron transport chain. The IM contains synthetic enzymes for components of all layers of the cell envelope together with proteins involved in secretion and chemosensing.

Capsule: Layer found loosely attached to the outside of many bacteria. Not essential and varies with species and growth conditions. It usually consists of simple or complex carbohydrates or, more rarely protein eg: Acetobacter - simple polysaccharide, cellulose; E. coli - complex polysaccharides made of amino sugars and sugar acids; Acinetobacter - regular array of protein subunits. Capsules are protective and keep both bacteriophages and macrophages at a distance. They also protect cells against desiccation.

Pilus (plural pili): Sex pili and common pili are both composed of protein monomers arranged helically. Sex pili are involved in binding male and female cells together for subsequent transfer of DNA during mating and are only produced by male cells. Only one or two sex pili per cell are made. Common pili (also called fimbriae; singular = fimbria) are made by both sexes and appear in higher numbers per cell. They are involved in adhesion to suitable surfaces or in floating - bacteria lacking common pili clump together and sediment.

Flagellum (plural flagella): Prokayotic flagella are quite different from those of eukaryotes. Bacterial flagella consist of a single filament made of helically arranged protein subunits. The basal structure in the IM acts as a rotor and the whole flagellar shaft rotates. Bacterial flagella are powered directly by the proton motive force, whereas eukaryotic flagella use ATP.

OVERVIEW OF METABOLISM

Growth Requirements: Cells need genetic information as well as raw materials (macro- & micro-nutrients), and must generate energy and reducing power as well as manufacturing cellular components.

a) Coding Capacity. E. coli has sufficient DNA to code for about 3000 genes. Roughly 1500 of these have been identified and somewhat over 1000 different proteins have been observed by 2 dimensional gel electrophoresis. About 2000 different metabolic reactions are known.

b) Macronutrients: C, H, O, N, S, P. The last three are often taken up as ammonia, sulfate and phosphate respectively.

c) Micronutrients: Inorganic ions. Potassium and magnesium are found in all cells. In contrast sodium and calcium are required by eukaryotes but not by most bacteria which in fact usually excrete them. The following trace elements are found in certain enzymes and cofactors:

Iron (Fe)	Cytochromes & other heme enzymes, Iron-sulfur proteins, FeMo cofactor, some ADH's
Zinc (Zn)	Many enzymes of nucleic acid metabolism, Alcohol dehydrogenase, Structural in cell envelope
Manganese (Mn)	Several enzymes, Photosynthesis
Copper (Cu)	Cytochrome oxidase, Plastocyanin
Cobalt (Co)	Vitamin B12
Nickel (Ni)	Methanogenesis, Hydrogenase
Selenium (Se)	Formate dehydrogenase
Molybdenum (Mo)	Nitrate reductase, Nitrogenase
Vanadium (V)	Alternative N-fixation systems
Tungsten (W)	Replaces Mo in certain organisms

Composition of a Typical Bacterial Cell

Bacteria are about 70% water. The other 30% - the dry weight - consists of:

Molecule	% Dry weight	Constituents
Protein	55	amino acids
Polysaccharide	9-10	sugars
Lipid	9	fatty acids, glycerol, phosphate
RNA	20	purines, pyrimidines, phosphate,
DNA	3	and pentoses
Small molecules	3-4	low molecular weight solutes and inorganic ions

Overall Macromolecular Composition of an Average E. coli Cell

Macromolecule	% of dry weight	Weight/cell x 10-15g	Molecular weight	Number per cell	Different kinds of molecule
Protein	55.0	155.0	4.0 x 104	2,360,000	1050
RNA	20.5	59.0
23 S rRNA		31.0	1.0 x 106	18,700	1
16 S rRNA		16.0	5.0 x 105	18,700	1
5 S rRNA		1.0	3.9 x 104	18,700	1
transfer		8.6	2.5 x 104	205,000	60
messenger		2.4	1.0 x 109	1,380	400
DNA	3.1	9.0	2.5 x 109	2	1
Lipid	9.1	26.0	705	22,000,000	4
Lipopolysaccharide	3.4	10.0	4346	1,200,000	1
Peptidoglycan	2.5	7.0	(904)	1	1
Glycogen	2.5	7.0	1.0 x 106	4,360	1
Total macromolecules Soluble pool	96.1 2.9	273.0 8.0
building blocks		7.0
metabolites, vitamins		1.0
Inorganic ions	1.0	3.0
Total dry weight	100.0	284.0
Total dry weight/cell		2.8 x 10-13g
Water at 70% of cell		6.5 x 10-13g
Total weight of one cell		9.3 x 10-13g

*Remember: 10^-3 gram = milligram, 10^-6 gram = microgram, 10^-9 gram = nanogram, 10^-12 gram = picogram, 10^-15 gram = femtogram

Strategy of Metabolism

1) Organisms convert raw materials into living matter by using energy from their environment. Chemotrophs oxidize chemical fuel molecules to release useful energy. Phototrophs use sunlight as an energy source. These two classes of organism depend on each other. Phototrophs produce organic material which is the food for chemotrophs. They also produce oxygen which is necessary for the oxidation of food molecules by chemotrophs. The chemotrophs break down organic materials so releasing carbon-dioxide which the phototrophs use during photosynthesis.

2) Biological energy is obtained from redox reactions. Chemotrophic bacteria oxidize organic molecules such as sugars or fatty acids in order to derive energy.

3) The free energy released by redox reactions is conserved either by high energy carrier molecules such as adenosine triphosphate (ATP) or as an energized state of the membrane - the Proton Motive Force (PMF). ATP and the PMF are interconvertible.

4) Biosynthesis requires reducing power as well as energy. The reducing power is required for the synthesis of many biomolecules as they are often highly reduced. NADH and NADPH are the universal carriers of reducing power. Reduced NADH is usually oxidized to give energy whereas reduced NADPH is used for reducing power in biosynthetic reactions. In E. coli about 80% of the glucose goes down the main, energy yielding pathways and about 20% down the side pathways which generate reducing power as NADPH.

5) Outline of catabolic pathways (see diagram). Energy Metabolism is sometimes divided into four stages:

Stage I	Breakdown of polymers to monomers. Most bacteria cannot take in polymers and require small molecules - i.e. sugars, fatty acids or amino acids.
Stage II	Conversion of monomers to central intermediates - in particular pyruvate and acetyl-CoA. Amino acids are normally only degraded if no sugars or fatty acids are available.
Stage III	a) Conversion of acetyl-CoA to CO2 by Kreb's cycle
	b) Production of reduced NADH by Kreb's cycle
Stage IV	a) Oxidation of NADH by electron transport chain to give PMF
	b) Interconversion of PMF and ATP
In addition we need:	a) Generation of reducing power (NADPH) by side pathways
	b) Anaplerotic pathways to replace essential intermediates consumed in biosynthesis (not shown in diagram)

Note that in the absence of oxygen cells cannot perform respiration so instead pyruvate and acetyl-CoA are converted to fermentation products. ATP is produced from glycolysis alone. (Some bacteria - but not higher organisms - can use alternative oxidising agents to respire anaerobically - discussed later.)