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Normally, dental plaque is formed by various of the Gram-positive lactic acid bacteria (usually among those known as dairy bacteria) when they are fed the disaccharide sucrose. These bacteria cleave the sucrose in half to a glucose molecule and a fructose molecule, and then they "eat" one of the products and polymerize the other. In this way, some species make dextrans* (poly-glucose among which are starches and cellulose) while others make levans (poly-fructose).

But what about Gram-negative bacteria, and about E. coli more specifically? In this vein, it is interesting that some students have found that a strain of E.coli produced copious amounts of slime when growing on the disaccharide lactose, while a mutant of that strain could not. The only difference between the two strains was that the second was unable to metabolise galactose, which is one of the two "halves" of lactose. (Strain #1 was lac⁺ gal⁺, while strain #2 was lac⁺ gal^-.)

If this E. coli process works like that in the lactic acid bacteria, the data suggest that #1 "eats" the galactose, and polymerizes the glucose making dextran; while #2 cannot make dextran otherwise it would have nothing to "eat."

It is highly likely that your local consultant might bring up the possibility that the E. coli is producing copious amounts of either capsule (aka K-antigen) or endotoxin (aka LPS or O-antigen), both of which contain high molecular weight polysaccharides. However, the suggested strains, below, are derivatives of a strain of E. coli that possesses neither capsule or LPS.

The Project

Determine the content of the slime: whether it is dextran (= glucosan) or galactosan.

How to do this. This is perhaps the hard part of the project: finding the analytical method for distinguishing dextran from galactosan. Here are a few suggestions. But remember that it is really up to you to figure out how to do it and then do it! Identification "could" be very easy if amylase only attacks dextran but not galactosan. (In the figure of starch, above, the sites of amylase attack are indicated by the arrows.) But your dextran might so happen to be cellulose, which amylase does not attack! (Suggested hint: "think aqueous solubilities.")

Also it would be useful to be able to determine that galactose specifically does or does not appear in the polymer. There are, of course, specific chemical tests for both glucose and for galactose.

But first, the polymers would have to be hydrolyzed down to the monosaccharide components without harming those components. Besides doing chemical tests to identify the monosaccharides, one could use physics by means of employing a polarimeter. Most sugars have very specific amounts of rotation per molar per distance. However, once you have hydrolyzed your polysaccharide, you then have the problem of determining its concentration. If you have a refractometer available you can, within seconds, determine the concentration with a precision of 3 decimal places. HOWEVER, neither polarimeters nor refractometers are commonly found in labs, and they are quite expensive. So you will probably need to revert to chemical and paper chromatographic methods.

Suggested E. coli Genotypes to be Used (Free from the E.coli Genetic Stock Center)
Syndey Brenner's Accession Number	Yale's Accession Number	Genotype	Relevant Phenotype
MO	4995	F- λ- e14- relA1 rspL150(strR), spoT1 (see note)	lac+ gal+
M26	4996	same as above except it is also galE15	lac+ gal-
Note: "F- " indicates this is a 'female' cell (i.e.: without an F fertility factor) "λ-" indicates that this cell is not lysogenized with phage λ "e14- relA1 rspL150(strR), spoT1" all indicate that these cells have various abnormal ribosome functions

Significance

Never do a project that has little significance! There are far too many significant projects to be done. So what is significant about this project? While the genome of E. coli has been sequenced, that does not mean we know what every gene does. In fact, we have elucidated fewer than a third of the 7,000 or so genes. And remember that gene operation is reflected in the cell's physiology - what the cell produces and how the cell changes with environmental changes. Far too little in known about the physiology of E. coli. The current fad or vogue is in DNA sequencing, but, as you see, it is not the end of the story of E. coli! Very little is known about how E. coli makes polysaccharides. This looks like a pretty good starting point. But - keeping focused - what is so important about knowing how E. coli makes polysaccharides? Two things:

1. It is because these polysaccharides are found in their exterior layers - clothing, if you need an analogy. It is from the appearance of this "clothing" that our bodies recognize these bacteria as being part of us, or aliens and needed to be conquered. This identification is fundamental to both how our body fights disease, and how we make vaccines because vaccines are often purified "clothing" of bacteria. (Sort of like having the bloodhound sniff the killer's clothing, and the bloodhound follows the trail and the killer is caught.)

2. While most types of E. coli are not pathogenic (in fact, most are good for you), several of its close relatives are very pathogenic. Shigella causes frequently fatal dysentery, and Salmonella causes occasionally fatal typhoid fever (pc: salmonellosis). So whatever you find out about E. coli in your experiments that will not get you sick, might apply to these relatives. Your work will be passed on to the experts who work in facilities that can safely handle these pathogens without getting themselves sick. You will have contributed to the first step in saving many lives. (By the way, it is an important concept for you to learn - the use of a model system (E. coli) for the study of something closely related.)

* The suffix "-an" indicates a polymer of the monosaccharide indicated in the main part of the word. Dextran is a polymer of dextrose** more modernly called glucose, while levan or levulan is a polymer of levulose** more modernly called fructose.

** When polarized light is passed through a solution of glucose, a polarimeter allows the viewer to see that the plane of the light's polarization is slowly rotated clockwise (to the right). Thus glucose is dextro-rotary, and was given the name dextrose. Levulose or fructose has the same story, but counter-clockwise.

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