The goal for this part of the course is to help you understand the basic ways that microbes "make a living" out there in the world. Because microbes are so small, it is through their physiologies that they affect the world around them. They don't use physical force to change the world but rather a more subtle form force - the forces of enzymatic reactions and metabolic byproducts including toxins etc.
Let's start by looking at the basics of how microbes make their livings.First, as with all living things, microbes are made up C, N, P, O, S, H, K, Fe, Mg, and Ca. In addition, microbes require some micronutrients, e.g. Mn, Zn, Co, Mo....... Many microbes don't require Na, which is required by most plants and animals.
Modes of microbial nutrition.
Microbes and all other living things can be classified based on how they obtain their carbon and energy. We have already touched upon some of these groups in lecture 5 and below is an expanded vocabulary of these terms
HeterotrophsChemoheterotrophs use organic compounds for energy and carbon (we are chemoheterotrophs as are the Fungi and many Bacteria and some Archaea).Autotrophs can fix carbon dioxide and turn it into organic molecules.Photoheterotrophs organisms that can use light for energy ("photo") and organic compounds ("hetero") for carbon (only some prokaryotes can do this)
Photoautotrophs use light energy to do this (plants, cyanobacteria and many other bacteria are photoautotrophs).Chemoautotrophs use chemical energy to fix CO2 (e.g. sulfur oxidizing and nitrifiers, there are no eukaryotic chemoautotrophs)
"Chemolithoautotrophs" is a term sometimes used for chemoautotrophs that use inorganic compounds for energy, but this is true of most chemoautotrophs so I will use the term chemoautotroph only.
Bacteria, Archaea and Fungi feed by absorbing chemicals from their environment. Everything that gets into the cell must enter via diffusion (e.g. O2, H2O, CO2) or via some sort of transport protein or channel. In contrast, most protozoa can phagocytize chunks of material (including whole bacterial cells) and digest them in vacuoles that act like little stomachs.Most of what we will discuss below applies to Bacteria, Archaea and Fungi.
Given that Bacteria, Archaea and Fungi take up all their food (and get rid of all their waste molecules) across their plasma membranes as individual molecules, it is not too surprising that they can NOT be any bigger than they are. To better understand the size limit for absorptive feeders we can do a little simple geometry.
Consider a coccoid cell of with a diameter of 1 µm.......
show formula to illustrate that surface area grows much more slowly than does volume as a sphere or cube gets bigger.
Microbes can partially overcome these constraints by being long and thin (fungi, spirochetes, actinomycetes etc.), by having prosthecae (e.g. Hyphomicrobiumand Caulobacter) or by having internal membrane-bound vesicles (true of most fungi and some bacteria) in which they can store nutrients or waste products. An example of a Bacterium that has large internal vacuoles is the giant (15 - 40 µm wide) chemoautotroph (H2S = e- donor; NO3- = e- acceptor) Thioplocathat forms huge mats (thousands of square kilometers) off the coast of Chile (see Fossing et al., 1995). These large Bacteria migrate up and down in the sediments, taking up nitrate (which is stored in their vacuoles) from seawater at the top of the sediments and then migrating down to zones rich in sulfides (the S can then be oxidized using the stored nitrate). These amazing creatures can also store elemental sulfur granuoles in their cytoplasm as inclusion bodies. Thus, they can carry around extra e- donor (S) and extra e- acceptor (nitrate) wherever they go......
Fossing, H., et al. 1995. Concentration and transport of nitrate by the mat-forming sulphur bacterium Thioploca. Nature, vol. 374: 713 - 715
.But regardless of their shapes they are taking up nutrients across their membranes.
Diffusion - small or uncharged molecules can diffuse across the plasma membrane.
Facilitated diffusion is just diffusion that utilizes a specific trans-membrane protein (a permease)
see Figure
Active Transport - See Figures
Group Translocation - see Figure (occurs only in some Bacteria)
How Microbes eat polymers and insoluble substrates:
Extracellular enzymesBacillusand extracellular amylase (show overhead)Siderophores are important in Fe uptake....(show complex structure)Cytophagaand extracellular cellulases (show Sporocytophagaon plant fiber)
Emulsifying agents or "biosurfactants" are extracellular compounds produced by microbes that eat oils.
The above section (Uptake of nutrients) is all about how microbes accumulate molecules from their environment - now let's look at what happens to these nutrients once they are inside the cell....
.Let's do non-photosynthetic reactions first..... Catabolic and anabolic reactions
(catabolic reactions)Compare to anaerobic respiration...... e.g. nitrate as the terminal e- acceptor:Oxidation/Reduction (redox) reactions
Catabolic reactions are Redox reactions. All biological oxidations are coupled to reductions.
oxidation is the loss of electrons (Ole-) Let's start with the simplest reaction to understand - Respiration
Kluyver and van Niel proposed that all respiratory reactions (aerobic and anaerobic) could be summarized using the simple formula:
AH2 + B -----> A + H2B
Let's apply this to the type of respiration that goes on in mitochondria and many other chemoheterotrophic microbes:
CH2O + O2 -----> CO2 + H2O
or for the specific case of glucose respiration
C6H12O6 + 6 O2 -----> 6 CO2 + 6 H2O
In this case we say that glucose (C6H12O6) is being oxidized to CO2 and O2 is being reduced to H2O. Glucose is the energy source and the electron donor and oxygen is the terminal electron acceptor.
Let's look at e- and H+ flow in a typical respiring microbe.....
Aerobic Respiration.
See notes from earlier in course. In Bacteria and Archaea the e- transport chain is located in the plasma membrane or in internal membrane systems. Remember also that the mitochondria of Eucarya are just Bacterial endosymbionts (see phylogenetic placement of mitochondria on tree of life) - so it is not too surprising that the e- transport chain of mitos and bacteria are so similar.....
Anaerobic Respiration.
An overview of anaerobic respiration would look just the same as aerobic respiration except that the terminal e- acceptor would be something like nitrate or sulfate (rather than Oxygen).
Compare anaerobic and aerobic respiration in organisms that can do both.....
show these processes in Paracoccus denitrificans (an Alpha Proteobacterium). This organism can completely denitrify nitrate all the way to N2.Let's look at a slightly simpler example, i.e. E. coli with and without oxygen. Note that in the absence of oxygen fewer protons are pumped (therefore fewer ATPs are ultimately made) and that the end products are nitrite and water (rather than oxygen and water). E. colican also carry out several fermentations (see below) when it runs out of O2 and NO3-.
Fermentation = All ATP production is via substrate-level phosphorylation
Organic compounds are e- donors and organic intermediates (= breakdown products) are e- acceptors. Because part of the original substrate acts as the e- dump, fermentations yield many fewer ATPs than respiration. see Fig.In nature one can find fermentative microbes wherever there is a paucity of terminal e- acceptors. Many microbes can also switch from respiration to fermentation in their own metabolisms. For example, when yeast cells are added to fruit juice, they initially carry out aerobic respiration and then switch to fermentation as the O2 runs out.
The diversity of fermentations
Fermentation of sugar to ethanol or lactic acid (you already saw these)Other Fermentations
Show figure "fate of pyruvate" (Fig. )
point out industrial products (e.g. butanol, isopropanol etc. etc....) and foods that are produced by fermentative microbes - e.g Propionibacterium produces the taste (propionic and acetic acids) and the holes (CO2 and H2) in Swiss cheese.
An example of a complex fermented food product....
Soya Sauce (shoyu) is produced via a succession of different physiological types of microbes. The process takes about 3 months to complete. The main stages of this succession are:
1) growth of Aspergillus (a fungus) and various bacteria (e.g. Micrococcus and Bacillusspp.) that digest the protein and polysaccharides (via extracellular enzymes, see above) in the soy beans and wheat.The final alcohol content of shoyu is 2-3% and the final lactic acid content is about 1%. Most of the flavor, however, is due to many different primary and secondary metabolites produced by the various organisms involved in the process. This is a big-time industrial process - there are presently over 25,000 different shoyu manufacturing facilities in Japan alone!...
2) later growth of Lactobacillus spp. which decrease the pH (via production of lactic and other acids as a result of fermentative pathways) of the mixture and finally...
3) Growth of acid-tolerant yeasts (e.g. Torulopsis) which carry out alcoholic fermentations.
Many fermentations don't involve sugars.... show Table.....
Examine a few fermentations in a little more detail...
some are quite complex:A neat variation on the theme of e- flow in fermentations is the "Stickland Reaction" as carried out by members of the genus Clostridium (strict anaerobes).
overall rxn.:
alanine + 2 glycine + ADP + Pi ----> 3 acetate + 3 NH4+ +ATP alanine is the e- donor (energy source) and glycine is the e- acceptor show Figure
Quick review of aerobic resp., anaerobic resp and fermentations.....
In terms of energy yield....
Aerobic resp. > Anaerobic resp. >>> Fermentations > Methanogenesis (see below) Give example of E. coli using O2 at the beginning of the small intestine and then switching to anaerobic resp. and finally fermentations as conditions get more anaerobic further down the digestive system...
This succession of e- acceptors also leads to a chemical stratification of e- acceptors in some anaerobic environments. For example, as you go deeper into the sediments at the bottom of a pond you would find that the O2 disappears first, followed by the nitrate, then the sulfate, until you get to a zone that is devoid of all good e- acceptors and you are left with fermentative organisms - and then when all fermentable substrates are used up you are in a zone where Methanogens (members of the Archaea; see tree of life overhead) make a living oxidizing H2 with CO2 as their e- acceptor. This very novel way of making a living (methanogenesis) can only occur in very reducing environments such as deep in the human colon or rumen of a cow (discuss flatulence and cow burps....).
Most of the metabolic pathways used by Methanogens are still not well understood but we know quite a bit about the Methanogens that use carbon dioxide and hydrogen to make methane. In general these organisms are using H2 as their e- donor and CO2 as their e- acceptor..
4 H2 + CO2 -----> CH4 + 2 H2O See overhead; note that Methanogens use a bunch of novel co-enzymes ("vitamins") in their metabolism. These co-enzyme are not found in any other life forms on earth. Methanogens (and most other Archaea) also do not have lipid bi-layer membranes or cell wall materials found in any other groups of organisms on the planet.
The acetoclastic Methanogens carry out a fermentation-type reaction:
CH3COOH -----> CH4 + CO2
End with summary of an anaerobic food web and show where each of the metabolisms discussed above fits in.....:
cellulose ----> sugar monomers ----> organic acids and alcohols ----> Acetate, H2 and CO2 ----> CH4 and CO2
We have already discussed how microbes take up nutrients (N, P, K etc.). For every type of nutrient taken up there are, of course, anabolic pathways for incorporating those nutrients into macromolecules. We don't have time to discuss all of the pathways (for there are thousands) but I do want to say a little more about nitrogen.....
Most Bacteria, Archaea and Fungi are capable of taking up N (usually passively and actively) in the form of nitrate or ammonium from the environment. These organisms can then use this N (e.g. see "nitrate assimilation" below) to make amino acids and other N-containing compounds used to build cellular materials (anabolism).
Before we move on,
let's put Nitrogen metabolism in a broader
perspective for a second and consider
the Global N cycle
and where some of the organisms we have been (and will be) discussing fit
in......
(draw N-cycle on board....)
...
..
.
N-fixation is the ability to utilize N2 from the atmosphere as an N source for anabolism. N-fixation is widespread in the Bacteria, being found in the Proteobacteria, i.e. Rhizobium, Azotobacter, Klebsiella (Micro. lec. 6), in the Gram + bacteria, e.g. Frankia and some Clostridium spp. (Micro lec. 7) and in many cyanobacteria (micro lec. 12). There are some N-fixers in the Archaea as well, e.g. Methanosarcina.(Micro lec. 8)No Eucarya have been found that fix N, although many eukaryotes depend on N from N-fixing prokaryotes, e.g. Legumes depend on Rhizobium
.
Why are there no N-fixing Eucarya?
N-fixation is a very expensive proposition. Figures 10.14 & 10.16 shows a simplified diagram of how the nitrogenase enzyme complex catalyzes the reduction of N2 to NH3. Note that 16 ATP and 8 e- are needed to make two molecule of NH3. Given that N-fixation is so expensive, it is not surprising that it only occurs in environments where N is limiting (e.g. rotting logs, guts of herbivores and many soils). N-fixing bacteria will shut off their N-fixing machinery when there are adequate supplies of ammonia or nitrate in the environment.
Show overheads of bacteroids and then draw the interconnections among N-fixation and the rest of the metabolism in Rhizobium......... (see handout)
Note that oxygen is delivered to the bacteroid by leghemoglobin. This molecule scavenges free oxygen and makes sure that it doesn't interfere with N-fixation (N-fixation is a very reducing process and the enzyme Nitrogenase is very sensitive to O2). All N-fixers have had to develop some adaptations to protect the N-fixing enzymes from O2. As another example, note that many N-fixing cyanobacteria have a special type of cell, the heterocyst, in which N fixation occurs. No photosynthesis or only cyclic e- flow (and therefore no O2 production) takes place in the heterocyst.
What does the sensitivity of nitrogenase to O2 and the presence of N-fixation in the Archaea and Bacteria tell you about when this process might have evolved?
Discuss how ruminants (deer, cows, camels etc.) get N when they are feeding on dead grass and sticks that have very little N (high C:N ratio).
N-fixation by gut symbionts is also how termites and many other insects get their N.
There are also reports that vegetarians have more N-fixing Bacteria in their colons than do people who eat a lot of meat. One study found very high levels of N-fixing Bacteria in the feces of a New Guinea tribe whose diet is almost 100% sweet potatoes (High C:N).
How do non-N-fixers assimilate N for anabolic purposes?
Remember that most Bacteria and Archaea (and all Fungi) don't fix N, but rather take up nitrate, ammonia or organic N, via transport proteins, just like they take up S, P etc.....
Example of nitrate assimilation (Fig. 10.13) .
Nitrifying Bacteria
These organisms are chemoautotrophs and hold a keystone position in the global nitrogen cycle (see handout and N-cycle figure). They are the organisms responsible for converting ammonia to nitrate. All known nitrifiers are gram - Proteobacteria (see Micro. lec. 6). The first step of nitrification is carried out by the ammonia oxidizers (Generic names all start with Nitroso e.g. Nitrosomonas, Nitrosospira) which convert ammonia to nitrite via the following overall redox reaction:
2 NH3 + 3 1/2 O2 -----> 2 NO2- + 3 H2O
The next step of nitrification is the conversion of nitrite to nitrate. The organisms that do this almost always live in close symbiotic relationships with the ammonia oxidizers (see handout). This is why nitrite (which is extremely toxic) almost never builds up in the environment. Nitrite oxidizers (Generic names all start with Nitro e.g. Nitrobacter convert nitrite to nitrate via the overall rxn.:
NO2- + 1/2 O2 -----> NO3-
Show some nitrifying bacteria....... Note the extensive internal membrane systems; many of the enzymes responsible for nitrification are concentrated in these membranes.
Denitrification
See anaerobic respiration of nitrate (above).
One last large metabolic groups that we will discuss are the sulfur oxidizers. Organisms that can oxidize sulfur are found in both the Archaea and the Eubacteria. These guys can oxidize all sorts of reduced S compounds with the most basic oxidation being complete aerobic respiration of hydrogen sulfide:.
H2S + 4 O2 ------> 2 H2SO4
The main groups of sulfur oxidizers are the unicellular guys (Most generic names start with Thio) and the filamentous critters which includes the famous Beggiatoa (lec. 6) that was first studied by Winogradsky (see lec. 2).
The sulfur oxidizers are found in any environment where reduced sulfur compounds are coming forth from the earth, e.g. hot springs, swamps, acid mine drainage and deep sea hydrothermal vents. In environments where there is little or no light, they are the dominant primary producers. Most of the sulfur bacteria use the Calvin cycle to fix CO2.
Some final thoughts on microbial physiology.....
Secondary Metabolism
Before we leave physiology behind I want to discuss one more topic, secondary metabolism. The last couple of lectures have concentrated on how organisms transform energy and utilize that energy to build cellular material. In contrast, secondary metabolic processes are metabolic pathways that are not involved in growth of cells (or energy harvesting) and often take place when cells have stopped growing. A good example is the production of antibiotics by bacteria and fungi. Figures 44.7 - 44.9 show the difference between primary and secondary metabolic products (metabolites).Show also figure of production of Bacitracin by Bacillus licheniformis just prior to endospore formation.
Can you think of an ecological explanation for why antibiotic production precedes endospore production in Bacillus licheniformis?