Bacterial Diversity
- there are an estimated 1000 - 10,000 different species of bacteria in a gram of soil.

What is a gram?
About the size of a sugar cube. There are probably more species of bacteria than bugs which used to be considered the most diverse group of organisms worldwide; there are probably microbial symbionts with each bug. Begin to look at this diversity but obviously cannot cover it all in a week. I will talk about some my favorites and some of the unusual.

Diversity of bacteria
Bibles which describe most of the species of bacteria known are Bergey's Manual of Systematic Bacteriology and The Prokaryotes.
Phylogenetic view (See Figure 16.1)
12 kingdoms of bacteria based on 16srRNA sequence. 16srRNA has become the benchmark for phylogentic analysis of bacteria. Why? because it seems to evolve relatively slowly so that evolutionary relationships can be determined based on sequence analysis.
16S rRNA analysis- based on DNA sequence analysis of the 16S gene. The sequences are compared with each other to determine the similarity between the sequences. Analysis of the sequences has revealed 3 domains in the 16S sequence: a highly conserved domain, a semi conserved domain, and a hypervariable domain. These domains are scattered throughout the 16S gene - in a mosaic fashion. The highly conserved domain shows very little differences between all eubacteria. The semiconserved domains show a high degree of similarity but not as high as the highly conserved domains. The semiconserved domains are conserved among members of the same phylogentic group. The hypervariable domains show a low degree of similarity between different species of bacteria examined.
12 kingdoms
First two are hyperthermophiles
Aquiflex and Hydrogenobacter are chemolithotrophs
Thermotoga are chemoorganotrophs
Green nonsulfur are thermophiles

At this point they lose thermophility as a characteristic
Deinococci and relatives - radiation resistant- relatives includes Thermus aquaticus - a important organism.
Spirochetes
Green sulfur bacteria - phototrophs
Bacteroides and Flavobacteria - strict anaerobes and aerobes
Planctomyces and relatives - lack peptidoglycan, oligotrophic aquatic
Chlamydiae - obligate intracellular parasite

These last three are what we will spend some time on.
Gram-positive bacteria
Cyanobacteria
Purple bacteria - or Proteobacteria which are broken down into alpha, beta, gamma, delta and epsilon.
These divisions are based on phylogenetic relationships derived from 16srRNA sequences.
Microbiologists like to remake the divisions to suit their interests. We will follow the text for simplicity.
Begin by talking about anoxygenic phototrophic bacteria. These include organisms from purple bacteria, green sulfur bacteria, green nonsulfur bacteria, and gram-positive bacteria.
Common features:
Possess bacteriochlorophyll
Phototrophic - Photophosphorylate - make ATP through light mediated reactions.
Remember there are bacteria - Cyanobacteria - that are photosynthetic too but they differ because they are oxygenic and have chlorophyll like higher plants. Also, they have two photosystems like higher plants whereas what we are talking about only have one photosystem.
These organisms grow phototrophically only under anaerobic conditions since oxygen represses synthesis of their pigments.
Can grow photoheterotrophically? Carbon is organic carbon and energy is sunlight energy to drive ATP synthesis.
Can grow chemoorganotrophically in the dark using an organic compound as an electron donor.
Obviously very diverse physiologically and ecologically.
Enrichments for isolating green and purple sulfur organisms
Found in anoxic zones of aquatic habitats where H₂S accumulates.
Enrichment includes basal salts medium, plus bicarbonate as a source of CO₂, vitamin B12, and a small amount of sodium sulfide as a source of reduced sulfur. Weak light intensities since these organisms live in poor light conditions.

Enrichment for purple nonsulfur bacteria is as above but with about 1/10 the amount of NaS - higher levels are toxic to these organisms- and an organic compound added as a source of carbon and/ or electron donor (if you leave out the sulfide).
Purple nonsulfur bacteria
phototrophs
photoheterotrophic
In the dark some grow fermentatively and some grow heterotrophically by respiration. Energetically very versatile.
Some are nitrogen fixing bacteria as well.
Purple sulfur bacteria
phototrophs
oxidize sulfide to elemental sulfur which might be deposited.
Few grow on organic carbon sources such as acetate or pyruvate.
Some grow as chemolithotrophs in darkness usning thiosulfate as an electron source.
Green sulfur and green nonsulfur bacteria
Green sulfur bacteria
strict anaerobes and obligately phototrophic
some can assimilate organic substances for phototrophic growth.
Green nonsulfur bacterium - Chloroflexus grows well as a photoheterotroph
Ecology of purple and green bacteria
large populations occur in stratified lakes where stable anoxic conditions prevail, hydrogen sulfide accumulates and light penetrates to the anoxic zone. Merometric lakes are perfect since the lake is permenantly stratified due to the denser water (saline) that sits on the bottom. Holomictic lakes seasonally stratify due to thermal gradients and the denser cold water sinks to the bottom. The primary productivity of the purple and green phototrophs may be greater than that of the higher algaes in small lakes. This could be significant to the carbon cycle in a lake.
Can be found in and around hot springs with sulfide especially Chloroflexus.
Chromatium a purple sulfur bacteria is frequently found in and around hot springs too.
Oxygenic phototrophs
Cyanobacteria
Can be divided up into five morphological types
i) unicellular
ii) colonial
iii) filamentous
iv) filamentous with heterocysts
v) branching
Heterocysts are for nitrogen fixation - a oxygen sensitive acitivity. The heterocysts are devoid of photosystem II where photolysis occurs and oxygen is released.
Simple physiologically
nitrate, ammonium or nitrogen fixation to satisfy their nitrogen requirements.
obligate phototrophs
photoheterotrophs if light is present

Some produce a neurotoxin that will affect animals.
some produce geosmin a chemical produced by actinomycetes as well. It gives soil that rich earthy smell.
Ecology
Widely distributed in terrestrial and aquatic systems. Found in the strangest places like in deserts as mats that dry up and revive when the short rains come. On the sides of granite buildings and tombstones. As symbionts with liverworts, ferns and cycads and are the photobionts in lichens.
Most certainly were the first oxygenic phototrophs responsible for creating the oxygenated air. Evidence suggests that they are over 3 billion years old.
Chemolithotrophs
What are they? obtain their carbon from carbon dioxide fixation and their energy and electrons from the oxidation of reduced inoganic molecules such as ammonia. These are aerobic organisms.
Nitrifiers - or nitrifying bacteria use reduced nitrogen as an electron and energy source. Historically these were the first chemolithotrophs studied by Winogradsky.
Carry out the process called Nitrification which is carried out in two step process
ammonia-oxidizing bacteria convert ammonia to nitrite; include Nitrosomonas
nitrite-oxidizing bacteria convert nitrite to nitrate; include Nitrobacter.
Methane may be oxidized by the ammonia oxidizing bacteria since the enzyme ammonia monooxygenase cannot distinguish between ammonia and methane.
Both the ammonia oxidizing and nitrite oxidizing bacteria use the electron transport chain to generate ATP as we have discussed. They also need to make NADPH which is done via the reversal of the electron transport chain at the expense of ATP.
Ecology - found where there is lots of ammonia around due to ammonification which occurs during the decomposition process. Large populations develop in streams and lakes that receive raw sewage due to the presence of large amounts of ammonia in the sewage. They occur in soils especially in neutral or alkaline habitats since nitrification leads to an acidification of the habitat.
Enrichment
selective medium using ammonia or nitrite plus bicarbonate as a source of carbon dioxide. Very slow to grow, so you might want to monitor the appearance or disappearance of of nitrite. Must take great care to ensure there are no chemoorganotrophs growing in your cultures. Also, trace amounts of organic material may be inhibitory to ammonia oxidizers so you might need to use an inorganic solidifying agent such as silica gel.
Hydrogen-oxidizing bacteria
Knallgas reaction 2H₂ +O₂ ---> 2H₂O to make ATP and as a source of electrons. Many, but not all, grow autotrophically. Includes gram-positive and gram-negative bacteria.
Most are facultative hydrogen-oxidizing bacteria and can grow chemoorganotrophically when warranted.
Making ATP via chemiosmotic mechanism using proton motive force established by the oxidation of hydrogen. Hydrogenases are responsible for the first step in the oxidation of hydrogen gas.
There may be two types of hydrogenases - soluble and membrane bound
Membrane bound hydrogenase is responsible for the formation of ATP
Soluble hydrogenase is responsible for the formation of reducing agents - NADH directly since the redox potential is more negative than the reduction of NAD⁺.
Most hydrogen oxidizing bacteria only have the membrane bound enzyme and need to reverse electron flow to make NADPH.
Ecology
Oxidization of hydrogen by Hydrogen oxidizing bacteria may not be too important since there is immense competition for hydrogen in the anoxic zone where hydrogen is formed by the fermentative organisms. This may explain why they are facultative chemolithotrophs.
Isolation of hydrogen oxidizing bacteria
microaerophiles - 10% oxygen not 20% as is found in air.
require nickel since hydrogenases require nickel.
mineral salts medium with nickel and incubate in 5% oxygen, 10% carbon dioxide, and 85% hydrogen.
Methanotrophs and methylotrophs - use one carbon compounds such as methane and methanol. Methane produced in anoxic environments by methanogens. Major gas from anoxic zones of lakes, the rumen, the mammalian intestinal tracts.
Methylotrophs use only one-carbon compounds.
Methanotrophs use methane and a few other single carbon compounds.
Methanotrophs are aerobes that oxidize single carbon molecules as an electron/energy source and a carbon source. Very diverse group of organisms. Widespread in aquatic and terrestrial habitats. Found where methane is present and meets oxygen - though reduced levels of oxygen since they are microaerophiles.
Isolated on a mineral salts medium with 80% methane and 20% air. Methanotrophs grow slowly and are usually pink.
Sulfate reducing bacteria
Sulfate reducing bacteria - these are not chemolithotrophs!!!! obligate anaerobic bacteria that use sulfate as an electron acceptor under anoxic conditions. Use various fermentation products as a source of electrons and energy - organic acids, fatty acids, alcohols, and hydrogen gas.
Very diverse group of organisms again they all have the same characteristics of sulfate reducers. 2 broad groups - nonacetate users that use lactate, pyruvate, ethanol, or certain fatty acids and acetate users which specialize in the oxidation of fatty acids particularly acetate.
Where would you find them? in anoxic aquatic and terrestrial habitats.
How would you isolate them? anoxic lactate-sulfate medium plus ferrous iron- might add thioglycolate or ascorbate to reduce the redox potential of the environment. Ferrous iron is there to bind up the sulfide which is toxic at elevated concentrations. Strict anaerobic techniques do not need to be observed though the medium should contain a reducing agent such as ascorbate and the plates should be incubated anoxic.
Shake tubes work well for the isolation of these organisms.
Common soil organisms
Pseudomonads -
rod shaped with polar flagella, aerobic, chemoorganotrophs, gram-negative and never fermentative.
phylogenetically scattered among the purple bacteria; includes Pseudomonas, Commamonas, and Burkholderia.
Nutritionally simple organisms. Use a wide range of substrates as a source of electrons and energy, but rarely if at all use polymers. Contain numerous inducible operons used in the degradation of simple, soluble energy sources.
Free living aerobic nitrogen fixing bacteria
Azotobacter, Azomonas, Azospirillum, and Beijerinckia are all free living nitrogen fixing bacteria.
Azotobacter - gram negative, obligately aerobic, large rods. Produce copious amounts of slime on plates.
Problem here - this is an obligate aerobe yet nitrogen fixation is sensitive to oxygen. How does Azotobacter deal with this? They have the highest respiratory rate measured for any living thing. By respiring they lower the oxygen level so that nitrogenase - the enzyme for nitrogen fixation- will function.
Chromobacterium - facultative aerobes that ferment sugars and grows aerobically on a variety of substrates.
C. violaceum is a bright purple pigmented organisms found in soil and water. The pigment, violacein, is a water soluble pigment that has antibiotic properties.
Gram-positive endospore forming rods
Bacillus
aerobic or facultative aerobic, catalase positive, superoxide dismutase positive. Found in soils where the spore is advantageous. Resistant to drying and heat and allows the organisms to survive famines. It germinates to quickly to take advantage of transitory nutrients.
Can be isolated by heating a soil suspension to 80o/C for 10 minutes. Grows on sugars, organic acids and alcohols. Produce extracellular enzymes to hydrolyze polysaccharides, nucleic acid, and lipids. Produce antibiotics too.
insecticide activity B. thuringiensis strains kill lepidopterans such as gypsy moth, tent caterpillar, some kill mosquitoes and black flies and others kill Colorado potato beetle.
Facultative aerobic gram-negative rods
Focus on enteric bacteria including Escherichia, Enterobacter, Shigella, Salmonella, Klebsiella, Arizona, Citrobacter, and Proteus.
Gram negative, non sporulating, rods, non motile or motile by peritrichous flagella, facultative aerobes, oxidase negative, ferment sugars to many end products.
Some are pathogenic to humans, animals. and plants.
Fermentative patterns: Mixed acids composed of acetic, lactic, and succinic acid plus ethanol, CO2 and H2 and no butanediol.
2,3-butanediol composed of butanediol plus ethanol, O2 and H2 produced.
Escherichia
Member of intestinal tract microflora in warm blooded animals and man.
Produce vitamin K
some strain are pathogenic including enteropathogenic strains which cause diarrhia and other symptoms.
Shigella very related to Escherichia. Commonly pathogenic to man causing gastroenteritis.
Salmonella again closely related to Escherichia and usually pathogenic to man. Cause typhoid fever and gastroenteritis.
Types by O antigen or somatic antigen - cell wall
H antigen flagella
Vi antigen outer polysaccharide
all used in epidemiology.
Proteus very motile and urease positive. Frequently found in urinary tract infections. Swarming colony due to motility. The colony grows in rings as the swarmers move out, settle, divide and produce new wave of swarmers.

Classification of Microorganisms

Why do we want to classify microorganisms?
Microorganisms are classified into taxa (taxonomic categories) to facilitate research, scholarship and communication. Biologists try to look for a natural classification system which are based on ancestral relationships. The hierarchy of the taxa reveals the evolutionary or phylogenetic relationships between microorganisms.

Five kingdom system
This system was proposed in 1969 by Robert Whittaker. This is the system most of us are familiar with. The kingdoms include i) Monera ( Procaryotae), the bacteria, ii) Protista, the unicellular Eukaryotes, iii) Fungi, iv) Plantae and v) Animalia.

Three domain system
This system was proposed in 1978 by Carl Woese. Molecular biology and biochemistry provide the basis of this newly proposed system. Based on distinct differences in the ribosomal RNA sequences two distinct taxons were discovered among the kingdom Monera. The three domains are Eubacteria, Archaea, and Eucarya. The kingdoms Animalia, Plantae, Fungi, and Protista are kingdoms in the domain Eucarya.

Taxonomic hierarchy
Domain, kingdom, phylum (division), class, order, family, genus and species. Species definition in Eucarya is relatively simple compared to bacteria since it relies on the sexual reproduction and isolation of individuals within a species. A bacterial species is defined are a population of cells with similar characteristics — either phenotypic or genotypic.

How are organisms named?
Binomial nomenclature where each organism has two names — a genus name and a specific epithet or species name. Both are always either underlined or italicized and the genus is always capitalized and the species is lowercase. Example Escherichia coli

Rules for naming bacteria are outlined by the International committee on Systematic Bacteriology. Descriptions of new species of bacteria are first published in International Journal of Systematic Bacteriology before they are incorporated into Bergey’s Manual of Systematic Bacteriology, the bible of systematic bacteriology.

Methods to classify and identify microorganisms

Bergey’s manual of determinative bacteriology is the bible for the identification of bacteria. You will find many tables of characteristics of identified and classified bacteria that appear in Bergey’s Manual of Systematic Bacteriology. In addition to the properties listed in Bergey’s, the source and habitat of microorganisms is also important to consider when identifying microorganisms.

Morphological characteristic
Useful for higher organisms but not so for microorganisms. Hundreds of different species of microorganisms are either rod or cocci shaped. Other morphological characteristics such as endospores and flagella are useful.

Staining
Gram and acid-fast staining are very useful in identifying microorganisms. Doctors can start prescribing antibiotics given the gram stain of a potential pathogen.

Biochemical tests
Enzymatic activities are widely used to differentiate and identify species of bacteria. Many different rapid, miniaturized testing systems have been commercialized. Examples include the API strips, Enterotubes, and BIOLOG. Each of these have a variety of tests that are done simultaneously on a single isolate. BIOLOG can do 95 different tests, API can do 20 tests and Enterotubes can do 14-17 different tests. Some of these have been somewhat automated and the results analyzed by computers.

Serology
Antibodies against specific microorganisms can be used for the identification of many microorganisms. Slide agglutination are used to rapidly identify potential pathogens using antiserum made against the pathogen. Positive identification is indicated by agglutination or clumping of the bacteria and negative is indicated by no clumping of the bacteria. Enzyme-linked immunosorbent assay (ELISA) is a rapid approach. Antibodies are coated on microtiter plates, bacteria are added, a second antibody with an enzyme linked to it is added, and a substrate is added. If the bacteria is recognized by the antibodies the enzyme will work on the substrate and you get a positive well. Another serological test is called Western blotting, where proteins of the patient are separated by electrophoresis and blotted onto a membrane. Antibodies with enzymes linked to them are used to "probe" the membrane with the patient’s proteins. If the protein of interest is there, the antibodies will bind and lightup with the substrate for the enzyme linked to the antibody.

Phage typing
Bacteriophage will lyse specific strains of bacteria. To determine the identity of a bacteria, it is plated out on a general medium. Before the bacteria grows up, drops of phage containing solutions are placed in various spots on the plate. If the cell is susceptible to the phage, the phage will lyse the cells resulting in clearing zones called plaques. By looking at the phage sensitivity patterns, you can identify the bacteria.

Amino acid sequencing
The amino acid sequence can be determined from any protein. If you compare the same protein from different bacteria, the more distantly related the bacteria are the more differences you will find in the amino acid sequences of the protein. The converse of this is that the more closely related the bacteria are the more similar the proteins will be.

Fatty acid profiles
The fatty acids of the membranes of bacteria are extracted and separated using a gas chromatograph. The fatty acids can be identified using internal known standards. The fatty acid profiles of a species is pretty constant so you can develop a data base to compare unknown bacteria with known strains of bacteria for identification. Commercial systems have been developed — Sherlock of MIDI for example.

DNA base composition
DNA base composition is usually expressed as percentage guanine plus cytosine. Related organisms that are genetically related - have many identical or similar genes - should have similar G+C %. Unrelated organisms predictably would have dissimilar G+C%, but they could also have similar G+C% by chance. Other types of analysis should be performed to support relationships.

DNA fingerprinting
DNA sequencing is becoming more of a reality but still is very costly and time consuming therefore it is not used routinely for determining relationships between organisms. But there is an approach that compares base sequences of organisms. Restriction enzymes - restriction endonucleases - cut DNA at specific sequences. For example the restriction enzyme EcoRI cuts DNA at G - AATTC (between the G and first A). DNA from two or more organisms can be digested and the resulting restriction fragments separated by electrophoresis. The patterns after electrophoresis can be compared to determine relatedness. Related organisms should have more similar patterns than unrelated organisms. This is routinely used in epidemiological studies of bacteria and viruses.

Ribosomal RNA sequencing
Sequencing the 16S ribosomal RNA gene. Used to determine the phylogenetic relationships among bacteria and determine diversity among communities of bacteria. Several advantages: All cells contain rRNA; Closely related species have fewer differences than distantly related species; rRNA genes are highly conserved compared to other gene sequences; rRNA sequencing doesn’t require culturing the organism. The gene can be obtained using the polymerase chain reaction.

Polymerase chain reaction
Specific sequences of DNA can be amplified using site specific DNA primers and a DNA polymerase called Taq polymerase. See figure 9.14 for details.

Nucleic acid hybridization
Double stranded DNA can be denatured either chemically or thermally. Thermally denatured DNA will reform native double stranded DNA if cooled slowly since the two strands are complements of each other. If you have DNA from two different organisms mixed and denatured the degree at which they will form a double stranded helix (hybridize) depends on their relatedness.

Southern blotting relies on the same principle. See figure 9.12 DNA is probed with a piece of DNA that is labelled either with an enzyme or radioactive nucleotide.

REVIEW Table 10.5

Dichotomous keys are used for identification of microorganisms based on data from analysis described above, especially biochemical type.

Cladograms
Tree like structure used to show evolutionary relationships.

Microbial Genetics

Bacteria of the same species are not genetically homogeneous - there is immense genetic diversity. In some cases more diversity than what is found between man and other mammals. Bacteria are good organisms to study molecular evolution and genetics.
Mutants - differ from their parents at the DNA sequence level. By definition they differ in their genotypes - the DNA sequence. Mutants may also differ from their parents in their phenotypes - their observable characteristics. Mutants may or may not have different phenotypes from their parents - there are many mutations that do not lead to observable differences between the wild-type and the mutant.
Mutants can be selectable or non-selectable. Selectable mutants have an advantage over the parental strain under certain environmental conditions. For example a spontaneous antibiotic resistant mutant would have a growth advantage over a wild-type sensitive strain in the presence of an antibiotic. Another example of mutants are formations of nutritional mutants called auxotrophs. There wild-type parents are called prototrophs. Auxotrophs are non-selectable mutants. Non-selectable mutants have no advantage over their parental strain and must be screened for by some sort of assay.
How would you select for auxotrophs? Recall they do not grow on minimal medium where they may have to make a specific amino acid. Neat little trick - add penicillin to a minimal medium minus the growth factor (e.g., amino acid). Penicillin will kill only the dividing cells such as the prototrophs, but will not kill the non-dividing cells. Now wash the cells free of the penicillin and spread the cells on minimal medium plus the growth factor. You will have both auxotrophs and a few prototrophs that escape the penicillin treatment.
Molecular basis of mutations
What are mutations? A heritable changes in base sequence of nucleic acid.
How do these heritable changes occur?
Point mutations - involve one or few bases.
Base-pair substitutions - can lead to (i) silent mutations, where the mutation doesn't lead to a different amino acid; (ii) nonsense mutations, where the mutation results in a stop codon and premature stopping translation; and (iii) missense mutations, where there is a change in the amino acid at that position. Some missense mutations lead to what are called temperature sensitive mutations. Temperature sensitive mutants (or conditionally lethal) are able to grow at a low temperature but are unable to grow at a higher temperature. Why? Because the protein effected is unable to fold properly at the high temperature and therefore cannot function properly whereas it is functional at the cool temperature. These are valuable for research purposes.

Microdeletion and Microinsertions
Both of these lead to frameshift mutations potentially. This could lead to a faulty protein depending on what region of the protein is effected.
Remember point mutations and micro deletions and insertions only affect the protein if they occur in the protein coding region of the gene.
Revertants
Defined as mutants that return to wildtype phenotype.
Two types of revertants: Same site revertants and second site revertants
Same site revertants is where a second mutation restores the site of the first mutation.
Second site revertants is where a mutation occurs at a different site in the DNA. These suppressor mutations restore the wild-type phenotype by compensating for the first mutation.
Deletions and insertions
large deletions and insertions may occur. Deletions are not revertable which makes them distinguishable from point mutations. Insertions may be due to insertion sequences such as transposons or insertion elements.
Interestingly, RNA genomes, such as found in viruses, may have a 1000 fold higher mutation rate than DNA genomes. This may be important in the evolution of new virulent forms of viruses.
Mutagens
agents that cause or induce mutations.
Chemical mutagen
base analogs resemble bases but cause errors in replication - examples inclue 5-bromouracil which represents thymine.
alkylating agents modify bases which leads to mutations - example nitrosoguanidine.
Both of these lead to basepair substitutions frequently.
Intercalating agents insert themselves between bases of the DNA and spread them apart causing microdeletions and insertions that lead to frameshift mutations - a common example is acridine orange.
Radiation
Nonionizing radiation - visible light is roughly between 400 and 700 nm wavelenghts. Below 400 nm down to 150 nm is ultraviolet wavelengths. The bases of DNA absorb these wavelengths because of the chemical nature of the nitrogenous bases. 260 nm ultraviolet is an excellent wavelength to kill bacteria because of the light absorbing properties of the bases. Pyrimidine dimers are formed by ultraviolet. The pyrimidine dimers interfere with DNA replication and can lead to basepair substitutions.
Ionizing radiation is defined by electromagnetic wavelengths that cause water and other substances to ionize. These wavelengths are in the region of xrays and gamma rays and are high energy. They produce hydroxyl radicals which react with macromolecules such as DNA and cause damage.
DNA repair induced mutations - Damaged DNA can induce a regulon that itself may cause mutations. The regulon is called SOS regulatory system.
RecA is activated by damaged DNA. RecA inactivates a repressor called LexA which allows an error-prone DNA repair mechanism to function.
Biological mutagens - transposons, insert elements, and viruses. These may insert themselves into random locations in the chromosome thus inactivating specific genes.
Ames test - a test to determine if a chemical is mutagenic, which may also be potentially carcinogenic. The Ames test makes use of various auxotrophs, such as histidine auxotrophs of Salmonella typhimurium with simple point mutation that use the error-prone pathways to repair DNA.
Test - the chemical is usually treated with an enzyme preparation of rat liver which modify the chemical much like your liver does. Many chemicals are not directly mutagenic/carcinogenic until they pass through the liver and are modified by liver enzymes.
The modified chemical is then absorbed onto a disc of filter paper much like you did when you looked at antimicrobial agents in the lab. This disc is laid on a plate with a lawn of the appropriate auxotroph and incubated overnight. Mutagens will increase the rate of back mutation from auxotroph to prototroph which will result in a high number of colonies growing around the disc compared to the proper controls.
Mutations are the primary source of genetic variation, but homologous or general recombination is another source. In recombination, one bacterium's DNA is integrated into another bacterium's genome. Remember that the only way this DNA is propagated is if it is integrated into the recipient's genome since the donor's DNA probably will not have an origin of replication.
How does one bacterium's DNA enter into another bacterium's cell?
Transformation - requires naked DNA in the environment
Transduction - requires virus mediated transfer
Conjugation - requires cell-cell contact and a conjugative-plasmid to mediate transfer
Transformation - one of the first microbial genetic experiments. Griffith showed that rough, avirulent forms of Streptococcus pneumoniae could be transformed into smooth, virulent forms of S. pneumoniae. Avery, MacLeod and McCarty showed that it was DNA that transformed the avirulent cells into virulent cells.
Requires free, naked DNA in the environment and competent cells which are cells that are capable of taking up DNA.
Naturally transformable cells (i.e., competent cells) are found in nature as well as in the laboratory which suggests that this is an important process in the evolution of new strains in nature. Azotobacter, Bacillus, Streptococcus, Haemophilus and Neisseria are naturally transformable. We can induce competence in the lab based on the treatment of cells at a certain growth phase that are treated with divalent cations and other chemicals.
Transduction - DNA is transferred from cell to cell by viruses.
Generalized transduction - random segments of the host bacterium's genome becomes part of the viruses genome. How? During the lytic cycle parts of the host bacterium's genome are accidentally packaged into the virus particle producing a transducing particle. The transducing particle may infect a new cell and genetic recombination occur to produce a new strain.
Specialized transduction - a specific region of the host chromosome becomes part of the viruses genome. The virus is usually a temperate virus - a virus that may integrate into the recipient's genome and replicate with it before causing lysis of the cell.
Phage integrates into a specific site in the host bacterium's genome, e.g., near the galactose genes. Upon induction of the lytic cycle, UV light perhaps, the virus packages either only its DNA or bits of its DNA and the host's DNA.
Many different species of bacteria are transducible including Escherichia, Pseudomonas, Salmonella, Staphylococcus, and Rhodobacter. Again, this process is probably important in nature.
Conjugation - cell to cell contact required to transfer DNA from one cell into another.
Before we talk about conjugation, we need to look at plasmids. We have mentioned them previously.
Plasmids are usually double stranded circular DNA that replicate independently of the host chromosome. They range in size from 1 kilobase to greater than 1000 of kilobases. They exist in cells as supercoiled DNA. There may be only a few copies of the plasmid to 100s of copies of a plasmid in a cell. The copy number is genetically controlled. Some cells contain several typse of plasmids. The coexistence of plasmids is genetically controlled by inc genes or the incompatibility genes. Plasmids of the same incompatibility group cannot reside in the same bacterium. Plasmids of a incompatibility group are genetically related and ones of different incompatibility groups are unrelated.
There are 1000s of plasmids that have been identified. They encode many different functions including (i) antibiotic resistance - so called R factors or plasmids, (ii) toxins and virulence factors such as colonization factors, hemolysins, enterotoxins (diarrhea), (iii) bacteriocins - agents that inhibit or kill closely related species or strains of the same species of bacteria. Generally bacteriocins are polypeptides, (iv) catabolic genes (e.g., naphthalene on the Nah plasmid) and (v) cryptic plasmids that have no known function.
Conjugative plasmids - plasmids that are transferred from one cell to another cell in replicative fashion - that is both cells contain the plasmid in the end. Not all plasmids are conjugative; the plasmid codes for the conjugation functions. A set of genes in a regions called the tra region code for the transmissibility of the plasmid.
Some conjugative plasmids integrate into the chromosome and mobilize the chromosome into recipient cells. Large segments of the chromosome may be mobilized from the donor cell to the recipient cell. The donor cell is called Hfr for high frequency of recombination.
Some plasmids move between a wide variety gram-negative bacteria - so called broad host plasmids. Some move between bacteria and other higher organisms such as plants.
Classic model - the F plasmid - F for fertility - in Escherichia coli.
Requires a donor cell or male and a recipient cell or female.
Genes on the conjugative plasmid in the donor cell code for a sex pili and if we are talking about the F plasmid the pili is called a F pili. Again sex pili of the male attaches to a specific receptor on the female cell and then begins to retract to bring the two cells into contact.
DNA transfer - (see Figure 9.23)
Rolling circle replication - a nick is introduced into the plasmid at OriT - origin of transfer. The 5´ end of the nick is passed into the female cell. At this point it is not real clear what happens. In the rolling circle replication, a primer is laid down on the plasmid and replication proceeds in the 5´ to 3´ fashion as usual and one strand is passed to the female which acts as a template to synthesize the complementary strand.
A second model suggests that a single strand is passed to the recipient and upon completion of the transfer a complementary strand is made to each of the strands in the donor and recipient.
Chromosome mobilization - movement of bits of chromosome by conjugation can occur with the aid of plasmids such as the F plasmid.
F-plasmid is an episome - a plasmid that can occur independent of the chromosome or integrated in the chromosome.
F⁺ cells are cells with the F plasmid unintegrated.
F^- cells are without the F plasmid and make good recipients and are referred to as female cells.
Hfr cells are cells with the F plasmid integrated into the chromosome. The plasmid integrates at specific sites called insertion sequences which represent sites of homology between the plasmid and the chromosome (See figure 9.25). Transfer of the DNA from an Hfr to a F^- cell is similar to the transfer of the unintegrated plasmid. Different Hfrs occur because there are a number of insertion sites in the chromosome.
F-prime are F plasmids that have excised from the chromosome and carry a portion of the chromosome with them during excision.
Results of transfer involving F plasmids
F⁺ mating with a F^- results in two F⁺.
Hfr mating with a F^- results in the donor being Hfr still but the female is usually still F^- since the entire F plasmid is not transferred to the female cell. In this mating usually the mating pair is broken before complete transfer of the donor chromosome and the F plasmid. Therefore the donor genes will not be detected unless they recombine with the recipient genes. More on this later.
F-prime mating with a F^- cell results in a conversion of the F^- cell to a F⁺ , F-prime or an Hfr.
Microbial geneticist can use the F plasmid to order genes on the chromosome. Using different, independent Hfrs with their different sites of integration of the F plasmid, the gene order and a genetic map can be determined. (See Figure 9.27).
How? Antibiotic sensitive Hfrs that are prototrophs are mated with antibiotic resistant recipients that are auxotrophs. Matings are allowed to occur for different periods of time. The mix is sheared to disrupt the mating pairs and this is plated out on appropriate minimal medium with the antibiotic. The recombinants are scored. Genes close to the origin of transfer are transfered to the recipient cell more frequently than more distal genes. (See Figure 9.28). The chromosome requires about 100 minutes for complete transfer.
Conjugative plasmids occur in other gram negative and positive cells. Conjugative transposons occur in in different species of gram positive bacteria. They may move between different species of gram positive bacteria. Leads to potential rapid evolution of new strains, e.g., antibiotic resistant strains.
Using mainly conjugation and transduction and some transformation, the gene order and position of over 1400 genes has been determined. Furthermore, several species of bacteria have had their genome sequenced to the bases.
Transposable elements: Transposons and insertion sequences
Transposable elements first found in maize by Barbara McClintock.
Transposons and IS elements both code for a transposase, which is essential for transposition, and terminal inverted sequences.
Insertion sequences are the simplest elements and only code for genes necessary for transposition.
Transposons- are more complex and code for other properties including antibiotic resistance. They may also be conjugative as well.