NAME

Total points 100

1.) Based on what you know from lecture and your assigned readings, describe the physical and chemical conditions on the primitive earth, and how it is believed that life developed from this "prebiotic soup." (8 points).

Note: in Fall 2002, we did not spend much time on this

Around 10 - 20 billion years ago (bya) the Big Bang happened; the temperature at that time was >1010 K. Our solar system formed around 5 bya; the temperature had dropped to ~ 103-104 K. The earth was hot, reducing, high CO2, little or no O2. The time period from 3.8 - 4.5 bya is referred to as the time of "chemical evolution," or protometabolism. It was a gaseous, hot, reducing environment consisting of NH2, CO2, CH4, some NH3, CO, H2S. Virtually no O2 was present, thus no ozone shielded the earth from irradiation. This was a time in which unknown chemical reactions generated self-replicating RNA which sustained the RNA world. These gaseous raw materials were abiotically transformed into amino acids and nucleotides upon receiving energy in the form of lightning or UV irradiation (as demonstrated by the Miller-Urey experiments). Somehow these monomers polymerized into macromolecules and underwent Darwinian selection at the level of chemistry - (i.e. based on their chemical properties, stability, ease of being replicated etc..). RNA is known to be catalytic, and thus it is believed that RNA represented the progenitor mechanism for biosynthesis. It is believed that RNA replication developed during this time of protometabolism. Eventually RNA-dependent protein synthesis developed to allow for Darwinian selection at the level of function (i.e what proteins did the RNAs encode). Other cellular components must have also arisen during this time including membrane components, cofactors etc... The first protocells appeared approximately 3.8 - 4.2 bya, and as the temperature dropped below 100˚C, liquid water was known to be present as evinced by the existence of sedimentary rocks dating to that time. Presumably, translational accuracy increased such that RNA sequence became linked to a given protein sequence (similar to our current state of affairs). The first fossil prokaryotes are dated to about 3.5 bya, and oxygen didn't reach current levels until ~ 2 bya even though oxygenic photosynthesis was believed to have arisen ~ 2.5 bya.

Note: all of this was not required for 8 points. However, this is a complete answer

2. There are currently approximately 5000 described species of bacteria. What is a bacterial species, or how do we currently define the term? Include both molecular and ecological considerations.(10 pts)

Note: in Fall 2002, we did not spend much time on this

Typically, microbiologists use polyphasic approach for species identification.

genetic definition - DNA:DNA hyvbridization at a level of 70% or greater with a DTm of 5˚C or less. or 16S rRNA identity of 97 % or greater. also, mole%GC GC content

physiological definition - C and N sources (and other nutrtients) used, metabolites, activities, or enzymes, etc...

ecological - role in ecosystem - niche - resources used, and products produced, aquatic vs. terrestrial, fresh vs. marine, oligotrophic vs. eutrophic etc....

misc.... morphology, growth rates, etc...

3.) What is meant by the phrase "Viable But Not Culturable (VBNC) bacteria? How do these differ from "nonculturable" and dead bacteria? Be sure to tell me whether these terms refers to cells that are metabolically active and reproducing. (6 points)

VBNC- metabolically active but not reproducing.

They can still uptake nutrients and synthesize cellular material, they are simply not dividing under the current conditions. Under different conditions, they can resume division and will then be culturable. VBNC bacteria may be in a dormant state or resting stage (i.e. spore), but this is not necessarily the case.

"Nonculturable" - metabolically active and reproducing.

We simply don't know how to culture them due to our lack of understanding of biotic and abiotic conditions governing their growth in the environment.

Dead - metabolically inactive and not reproducing....i.e. dead!

4.) There have been several studies in the past few years which have demonstrated that iron is a limiting nutrient for phytoplankton in the open ocean. In 1999, an iron-fertilization experiment was conducted where the dissolved iron concentration was raised to 4 nM over an area of ~ 70 km². This iron enrichment caused a huge, temporary bloom of phytoplankton. Circle the graph below that best indicates the probable effect of iron on the growth of phytoplankton? Assume the normal open ocean iron concentration to be 1 nM. In 1-2 sentences, tell me why you chose this graph(7 pts). (3 for choosing graph, 4 for explanation)

As soon as you add a limiting nutrient, growth rate will increase. Since we know the bloom occurred when 4 nM iron was added, we assume the growth rate would have been higher at 4 nM vs. 1 nM, thus the bottom right curve is not correct.

Fall 02 folks...expect growth related questions including curves.

5.) Many scientists believe that if we can trigger massive, long lasting phytoplankton blooms (via iron fertilization), that will help ease global warming since the phytoplankton will consume CO₂ (one of the primary greenhouse gases contributing to global warming). Last week in the journal Nature, the first report of creating a long lasting large-scale bloom was reported. They did see a decrease in CO₂ that was attributed to the increased level of photosynthesis, and the carbon (formerly in the CO₂), is now present in the phytoplankton biomass. Is there anything else these researchers should be concerned about? (hint: think about where this carbon may eventually end up). Explain your answer.(6 pts)

Since the C is currently tied up in a plankton bloom, it is probable that something will eventually become limiting for the phytoplankton and lead to their death. Once that happens, dead plankton biomass will serve as food for the aerobic heterotrophic bacteria (remember eutrophication). These "guys" will use up the O2 while respiring plankton remains...thus using up the O2 and producing CO2 as a waste product. Some of the plankton biomass may settle in the ocean sediments, and be a source of organic material for anaerobic processes such as: fermentation, and methanogenesis. Methane is a more potent greenhouse gas than is CO2. The point is - that it is hard to say what will happen until we take long term measurements.

6.) From your reading, and from lectures, you know that the 16S ribosomal RNA is one of the preferred methods for gauging prokaryotic diversity and phylogenetic relationships. List the features that make rRNA (16S or 23S) the molecule of choice for molecular microbial ecologists? (8 points)

1.) of profound importance in all organisms

2.) conservation of function dictates conservation of structure and sequence

3. different positions in sequence change at different rates.

4.) abundant and easy to isolate.

5.) 16S and 23S are relatively large and consist of many domains.

6.) no lateral transfer

7.) Suppose you get a job working for Pacific Lumber, and in response to their criticisms received from the community and from environmental groups, they are trying to investigate the effects of clearcutting on the forest ecosystem. Your tasks are to (1) investigate the effects of clearcutting on microbial diversity, and (2) convince the public why this (task #1) is a necessary thing to do. How would you go about fulfilling each of these objectives. When answering part 1 - make sure to be specific about experimental details when possible. Assume you have access to (properly preserved) forest soil samples before and after clearcutting events. (15 points)

Several approaches to part 1. - where many of you lost points is that you wrote down a bunch of methods w/o telling the information you get from each. An easy (though time consuming approach - isolate DNA from pre- and post- clear cut samples--> amplify 16 S rRNA genes, clone and sequence. This will be the best estimate of who is there before and after clearcut. You could also do crude estimates of diversity as was discussed in lecture.

Part 2 - why is microbial diversity important - create/maintain atmosphere (weather and clouds)

- unique metabolic activities (N2 fixation)

- key role in nutrient/energy recycling

- truly the base of the food web

- obligate symbionts

- detoxification of hazardous materials

- teach us about life at the extremes

- cool - big $ - products. Discovery of Taq polymerase lead to "chemical prospecting"

8.)Suppose that one of things you do in a crude investigation of diversity is to do a Cot curve with DNAs from both pre/ and post-clearcutting. What would your conclusions be based on this result? (5 points)

Conclusions:

Post clear cut shows an apparent increase genomic complexity vs preclearcut. This increased Cot value corresponds to an increase in diversity.

9.) Distinguish between the primary roles of prokaryotes, algae and fungi in microbial ecosystems. (9 pts)

proks - key role in nutrient/energy recycling, truly the base of the food web

obligate symbionts, decomposition

algae - primary production - especially in aquatic ecosystems (important symbioses (lichens)

fungi - primary decomposition especially lignin and cellulose. important symbioses (mycorrhizae)

10.) Within a day of the Amoco Cadiz oil spill off the coast of France in 1978, culturable hydrocarbon-degrading populations increased by several orders of magnitude Similarly, shortly after the Exxon Valdez spill in Alaska, it was reported that culturable hydrocarbon degraders increased by as much as four orders of magnitude. There are (at least) two distinct means by which these increases could occur. Discuss these, and tell me how you would distinguish which of these events was most likely. (12 points)

The oil spill provided an influx of nutrients that (1) would represent a "new food source" to those who could already use the compounds contained in the oil and (2) a selective pressure for those who do not possess the genetic capacity (to degrade the hydrocarbon HC) to acquire the genes.

Two possible schemes could explain the increased numbers of culturable HC degraders.

1. Increase in a single or few species (populations) possessing the ability to degrade hydrocarbons.

2. Transfer of the genes that allow degradation of the compounds to many organisms within the environment. This can occur by any of the methods we discussed in class.

remember - we're only talking about culturable HC degraders.

To test - isolate HC-degraders on enrichment media containing HC as sole C source - as was described in the question. Presumably, since the question indicates you had an increase in the culturable HC degraders, you have access to samples before and after the spill. Also plate samples on non-HC media - do you see differences in (numbers and morphologies) of total culturable before and after. Determine species of post-spill isolates by 16S sequencing, traditional taxonomy etc....Alternatively, you could do some type of genetic fingerprinting (via PCR) to screen isolates. Do you find a wide morphological and biochemical diversity in the isolates? If you do find a wide diversity - do you find the same genes for HC degradation in all the organisms. If you find only one ( or a few) species (with higher numbers), option 1 is more likely.

11.) Based on what you know about protozoan grazing on bacteria and lytic bacteriophage infections - Discuss the relative effects of a non-prey-specific flagellate and a lytic bacteriophage on a microbial ecosystem of low prokaryotic diversity? and an ecosystem of high diversity? (6 points)

Although this is a complete answer - near full credit was given for far less info.

I was basically looking for general descriptions about the control of microbial populations through viral parasitism and meiofaunal (i.e. protozoan or dinoflagellate) grazing. We talked about all of these features in lecture.

Most of you realized that (in general), low diversity ecosystems are less stable or flexible than high diversity systems. In a high diversity ecosystem many species/genera are present with lower numbers of each representative group, and no one group is dominant in number. Eliminating a species or genera will not usually destroy the ecosystem since another will step in to assume the role. In a low diversity ecosystem, there are less species/genera represented, but there are often one or a few species (or genera) which dominate(s) the ecosystem with respect to their total numbers. Elimination of even an apparently minor player can be dramatic since there is less flexibility (genomic plasticity) to assume the role. Thus, both the phage and the dinoflagellate will probably have less of a pronounced effect on high diversity (vs. low diversity) ecosystems. In a high diversity ecosystem, we may (generally) expect that protozoa/dinoflagellates would have a greater impact than lytic bacteriophages (see below).

However, there are some important factors to consider. Protozoa have very high feeding rates and can consume ~ 25 - 100% of the daily bacterial production. They do not appear to be sensitive to the physiologic status of their prey. Also, since they are motile, they can move to where the prey is. Protozoa/dinoflagellates have a threshold feeding density of ~ 106 bacteria/ml (below which they will not feed). In this example, they are not prey specific, and therefore will feed on whomever is plentiful even if it is not a dominant player in terms of its total numbers. Thus, the figure of 106 bacteria does not necessarily pertain to cells of a given species. That means that they could theoretically wipe out a species (or significantly decrease the numbers) of organisms which they found to be tastier than the others. Obviously this could have a profound effect if the organism had an important unique role an an ecosystem (i.e. H2 production).

Viruses are not motile, and they are often very strongly sorbed to particles (especially in terrestrial environments). They are obligate symbionts and they are dependent on their host cell for growth and reproduction. In this example, it is a lytic virus, thus lysogeny is not an option. They have a threshold infection density of ~ 104 susceptible host bacteria/ml. Viruses are very host specific. In a high diversity ecosystem, they may have a better opportunity of finding their ideal host, but the hosts may be in too low a number for the virus to initiate infection. In a low diversity ecosystem they may have a dramatic effect on a particular microorganism who is dominant in terms of numbers, and this could clearly have a significant impact as the ecosystem adjusts to the newly vacated niche. When a lytic virus does infect susceptible cells, it has an exponential effect on bacterial cell density since each infected cell will serve to liberate several (often 100's - 1000's) more infective virions upon lysis -each of which can now infect a new cell. Phage infection is very sensitive to the physiologic status of the host cells. Viruses will only infect if the host cells are nonsorbed, actively growing, healthy etc... In natural environments we know that nutrients are often limiting, and bacteria are growing at far below their optimal growth rates. Thus, we tend to find viruses in situations in which there are high bacterial population densities (i.e. rivers receiving wastewater effluent). In situations where bacterial population densities and growth rates are high, viruses have a much greater potential than do protozoa for quickly leading to catastrophe in an ecosysytem . However, the requirements which permit such a catastrophic viral event from crashing an ecosystem are stringent, and hence the probability is lessened. Protozoa/dinoflagellates can have a greater effect (than viruses) in situations where densities are lower and microorganisms are growing at suboptimal rates.

12.) Some of the methodological limitations in microbial ecology have to do with sampling - spatial variability, temporal variability, and sampling methods altering the in situ context of the microorganisms. Discuss these limitations with respect to obtaining (and interpreting information from) soil samples. ( 8 pts)

When you take a sample you face the following issues:

1. structural disturbance of environment

2. remove sample from landscape and place in container

3. transport

4. storage under conditions unlike the field

5. drying/mixing of samples

6. processing - does it destroy the in situ distribution

7. measurements - how do you do it.

w/in microorganisms:

a. light catalyzed reactions are instantaneous

b. enzymatic reactions occur 10-6 - 10-3 sec.

c. DNA replication ~ 40 minutes

d. sporulation (encystment) w/in 1-3 hours

soils- microenvironments on level of µm scale - differences in pH, O2, chemical concentration, [CO2] , water etc...Also, materials sorb (bind) strongly to clays and may not permeate through i.e. 1 mm soil particle. You never know if you take a 1 g sample of soil, that it will contain the same chemical, physical and biological constituents as one 5 inches away. Also microorganism are not uniformly distributed - they are patchy and form microcolonies within soil aggregates. Time of day will play a key role for the distribution and activities of photosynthetic organisms (or light sensitive organisms). Also the time scales for many bacteria are very small (see above) - so if you're looking for a point in their life cycle it may be hard to predict when that will come. Weather has profound effects - water, temp, nutrient loading from run-off, pH, redox etc... All this must be considered.

I could go on ...but I think you got the point. microenvironments are the key - and that you define what conditions can vary over what type of distance scale.

Also covered in Fall 02 – populations/community interactions. Stuff from lab expts and lectures will also be included on exam.

YAY....HURRAY...YIPPY-I-O-I-A!