Step 5] New Module... · Web viewChapter Overview 1.1 WHAT IS ENVIRONMENTAL MICROBIOLOGY? The study of how microorganisms affect the earth and its atmosphere is called environmental

Introduction to Environmental MicrobiologyENV 411

Chapter 1

Chapter Overview

1 |I n t r o d u c ti o n t o e n v i r o n m e n t a l m i c r o b i o l o g y

Introduction to Environmental Microbiology1

INTRODUCTION TO ENVIRONMENTAL MICROBIOLOGY & MICROBES

Environmental Microbiology

Significance of Environmental Microbiology

Microbial cell, it structure and function

Microbial growth

Microbial metabolism

Chapter 1 Introduction to Environmental MicrobiologyENV 411

1.1 WHAT IS ENVIRONMENTAL MICROBIOLOGY?

The study of how microorganisms affect the earth and its atmosphere is called

environmental microbiology or microbial ecology. The study of the relationships that exist

between microorganisms and the environment. The study of relationship of microorganisms

with themselves and with their surroundings.

1.2 TRAITS OF MICROORGANISMS AND IMPACT ON BIOSPHERE

Microorganisms’ unique combination of traits and their broad impact on the biosphere

Traits of microorganisms Ecological consequences of traits

Small size Geochemical cycling of elements

Ubiquitous distribution throughout

the earth’s habitats

Detoxification of organic pollutants

High specific surface areas Detoxification of inorganic pollutants

Potentially high rate of metabolic

activity

Release of essential limiting nutrients from

the biomass in one generation to the next

Physiological responsiveness

Genetic malleability

Maintaining the chemical composition of

soil, sediment, water and atmosphere

2 | i n t r o d u c ti o n t o e n v i r o n m e n t a l m i c r o b i o l o g y

LEARNING OBJECTIVES:After studying this chapter, you should be able to:

1. Define environmental microbiology

2. Understand the impact of microorganisms on the biosphere

3. Describe the microbial cell, its structure and function.

4. Explain differences in cell walls, cytoplasmic membrane of Bacteria, Archaea & Eucarya

5. Explain the different types of transport across cytoplasmic membrane

6. Elaborate the microbial growth, development and characteristics of spores.

7. Differentiate the different types of microbial metabolism.


Chapter 1

Potential rapid growth rate

Unrivaled nutritional diversity

Unrivaled enzymatic diversity

required by other forms of life

1.3 MICROBIAL CELL, ITS STRUCTURE AND FUNCTION

Based on ribosomal RNA sequence comparisions (16S, 23S). 3 basic groups or

domains established (domains are a higher order than kingdoms, ie are superkingdoms).

The 3 domains (i) Bacteria, (ii) Archaea and (iii) Eucarya. 3 domains are related to each

other; progenote = hypothetical ancient universal ancestor of all cells. Natural relationships

amongst cells established (phylogeny).

Microbes have different shapes and is of advantage: Cell wall establishes the shape of a

microbial cell but environmenta conditions can change it

Shapes include: (i) Spheres called cocci (greek = berry) can divide once in one axis

to produce diplococci (Neisseria gonnorrhoeae, N. meningitidis), or more than once

to produce a chain (Streptococcus pyogenes), divides regularly in two planes at right

angles to produce a regular cuboidal packet of cells (xxx) or in two planes at different

angles to produce a cluster of cells (Staphyloccus aureus). (ii) Cylinders called rods

or bacilli (Latin bacillus = walking stick). (iii) Spiral or spirilli (Greek spirillum = little

coil)

Shape offers an advantage to the cell: (i) Cocci: more ressistant to drying than rods

(ii) Rods: More surface area & easily takes in dilute nutrients from the environment.

(iii) Spiral: Corkscrew motion & therefore less ressistant to movement (iv) Square:

Assists in dealing with extreme salinities.

Microbes are small but this feature is crucial : Nutrients and wastes are transported in and

out the cell via the cytoplasmic membrane. The rate of transport determines the metabolic

rates and therefore the growth rates of microbial cells The smaller the size, the larger the

surface area of the cytoplasmic membrane to volume and therefore the faster is it's potential

growth rate. This can be seen as follows:



radius (r) of cell A = 1um radius (r) of cell B = 2um

Surface area (SA) of cell = 4pir2 12.6um2 50.3um2

Volume (V) of cell = 4/3pir3 4.2um3 33.5um3

Ratio of SA to V 3 1.5

Features of bacterial, archaeal and eucarya cells

This section deals with the structure and functions of cells. Cells are of three types as

described above (Bacteria, Archaea & Eucarya) and the description below provides

similarities and differences amongst these cell types.

Diagrammatic representation of cells

Cell walls are external structures that shape and protect cells

a. Bacterial Cell Walls:All the members of domain Bacteria, with the exception of the genera Mycoplasma,

Ureaplasma, Spiroplasma, and Anaeroplasma contain cell walls Cell walls are



Chapter 1

chemically peptidoglycans ie peptides (short amino acids chains) and glycans

(sugars); peptidoglycans are a.k.a. mureins, mucopeptide.

o Glycans: are modified sugars viz, N-acetyl muramic acid (NAM or M) & N-

acetly glucose amine (NAG or G).M and G are linked to each other by a beta

1, 4 glycosidic bond & alternate to form the wall backbone. Lysozyme (an

enzyme produced by organisms that consume bacteria, and normal body

secretions such as tears, saliva, & egg white = protect against would-be

pathogenic bacteria) digests beta 1,4 glycosidic bonds. Lysozyme lyses

growing or non growing cells but cell wall-less microbes are not affected High

osmotic pressure in high solute concentrations prevents lysis of Gram +ve &

Gram -ve cells when treated with lysozyme:

spheroplasts = part of cell wall removed (Gram -ve)

protoplasts = complete removal of cell wall (easier for Gram +ve)

o Peptides: Short peptides (4 amino acids, tetrapeptides) attached to M. Some

of the amino acids are only found in cell walls & not in other cellular proteins

(D- amino acids, eg D-alanine & diaminopimelic acid, DAP). Tetrapeptides

chains are cross linked (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 stoping

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 walss and ultimately death.

Glycans and peptides therefore forms 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.



Differences Between Gram-positive And Gram-negative Bacterial Cell Walls

Gram-positive wall Gram-negative wall

Peptidoglycan Thick layer Thin layer

Peptidoglycan tetrapeptide Most contain lysine All contain diaminopimelate

Peptidoglycan cross linkage Generally via pentapeptide Direct bonding

Teichoic acid Present Absent

Teichuronic acid Present Absent

Lipoproteins Absent Present

LPS Absent Present

Outer Membrane Absent Present

Periplasmic Space Absent Present

b. Archaeal Cell Walls: Archaeal cells have more variations in their cell wall chemistries, and some do not

contain cell walls (eg Thermoplasma). Methanobacterium sp. contain glycans

(sugars) and peptides in their cell walls:

o Glycans: are modified sugars viz, N-acetyl talosaminouronic acid (NAT or T)

& N-acetly glucose amine (NAG or G). T and G are linked to each other by a

beta 1, 3 glycosidic bond & alternate to form the cell wall backbone.

Lysozyme (an enzyme produced by organisms that consume bacteria, and

normal body secretions such as tears, saliva, & egg white = protect against

would-be pathogenic bacteria) cannot digest beta 1,3 glycosidic bonds.

o Peptides: 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.



Chapter 1

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. & Methanococcus sp. cell walls

are exclusively made up of proteins subunits.

c. Eucaryal Cell Walls:

Cell walls of algae have a variety of different cell wall types and include cellulose,

calcium carbonate, silcone dioxide, proteins and even polysaccharides. The cell walls

of fungi are made up of chitin (a nitrogen-containing polysaccharide) and is similar to

that found in the exoskeletons of arthropods & crabs . Protozoa do not have a true

cell wall. In some species, silicon dioxide, calcium carbonate or strontium sulfate are

found but do not provide the cell wall with a protective function.

d. Glycocalayx, Capsules, Slime Layers & S layers:

Various external structures which have different functions surround the bacterial cell

wall, and are collectively called glycocalyx. Glycocalyx varies in different species:

Capsules

Are thick & rigid structures which exclude stain.

Adhere externally to the to cell walls

Negative stain allows capsules to be observed.

Chemically polysaccharides. Found in pneumonia

causing pathogens such as Streptococcus pneumoniae,

Haemophilus influenzae & Klebsiella pneomoniae.

Chemically D-glutamic acid found in some Bacillus sp.

Capsulated variants of a species are pathogenic whereas

non-capsulated variants of the same species are non-

pathogenic. Capsules protect against phagocytosis by

human white blood cells.



Slime layers

Similar in composition to capsules but are not as tightly

bound to the cell wall.

Protects cells against dehydration and a loss of nutrients.

S layer

Some bacteria have a crystalline protein layer called a S

layer.

Found outside the cell walls of some species of Gram-

negative, Gram-positive Bacteria, and outside the cell

membranes of some Archaea.

Function is unknown.

Cytoplasmic membranes are involved in transport of molecules

(A) Structural & Biochemical Diversity

Thickness 4-5nm

Regulates flow of molecules in and out of the cell but is a differentially permeable

barrier- movement across the membrane is selectively restricted (structure &

chemistry is key to this)

Small, neutrally charged molecules (H2O, O2 & CO2) easily transportable but large

molecules & ions (glucose) or small charged atoms (protons, H+) require specific

transport systems.

Provides increased surface area to volume & is very important to small cells

Bilayered structural backbone are the phospholipids (bacteria & eucaryotes only);

forms a separation barrier with water inside and outside the cell

"Fluid mosaic model": Proteins are integrated into the lipid layer and both "float"

laterally in the membrane ie are in dynamic rather than static state (lipids float more

than proteins)

o Peripheral proteins: confined to the membrane surface

o Integral proteins: partially / completely buried & may span the entire

membrane

Distribution & properties of proteins on each side of the layer are different & therefore

the functions of the 2 layers are different

The structure and chemical properties of archaeal, bacterial and eucaryotic

membranes are "phylogenetically" distinct



Chapter 1

Characteristics of Bacterial Eucaryotic Archaeal cytoplasmic membranes

Characteristics Bacteria Eucaryotic Archaea

Protein content High Low High

Lipid composition

Phospholipid Phospholipids Sulfolipids, glycolipids, nonpolar isoprenoid lipids, phospholipids

Lipid structure Straight chain Branched Straight chain

Lipid linkage Ester linked(1) Ester linked Ether linked (di& tertaethers)

Sterols Absent(2) Present Absent

(1) Aquifex pyrophilus contains phospholipids & ether linked lipids (2) Cell wall-less bacteria (Mycoplasma, Ureaplasma, Spiroplasma, Anaeroplasma) contain sterols



A. Bacterial cytoplasmic membranes:

i. Phospholipids: (structure, functions & utility)

Made of phospholipids - a phosphate group joined to 2 fatty acids by glycerol

(glycerol diester); oleate, stearate The phosphate group is -vely charged & is therefore

hydrophilic ("water loving")- exposed to cell wall & cytoplasm

The fatty acid group is nonpolar & therefore hydrophobic ("afraid of water")- exposed

within the internal membrane matrix Electron micrographs of thin sections of bacteria

cells show a pair of electron dense dark railroad track-like appearance (hydrophilic

portion) & electron light middle layer (hydrophobic) Form a bilayer due to hydrophobic /

hydrophilic interactions (spontaneous aggregation)- contributes to flow of molecules.

Phospholipid composition varies with species & environmental conditions. Psychrophiles:

high proportion of unsaturated fatty acids enhance membrane fluidity (saturated fatty

acids pack together more tightly & produce a rigid less-fluid membrane). Bacteria can be

identified on phospholipid composition (computerized databanks available) but cells have

to be grown under standard conditions (Why?)

ii. Protein

Are in dynamic state and distribution is according to the fluid mosaic model

Function Location in Membrane Example

Energy transformation

Inside membrane ATPase F1

Transport of molecules

Inside membrane HPr

Protein export Inside membrane Docking protein

Association of DNA with membrane

Inside membrane DNA binding protein



Chapter 1

Transport of molecules

Both sides Permease

Chemotaxis Both sides Methylase-accepting chemotaxis proteins

Electron & proton transport

Both sides Flavoproteins

Flagellar activity Outside surface M protein (basal body of flagella)

Penicillin-binding proteins

Outside surface Cell wall biosynthesis

B. Archaeal cytoplasmic membranes:

Structure fundamentally different to bacterial & eucaryotic membranes Glycerol

molecules may be linked: (i) to a phosphate group (similar to bacteria & eucaryotes) and / or

(ii) to a sulfate and carbohydrates (unlike bacteria & eucaryotes) & therefore phospholipids

are not the structural lipids. Lipids are hydrocarbons (isoprenoid hydrocarbons) not fatty

acids, are branched (straight chain in bacteria & eucaryotes) and linked to glycerol by ether

bonds (ester linked in bacterial & eucaryotes). Lipids are diverse in structure:

o Glycerol diether (Glycerol + C20 hydrocarbons)- Bilayered membrane

o Glycerl tetraether (Glycerol + C40 hydrocarbons)- Monolayered membrane

o Mixture of di- & tetra- Mono /Bi layered membrane

o Cyclic tetraethers (Glycerol + > C40)- maintain the 4-5nm membrane thickness

Diversity of membranes is related to the diverse habitats that archaea live in

o Sulfolobus (90oC, pH 2)- branched chain C40 hydrocarbons. Branched chains increase

membrane fluidity (unbranched & saturated fatty acids limit sliding of fatty acid

molecules past one another)- required for growth at high temperatures (upto 110oC,

hyperthermophies)

o Halobacterium (saturated salts)-

o Thermoplasma- high temperature, cell wall-less archaea



C. Eucaryal cytoplasmic membranes:

Phospholipids similar to bacterial membranes but terols make upto 25% of the lipids

(cholestrol in humans,ergosterol in fungi). Polyene antibiotics (eg nystatin, candicidin)

targets sterols & has more affinity for ergosterol than cholesterol (more effective against

fungi rather than human cells)

Transport Across Cytoplasmic membrane Membranes must selectively regulate transport of materials and waste ie semipermeable &

several mechanisms are available for this: (i) Pass directly enter thro' the lipid layer or via

proteins , (ii) Altered / modified as it passes thro' , (iii) Process requires cellular energy and

(iv) Solutes are concentrated against a gradient.

Passive Processes: Transport does not require energy & include diffusion, osmosis and

facilated diffusion

Diffusion: Unassisted movement of molecules from a higher concentration to lower

concentration (concentration gradient) until equilibrium is reached is called passive diffusion.

Rate of diffusion depends on membrane permiability & solute concentrations. Some solutes

after moving into the cell binds with some other proteins or are metabolically transformed.

Therefore concentration is not built up in the cell & the diffusion process continues at a faster

rate. Passive diffusion is slow eg glucose and tryptophan have diffusion rates of 1/10,000

that of water, & not enough for cellular growth & reproduction.

Osmosis : Process by which water croses the membrane in response to concentration

gradient of the solute 30 minutes at 70o C . Water moves from a region of low solute

concentration to high solute concentration.

o isotonic- solute conc. outside the cell = solute conc. inside the cell

o hypertonic- solute conc. is higher than that inside the cell; water flows out causing the

cell to shrink, plasmolysis

o hypotonic- reverse of hypertonic; water will flow into the cell & the cell will burst

Usually water moves into the cell as cytoplasm has solutes resulting in increased pressure

on the membrane- osmotic pressure. Cells can lyse due to osmotic shock but have

developed strategies to protect against this (see shock-sensitive proteins later)



Chapter 1

Facilitated Diffusion: Enhanced rate of diffusion found mainly in eucaryotic cells but rarely

in bacteria & archaea (glycerol is the only known substrate that undergoes facilitated

diffusion in some bacteria). Facilitator proteins (membrane proteins) selective increase the

permeability of the membrane for certain solutes. Facilitator proteins are very specific & act

as carriers ie solutes bind to the facilitator protein changing its 3D properties. This change in

shape allows the solute to be carried across the membrane.

Active Energy-linked transport processes Require energy for transport and the processes include active transport, group translocation,

binding protein transport and cytosis

a. Active Transport:

Active transport requires energy but the molecule is not modified during transport.

Transport occurs against concentration gradients

Permeases are very specific membrane protein transport carriers .Uniporters- carry one

substance at a time .Cotransporters- carry more than one type of substance . Symporter-

Two substances carried in the same direction simultaneously [(eg lactose & proton (H+)]

Antiporter- Substances are transported across the membrane in opposite directions (eg Na+

are pumped outside the cell at the same time H+ are transported inside the cell).

Protonmotive force (PMF): Energy for active transport in bacteria (oxidative

phosphorylation) in archaea, algae, mitochondria & chloroplasts generally comes from PMF.

PMF force is essential. Various metabolic activities produce protons (H+) and these are

translocated outside the cell. Higher concentrations & an increase in positive charge

outside the cell favours movement of protons back into the cell but cannot do so on their

own. Uncharged molecules (eg amino acids & sugars) are usually transported into the cell

with protons The various means by which PMF is produced will be discussed later

Sodium-potassium pump: A gradient between Na+ & K+ similar to protonmotive force &

known as sodium-potasium pump . Found in many eucaryotes.Three Na+ are pumped out of

the cell and two K+ are pumped into the cell by Na+-K+ ATPase enzyme; ATP is expanded.

Unequal distribution of positive ion with a higher Na+ conc. outside the cells and a higher

conc. of K+ inside the cells; leads to a powerful electrochemical gradient used for active

transport (eg symport protein binds both Na+ and glucose for transport therebye lowering

Na+ conc gradient across the membrane)



b. Group translocation- Phosphoenol pyruvate: Phosphotransferase system (PEP:PTS).

Transported substance is chemically altered during passage thro' the membrane by the

addition of phophate . Carbohydrates, fatty acids, some nucleic acid building blocks. In E.

coli, glucose outside the cell is phosphorylated during transport (G6-P) into the cell.

Metabolism almost instantaneous once inside (couples energy resources efficiently thro

transport & initiation of energy-generating metabolism. Concentration gradient of glucose is

prevented (not in the same chemical state). Prokaryotic specific; in anaerobes, facultative

anaerobes but not in aerobes (active transport occurs).

c. Binding protein transport

Specialized transport system associated with the outer membrane of Gram negative

bacteria only. Periplasmic space(periplasm, periplasmic gel) is the space between the outer

membrane & the cytoplasmic membrane. There is interplay between porins, binding

proteins, permeases & transport proteins, eg maltose transport in E. coli . Binding protein

transport is also called shock-sensitive transport (cells that are osmotically shocked loose

the transport proteins of the periplasm).

d. Cytosis- Eucaryotic specific transport

A transport process in which a substance is engulfed by the cell membrane to form a

vesicle. Cytosis requires energy:

o Endocytosis- movement into the cell

o Exocytosic- movement out of the cell

o Phagocytosis- engulfing by a cell of a smaller cell or a particle (protozoa, Amoeba)

o Pinocytosis- cell engulfs liquid

o Receptor-mediated endocytosis- receptor binds to a substance and assist in

transport (viruses and host cells)

Sites of cellular energy transformations where ATP is generated. ATP generation &

utilization is a central metabolic activity. The location & structures involved in cellular-energy

generating reactions will be discussed here (i) Some reactions occur in the cytoplasm, (ii)

Some cell membrane structures are a key to generate cellular energy. Two mechanisms for

generating cellular energy; (i) Substrate level phosphorylation and (ii) Chemiosmosis:

Movement of microbial cells : (i) Flagella / Cilia, (ii) Axial Filament, (iii) Gas vacuoles, (iv)

Magnetosomes, (v) Pseudopodia and (vi) Chemotaxis, magnetotaxis and phototaxis.



Chapter 1

Microbes do not die? There are structures to ensure survival

Ordinary microbes are killed by minor stresses eg chilling, antibiotics, disinfectants but cells

with protecive bodies, namely endospores and cysts ressist such stresses. In most cases,

the cells that produce endospores and cysts are a part of the soil microflora. Soil heats &

dries in summer but is periodically flooded by rain -- harsh fluctuating environment.

a. Endospores:

Historical Developement & Importance

100's of species mainly of the genera Bacillus (aerobic rods, facultative anaerobes), and

Clostridium (anaerobic rods); Few others include Sporosarcina (aerobic cocci),

Desulfotomaculum (anaerobic rods, sulfate-reducers) . Food industries (canning, milk

etc) heat treat products to reduce microbial spoilage & kill pathogens; spore-formers are

a problem (swelling of tins; putrification of meat etc). Mainly found in soils --> vegetables

--> meat where spores germinate to produce toxins (eg veg / meat salad stored

improperly prior to use; wooden choping boards prefered over synthetic) . Mainly found

in soils --> infect wounds (problem with farm associated workers)

Some strains were being developed for biological warfare eg B. anthracis (anthrax).

Some strains produce important biopesticides (biotechnology) eg B. thuringiensis var.

israelensis produces toxic proteins against mosquito & blackfly larvae. Commercial

variants available which produce toxins towards slightly different insect pests eg

Thuricide, Teknar, M-one. Spore which can germinate have been found from structures

7200 year old temples have been found and recently from GI tract of a bee preserved in

amber (1 million years old)

Distribution

Bacteria Fungi

Present in some genera Present

Protective & dispersal function Reproductive function

Endospores Endo- or Exo- spores

One per cell but C. disporicum=2; C.

polypendens=5Numerous

Size : Larger (distends the cell) or smaller than the cell



Shape : (i) Cylindrical, (ii) Ellipsoidal (iii) Spherical

Location : (i) Central, (ii) Terminal, (iii) Sub-terminal

Cells with endospores can be identified by spore-staining: (i) B. megaterium,an

aerobe: Small cylindrical sub-terminal spores, (ii) C. tetani, an anaerobe: Large

(distend) spherical terminal spores .

Heat ressistance

Endospore-forming cellTime required to kill a suspension in

boiling water (100oC)

B. anthracis 1-2 min (not very heat ressistant)

C. botulinum 2-6 hours

C. tetani 1-3 hours

E. coli & S. aureus (non-endospore formers)

30 minutes at 70o C

Spore structure

Spores are formed during unfavourable growth conditions & germinate under

favourable conditions. The spore can be differentiated into 4 distinct parts: Core: Nucleic

acids, ribosome, low levels of enzyme activity, Calcium dipicolonic acid (CDPA) & low

water content. Low level of metabolic activity .Two wall like layers:

Cortex: Surrounds the core, mainly electron light peptidoglycan

Coat: Surrounds the cortex, mainly protein

Exosporium: The outer most thin layer

Mechanism of heat ressistance

Physical (sporecoat): Ressistance to staining demonstrates imperability & therefore

ressistant to dehydration & effects of toxins (multilayered thick peptidoglycan) . Chemical

(core): Low water content (15% instead of 80% found in cells) makes prteins & nucleic

acids more ressistant. CDPA complexes with proteins & other labile components &

makes them more ressistant. Medium lacking calcium or mutant strains that do not form

CPDA produce less "tolerant" spores.

b. Cysts



Chapter 1

Ressistant to dehydration but not to heat and hence unlike spores. Deposition of layers &

layers of cell wall around the cell rather than within the cell as in case of spores. Azotobacter

(free living nitrogen fixing bacterium found in soil) and Myxbacteria .Involved in nitrogen

fixation and protection.

Cellular storage of genetic information

i. Bacterial & archaeal chromosome

Usually a single circular chromosome (Streptomyces & Borrelia = linear, Rhodobacter

sphaeroides = 2 separate chromosomes). "Naked DNA" - not membrane bound (nucleoid

region). Negatively supercoiled (highly twisted)- can expand to 1mm in length uncoiled

(length of a "typical" bacterium is a few micrometers and not associated with histone proteins

(histones responsible for eucaryotic DNA coiling) but histone-like proteins found. Genome

size extremely heterogenous, determined in nucleotide base pairs (bp).

Microbe Characteristics Size (Mb) Sequence information

Mycoplasma genitalium No cell wall, bact 0.58

Haemophilus influenzae bact pathogen 1.83

Helicobacter pylori bact pathogen

Neisseria meningitidis bact pathogen

Escherichia coli GI bact 4.4

Thermotoga maritima bact thermo

Archaeoglobus fulgidus archaea thermo

Pyrodictium occultum archaea thermo

Methanococcus

jannaschiiarchaea therm

G+C content between 28% to 72%

Cell division (binary fission) & DNA duplication are synchronised. DNA duplication is

slower than cell division & therefore new rounds of DNA synthesis are initiated by the

cell even though the previous copy has not fully replicated. A cell can carry one full

copy & several partial copies.

ii. Plasmids

small, circular, self replicating extrachromosomal genetic elements- >= 1



the genetic information supplements the chromosomal genetic information

o antibiotic ressistance

o tolerance to toxic metals

o production of toxins

o mating capabilities

genetic information is 1 - 5% of chromosomal DNA information but means 0% or

100% survival eg antibiotic ressistance

classfied on the basis of its function

o "mating" plasmids - F (fertility) factor

o antibiotic, metal ressistance- R (ressistance) factor

benefits & hazards

o multiple drug ressistant pathogens

o genetic engineering- cloning & expression of useful substances

iii. Nucleus & chromosomes of Eucarya cells

linear chromosomes associated with chromatins; chromatins are histone proteins

(basic proteins) around which DNA coils (~ 200 nucleotides/histone) to form

nucleosomes- "beads on a string" under EM

chromosomes are located in the nucleus

o nucleus is separated from the cytoplasm by pore containing nuclear

membrane (double layered bilayered membrane)

o more processing of the DNA is needed before it can be expressed & hence

this type of separation is necessary

usually greater than 1 different sized chromosomes present

Dinoflagellate algae is an evolutionary link between eucaryotes and bacteria-

o DNA inside nucleus (similar to eucaryotes)

o not histone associated ie not coiled like eucaryotic chromosomes

o DNA arangement is similar to that of bacterial DNA (nucleoid region)

Information flow in cells: the role of ribosomes



Chapter 1

DNA -------> RNA (tRNA, mRNA, rRNA)-------->proteins

~10,000 ribosomes in archaea & bacteria depending on growth rates but many more in

eucarya. Measured in Svedgurg (S) units: rate of sedimentation in an ultracentrifuge

dependent on shape & size. Bacteria & Archaea: (i) 70S (30S = 21 proteins, 16SrRNA

[~1542 nucleotides] + 50S = 34 proteins, 23S rRNA [2900 nucleotides, 5S [120

nucleotides]) -- Phylogeny. (ii) Similar 70S but differences exist in protein composition

archaea are not sensitive to antibiotics that inhibit bacterial protein synthesis

tetracycline, erythromycin, chloramphenicol

diptheria toxin & anisomycin affects ribosomes of archaea but not bacterial

Eucarya:

o 80S (40S = 18SrRNA, 60S = 25 to 28S rRNA, 5.8S rRNA)

o synthesised in the nucleolus & transported via nuclear pores into cytoplasm

o Primitive protozoa Giardia contains 70S

o mitochondria & chloroplast contain 70S; rRNA sequence shows similarity to

noncultured archaea & Rickettsia (proteobacteria) endosymbiotic theory.

Differences in 70S & 80S can be targeted for treatment of animal / plant diseases

o Streptomycin & Erythromycin bind & alter 70S shape of bacteria not eucaryotes

Storage of materials

1. Inclusion bodies of bacteria

Bacteria store chemicals under certain conditions. eg, increased carbon availability but

not inadequate nitrogen-containing compounds for protein synthesis available. Not

separated by membranes & display differential solubility.

o Nutrient reserves synthesised by the cell: poly-beta-hydroxybutyrate (PHB)

o Energy reserves: inorganic polyphosphates (volutin, metachromatic granules) for

ATP synthesis; viewable after staining by light microscopy

o Metabolic deposits: Sulfur deposited as a result of metabolism (photosynthetic

bacteria)

2. Membrane bound organelles in Eucarya

Endoplasmic reticulum



Golgi apparatus

Lysosomes

Microbodies

Vacuoles

Cytoskeletal network

Cell surface structures involved in attachment

Glycocalyx: Bind cells togethr forming multicellular aggregates. In some cases the bacterial

cells adhere to solid surfaces using these structures.

o Some pathogenic bacteria adhere to animal tissues

o Some aquatic bacteria adhere to rocks

o Some are involved in plaque formation leading to dentall caries

Fimbrae: Not all bacteria posses fimbrae -- it is an inherited trait

Arise from the cytoplasmic membrane or just below the membrane

Can be mistaken for flagella but are not involved in motility

Much shorter and more numerous than flagella

Adhesion functions which enables cells to form a pellicle on liquid surfaces

Pili: Similar to fimbrae but longer and fewer; sometimes only one per cell. Three functional types

of bacterial pili:

o Act as receptors sites for some attachment of some phages ie phage infection

o Act as sex pilus for bacterial conjugation processes (F aka Fertility pili of E. coli)

o Attachment for pathogenic bacteria to human tissues (Neisseria gonorrhoeae)

1.4 MICROBIAL GROWTH



Chapter 1

Microbial growth may be described as occurring in different ways under different

circumstances. Increase in both population size and population mass: (i) Microbial

populations tend to increase in number and in cell mass simultaneously. (ii) Increase in cell

number and increase in cell population mass both usually occur in a measurably coordinated

fashion.

Note that, for bacteria, while the cell population and population mass typically increase with

time (with growth), over the course of population growth individual cells actually cycle

through increases and decreases in cell mass (i.e., growth, division, growth, division,

growth . . .). Bias toward cell number:

i. When a microbiologist speaks of microbial growth it is usually increase in cell number

that she is after.

ii. The reason for this bias is that a typical microbiologist is more interested in population

characteristics than in the characteristics of individual cells, or both (since the

characteristics of individual cells tend to be studied, by necessity, within the context of

populations of cells).

iii. Consequently, there is a tendency for microgiologists to follow microbial growth as

populations rather than following the growth of individual cells, and therefore

microbiologists tend to be more interested in population sizes than the size (mass) of any

indvidual cell. Futhermore, the typical measurement of microbial growth will be done over

the span of more than one microbial generation.

a. Increase in cell number : An increase in cell number is an immediate consequence of cell division. Because most

bacteria grow by binary fission, doubling in cell number usually occurs at the same rate that

individual cells grow and divide.

b. Increase in cell mass Doubling in size: Individual cells of many species double in size between divisions. Cell

mass thus increases at the same rate as cell number. The implication of this is that while

increase in cell number may be emphasized while considering microbial growth, increases

(and decreases) in individual cell masses are also occurring, though these increases and

decreases ballance each other out such that the average cell size tends to remain constant

under constant conditions.


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Anabolic process: The increase in mass is a consequence of anabolism. For anabolism to

occur a cell must be situated in an environment that supplies all necessary nutrients and

which physically falls into a range in which growth can occur.

c. Binary fission i. Procaryotic cell division:

Binary fission is the process by which most procaryotes replicate. Binary fission

generally involves the separation of a single cell into two more or less identical daughter

cells, each containing, among other things, at least one copy of the parental DNA.

ii. Stepwise process:

The first steps of binary fission include cell elongation and DNA replication. The cell

envelope then pinches inward, eventually meeting. A cross wall is formed and ultimately two

distinct cells are present, each essentially identical to the original parent cell.See illustration

below.


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Chapter 1

d. Generation time [doubling time]

Procaryotic cell division: A bacterial generation time is also know as its doubling time.

Doubling time is the time it takes a bacterium to do one binary fission starting from

having just divided and ending at the point of having just completed the next division.

Generation times vary with organism and environment and can range from 20 minutes

for a fast growing bacterium under ideal conditions, to hours and days for less than ideal

conditions or for slowly growing bacteria.

e. Standard bacterial growth curve The standard bacterial growth curve describes various stages of growth a pure culture of

bacteria will go through, beginning with the addition of cells to sterile media and ending

with the death of all of the cells present. The phases of growth typically observed

include: (i) lag phase, (ii) exponential (log, logarithmic) phase, (iii) stationary phase and

(iv) death phase (exponential or logarithmic decline).

In standard bacterial growth curves one keeps track of cell growth by some measure or

estimation of cell number.


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Exponential [log or logarithmic] growth (phase)

Back-to-back divisions: Exponential growth is a physiological state marked by back-to-

back division cycles such that the population doubles in number every generation time.

Note that during exponential growth there is no change in average cell mass, though

individuals cells are constantly changing in mass as they increase in mass, then divide

thus rapidly decreasing in mass (while increasing in number).

The algebra of exponential growth: Note that during exponential growth the number of

cells present at any given time is a multiplicative function of the number of cells present

at a previous time. Under constant conditions the multiplicative increase in cell number

consequently is constant for any given interval of the same duration.

If a log phase culture goes from 2 cells to 4 cells during a 20 minute interval, then the

culture will go from 4 cells to 8 cells during the next 20 minutes. If a log phase culture

goes from 2 cells to 6 cells during a 60 minute interval, then the culture will go from 6

cells to 18 cells during the next 60 minutes. If during exponential phase there are 10 cells

present at time 0, and 100 cells present at time 200, then at time 400 there will be

10,000 (100 * 100) cells present.

Lag phase

Lag in division: Upon a change in environment (especially from a rich environment to a

poor environment), or when going from stationary phase to exponential phase, there is a

lag before division resumes. For example, stationary phase Escherichia coli placed in an

excess of sterile broth will go through a lag phase during which they increase in cell size

but do not divide. They will divide only once they have reached the size of a cell which is

about to divide during exponential growth under those conditions. During this time a

culture is said to be in lag phase.

Increase in mass: During lag phase cells increase in mass but do not divide. In other

words, there is no change in number, but an increase in mass.

"The length of the lag phase is determined in part by characteristics of the bacterial

species and in part by conditions in the media---both the medium from which the

organisms are taken and the one to which they are transferred. Some species adapt to


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Chapter 1

the new medium in an hour or two; others take several days. Organisms from old

cultures, adapted to limited nutrients and large accumulated wastes, take longer to

adjust to a new medium than do those transferred from a relatively fresh, nutrient-rich

medium." (p. 138, Black, 1996)

Stationary phase

Stationary phase is classically defined as a physiological point where the rate of cell

division equals the rate of cell death, hence viable cell number remains constant.

No cell division: Note that when cell division = 0 and cell death = 0, then the rate of cell

division = rate of cell death. In other words, when cells stop dividing but have not yet

started dying they are in stationary phase.

A way to distinguish these possibilities is to compare viable count with total count. If both

total counts and viable counts don't change then you know that there is both no cell

division and no cell death. If total count increases while viable counts remain constant,

then you know that you are observing a true balance between ongoing cell division and

cell death.

Physiological adaptation to cell excess: Stationary phase usually occurs when cell

concentration is so great and that some aspect of the environment is no longer able to

serve the requirements of exponential growth. Stationary phase is a time of significant

physiological change and particularly involves the physiological adaptation of cells to

survival through periods of little growth.

Cell death

In single celled microorganisms cell death is the point at which reinitiation of division is

no longer possible. Qualified definition:

i. Note that the concept of cell death is actually dependent on how one attempt to

reinitiate growth.

ii. Particularly, there are ways to gently revive some microbes from physiological states

that would result in permanent lack of growth in other growth environments.

An analogous situation would be a person with an injury that is inevitably fatal in a third-

world hospital, but readily treated in a first-world hospital.


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Example: seeds: Another analogy is with a plant seed. You can try to sprout it in all kinds

of environments but not all will work out in the seed's favor. You may end up killing the

seed by allowing it to attempt to germinate in the wrong environment. The more

degraded is the seed prior to planting, the greater the likelihood that germination will not

successfully occur unless you take great care to make sure sprouting conditions are as

close to ideal as you can make them.

Death phase [logarithmic decline, exponential decline]

Death phase is a physiological point at which cell deaths exceed cell births. More

specifically, viable count declines. "During the decline phase, many cells undergo

involution---that is, they assume a variety of unusual shapes, which makes them difficult

to identify." (p. 140, Black, 1996

Endospore [spore, sporulation, sporogenesis, activation, germination]. Tough, dormant

state: A very tough, dormant form of certain bacterial cell that is very resistant to

desiccation, heat, and a variety of chemical and radiation treatments that are otherwise

lethal to non-endospore bacterial cells. At least part of the toughness associated with a

spore is found in its very tough outer layers, called a coat. Only some bacteria produce

endospores. Endospores of some bacteria can last so long under proper conditions that

various endospores found in such things as Egyptian mummies are likely the oldest

living things.

Sporulation and sporogenesis:

Sporulation and sporogenesis refer to the formation of endospores by vegetative (i.e.,

growing) cells. The endospore is actually the intracellular product of sporogenesis. A

spore is an endospore which has been released from a cell, i.e., it exists is a free state.

In bacteria the formation of a spore is not considered to be an act of reproduction.

Indeed, the formation of the endospore is directed by the DNA that will ultimately be

found in the spore, and the sister DNA found in the vegetative part of the cell ultimately is

destroyed.

The first step of germination often requires some kind of coat traumatizing insult such as

high temperature or low pH. The transformation from the endospore state to the

vegetative state. The key thing to worry about with endospores is that they are capable


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Chapter 1

of germinating despite harsh treatment, and thus can potentially produce actively

replicating cells where there may have been none previously prevent. Of those bacteria

on your list, the following are spore formers (note that all are gram-positives):

i. Bacillus anthracis

ii. Bacillus subtilis

iii. Clostridium botulinum

iv. Clostridium perfringens

v. Clostridium tetani

1.5 MICROBIAL METABOLISM

Based on their modes of metabolism, the procaryotes are much more diverse than all

eucaryotes, and the real real explanation for "microbial diversity" rests fundamentally on

some aspect procaryotic metabolism, especially with regards to energy-generating

metabolism and synthesis of secondary metabolites.

Microbial diversity translates to metabolic diversity. The procaryotes, as a group, conduct

all the same types of basic metabolism as eucaryotes, but, in addition, there are several

types of energy-generating metabolism among the procaryotes that are non existent in

eucaryotic cells or organisms. These include: (i) Unique fermentation pathways that

produce a wide array of end products. (ii) Anaerobic respiration: respiration that uses

substances other than O2 as a final electron acceptor.

Lithotrophy: use of inorganic substances as sources of energy

Photoheterotrophy: use of organic compounds as a carbon source during bacterial

photosynthesis

Anoxygenic photosynthesis: uses special chlorophylls and occurs in the absence of

O2

Methanogenesis: an ancient type of archaean metabolism that uses H2 as an energy

source and produces methane.


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Light-driven nonphotosynthetic energy production: unique archaean metabolism

that converts light energy into chemical energy; occurs in the archaea (extreme

halophiles).

Unique mechanisms for autotrophic CO2 fixation, including primary production on

anaerobic habitats

What is metabolism?

The term metabolism refers to the sum of the biochemical reactions required for energy

generation and the use of energy to synthesize cell material from small molecules in the

environment. Hence, metabolism has an energy-generating component, called

catabolism, and an energy-consuming, biosynthetic component, called anabolism.

Catabolic reactions or pathways produce energy as ATP, which can be utilized in anabolic

reactions to build cell material from nutrients in the environment. The relationship between

catabolism and anabolism is illustrated in Figure 1 below.

Figure : The relationship between catabolism and anabolism in a cell. During catabolism, energy is changed from one form to another, and keeping with the laws of thermodynamics, such energy transformations are never completely efficient, i.e., some energy is lost in the form of heat. The efficiency of a catabolic sequence of reactions is the amount of energy made available to the cell (for anabolism) divided by the total amount of energy released during the reactions.



Chapter 1

Metabolism is usually visualized as as a series of biochemical reactions mediated by

enzymes, referred to as a metabolic pathway. Catabolic pathways lead to end products,

which are "waste products" and result in the generation of energy which is temporarily

conserved as adenosine triphosphate (ATP). In heterotrophs, the most common catabolic

pathways are the Emden-Meyerhof pathway for degradation of sugars as energy sources

(glycolysis and the tricarboxylic acid cycle (TCA cycle), which can be linked to the further

degradation of almost any organic compound and further leads to the synthesis of ATP.

Model of a catabolic pathway. Each reaction in the pathway is mediated by a specific enzyme. s x y z

sugar--------> X--------> Y--------> Z--------> Intermediate + ATP

Anabolic pathways utilize ATP to provide energy for the synthesis of the monomeric

compounds that are required for the manufacture of the small molecules needed in cells,

i.e., carbohydrates, lipids, amino acids, nucleotides, vitamins, etc.

Model of an anabolic pathway. Each reaction in the pathway is mediated by a specific enzyme.

a b c d

Intermediate + ATP--------> A--------> B--------> C--------> Final product

ATP

During catabolism, useful energy is temporarily conserved in the "high energy bond" of ATP - adenosine triphosphate. No matter what form of energy a cell uses as its primary source,

the energy is ultimately transformed and conserved as ATP. ATP is the universal currency

of energy exchange in biological systems. When energy is required during anabolism, it may

be spent as the high energy bond of ATP which has a value of about 8 kcal per mole.

Hence, the conversion of ADP to ATP requires 8 kcal of energy, and the hydrolysis of ATP to

ADP releases 8 kcal.



The structure of ATP. ATP is derived from the nucleotide adenosine monophosphate (AMP) or adenylic acid, to which two additional phosphate groups are attached through pyrophosphate bonds (~P). These two bonds are energy rich in the sense that their hydrolysis yields a great deal more energy than a corresponding covalent bond. ATP acts as a coenzyme in energetic coupling reactions wherein one or both of the terminal phosphate groups is removed from the ATP molecule with the bond energy being used to transfer part of the ATP molecule to another molecule to activate its role in metabolism. For example, Glucose + ATP -----> Glucose-P + ADP or Amino Acid + ATP ----->AMP-Amino Acid + PPi.

NAD

Another coenzyme commonly involved in metabolism, derived from the vitamin niacin, is the

pyridine nucleotide, NAD (Nicotinamide Adenine Dinucleotide). The basis for chemical

transformations of energy usually involves oxidation/reduction reactions. For a biochemical

to become oxidized, electrons must be removed by an oxidizing agent. The oxidizing agent

is an electron acceptor that becomes reduced in the reaction. During the reaction, the

oxidizing agent is converted to a reducing agent that can add its electrons to another

chemical, thereby reducing it, and reoxidizing itself. The molecule that usually functions as

the electron carrier in these types of coupled oxidation-reduction reactions in biological

systems is NAD and its phosphorylated derivative, NADP. NAD or NADP can become

alternately oxidized or reduced by the loss or gain of two electrons. The oxidized form of

NAD is symbolized NAD; the reduced form is symbolized as NADH2. The structure of NAD

is drawn below.



Chapter 1

The Structure of NAD. (a) Nicotinamide Adenine Dinucleotide is composed of two nucleotide molecules: Adenosine monophosphate (adenine plus ribose-phosphate) and nicotinamide ribotide (nicotinamide plus ribose-phosphate). NADP has an identical structure except that it contains an additional phosphate group attached to one of the ribose residues. (b) The oxidized and reduced forms of of the nicotinamide moiety of NAD. Nicotinamide is the active part of the molecule where the reversible oxidation and reduction takes place. The oxidized form of NAD has one hydrogen atom less than the reduced form and, in addition, has a positive charge on the nitrogen atom which allows it to accept a second electron upon reduction. Thus the correct way to symbolize the reaction is NAD+ + 2H----->NADH + H+. However, for convenience we will hereafter use the symbols NAD and NADH2.

ATP Synthesis

The objective of a catabolic pathway is to make ATP, that is to transform either chemical

energy or electromagnetic (light) energy into the chemical energy contained within the high-

energy bonds of ATP. Cells fundamentally can produce ATP in two ways: substrate level phosphorylation and electron transport phosphorylation.

Substrate level phosphorylation (SLP) is the simplest, oldest and least-evolved way to

make ATP. In a substrate level phosphorylation, ATP is made during the conversion of an

organic molecule from one form to another. Energy released during the conversion is

partially conserved during the synthesis of the high energy bond of ATP. SLP occurs during

fermentations and respiration (the TCA cycle), and even during some lithotrophic

transformations of inorganic substrates.



Three examples of substrate level phosphorylation. (a) and (b) are the two substrate level phosphorylations that occur during the Embden Meyerhof pathway, but they occur in all other fermentation pathways which have an Embden-Meyerhof component. (c) is a substrate level phosphorylation found in Clostridium and Bifidobacterium. These are two anaerobic (fermentative) bacteria who learned how to make one more ATP from glycolysis beyond the formation of pyruvate.

Electron Transport Phosphorylation (ETP) is a much more complicated affair that evolved

long after SLP. Electron Transport Phosphorylation takes place during respiration,

photosynthesis, lithotrophy and possibly other types of bacterial metabolism. ETP requires

that electrons removed from substrates be dumped into an electron transport system (ETS)

contained within a membrane. The electrons are transferred through the ETS to some final

electron acceptor in the membrane (like O2 in aerobic respiration) , while their traverse

through the ETS results in the extrusion of protons and the establishment of a proton motive force (pmf) across the membrane. An essential component of the membrane for

synthesis of ATP is a membrane-bound ATPase (ATP synthetase) enzyme. The ATPase

enzyme transports protons, thereby utilizing the pmf (protons) during the synthesis of ATP.

The idea in electron transport phosphorylation is to drive electrons through an ETS in the

membrane, establish a pmf, and use the pmf to synthesize ATP. Obviously, ETP take a lot

more "gear" than SLP, in the form of membranes, electron transport systems, ATPase

enzymes, etc.



Chapter 1

A familiar example of energy-producing and energy-consuming functions of the bacterial

membrane, related to the establishment and use of pmf and the production of ATP, is given

in the following drawing of the plasma membrane of Escherichia coli.

The plasma membrane of Escherichia coli. The membrane in cross-section reveals various transport systems, the flagellar motor apparatus (S and M rings), the respiratory electron transport system, and the membrane-bound ATPase enzyme. Reduced NADH + H+ feeds pairs of electrons into the ETS. The ETS is the sequence of electron carriers in the membrane [FAD --> FeS --> QH2 (Quinone) --> (cytochromes) b --> b --> o] that ultimately reduces O2 to H2O during respiration. At certain points in the electron transport process, the electrons pass "coupling sites" and this results in the translocation of protons from the inside to the outside of the membrane, thus establishing the proton motive force (pmf) on the membrane. The pmf is used in three ways by the bacterium to do work or conserve energy: active transport (e.g. lactose and proline symport; calcium and sodium antiport); motility (rotation of the bacterial flagellum), and ATP synthesis (via the ATPase enzyme during the process of oxidative phosphorylation or electron transport phosphorylation). Heterotrophic Types of Metabolism

Heterotrophy (i.e., chemoheterotrophy) is the use of an organic compound as a source of

carbon and energy. It is the complete metabolism package. The cell oxidizes organic

molecules in order to produce energy (catabolism) and then uses the energy to synthesize

cellular material from these the organic molecules (anabolism). We animals are familiar with

heterotrophic metabolism. Fungi and protozoa are all heterotrophs; many bacteria, but just a

few archaea, are heterotrophs, Heterotrophic fungi and bacteria are the masters of

decomposition and biodegradation in the environment. Heterotrophic metabolism is driven

mainly by two metabolic processes: fermentations and respirations.

Fermentation



Fermentation is an ancient mode of metabolism, and it must have evolved with the

appearance of organic material on the planet. Fermentation is metabolism in which energy is

derived from the partial oxidation of an organic compound using organic intermediates as electron donors and electron acceptors. No outside electron acceptors are involved;

no membrane or electron transport system is required; all ATP is produced by substrate level phosphorylation.

By definition, fermentation may be as simple as two steps illustrated in the following model.

Indeed, some amino acid fermentations by the clostridia are this simple. But the pathways of fermentation are a bit more complex, usually involving several preliminary steps to prime

the energy source for oxidation and substrate level phosphorylations.

Model fermentation. L. The substrate is oxidized to an organic intermediate; the usual oxidizing agent is NAD. Some of the energy released by the oxidation is conserved during the synthesis of ATP by the process of substrate level phosphorylation. Finally, the oxidized intermediate is reduced to end products. Note that NADH2 is the reducing agent, thereby balancing its redox ability to drive the energy-producing reactions. R. In lactic fermentation by Lactobacillus, the substrate (glucose) is oxidized to pyruvate, and pyruvate becomes reduced to lactic acid. Redox balance is maintained by coupling oxidations to reductions within the pathway. For example, in lactic acid fermentation via the EmbdenMeyerhof pathway, the oxidation of glyceraldehyde phosphate to phosphoglyceric acid is coupled to the reduction of pyruvic acid to lactic acid.

In biochemistry, for the sake of convenience, fermentation pathways start with glucose. This

is because it is the simplest molecule, requiring the fewest enzymatic ( catalytic) steps, to

enter into a pathway of glycolysis and central metabolism.

In the bacteria there exist three major pathways of glycolysis (the dissimilation of sugars):

the classic Embden-Meyerhof pathway, which is also used by most eucaryotes, including

yeast (Saccharomyces): the heterolactic pathway used by lactic acid bacteria, and the



Chapter 1

Entner-Doudoroff pathway used by vibrios and pseudomonads, including Zymomonas.

Although the latter two pathways have some interesting applications in the manufacture of

dairy products and alcoholic beverages, they will not be discussed further in this section..

The Embden-Meyerhof Pathway

This is the pathway of glycolysis most familiar to biochemists and eucaryotic biologists, as

well as to brewers, breadmakers and cheese makers. The pathway is operated by

Saccharomyces to produce ethanol and CO2. The pathway is used by the lactic acid bacteria

to produce lactic acid, and it is used by many other bacteria to produce a variety of fatty

acids, alcohols and gases. Some end products of Embden-Meyerhof fermentations are

essential components of foods and beverages, and some are useful fuels and industrial

solvents. Diagnostic microbiologists use bacterial fermentation profiles (e.g. testing an

organism's ability to ferment certain sugars, or examining an organism's array of end

products) in order to identify them, down to the genus level.



The Embden Meyerhof pathway for glucose dissimilation. The overall reaction is the oxidation of glucose to 2 pyruvic acid. The two branches of the pathway after the cleavage are identical.

The first three steps of the pathway prime (phosphorylated) and rearrange the hexes for

cleavage into 2 triodes (glyceraldehyde phosphate). Fructose 1,6-diphosphate aldolase is

the key (cleavage) enzyme in the E-M pathway. Each triose molecule is oxidized and

phosphorylated followed by two substrate level phosphorylations that yield 4 ATP during the

drive to pyruvate. Lactic acid bacteria reduce the pyruvate to lactic acid; yeast reduce the

pyruvate to alcohol (ethanol) and CO2 as shown in Figure below.



Chapter 1

(a) The Embden Meyerhof pathway of lactic acid fermentation in lactic acid bacteria (Lactobacillus) and (b) the Embden Meyerhof pathway of alcohol fermentation in yeast (Saccharomyces). The pathways yield two moles of end products and two moles of ATP per mole of glucose fermented. The steps in the breakdown of glucose to pyruvate are identical. The difference between the pathways is the manner of reducing pyruvic acid, thereby giving rise to different end products.

Besides lactic acid, Embden-Meyerhof fermentations in bacteria can lead to a wide array of

end products depending on the pathways taken in the reductive steps after the formation of

pyruvic acid. Usually, these bacterial fermentations are distinguished by their end products

into the following groups.

a. Homolactic Fermentation. Lactic acid is the sole end product. Pathway of the

homolactic acid bacteria (Lactobacillus and most streptococci). The bacteria are used

to ferment milk and milk products in the manufacture of yogurt, buttermilk, sour

cream, cottage cheese, cheddar cheese, and most fermented dairy products.



b. Mixed Acid Fermentations. Mainly the pathway of the Enterobacteriaceae. End

products are a mixture of lactic acid, acetic acid, formic acid, succinate and

ethanol, with the possibility of gas formation (CO2 and H2) if the bacterium

possesses the enzyme formate dehydrogenase, which cleaves formate to the gases.

c. Butanediol Fermentation. Forms mixed acids and gases as above, but, in addition,

2,3 butanediol from the condensation of 2 pyruvate. The use of the pathway

decreases acid formation (butanediol is neutral) and causes the formation of a

distinctive intermediate, acetoin. Water microbiologists have specific tests to detect

low acid and acetoin in order to distinguish non fecal enteric bacteria (butanediol

formers, such as Klebsiella and Enterobacter) from fecal enterics (mixed acid

fermenters, such as E. coli, Salmonella and Shigella).

d. Butyric acid fermentations, as well as the butanol-acetone fermentation (below),

are run by the clostridia, the masters of fermentation. In addition to butyric acid, the

clostridia form acetic acid, CO2 and H2 from the fermentation of sugars. Small

amounts of ethanol and isopropanol may also be formed.

e. Butanol-acetone fermentation. Butanol and acetone were discovered as the main

end products of fermentation by Clostridium acetobutylicum during the World War I.

This discovery solved a critical problem of explosives manufacture (acetone is

required in the manufacture gunpowder) and is said to have affected the outcome of

the War. Acetone was distilled from the fermentation liquor of Clostridium

acetobutylicum, which worked out pretty good if you were on our side, because

organic chemists hadn't figured out how to synthesize it chemically. You can't run a

war without gunpowder, at least you couldn't in those days.

f. Propionic acid fermentation. This is an unusual fermentation carried out by the

propionic acid bacteria which include corynebacteria, Propionibacterium and

Bifidobacterium. Although sugars can be fermented straight through to propionate,

propionic acid bacteria will ferment lactate (the end product of lactic acid

fermentation) to acetic acid, CO2 and propionic acid. The formation of propionate is a

complex and indirect process involving 5 or 6 reactions. Overall, 3 moles of lactate

are converted to 2 moles of propionate + 1 mole of acetate + 1 mole of CO2, and 1

mole of ATP is squeezed out in the process. The propionic acid bacteria are used in



Chapter 1

the manufacture of Swiss cheese, which is distinguished by the distinct flavor of

propionate and acetate, and holes caused by entrapment of CO2.

The Embden-Meyerhof pathway for glucose dissimilation (Figure 8), as well as the TCA

cycle discussed below (Figure 10), are two pathways that are at the center of metabolism in

nearly all organisms. Not only do these pathways dissimilate organic compounds and

provide energy, they also provide the precursors for biosynthesis of macromolecules that

make up living systems. These are sometimes called amphibolic pathways since the have

both an anabolic and a catabolic function.

Respiration

Compared to fermentation as a means of oxidizing organic compounds, respiration is a lot

more complicated. Respirations result in the complete oxidation of the substrate by an

outside electron acceptor. In addition to a pathway of glycolysis, four essential structural or

metabolic components are needed:

i. The tricarboxylic acid (TCA) cycle (also known as the citric acid cycle or the Kreb's

cycle): when an organic compound is utilized as a substrate, the TCA cycle is used

for the complete oxidation of the substrate. The end product that always results from

the complete oxidation of an organic compound is CO2.

ii. A membrane and an associated electron transport system (ETS). The ETS is a

sequence of electron carriers in the plasma membrane that transports electrons

taken from the substrate through the chain of carriers to a final electron acceptor.

The electrons enter the ETS at a very low redox potential (E'o) and exit at a relatively

high redox potential. This drop in potential releases energy that can be harvested by

the cells in the process of ATP synthesis by the mechanisms of electron transport phosphorylation. The operation of the ETS establishes a proton motive force (pmf)

due to the formation of a proton gradient across the membrane.

iii. An outside electron acceptor ("outside", meaning it is not internal to the pathway,

as is pyruvate in a fermentation). For aerobic respiration the electron acceptor is

O2, of course. Molecular oxygen is reduced to H20 in the last step of the electron

transport system. But in the bacterial processes of anaerobic respiration, the final



electron acceptors may be SO4 or S or NO3 or NO2 or certain other inorganic

compounds, or even an organic compound, such as fumarate.

iv. A transmembranous ATPase enzyme (ATP synthetase). This enzyme utilizes the

proton motive force established on the membrane (by the operation of the ETS) to

synthesize ATP in the process of electron transport phosphorylation. It is believed

that the transmembranous Fo subunit is a proton transport system that transports

2H+ to the F1 subunit (the actual ATPase) on the inside of the membrane. The 2

protons are required and consumed during the synthesis of ATP from ADP plus Pi.

See Figure 6 -the membrane of E. coli. The reaction catalyzed by the ATPase

enzyme is ADP + Pi + 2 H+ <----------> ATP. (It is important to appreciate the

reversibility of this reaction in order to account for how a fermentative bacterium,

without an ETS, could establish a necessary pmf on the membrane for transport or

flagellar rotation. If such an organism has a transmembranous ATPase, it could

produce ATP by SLP, and subsequently the ATPase could hydrolyze the ATP,

thereby releasing protons to the outside of the membrane.)

The diagram below of aerobic respiration integrates these metabolic processes into a

scheme that represents the overall process of respiratory metabolism. A substrate such as

glucose is completely oxidized to to CO2 by the combined pathways of glycolysis and the

TCA cycle. Electrons removed from the glucose by NAD are fed into the ETS in the

membrane. As the electrons traverse the ETS, a pmf becomes established across the

membrane. The electrons eventually reduce an outside electron acceptor, O2, and reduce it

to H20. The pmf on the membrane is used by the ATPase enzyme to synthesize ATP by a

process referred to as "oxidative phosphorylation".



Chapter 1

Model of Aerobic respiration.

The overall reaction for the aerobic respiration of glucose is

Glucose + 6 O2 ----------> 6 CO2 + 6 H2O

In a heterotrophic respiration, glucose is dissimilated in a pathway of glycolysis to the

intermediate, pyruvate, and it the pyruvate that is moved into the TCA cycle, eventually

becoming oxidized to 3 CO2. Since 2 pyruvate are formed from one glucose, the cycle must

turn twice for every molecule of glucose oxidized to 6 CO2. The TCA cycle (including the

steps leading into it) accounts for the complete oxidation of the substrate and it provides 10

pairs of electrons (from glucose) for transit through the ETS. For every pair of electrons put

into the ETS, 2 or 3 ATP may be produced, so a huge amount of ATP is produced in a

respiration, compared to a fermentation.

The TCA cycle is an important amphibolic pathway, several intermediates of the cycle may

be withdrawn for anabolic (biosynthetic) pathways



The tricarboxylic acid (TCA) or Kreb's cycle. Also called the citric acid cycle because citric acid is one of the first intermediates formed during the cycle. When an organic compound is utilized during respiration it is invariably oxidized via the TCA cycle. Combined with the pathway(s) of glycolysis (e.g. Embden-Meyerhof) TCA is central to the metabolism of all heterotrophic respiratory organisms.....worth memorizing if you are a biologist.

Anaerobic Respiration

Respiration in some procaryotes is possible using electron acceptors other than oxygen (O2).

This type of respiration in the absence of oxygen is referred to as anaerobic respiration.

Although anaerobic respiration is more complicated than the foregoing statement, in its

simplest form it represents the substitution or use of some compound other than O2 as a final electron acceptor in the electron transport chain. Electron acceptors used by



Chapter 1

procaryotes for respiration or methanogenesis (an analogous type of energy generation in

archaea) are described in the table below.

Electron acceptors for respiration and methanogenesis in procaryotes

electron acceptor

reduced end product name of process organism

O2 H2O aerobic respirationEscherichia, Streptomyces

NO3 NO2, NH3 or N2anaerobic respiration: denitrification

Bacillus, Pseudomonas

SO4 S or H2Sanaerobic respiration: sulfate reduction

Desulfovibrio

fumarate succinateanaerobic respiration:

using an organic e- acceptor

Escherichia

CO2 CH4 methanogenesis Methanococcus

Biological methanogenesis is the source of methane (natural gas) on the planet. Methane

is preserved as a fossil fuel (until we use it all up) because it is produced and stored under

anaerobic conditions, and oxygen is needed to oxidize the CH4 molecule. Methanogenesis is

not really a form of anaerobic respiration, but it is a type of energy-generating metabolism

that requires an outside electron acceptor in the form of CO2.

Denitrification is an important process in agriculture because it removes NO3 from the soil.

NO3 is a major source of nitrogen fertilizer in agriculture. Almost one-third the cost of some

types of agriculture is in nitrate fertilizers The use of nitrate as a respiratory electron acceptor

is usually an alternative to the use of oxygen. Therefore, soil bacteria such as Pseudomonas

and Bacillus will use O2 as an electron acceptor if it is available, and disregard NO3. This is

the rationale in maintaining well-aerated soils by the agricultural practices of plowing and

tilling. E. coli will utilize NO3 (as well as fumarate) as a respiratory electron acceptor and so it

may be able to continue to respire in the anaerobic intestinal habitat.



Sulfate reduction is not an alternative to the use of O2 as an electron acceptor. It is an

obligatory process that occurs only under anaerobic conditions. Methanogens and sulfate

reducers may share habitat, especially in the anaerobic sediments of eutrophic lakes such

as Lake Mendota, where they crank out methane and hydrogen sulfide at a surprising rate.

Anaerobic respiring bacteria and methanogens play an essential role in the biological cycles

of carbon, nitrogen and sulfur. In general, they convert oxidized forms of the elements to a

more reduced state. The lithotrophic procaryotes metabolize the reduced forms of nitrogen

and sulfur to a more oxidized state in order to produce energy. The methanotrophic bacteria,

which uniquely posses the enzyme methane monooxygenase, can oxidize methane as a

source of energy. Among all these groups of procaryotes there is a minicycle of the elements

in a model ecosystem.

Lithotrophic Types of Metabolism

Lithotrophy is the use of an inorganic compound as a source of energy. Most lithotrophic

bacteria are aerobic respirers that produce energy in the same manner as all aerobic

respiring organisms: they remove electrons from a substrate and put them through an

electron transport system that will produce ATP by electron transport phosphorylation.

Lithotrophs just happen to get those electrons from an inorganic, rather than an organic,

compound.

Some lithotrophs are facultative lithotrophs, meaning they are able to use organic

compounds, as well, as sources of energy. Other lithotrophs do not use organic compounds

as sources of energy; in fact, they won't transport organic compounds. CO2 is the sole

source of carbon for the methanogens and the nitrifying bacteria and a few other species

scattered about in other groups.

Most lithotrophs get their carbon from from CO2 and are thus autotrophs and are properly

referred to as lithoautotrophs or chemoautotrophs. The lithotrophs are a very diverse

group of procaryotes, united only by their ability to oxidize an inorganic compound as an

energy source.

Lithotrophy runs through the Bacteria and the Archaea. If one considers methanogen

oxidation of H2 a form of lithotrophy, then probably most of the Archaea are lithotrophs.



Chapter 1

Lithotrophs are usually organized into "physiological groups" based on their inorganic

substrate for energy production and growth (see Table 2 below).

Physiological groups of lithotrophs

physiological group energy source oxidized end product organism

hydrogen bacteria H2 H2O Alcaligenes, Pseudomonas

methanogens H2 H2O Methanobacterium

carboxydobacteria CO CO2 Rhodospirillum, Azotobacter

nitrifying bacteria* NH3 NO2 Nitrosomonas

nitrifying bacteria* NO2 NO3 Nitrobacter

sulfur oxidizers H2S or S SO4 Thiobacillus, Sulfolobus

iron bacteria Fe ++ Fe+++ Gallionella, Thiobacillus

* The overall process of nitrification, conversion of NH3 to NO3, requires a consortium of microorganisms.

The hydrogen bacteria oxidize H2 (hydrogen gas) as an energy source. The hydrogen

bacteria are facultative lithotrophs as evidenced by the pseudomonads that fortuitously

possess a hydrogenase enzyme that will oxidize H2 and put the electrons into their

respiratory ETS. They will use H2 if they find it in their environment even though they are

typically heterotrophic. Indeed, most hydrogen bacteria are nutritionally versatile in their

ability to use a wide range of carbon and energy sources. the bacterial electron transport

system.

The methanogens used to be considered a major group of hydrogen bacteria - until it was

discovered that they are Archaea. The methanogens are able to oxidize H2 as a sole source

of energy while transferring the electrons from H2 to CO2 in its reduction to methane.

Metabolism of the methanogens is absolutely unique, yet methanogens represent the most

prevalent and diverse group of Archaea. Methanogens use H2 and CO2 to produce cell

material and methane. They have unique enzymes and electron transport processes. Their

type of energy generating metabolism is never seen in the Bacteria, and their mechanism of

autotrophic CO2 fixation is very rare, except in methanogens.



The carboxydobacteria are able to oxidize CO (carbon monoxide) to CO2, using an enzyme

CODH (carbon monoxide dehydrogenase). The carboxydobacteria are not obligate CO

users, i.e., some are also hydrogen bacteria, and some are phototrophic bacteria.

Interestingly, the enzyme CODH used by the carboxydobacteria to oxidize CO to CO2, is

used by the methanogens for the reverse reaction - the reduction of CO2 to CO - in their

unique pathway of CO2 fixation.

The nitrifying bacteria are represented by two genera, Nitrosomonas and Nitrobacter.

Together these bacteria can accomplish the oxidation of NH3 to NO3, known as the process

of nitrification. No single organism can carry out the whole oxidative process. Nitrosomonas

oxidizes ammonia to NO2 and Nitrobacter oxidizes NO2 to NO3. Most of the nitrifying bacteria

are obligate lithoautotrophs, the exception being a few strains of Nitrobacter that will utilize

acetate. CO2 fixation utilizes RUBP carboxylase and the Calvin Cycle. Nitrifying bacteria

grow in environments rich in ammonia, where extensive protein decomposition is taking

place. Nitrification in soil and aquatic habitats is an essential part of the nitrogen cycle.

Lithotrophic sulfur oxidizers include both Bacteria (e.g. Thiobacillus) and Archaea (e.g.

Sulfolobus). Sulfur oxidizers oxidize H2S (sulfide) or S (elemental sulfur) as a source of

energy. Similarly, the purple and green sulfur bacteria oxidize H2S or S as an electron donor

for photosynthesis, and use the electrons for CO2 fixation (the dark reaction of

photosynthesis). Obligate autotrophy, which is nearly universal among the nitrifiers, is

variable among the sulfur oxidizers. Lithoautotrophic sulfur oxidizers are found in

environments rich in H2S, such as volcanic hot springs and fumaroles, and deep-sea thermal

vents. Some are found as symbionts and endosymbionts of higher organisms. Since they

can generate energy from an inorganic compound and fix CO2 as autotrophs, they may play

a fundamental role in primary production in environments that lack sunlight. As a result of

their lithotrophic oxidations, these organisms produce sulfuric acid (SO4), and therefore tend

to acidify their own environments. Some of the sulfur oxidizers are acidophiles that will grow

at a pH of 1 or less. Some are hyperthermophiles that grow at temperatures of 115

degrees C.

Iron bacteria oxidize Fe++ (ferrous iron) to Fe+++ (ferric iron). At least two bacteria probably

oxidize Fe++ as a source of energy and/or electrons and are capable of lithoautotrophic

growth: the stalked bacterium Gallionella, which forms flocculant rust-colored colonies



Chapter 1

attached to objects in nature, and Thiobacillus ferrooxidans, which is also a sulfur-oxidizing

lithotroph.

Lithotrophic oxidations. These reactions produce energy for metabolism in the nitrifying and sulfur oxidizing bacteria.

Phototrophic Metabolism

Phototrophy is the use of light as a source of energy for growth, more specifically the

conversion of light energy into chemical energy in the form of ATP. Procaryotes that can

convert light energy into chemical energy include the photosynthetic cyanobacteria, the

purple and green bacteria, and the "halobacteria" (actually archaea). The cyanobacteria

conduct plant photosynthesis, called oxygenic photosynthesis; the purple and green

bacteria conduct bacterial photosynthesis or anoxygenic photosynthesis; the extreme

halophilic archaea use a type of nonphotosynthetic photophosphorylation mediated by a

pigment, bacteriorhodopsin, to transform light energy into ATP.

Biosynthesis

The pathways of central metabolism (i.e., glycolysis and the TCA cycle), with a few

modifications, always run in one direction or another in all organisms. The reason - these

pathways provide the precursors for the biosynthesis of cell material. When a pathway, such

as the Embden-Meyerhof pathway or the TCA cycle, functions to provide energy in addition

to chemical intermediates for the synthesis of cell material, the pathway is referred to as an



amphibolic pathway. Pathways of glycolysis and the TCA cycle are amphibolic pathways

because they provide ATP and chemical intermediates to build new cell material. The main

metabolic pathways, and their relationship to biosynthesis of cell material, are shown in

Figure 25 below.

Biosynthesis or intermediary metabolism is a topic of biochemistry, more so than

microbiology. It will not be dealt with in detail here. The fundamental metabolic pathways of

biosynthesis are similar in all organisms, in the same way that protein synthesis or DNA

structure are similar in all organisms. When biosynthesis proceeds from central metabolism

as drawn below, some of the main precursors for synthesis of procaryotic cell structures and

components are as follows.

Polysaccharide capsules or inclusions are polymers of glucose and other sugars.

Cell wall peptidoglycan (NAG and NAM) is derived from glucose phosphate.

Amino acids for the manufacture of proteins have various sources, the most

important of which are pyruvic acid, alpha ketoglutaric acid and oxalacetic acid.

Nucleotides (DNA and RNA) are synthesized from ribose phosphate. ATP and

NAD are part of purine (nucleotide) metabolism.

Triose-phosphates are precursors of glycerol, and acetyl CoA is a main precursor

of lipids for membranes

Vitamins and coenzymes are synthesized in various pathways that leave central

metabolism. In the example given in Figure 24, heme synthesis proceeds from the

serine pathway, as well as from succinate in the TCA cycle.



Chapter 1

The main pathways of biosynthesis in procaryotic cells

Written and Edited by KennethTodar University of Wisconsin-Madison Department of Bacteriology. All rights reserved.


mailto:[email protected]


At this point you should be able to:

Understand the impact of microorganisms on the biosphere

Explain differences in cell walls, cytoplasmic membrane of Bacteria, Archaea & Eucarya

Explain the different types of transport across cytoplasmic membrane

Elaborate the microbial growth, development and characteristics of spores.

Differentiate the different types of microbial metabolism.

Identify the role of microbes in the following sectors:

Food

Water

Air

Soil


Define environmental microbiology

Describe the microbial cell, its structure and function.


Chapter 1

PART A: DEFINITIONPlease define the following terms:

Prokaryote

Eukaryote

Gram positive/Gram negative

Binary fission

Endospore

Lag phase

Exponential phase

Stationary phase

Death phase

Catabolism

Anabolism

Respiration

Aerobic respiration

Anaerobic respirartion

TCA pathway

Fermentation

Emden-Meyerhof pathway

Lithotrophic Metabolism

Phototrophic Metabolism

Metanogens

Nitrification

Denitrification



PART B: SHORT ANSWERAnswer the following questions:

1. Differentiate structurally between eukaryote and prokaryote.

2. Differentiate between gram negative and gram-positive bacteria

3. Explain microbial cell growth.

4. List the phases of bacterial growth

5. Name the different microbes involved:

i. Nitrification

ii. Denitrification

iii. Methanogenesis

6. State the importance of nitrifiers , denitrifiers and methanogens.

7. Differentiate TCA and Emden-Meyerhof pathways

8. Identify the energy source in lithotrophic and phototrophic metabolisms.



Chapter 1


STUDY NOTES:

Documents

Step 5] New Module... · Web viewChapter Overview 1.1 WHAT IS ENVIRONMENTAL MICROBIOLOGY? The study of how microorganisms affect the earth and its atmosphere is called environmental