Microbiology, Lec 13.
Anabolic reactions in cells are almost always linked to catabolic reactions (via ATP, reducing power and common pathways). Remember that ATP and NADH are also needed to build macromolecules and that these come from catabolic processes or from photosynthesis in photosynthetic organisms.
We have already discussed how microbes take up nutrients (N, P, K etc.; lec. 10). 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). There are some Bacteria and Archaea, however that have the ability to also utilize N2 from the atmosphere as an N source for anabolism - we call this process N-fixation.
N-fixation is widespread in the Bacteria, being found in the Proteobacteria, i.e. Rhizobium, Azotobacter, Klebsiella (lec. 6), in the Gram + bacteria, e.g. Frankia and some Clostridium spp. (lec. 7) and in many cyanobacteria (lec. 12). There are some N-fixers in the Archaea as well, e.g. Methanosarcina.No Eucarya have been found that fix N, although many eukaryotes depend on N from N-fixing prokaryotes (e.g. Legumes depend on Rhizobium).
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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 (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) .
Now that we understand catabolism and anabolism in typical bacteria let's look at some not so typical (and not so well understood) organisms. In what follows we will discuss both anabolic and catabolic reactions.
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 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.
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...Before we move on,
let's put N- metabolism in a broader perspective for a second and consider
the Global N cycle
and where some of the organisms we have been discussing fit in......
(draw N-cycle on board....)
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Methanogenesis
Methanogens (Archaea, lec. 8) live in very reducing environments where e- acceptors like nitrate and sulfate have been depleted and where even fermentable substrates have been all used up (see handout). Most of the metabolic pathways used by these guys 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.......
The acetoclastic methanogens carry out a fermentation-type reaction:
CH3COOH -----> CH4 + 2 CO2
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Acetogenic Bacteria
These chemoautotrophs are anaerobic bacteria, most of which are gram + (e.g. Clostridium aceticum and Acetobacterium woodii). In addition to carrying out the reaction shown below they can also ferment sugars to produce acetic acid. (don't confuse these guys with the gram - acetic acid or vinegar Proteobacteria, lec. 6). But the reaction the Acetogens are known for is the production of acetic acid via anaerobic respiration of hydrogen (with CO2 as the e- acceptor):
4 H2 + 2 CO2 -----> CH3COOH + 2 H2O
Note that this reaction is quite similar to the methanogenic reaction we discussed above, except that acetate is the end product rather than methane. There are other similarities between the acetogens and methanogens.
Acetobacterium woodii is one bacterium that requires sodium for growth. It uses an NA+ (instead of H+) membrane gradient to generate ATP.
Sulfur Oxidizers.
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.
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?