Oklahoma State University

Gene Regulation

Need to regulate expression - "waste not". Enzymatic activity and transcription/translation are energy consuming processes. Generally, bacteria do not expend energy unnecessarily.

How do bacteria regulate gene expression?
1. control the activity of preexisting enzymes
2. Control transcription/translation

Controlling activity of enzymes - this approach allows cells to respond rapidly to their environment.

Product inhibition - enzymes participate in reactions that can go in either direction. If the concentration of the product builds up the activity of the enzyme may be reversed from product to substrate.

Feedback inhibition - usually occurs in pathways from substrate A to product F. Product F may inhibit some key enzyme near the beginning of the pathway. Again, as the concentration of the product builds up, the pathway is inhibited.

How? The key enzyme is an allosteric enzyme with two important sites: the active site and the allosteric site. The allosteric site binds reversibly an effector molecule (e.g., product) that causes a conformational change in the active site so that the substrate can no longer enter the active site effectively.

Transcriptional/translational control or regulating protein synthesis.
simplest of them

Enzyme repression - an endproduct of a pathway represses the synthesis of the enzymes. Found in the biosynthesis of amino acids and nucleotides. The substance that represses transcription is called a corepressor. The corepessor binds to a repressor protein, an allosteric protein, and causes a conformational change in the repressor such that it binds to the operator region, a region close to the promoter region.

Enzyme inductions - synthesis of the enzymes only if substrate is available. Frequently associated with catabolism of carbon and energy sources. The substance that induces transcription is called an inducer. A repressor protein binds to the operator and blocks transcription when the inducer is absent. When the inducer is present and binds to the repressor, the repressor cannot bind to the operator and transcription proceeds. Notice the underlying mechanism is for the repressor to inhibit transcription and the repressor activity is effected by a small molecule. Inhibition by a repressor in general is called negative control.

In both cases they work on the need basis! don’t need repress; do need induce!

Constitutive enzymes are enzymes that have little or no control. Their synthesis is pretty much level throughout the life cycle of the cell. These are house hold type enzymes that are required for the daily maintenance of the cell.

What if a bacteria is faced with two different carbon sources, e.g., glucose and lactose? Perhaps one carbon source is better than the other. What would a bacteria do? Not express the genes necessary to metabolize the poorer carbon source is a good idea? Repress these genes somehow.

Exactly what we will look at - a phenomenon called catabolite repression.

Catabolite repression or Glucose effect - is defined where a simple energy source is preferentially used over a more complex energy source. The utilization of the one energy source, in this case glucose, represses the genes of many unrelated enzymes.

What does the phenomenon look like?

One consequence is what is called diauxic growth. In diauxic growth the organism grows on the preferred substrate and then switches to metabolize the alternative substrate.

For example inoculate a flask of medium containing glucose and lactose with Escherichia coli. E. coli will grow on the glucose until it runs out of glucose. There will be a brief cessation of growth - a lag period- and then the cells will grow on lactose as a carbon and energy source.

Operon model for gene expression - Lac operon model from Francois Jacob and Jacques Monod. Operon consists of a promoter, operator and the structural genes in this case lac z, y and a which code for beta galactosidase, a permease and transacetylase respectively. Near the operon is a gene called lacI which codes for a repressor for the lac operon.

When lactose is absent, the repressor molecule binds to the operator region and nearly completely shuts down transcription of the the operon by RNA polymerase. This is analogous to the inducible operon we talked about earlier.

When lactose is present, some of the lactose is converted to allolactose, the inducer of the operon. Allolactose binds to the repressor and inactivates it such that it will not bind to the operator region. Therefore, RNA polymerase can transcribe the operon.

What happens if glucose and lactose are both present in the medium?

What was happening during growth on glucose? - Catabolite repression. Catabolite repressed operon require an allosteric protein called catabolite activator protein or CAP to bind to the DNA before RNA polymerase will bind and transcription will occur. CAP will bind to the CAP binding site of the DNA if and only if it has bound cyclic AMP. In the absence of cAMP, CAP will not bind to catabolite repressed operons. Glucose inhibits the synthesis of cAMP in a complex manner.

So what is happening in our culture of E. coli? The presence of glucose is keeping the levels of cAMP low, which does not allow CAP to bind to the lactose operon and therefore the lac genes are not transcribed or translated. There are many other operons that are controlled by cAMP levels and CAP.

What then is required for the lac operon to be expressed?

1. cAMP bound to CAP which binds to the CAP binding site to activate transcription of the operon.

2. There must be an inducer such as allolactose, which binds to the repressor and prevents it from binding to the operator region. An example of negative control since there is a repressor regulating transcription.

 

Molecular Genetics

Genetics is defined as the mechanism by which traits are passed on from one organism to another and how they are expressed.
How does information flow? Central dogma of molecular biology
Replication - to ensure that following generations acquire genetic material
Transcription - intermediate process between DNA and protein synthesis. Generates mRNA, rRNA and tRNA.
Translation - messenger RNA is translated by ribosomes into a polypeptide based on genetic code of the nucleic acids. Genetic code is made up of "words" that are 3 bases long called a codon.
Reiterate - central dogma of molecular biology is that genetic information flows from DNA through RNA to protein.
Genetic information - genetic information is stored primarily in DNA.
What is DNA? DNA is composed of four nucleotides adenine, guanine, cytosine and thymine. Each nucleotide is composed of a nitrogenous base, deoxyribose, and phosphate groups. DNA is a polynucleotide molecule where the backbone is a sugar phosphate backbone and the nitrogenous bases stick out like rungs on a ladder. The phosphate linkage is called a phosphodiester linkage between the 5 prime phosphate group of one sugar and the 3 prime hydroxyl group on the second sugar.
DNA is a double helix where two polynucleotide strands that are complementary are held together by hydrogen bonds. Specifically adenine and thymine complement each other and guanine and cytosine complement each other. The two strands are oriented in an antiparallel fashion meaning that one strand runs 5 prime to 3 prime and the complement is 3 prime to 5 prime. The DNA is not a ladder but rather has a twisted about it which gives it it's helical nature.
The amount of DNA in an microorganism may be in the thousands of kilobase pairs. E. coli has about 4700 kilobase pairs which is about 1.5 millimeter in length if it were linear but bacterial chromosomes are circular.
Problem still getting all of that DNA in a cell. How do they pack the DNA in a cell? The answer is as a supercoiled molecule. Supercoiled DNA is double stranded DNA further twisted in what is either negatively or positively supercoiled. Most DNA in nature is negatively supercoiled. Supercoiled DNA is only stable if the two strands are intact. If one strand is nicked, the supercoil tension is relaxed and the DNA is in a relaxed state.
How is DNA supercoiled in bacteria? A enzyme called DNA gyrase which introduces the supercoils. DNA gyrase is one topoisomerase enzyme which change the topology of DNA - specifically topoisomerase II.
Topoisomerase I removes supercoils from DNA. A nick is made in one of the two strands and the supercoil relaxes. In bacteria, like eukaryotic cells, a single nick does not cause the entire chromosome to become relaxed since there are some 50 domains of supercoil which are independent of each other. The level of supercoiling is balanced between the activity of the two topoisomerases.
Genetic elements includes chromosome and other elements.
Chromosome - single circular double stranded helix that codes for indispensable functions - so called house keeping genes
Plasmid - small cirular double stranded helix. Code for important properties for the cell such as antibiotic resistance. There may be more than one type of plasmid in a cell.
Transposable elements - pieces of DNA that can move about the chromosome. Three types of transposable elements:
insertion sequences that carry no genetic information other than what is required to move.
Transposons - which carry other genes in addition to what is required to move.
Some special viruses -
DNA Replication - making a second copy of the chromosome before the cell divides.
Beginning of replication - in a circular DNA molecule there is a single origin of replication (oriR) which is where replication begins. The origin of replication opens up and DNA replication begin on the two single strands and as the two strands are separated a replication fork is formed and proceeds down the DNA. Usually bidirectional replication occurs.

leading strand - newly synthesized strand that grows towards the replication fork. Replication begins with a small stretch of RNA - a primer - laid down by primase. DNA polymerase III catalyzes the bonding between the 3 prime hydroxyl group of the primer and the 5 prime phosphate group of the incoming nucleotide with the concomitant hydrolysis of the terminal two phosphate groups. This leads to 5´ to 3´ extension of the newly synthesized strand.
At the replication fork, a helicase unwinds the DNA to expose the single strand. Helicases are ATP-dependent enzymes that hydrolyze ATP as they move in advance of the replication fork. Single stranded binding proteins stabilize single stranded DNA before it forms double stranded DNA.
lagging strand - because DNA is antiparallel, this strand is synthesized discontinuously. What do we mean by discontinously? Synthesis of the lagging strand occurs in starts and stops. Why because the 3 prime hydroxyl group is pointed away from the replication fork. As the replication fork opens up, primase lays down 11 bases and then DNA polymerase III lays down nucleotides until it reaches the beginning of another short segment called Okazaki fragments which are about 1000 bases long. Here Pol III falls off and Pol I removes the primer sequence and lays down deoxynucleotides until it has removed all of the ribonucleotides of the primer and then it falls off. DNA ligase forms a bond between the two new fragments.
The fidelity of replication is remarkable. About one error in between 10 8 and 10 11 base pairs. Errors are corrected by the proofreading activity of PolIII. Pol III has 3 prime to 5 prime exonuclease activity to remove any mistakened nucleotides that tries to make an incorrect insertion.
Transcription
The synthesis of RNA from DNA template. There are three different RNA molecules: messenger RNA, ribosomal RNA, and transfer RNA. There are three key differences between RNA and DNA: 1. ribose instead of deoxyribose; 2. uracil instead of thymine; 3. RNA is usually single stranded.
RNA polymerase - the enzyme that catalyzes the formation of RNA by transcribing DNA into RNA. Requires a DNA template, ribonucleotides ATP, GTP, CTP and UTP. Elongation is 5 prime to 3 prime just like DNA synthesis. No need for a primer unlike DNA synthesis.
Template DNA is usually double stranded DNA but only one of the two strands is transcribed for any gene.
RNA polymerase composed of beta, beta prime, two alpha subunits plue a sigma subunit. The core enzyme is beta, beta prime and the two alpha subunits.
RNA polyerase must start at the proper site of each gene to generate a complete correct transcript. RNA polymerase binds at the promoter site to orient the enzyme at the correct start position. It is the sigma factor of the polymerase that recognizes the promoter region.
Features of the promoter sequence of DNA - Two highly conserved sequences in the promoter sequence of many different genes. -10 region or the Pribnow box has a sequence of TATAAT and the -35 region has the sequence TTGACA. Again these sequences are highly conserved but not perfectly conserved among all genes.
Features of transcription terminators - Some terminators are a result of secondary structures in the RNA transcript that are stem loops followed by runs of uracil. A second terminator is a GC rich region followed by a AT rich region. A third type of termination is due to an extrinsic factor called Rho which binds to the RNA and moves toward the DNA/RNA polymerase complex and when the RNA polymerase stalls at a rho dependent site, rho causes RNA polymerase and RNA to leave the DNA.
Features of messenger RNA - mRNA is unstable unlike tRNA or rRNA. In prokaryotes mRNA often codes for more than one polypeptide but 2 or more. This is referred to as polycistronic mRNA.
Features of transfer RNA - have two important features: 1. carry an amino acid and 2. recognize a sequence on the messenger RNA - the codon.

tRNA structure - short single stranded RNAs of about 73-93 nucleotides in length. There are many unusual nucleotides in tRNAs which are a result of post-transcriptional modifications of the tRNA. The tRNA folds back upon itself to have secondary structure due to internal base pairing. Often drawn to look like a clover leaf which is slightly missleading.
One loop of the tRNA is very important - the anticodon loop -which is where you find the anticodon. The three nucleotides of the anticodon recognize the three nucleotides of the codon in the mRNA molecule.
The other important end of the tRNA is the 3 prime end which is alway CCA where the amino acid attaches to the terminal adenine nucleotide via an ester linkage.
how is the correct amino acid attached to the correct tRNA? There are key regions of the tRNA like the anticodon region and the 5 prime end of the tRNA that are important in the recognition of the correct tRNA by the correct aminoacy-tRNA synthetase protein.
First reactions involves the activation of the amino acid by a reaction with ATP to form aminoacyl - AMP which remains bound to the synthetase protein. A correct tRNA enters this complex and the amino acid is transferred to the tRNA to form a charged tRNA - aminoacyl-tRNA. The aminoacyl-tRNA participates in translation.
Translation - the synthesis of a polypeptide. Ribosomes are the site of protein synthesis. Ribosomes are composed of two subunits - 30S and 50S subunits. Each of these are composed of ribosomal RNA and proteins. For example the 50S subunit is composed of a 5S and 23S rRNA and about 34 different proteins.
Protein synthesis can be broken down into initiation, elongation, and termination. These are ongoing processes that are continuously going on.
Initiation of protein synthesis.
30S subunit, mRNA, formylmethionine tRNA and initiation factors are required. These components form a complex near the 5prime end of the mRNA at a site called the Shine-Delgarno sequence which interacts with the 16S rRNA to ensure that the ribosome is starting at the beginning of a gene. The 50S subunit then joins the complex to form a complete ribosome. The first codon of the mRNA is usually AUG - the start codon for translation. This codon is recognized by formyl-methionine tRNA. The formyl group and maybe even the methionine amino acid may be removed later.
Elongation - Important features of the 50S subunit are two sites called the A site and P site. The A site is the accepting site where incoming amino acids dock with the ribosome. The P site is the peptide site where the growing peptide is attached to a tRNA. Initially f-met-tRNA occupies the P site and the A site is empty. A respective charged tRNA comes and occupies the A site - Which charged tRNA? that depends on the exposed codon in the A site. Now with both P and A site occupied a peptide bond forms between the carboxyl group of the P site amino acid and the amine group of the A site amino acid. Hydrolysis of the aminoacyl-tRNA bond is used to drive the peptide bond formation. Peptidyl transferase is responsible for forming the peptide bond. Now the peptide is attached to the tRNA in the A site. The peptide-tRNA must now move to the P site in a process called translocation which expends another GTP and a new codon is exposed in the A site. The empty tRNA is moved to what appears to be a third site in the ribosome called the E site. This continues on until the ribosome reaches a specific termination signal.
Termination - there are three codons that are stop or nonsense codons - UAA, UAG, and UGA. There are no tRNAs that recognize these codons but rather release factors recognize these codons and come in a cleave the peptide from the tRNA and cause the ribosome to dissassociate.
Energy requirements - 4 high energy phosphate bonds are hydrolyzed per amino acid incorporated into the peptide. 2 bonds are consumed by the activity of the aminoacy-tRNA synthetase activity, 1 bond when the charged tRNA enters the A site and 1 bond hydrolyzed for the translocation process. Point is that protein synthesis is energy demanding and we will see how it is controlled later.
Secreted proteins - proteins that must pass out the cytoplasmic membrane into the periplasmic space or extracellular. These proteins have an extra 15-20 amino acids on the N-terminal portion of the polypeptide. These amino acids are referred to as the signal sequence. The signal sequence is usually rich in hydrophobic amino acids to help it insert into the cytoplasmic membrane. Once secreted, the signal sequence is cleaved by specific peptidases in a posttranslational modification.
Antibiotics - protein synthesis is a great target for antibiotics to control growth of the bacteria. Specifically, antibiotics that interact with the ribosomes are medically useful. Streptomycin inhibits initiation, puromycin, chloramphenicol, cycloheximide, and tetracycline inhibit elongation. Puromycin will compete with incoming amino acids for the A site. Chloramphenicol inhibits peptide bond formation.
Because of the differences between eukaryotic and prokaryotic ribosomes, drugs can be used to stop one group and not the other. Streptomycin and chloramphenicol inhibit prokaryotes and cycloheximide inhibits eukaryotes.
Genetic code - recall that three nucleotides in the mRNA codes for a specific amino acid and these three nucleotides are called a codon. If there are 4 possible bases for each position of a codon then there are 4e3 power possible codons or 64 codons. There are only 20 or so amino acids though. What does this mean? More than one codon may code for a specific amino acid - but no codon codes for more than one amino acid! This is referred to as degeneracy or redundancy.
Significance of degeneracy is that I) there is more than one tRNA for some amino acids and ii) a single tRNA may pair with more than one codon in what is called the wobble effect. There is not a tRNA for every codon, so some tRNAs recognize more than one codon. How? the first two bases are complementary but the third base may be a mismatch but still the correct amino acid is incorporated into the polypeptide.
Open reading frames - it is essential that the ribosome starts at the right codon to initiate the polypeptide. There are many safegards to ensure that this is so. Recall that most polypeptides begin with f-met which is coded for by AUG. This is the start codon and sets the ribosome in the right reading frame.
Another feature of the genetic code is that it is universal. Essentially the same between Escherichia coli and human beings. Your book talks about some exceptions to the universality of the genetic code. But still, these exceptions appear to have evolved from the universal code. In some instances stop codons have been assigned an amino acid.

Microbial Growth Control
Talk about various methods to control the growth of bacteria, e.g., on food to prevent spoilage.
Begin with physical methods to control growth and then look at chemical methods.
Heat Sterilization - Two important parameters: How long? and What temperature? Both of these can vary depending on the sensitivity of the microorganisms to temperature. Temperature effects are obvious when you look at what is called decimal reduction time - which is the time required to reduce the population by 10 fold at a given temperature.
Thermal Death time - is the time required to kill all bacteria in a sample at a specific temperature. Need to standardize the population size of your sample before determining the thermal death time.
Heat killing is most effective if the heat is a moist heat. Dry heat killing requires higher temperatures for longer periods of time.
Heating must kill spores which are heat resistant structures. The environment the spores are in affects their sensitivity to heat killing. For example a solution rich in sugars and proteins increases spores resistance to heat killing. Also the amount of water in the spores affects their sensitivity - the more water the more sensitive.
The autoclave - 15 lbs/sq. inch which yields a temperature of 121 oC for 10 -15 minutes.
Not all materials can withstand autoclaving, e.g., milk, so they are pasteurized. Pasteurization is a mild heating to kill sensitive spoilage bacteria. Milk is heated to 71oC for 15 seconds in a continuous flow system.
Electromagnetic irradiation - microwave, UV, X-rays, gamma rays, and electrons are all forms of electromagnetic radiation. They all work in different ways, for example microwaves heat up the sample, UV breaks the DNA up causing death.
Ionizing radiation - electromagnetic radiation that produces electrons, hydroxyl radicals, and hydride radicals. These products react with macromolecules such as DNA and proteins which are detrimental to the cell. Common sources of ionizing radiation are 60Co and 137Cs. Radiation is becoming accepted for foods.
Filter sterilization - useful for sterilizing heat labile liquids. The filters have pores of specific sizes down to 0.2 micron in diameter.
Three types of filters
Depth filters - routine filters made of paper, asbestos or glass fibers. Just a mat of the fibers.
Membrane filters - cellulose acetate or cellulose nitrate materials. Contains lots of tiny holes that act live sieves to retain bacteria.
Nucleopore filters - polycarbonate filters. Very uniform pore sizes. Great for microscopy work since the filters are flat in nature.
Chemical growth control - chemical agents that either kill or inhibit growth. Bacteriocidal agents kill bacteria and bacteriostatic agents inhibit growth.
Bacteriostatic agents usually target a cellular process such as protein synthesis where they inhibit ribosomes
Bacteriocidal agents kill cells but do not cause lysis of cells. These agents bind tightly to their target.
Bacteriolytic agents kill cells by lysis.
Minimal inhibitory concentration - concentration of chemical agent that inhibits growth. Can determine it using a series of dilutions of the chemical introduced into tubes with growth medium and inoculated. This can be standardized so that results can be compared. Agar diffusion method is used where a disc is loaded with the chemical and placed on a plate with the bacteria growing. Results in a zone of inhibition around the disc with the chemical.
Antiseptics and disinfectants
Antiseptics are chemicals sufficiently non-toxic to use on living tissue. Includes silver nitrate, iodine, alcohol (70%), and hydrogen peroxide.
Disinfectants are chemicals that kill microorganisms and are used on inanimate objects. Includes copper sulfate, mercuric chloride, chlorine gas, ethylene oxide and ozone.
Growth factor analogs - chemotherapeutic agents that can be taken internally and mimic a growth factor needed for normal growth.
Sulfa drug - a growth factor analog that mimics p-amino benzoic acid which is part of the vitamin folic acid. Sulfanilamide inhibits bacteria since they synthesize their own folic acid whereas higher animals obtain it from the food.
Other growth factor analogs block DNA metabolism such as nucleotide synthesis.
Antibiotics - chemicals that are produced by bacteria that kill or inhibit other bacteria.
Gram positive bacteria are more sensitive to antibiotics than Gram negative bacteria generally. Broad spectrum antibiotics act on both types of bacteria.
Targets of antibiotics
cell wall synthesis
cytoplasmic membrane
protein and nucleic acid biosynthesis
[beta]-lactam antibiotics includes penicillin, cephalosporins and cephamycins. First one discovered was penicillin G effective against Gram positive cells. Gram negative bacteria are impermeable to this drug. Other penicillins are effective against gram negative bacteria for example carbenicillin and ampicillin.
Mode of action - inhibit cell wall synthesis specifically the transpeptidation reaction between adjacent glycan chains. Works only of growing cells which are actively synthesizing peptidoglycan layers.
Aminoglycosides - are amino sugars bonded by glycosidic linkages. Include streptomycin, kanamycin, neomycin, and gentamicin. Used against gram negative bacteria. Not commonly used today.
Mode of action - inhibit protein synthesis at the 30S subunit of the ribosome.

Macrolide antibiotics - lactone rings connected to sugars. Includes erythromycin.
Mode of action - inhibits protein synthesis at the 50S subunit of the ribosome.
Tetracylines - first of the broad spectrum antibiotics.
Mode of action - inhibits protein synthesis. Used on poultry feed.

Microbial Growth

Microbial growth is usually studied as a population not an individual. Individual cells divide in a process called binary fission where two daughter cells arise from a single cell. The daughter cells are indentical except for the occassional mutation.
Binary fission requires:
cell mass to increase
chromosome to replicate
cell wall to be synthesized
cell to divide into two cells
Exponential growth
Exponential growth is a function of binary fission since at each division there are two new cells. The time between divisions is called generation time or the doubling time since this is the time for the population to double. These can range from minutes to days depending on the species of bacteria.
Growth rate is the change in cell number or mass per unit time.
What do we mean by exponential growth? A population doubles each generation is exponential growth.
Graphically on arithmetic coordinates the graph takes the shape of a J - a curve with ever increasing slope - growth rate. Plotted on semilogarithmic paper, where the Y axis is logarithmic and the X axis is arithmetic, you get a straight line.

Generation times - N = No2n where No is the original number of cells and n is the number of generations. g, generation time, equals t/n, time divided by generation.
How do you calculate n?
N = No2n
log N = log No + nlog2
log N - log No = n log2
n = [log N - log No] / log2
n = [log N - log No] / 0.301
We also know that the slope of the semilog line equals 0.301 divided by the generation time.
Batch culture of bacteria
Culturing bacteria in a Erlenmeyer flask where you simply inoculate it and let the bacteria grow.
There are 4 phases of growth in batch culture.
Lag phase - A newly inoculated culture usually does not begin growing immediately but rather after of period of no growth which is referred to as the lag phase.
Conditions that lead to a lag phase -
inoculum which is in stationary phase inoculated into the same medium
inoculum which is damaged but not killed inoculated into the same medium
inoculum transfered from rich to poor medium
Why is there a lag phase? the cells are tooling up for growth.
Stationary cells have probably depleted essential requirements and they need to be resynthesized.
Damaged cells need to repair before they can grow
Transfered cells need to synthesize new enzymes required for growth in the poor medium.
When is a lag phase not necessary?
When active cells are transfered back to the same medium.
Exponential phase - a consequence of each cell dividing to form two cells. Usually the phase with the greatest rate of increase in the population size. The rate is influenced by the environmental conditions such as temperature, aeration, and composition of medium.
Stationary phase - realize that a bacterium - a single cell - with a generation time of 20 minutes would produce a population with the weight of 4000 times the earth after 48 hours. Wow A bacterium weighs about 10-12 gram.
What happens to stop this?
There are factors that limit population growth -
1. intraspecific competition for nutrients which are running out as the culture ages.
2. Build up of toxic metabolites
All of this leads to a stationary phase in which the growth rate of the population is zero.
Death phase - after stationary phase the cells may remain alive for a long period of time or begin to die off as in a death phase. The cells may begin to lyse as they die and other viable cells may grow on the remains of the lysed cells in what is called cryptic growth.
Now remember that we are talking about a population - not a single cell.
How do we measure growth? -
Direct microscopic counts - use the microscope and a slide with a grid engraved on it. A coverslip and placed over the grid which captures a known volume of liquid.
Problems with direct microscopic counts
dead cells are difficult to distinguish
small cells are difficult to see
method not suitable for dilute samples
Viable counts - count only cells that are able to divide and form offspring. Referred to as plate counts or colony counts. Assumption each viable cell gives rise to a colony.
spread plates and pour plates
Dilutions - to cover a cell density that ranges from 30 - 300 colony forming units per plate.
Problems
Not all species of bacteria will form colonies on any particular medium.
small colonies are not counted
Despite problems, it is still widely used in ecology, food microbiology, medical microbiology, and dairy microbiology.
Turbidity - cell suspensions look cloudy because each cell scatters light as it passes through a suspension of cells. Take advantage of the light scattering properties of a suspension using a spectrophotometer which measures unscattered light as it passes through. The scatter is proportional to cell number (density of cells) up to high density cultures because cells begin to cause rescatter the light back into the path of unscattered light. Therefore the optical density is not linear at high density suspensions.
Need to develop a standard curve between OD and cell numbers (viable counts).
Continuous culture
In a batch culture, culture conditions are dynamic since there are a fixed amount of nutrients and the number of cells is changing. Continuous culture has been developed to maintained constant conditions. Continuous cultures are flow through cultures where there is input and output and the conditions reach an equilibrium where the cell number and nutrient concentration remains constant at steady state.
Chemostat - common continuous culture devise where the concentration of nutrients and dilution rate are controlled. The dilution rate controls the growth rate of the bacteria and the nutrient concentration controls the growth yield of the culture (see figure 5.11)
At low dilution rates the cells are starving and dying and the culture may washout. At higher dilution rates the cells may wash out also if the dilution rate is greater than their growth rate.
Cell density is controlled by nutrient concentration in the input. Keeping the dilution rate constant and increasing the nutrient concentration results in a larger population with the same growth.
Environmental factors -
Temperature - as temperature increases, the growth rate increases until a point at which the population dies.
Minimum temperature - below growth does not occur may be due to the stiffening of the cytoplasmic membrane.
optimum temperature where the growth rate is maximum
Maximum temperature- above which growth does not occur which reflects when proteins may be denatured, nucleic acids and other cellular components are irreversibly damaged.

Classification of bacteria based on temperature optimum
psychrophiles - low temperature optima <15 C - may even be killed by brief warming or thawing.
mesophiles - midrange temperature optima 25 - 40 C
thermophiles - high temperature optima 45 - 80 C
hyperthermophiles - very high temperature optima >80 C
Psychrophiles
open ocean water is between 1 and 3 C.
Artic and Antartic regions are cold.
Adaptation
membranes rich in unsaturated fatty acids
Thermophiles
hot springs all over the world
fermenting compost
Adaption
thermostable proteins with usually a few changes in the amino acid sequence when compared to a mesophile's protein
saturated fatty acids in their membranes
Biotechnology
Taq DNA polymerase from Thermus aquaticus.
Acidity and alkalinity
Most environments are between 5 and 9 and optima are between these values.
Acidophiles
live at low pH
Obligate acidophiles such as Thiobacillus.
cytoplasmic membrane actually dissolves and the cell lysis at more neutral pH.
Alkaliphiles
live at high pH such as soda lakes and carbonate soils.
Important to biotechnology since they have hydolytic proteases that function at alkaline pH and are used in household cleaners.
Water availability - bacteria need water as a solvent.
Water availability is expressed as water activity - how much water is available. Solutes and surfaces affect water activity - both decrease it. Water moves from high water activity values to lower values in the process of osmosis. Different bacteria have different tolerances towards low water activities.
Halophiles require 1-6% for mild halophiles and 7-15% salt for moderate halophiles. Extreme halophiles require 15-30% salt.
Osmophiles grow in sugary solutions and xerophiles grow in dry environments.
As the water activity or water potential drops, bacteria will lose water to their environment unless they stop it. Compatible solutes are solutes produced by the bacteria to counteract the effects of more negative water potentials. Compatible solutes include amino acids, sugars, and alcohols such as glycerol.
Oxygen -
Aerobes require oxygen up to 21% as in air.
Microaerophilic bacteria require reduced levels of oxygen
strict or obligate anaerobes require the absence of oxygen.
Anaerobic culture conditions - add reducing reagents such as thioglycolate, bubble nitrogen gas through your solutions to remove oxygen after autoclaving, add a dye such as resazurin to indicate when oxygen is penetrating, use an anaerobic jar with an atmosphere containing hydrogen gas and carbon dioxide.
Why go to such great troubles for the strict anaerobes? Because they contain lots of flavins which react with oxygen to produce toxic oxygen species that are very reactive.
Oxygen species - singlet oxygen which the valence electrons become highly reactive and oxidize organic matter readily.
Superoxide anion, hydrogen peroxide, and hydroxyl radical which are inadvertant byproducts during respiration. These can all damage cell macromolecules by oxidation processes.
Measures to counter these toxic oxygens - catalase degrade hydrogen peroxide to oxygen and water.
peroxidases - destroys hydrogen peroxides too but requires NADH.
super oxide dismutase produces hydrogen peroxide from super oxides.
Aerobes and facultative aerobes generally contain catalase and super oxide dismutase.