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18/02/2011

Sizing Up the UncultivatedMajority

A case for Problem Based Learning I

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Sizing Up the Uncultivated MajorityA case for Problem Based Learning I

Background presentation

Biological nitrogen fixation, the conversion of atmospheric nitrogen to ammonia for biosynthesis, is

exclusively performed by a few bacteria and archaea. Despite the essential importance of biological

nitrogen fixation, it has been impossible to quantify the incorporation of nitrogen by individual

bacteria or to map the fate of fixed nitrogen in host cells.Coupling the identity of microbes with their

activity in the environment remains an important gap in our ability to explore microbial ecology. Thedevelopment of techniques to quantify the metabolic activity of single microbial cells has been

especially challenging, mostly due to their small size.

Bacteria and archaea responsible for biological nitrogen fixation can be found in free-living form or in

symbiosis with algae, higher plants, and some animals. Although these microbes are a critical part of

the global nitrogen cycle, there has previously been no means to evaluate this fixation process at

subcellular resolution.

Wood and woody plant materials are abundant in the biosphereand are important nutrient sources for

a variety of fungi and microorganisms. Yet few animals are ableto feed primarily on wood. Although

rich in carbon, wood typically contains two orders of magnitude less nitrogen per unit of carbon than

does animal tissue. Animals using wood as food must therefore obtain other sources of combined

nitrogen for biosynthesis. For example, wood-eating termites are thought to supplement their diet with

nitrogenous compounds produced by nitrogen-fixing bacteria inhabiting their gut. This conclusion is

supported by observations that a variety of nitrogen-fixing bacteria have been cultivated from termite

guts or detected by culture-independent methods, and that substantial rates of nitrogen fixation have

been measured in association with termite guts and intact termite colonies. Direct measurement of

nitrogen fixation by individual bacteria and of nitrogen use by host cells, however, has remained

impossible. Nitrogen fixation has also been detected in intact specimens of wood-eating marine

bivalves of the family Teredinidae (commonly known as shipworms), but the site of fixation and the

identity of the nitrogen-fixing microorganisms have not been previously determined. Although

conspicuous communities of nitrogen-fixing bacteria have not been found in the gut of shipworms, as

they have in termites, dense populations of intracellular bacterial symbionts have been observed incells (bacteriocytes) in a region of shipworm gills known as the gland of Deshayes. Moreover, a

bacterium(Teredinibacterturnerae) capable of fixing nitrogen gas in pure culture has been isolated

from the gills of numerous shipworm species, and its presence in the gill symbiont community of the

shipworm Lyroduspedicellatushas been confirmed by in situ hybridization and quantitative

polymerase chain reaction analysis. These observations raise the questions of whether bacterial

symbionts within the gills ofL. pedicellatuscan fix nitrogen and whether this fixed nitrogen is

supplied to the host.

Main background ideas

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In the biogeochemical cycle of nitrogen, nitrogen fixation is paramount, but only some bacteria

and archaea are able to fix atmospheric N2.

Wood is an abundant source of organic carbon which can be used to feed animals.

Wood-eating organisms rely on a food source which contains a nitrogen-to-carbon ratio which is

about one hundred times lower that the ratio found in their tissues.

Nitrogen fixing organisms are bacteria and archea. They might live free in the environment or in

symbiosis with other organisms.

Shipworms are mollusk bivalves that rely on wood as their primary food source. Shipworms do

not have conspicuous communities of nitrogen-fixing bacteria in their guts as thermites do.

Shipworms have a region on their gills called Deshayes gland where they have special cells called

bacteriocytes which are full of intracellular symbiont bacteria.

FISH and qPCRave been used to determine the bacterial nature of the symbionts

Main questionCan we couple the identity of microbes with their activity in the environment?

Can we determine whether symbionts in bacteriocytes are responsible for nitrogen fixation and supplyof organic nitrogen to the worm?

Background insights

Here the student must gather information of the key points in order to build a clearer picture

of the topic under investigation. One possible way is to get insight of the main background

ideas.

The biogeochemical cycle of

nitrogenA classical view of the cycle considers

five main forms of nitrogen compounds

and links them in a symmetric manner

(Fig. 1). However, if we take into

consideration the diversity of organisms

that are able to participate in the

different transformations, we soon

realize that while animals and a great

deal of saprophytic microorganisms are

able to convert organic nitrogen intoinorganic NH4+, and many genera are

able to convert nitrate to organic

nitrogen, as for example plants (Fig.2),

few genera are responsible for

nitrification and other few genera are

responsible for denitrification or

nitrogen gas production via

anammoxreation. Taking into account

only these reactions we would assist to a

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Fig. 1

Fig. 2

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gradual decrease of assimilable and organic nitrogen pools. Thus, atmospheric nitrogen

fixation is a key process to:

Avoid assimilable nitrogen depletion

Ensure access to atmospheric nitrogen pools.

Signing agreements with bacteria for a mutualistic relationship might be a good deal for many

species to ensure a proper organic nitrogen supply.

ShipwormShipworms are not worms at all, but rather a group of unusual

saltwater clams with very small shells, notorious for boring into

(and eventually destroying) wooden structures that are immersed in

sea water, such as piers, docks and wooden ships. Sometimes called

"termites of the sea", they are marine bivalve mollusks

(Eulamellibranchiata) in the family Teredinidae, also often known

as Teredo Worms.

When shipworms bore into submerged wood, bacteria

(Teredinibacterturnerae strain ATCC 39867 / T7901) in a special

organ called the gland of Deshayes allow them to digest cellulose.

The excavated burrow is usually lined with a calcareous tube.

Shipworms have slender worm-like forms, but nonetheless possess

the characteristic structures of bivalves. The valves of the shell of

shipworms are small separate parts located at the anterior end of

the worm, used for excavating the burrow.

Fig. 4.(1) Shell; (2) Foot; (3) Cephalic hood; (4) Mantle collar; (5) Excurrent siphon;(6) Incurrent siphon; (7) Pallet.

FISH

The ribosomal-RNA (rRNA) approach to microbial evolution and ecology has become an integral partof environmental microbiology. Based on the patchy conservation of rRNA, oligonucleotide probes can

be designed with specificities that range from the species level to the level of phyla or even domains.

When these probes are labelled with fluorescent dyes or the enzyme horseradish peroxidase, they can

be used to identify single microbial cells directly by fluorescence in situ hybridization.

rRNA molecules are well suited for the identification of Bacteria and Archaea for several reasons. First,

all cells require ribosomes for translation. Each prokaryotic ribosome contains one 16S rRNA of

approx1,600 nucleotides in the SSU, and one 23S rRNA of approx3,000 nucleotides and one 5S rRNA

of approx120 nucleotides in the large subunit (LSU). As each cell contains many ribosomes, these

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Fig. 3

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target molecules are naturally amplified to numbers that can range from a few hundred to 100,000 per

cell.

Second, the evolutionary conservation of rRNA sequences is patchy, but is generally much higher than

that of most protein-encoding genes. As the evolutionary pressure is on the RNA product, there is no

rapidly evolving third-codon position. This enables the design of oligonucleotide probes for large

taxonomic entities, such as domains, phyla, classes or orders.

Third, targeting rRNA for

identification links microbial ecology

and microbial evolution, a concept

that was first promoted by Norman

Pace and colleagues14 in 1986. In

this system, categorization is no

longer based on morphology (for

example, rods versus cocci) or

physiology (for example, aerobes

versus facultative anaerobes versusstrict anaerobes), but rather is based

on the three domains, Archaea,

Bacteria and Eukarya, and phyla and

classes, such as the Alpha-, Beta- and

Gammaproteobacteria.

The treatment that takes place before hybridization is crucial for a quantitative FISH assay. The task is,

on the one hand, to preserve the integrity and shape of all cells and prevent cell loss through lysis,

while on the other hand, permeabilizing as many cells as possible to allow the labelled oligonucleotides

to diffuse to their intracellular rRNA target molecules. The challenge is that the cell-wall composition

of bacteria and archaea is diverse. Formaldehyde and ethanol continue to be the main fixatives used,

but there is still no standard permeabilization protocol for all microbial cells. Empirical optimizationsoften consider the specific composition of the cell wall and can include modifications such as

enzymatic digestion of thick peptidoglycan

layers by lysozyme, digestion of

proteinaceous cell walls by proteases, the

removal of wax by solvents, the use of

detergents and even short-term

incubations in hydrochloric acid.

In figure 5 there is an example of

fluorescence microscopy, using FISH

technology of the interaction between

Microalgae growth-promoting bacteria

(Azospirillum) immobilized in alginate

bead with native bacteria of the

wastewater. (Green = genusAzospirillum;

Red = Domain bacteria; Yellow = Super-

imposition of the two images [producing

yellow cells] specifically identifying

Azospirillum)

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Analysis of the main question

This section is devoted to the analysis of the main question. Here we will try to propose and discuss the

methods that can be used to shed light into the question of whether the bacteria

Teredinibacterturnerae located in the gland of Deshayes of the shipworm Lyroduspedicellatusis

responsible for the fixation of N2 and the transfer of nitrogen to the mollusk.

Then, we will evaluate the statistical implications of the data sampled from the experiments.

Experimental proceduresIn the classroom we have proposed the following experimental procedures.

First proposalOne way to see if bacteria within the consortium are fixing N2 is to grow the shipworm in a medium

which does not contain any source of nitrogen except N2. Thus no organic nitrogen, no nitrates or

nitrites, no ammonia should be present.

First we should analyze, what kind of questions can we really answer with this experimental design?

Logical analysis of the proposalAs nitrogen is vital for the growth and maintenance of Lyrodus:

1. if, after several weeks of experiment, the mollusk is dead or decaying we can conclude that:

Lyrodus is not able to fix atmospheric nitrogen. This is in agreement with the observation thatno animal has been found to fix N2.

Whether Teredinibacteris able of fixing nitrogen or not, it does not provide any usable form of

nitrogen toLyrodus.

1. if, after several weeks of experiment, the mollusk is alive or even has been growing, we can

conclude that:

The symbiosis between the mollusk and bacteria is able of fixing nitrogen.

Thus, this experiment is only able to answer one question:

Is the symbiosis betweenLyrodus and Teredinibacterable to fix nitrogen?

Conclusions

At the beginning we hypothesized that this symbiosis should fix nitrogen because the food source of

the shipworm has an average nitrogen concentration much lower than nitrogen concentration in the

mollusk tissues. However we did not have direct experimental evidence. This experiment would, thus,

provide the experimental evidence for this hypothesis, but nothing else and obviously nothing relevant

for our main question.

Second proposal

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Isolating and maintaining in a culture media the gill tissue containing both animal cells and bacterial

cells. Then, cultivating them in a medium free of any form of assimilable nitrogen other than N2.

Logical analysis of the proposalThis experiment is rather similar to the one first proposed but it restricts the analysis to the physiology

of gills. As nitrogen is vital for the growth and maintenance of Lyrodus,

1. if, after several weeks of experiment, the tissue is dead or decaying we can conclude that:

The tissue is not able to fix atmospheric nitrogen. This is in agreement with the observation

that no animal has been found to fix N2.

Whether Teredinibacteris able of fixing nitrogen or not, it does not provide any usable form of

nitrogen to the tissue.


conclude that:

The symbiosis between the mollusk and bacteria is able of fixing nitrogen.

Thus, this experiment is only able to answer two questions:

Is the symbiosis betweenLyrodus and Teredinibacterable to fix nitrogen? And, is the nitrogen

fixation process performed at least in the gills?

However, care must be taken if the results are negative because growing a tissue outside the host is not

straightforward. Thus, the appropriate controls should be put in place.

Conclusions

At the beginning we hypothesized that this symbiosis should fix nitrogen because the food source of

the shipworm has an average nitrogen concentration much lower than nitrogen concentration in the

mollusk tissues. However we did not have direct experimental evidence. This experiment would, thus,

provide the experimental evidence for this hypothesis, but nothing else and obviously nothing relevantfor our main question.

Third proposalTreating the shipworm with antibiotics to bleach it. A bleached shipworm would be cultivated in a

medium with N2 as the sole source of nitrogen.

Logical analysis of the proposalAs nitrogen is vital for the growth and maintenance of Lyrodus,

1. if, after several weeks of experiment, the animal is dead or decaying we can conclude that:

The symbiosys is mutualistic and the mollusk depends on the bacteria for its survial.

No conclusion can be achieved on whether the key point is nitrogen fixation. It could well be

cellulose processing, or the synthesis of a key component.


conclude that:

The wormship itself is able to fix nitrogen which does not exclude the possibility that part of

the nitrogen in the mollusk tissues comes from the bacteria. However, this colud be a very nice

way of demonstrating the ability of the mollusk to fix nitrogen..

Thus, this experiment is only able to answer two questions:

Is the symbiosis betweenLyrodus and Teredinibactera form of mutualism?

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Is the mollusk, by itself, able to fix N2?

Conclusions

This experiment is relatively poor in terms of providing evidence on the physiology of the symbiotic

association. However, if the bleached mollusk is able to grow on N2, then we could demonstrate for the

first time in the history that an animal is able to fix atmospheric nitrogen.

Fourth proposalGrowing the shipworm toghether with its symbiotic partner in a medium containing only N2 as a

source of nitrogen. If we use a isotope of nitrogen we can later follow the incorporation of nitrogen in

the different tissues of the shipworm.

Logical analysis of the proposal

In order to explore this hypothesis we have to find the right tools. The two critical points are:

The type of isotope to be used.

The technique to measure the incorporation of isotopes in the tissues.

Isotopes

Nitrogen has got 17 isotopes (http://en.wikipedia.org/wiki/Isotopes_of_nitrogen) 15 of them are

radiactive and two of them are stable. Using radiactive isotopes have proved to be very useful for

matabolic studies. However the half-life of the isotopes is very important. 13N is the one having the

longest half-life and it is only of 10 minutes. Thus we can only work with this isotope if:

We have a cyclotron in the lab for producing the isotope just on time.

The process that we study last less than ten minutes.

As the fixation and further transfer of organic nitrogen to the host tissue might last longer than ten

minutes, 13N is probably not the isotope of choice.

15N is a stable isotope which is very scarce in nature. The natural ratio 15N/14N is in the order of

0.00367.

Detection methods

In order to detect stable isotopes we can use two approximations:

Differential centrifugation. The tissues that have incorporated 15N will be denser than those

that have natural proportions of this isotope. Thus, centrifugation could be a technique of

choice. The only problem is sensibility. Would differential centrifugation be sensible enough

to discriminate between 15N rich and 15N poor tissues? We could perform some preliminary

assays, but taking into account that nitrogen is only a minor component of proteins and that

proteins are a minor component of the wheight of a tissue, which is overhemingly dominated

by water, the influence of the incorporation of15N into the experimental tissues on the total

density of the sample could be too weak to be detected.

Mass spectrometry. Mass spectrometry is the technique of choice when we want todiscriminate between two isotopes that share common chemical properties, but the problem ishow to make direct mesures on an intact tissue. Multi-isotope imaging massspectrometry(MIMS) is a technique developed

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http://en.wikipedia.org/wiki/Isotopes_of_nitrogenhttp://en.wikipedia.org/wiki/Isotopes_of_nitrogen

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Conclusions

This experiment is relatively poor in terms of providing evidence on the physiology of the symbiotic

association. However, if the bleached mollusk is able to grow on N2, then we could demonstrate for the

first time in the history that an animal is able to fix atmospheric nitrogen.

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http://pubs.acs.org/cen/news/85/i38/8538notw8.html

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