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.