Introduction

The modern taxonomy of cyanobacteria is complicated by three sets of more or less incompatible data. They are:

1) Knowledge of wide diversity of cyanobacteria in nature, based on traditional morphological criteria. The diversity of morphotypes (“traditional species”) is described only partly in identification manuals.

2) Still fragmentary data, based on biochemical and molecular methods and evaluations, which are considered often not to be conform with descriptions of phenotype taxonomy.

3) The old idea survives, that all cyanoprokaryotes can occur elsewhere.

Problems of modern cyanobacterial taxonomy

The molecular approach was introduced in cyanobacterial taxonomy particularly by Stanier’s team about 30 years ago

The first attempt to synchronize the modern molecular data with phenotype cyanobacterial taxonomy was published by Anagnostidis and Komárek , who revised particularly the

phenotype generic diagnostic characters, respecting the up to date knowledge of cytomorphological variability, as well as modern molecular results.

The recent molecular analyses confirmed perfectly phenotypically revised stable taxa on the generic level.

Summary of the main current problems

· Diversification and speciation processes.· Relations between genotype (molecular) and stable

phenotype characters. Molecular evaluation must be combined with evaluation of phenotype and ecological characters.

· Recognition of life cycles, ecological plasticity (and limits) and morphological variation in natural populations and in culture.

· Definition of the conventional criteria of “species” (and other taxonomic categories); this problem is connected with numerous “formal” questions (typification, naming, etc.). It is necessary to tend to such definition, which can be designated as evolutionary and phenotypically well characterised natural units, recognisable in nature and/or in culture.

Diversity in cyanobacteria genera

Synechocystisspherical cells division of cells in two planes parietal position of thylakoids (in allspecies( Cyanobium-widely oval cellsdivision of cells in one planeparietal position of thylakoids Synechococcuscylindrical, rod- like cells division of cells in one planeparietal position of thylakoids (few to

many)

Diversity in cyanobacteria genera

Cyanobacterium- widely cylindrical to oval cells- division of cells in one plane- - lengthwise position of

thylakoids (numerous!) Cyanothece- widely oval to cylindrical-oval

cells- division of cells in one plane- radial position of thylakoids- (numerous!)

Diversity in cyanobacteria genera

Diversity in cyanobacteria genera

Diversity in cyanobacteria genera

Diversity in cyanobacteria genera

Diversity in cyanobacteria genera

Sequencing 16s rDNA from individual organisms has given us the ability map phylogenetic relationships between single celled organisms based upon how similar their 16s genes are.

Thanks to Carl Woese

Molecular systemetics based upon 16s rRNA sequences

(thanks to Carl Woese, the father of modern microbial systematics

The 16S rRNA gene is ideal because much of it is conserved due to structural constraints but some of it is not. This allows for variability in the gene sequence useful in making phylogenetic comparisons.

Because the gene is only about 1550 bp in length and it portions of it are conserved, it is possible to construct primers that will amplify this gene in a major portion of the organisms present in any given environment.

After PCR amplification, it is possible to sequence the gene and make comparisons with other members of the microbial community or members of its own species.

16SrRNA Molecule

16S Ribosomal RNA: ("the molecule" of modern genomics)

The section (or gene) that codes for ribosomal RNA in a cell is the 16S rRNA gene (or rDNA gene).

Every cell has a 16S rRNA gene as the ribosomal RNA (rRNA) is an integral part of the ribosome, which is responsible for making cellular proteins.

This gene is only about 1550 bp in length in prokaryotes and every cell has from one to more than 25 copies of this gene.

The 16S rRNA molecule folds itself into a shape that must fit like a puzzle piece with other molecules and consequently, some structural elements must be maintained.

Therefore, some sections of the sequence may vary and others may not.

What is DNA Extraction

What is DNA Extraction?

DNA Extraction is the removal of deoxyribonucleic acid (DNA) from the cells or viruses in which it normally resides.

What is it used for?

Extraction of DNA is often an early step in many diagnostic processes used to detect bacteria and viruses in the environment as well as diagnosing disease and genetic disorders.

How does it work?

Break open (lyses) the cells or virus containing the DNA of interest-This is often done by sonicating or bead beating the sample. Vortexing with phenol (sometimes heated) is often effective for breaking down protienacious cellular walls or viral capsids. The addition of a detergent such as SDS is often necessary to remove lipid membranes.

DNA associated proteins, as well as other cellular proteins, may be degraded with the addition of a protease. Precipitation of the protein is aided by the addition of a salt such as ammonium or sodium acetate. When the sample is vortexed with phenol-chloroform and centrifuged the proteins will remain in the organic phase and can be drawn off carefully. The DNA will be found at the interface between the two phases.

How does it work?

DNA is the precipitated by mixing with cold ethanol or isopropanol and then centrifuging. The DNA is insoluble in the alcohol and will come out of solution, and the alcohol serves as a wash to remove the salt previously added.

Wash the resultant DNA pellet with cold alcohol again and centrifuge for retrieval of the pellet.

After pouring the alcohol off the pellet and drying, the DNA can be re-suspended in a buffer such as Tris or TE.

Presence of DNA can be confirmed by electrophoresing on an agarose gel containing ethidium bromide, or another fluorescent dye that reacts with the DNA, and checking under UV light.

DNA Replication

What is PCR?

Sometimes called "molecular photocopying," thepolymerase chain reaction (PCR) is a fast andinexpensive technique used to "amplify" - copy – smallsegments of DNA. Because significant amounts of asample of DNA are necessary for molecular and geneticanalyses, studies of isolated pieces of DNA are nearlyimpossible without PCR amplification. Often heraldedas one of the most important scientific advances inmolecular biology, PCR revolutionized the study of DNA tosuch an extent that its creator, Kary B. Mullis, was awardedthe Nobel Prize for Chemistry in 1993.

What is it used for?

Once amplified, the DNA produced by PCR can be used in many different laboratory procedures such as - Most mapping techniques in the Human Genome Project rely on PCR.

PCR is intigral in a number of new laboratory and clinical techniques, including DNA fingerprinting (think CSI and catching criminals).

Diagnosing disease and genetic disorders. Detection of bacteria and viruses in the environment. Analysis of microbial communities.

How does it work?

To amplify a segment of DNA using PCR, the sample is first heated so the DNA denatures, or separates into two pieces of single-stranded DNA.

Next, an enzyme called "Taq polymerase" synthesizes - builds - two new strands of DNA, using the original strands as templates.

This process results in the duplication of the original DNA, with each of the new molecules containing one old and one new strand of DNA.

Then each of these strands can be used to create two new copies, and so on, and so on.

The cycle of denaturing and synthesizing new DNA is repeated as many as 30 or 40 times, leading to more than one billion exact copies of the original DNA segment. The entire cycling process of PCR is automated and can be completed in just a few hours.

It is directed by a machine called a thermocycler, which is programmed to alter the temperature of the reaction every few minutes to allow DNA denaturing and synthesis.

PCR Procedure

For each reaction: To a 0.2 ml PCR microfuge tube add 10 ml of Eppendorf

2.5X MasterMix which contains: · sterile water · MgCl 2 · PCR buffer · nucleotides (also called dNTPs) · Taq DNA polymerase 5 µl extracted DNA (DNA from glow and no glow

bacteria) 5 µl of the forward primer 5 µl of the reverse primer

PCR Procedure

Procedure 1. Label the microfuge tube glow/ no glow 2. Using the micropipet with a clean tip, add 5 µl extracted DNA

to the appropriate tube. 3. After adding the DNA, add 10µl master mix and 5 µl of each of

the primers to the tubes. If necessary, gently tap your tube on the counter to get all of the liquid to the bottom of the tube.

4. Place your tube into the thermocycler to run the 'GFP' program. This program is 30 cycles of:

            · 94°C for 30 seconds             · 55°C for 45 seconds             · 72°C for 1.5 minutes 5. After the cycles are complete, PCR reactions can be

refrigerated or prepared for electrophoresis.

PCR Procedure

A gel box is used to separate DNA in an agarose gel with an electrical charge. When the red and black leads are plugged into a power supply the DNA migrates through the gel toward the positive charge due to the net negative charge of the molecule. Different sized pieces of DNA move at different rates, withhe larger pieces moving more slowly through the porus medium, thereby creating a size separation that can be differentiated in a gel.

Gel Electrophoresis

Presence of DNA can be confirmed by electrophoresing on an agarose gel containing ethidium bromide, or another fluorescent dye that reacts with the DNA, and checking under UV light.

Gel Electrophoresis