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How do Bacteria Know That Food is Near?

Gypsy Achong
Civil & Environmental Engineering
Stanford University
May 2001

The contamination of soils and groundwater as a result of leaking underground gasoline storage tanks is a widespread problem. Since known and suspected carcinogens are present in gasoline and because gasoline affects the taste and smell of drinking water, gasoline spills that occur near potable water supplies must be cleaned up.

Underground gasoline spills are expensive to clean up using traditional methods since they involve pumping groundwater to the surface for treatment or excavating contaminated soils for incineration. Fortunately, bacterial communities able to “eat” the gasoline are ubiquitous in soil environments and offer environmental engineers a potential, and hopefully cheaper, alternative to traditional treatment methods. This alternative, called bioremediation, refers to clean-up methods that depend on biological rather than physical or chemical processes. However, in order to rely on bioremediation for the clean up of a contaminated site, we need to know that this method will work. There are two crucial prerequisites for the bioremediation of gasoline-contaminated sites to be successful: the presence of bacteria able to degrade the spilled gasoline and environmental conditions that allow the bacteria to “eat” the gasoline. Gasoline degradation can occur under two conditions: in the presence of oxygen, or aerobically, and in the absence of oxygen, or anaerobically. My research will determine the environmental conditions necessary for gasoline degradation under anaerobic conditions.

The most toxic components of gasoline are benzene, toluene, ethylbenzene and the three xylenes: o-xylene, m-xylene, and p-xylene, known as BTEX. These chemical compounds are also the most soluble components of gasoline and readily dissolve in groundwater at the spill site. They then move away from the contaminated site with the groundwater flow and contaminate drinking water supplies. This contamination is of great concern because benzene is a known carcinogen, and toluene, ethylbenzene and the xylenes are suspected carcinogens. Some bacteria are able to use BTEX to grow under aerobic conditions, i.e. in the presence of oxygen, in the same way that humans burn food with oxygen. These bacteria quickly use all of the dissolved oxygen and, since the solubility of oxygen in water is low, the groundwater becomes anaerobic, or oxygen-free. In the last ten years, a few bacteria capable of using some of the BTEX compounds in the absence of oxygen have been discovered. Instead of using oxygen, these bacteria are able to use simple compounds that are dissolved in the ground water, including nitrate and sulfate. Great advances have been made in understanding how toluene, m-xylene and ethylbenzene are degraded without oxygen. I will study how these pathways are regulated in response to environmental signals, such as the presence or absence of oxygen.

Enzymes are proteins that catalyze, or speed up, reactions. They are encoded on DNA and the region of DNA that encodes an enzyme is called a gene. Think of a gene as the instruction manual for making an enzyme. When the gene for an enzyme is expressed, the enzyme is produced. Bacteria alter gene expression in response to their environment because they usually live in a natural environment where nutrients are scarce and must live meagerly in order to survive. Therefore, they only make cell components when they need them. On the other hand, they need many different genes so that they can live on different food sources depending on what is available at a given time. In response to an environmental signal, some regulatory proteins bind DNA to change the “direction” of a cell by changing which genes are expressed, or “turned on.” Bacteria have developed complex regulatory systems to express only the genes that are necessary at a particular time. These regulatory systems continuously observe the environment and alter gene expression to maximize the chance of that bacterium's survival. Regulatory systems may be quite complex because they measure and integrate all of the important signals into a final output signal: the expression or repression of genes. Anaerobic toluene and m-xylene degradation probably involves fairly complicated regulation because several environmental factors control gene expression.

Why is the regulation of anaerobic toluene and m-xylene degradation potentially complex? An enzyme, called benzylsuccinate synthase, which is destroyed by oxygen, catalyzes the first step in toluene and m-xylene anaerobic degradation. Remember that bacteria are extremely energy-conscious organisms. In order to conserve energy, the genes encoding benzylsuccinate synthase should not be turned on in the presence of oxygen. So what happens if oxygen is absent? In such case the bacterium should only express the benzylsuccinate synthase genes if toluene and/or m-xylene are present. Otherwise, the bacteria would spend a lot of energy preparing benzylsuccinate synthase but the enzyme, once prepared, would have nothing to work with to make the energy investment worthwhile. So let's assume that oxygen is absent and toluene is available, then some oxygen substitute, in this case nitrate, must also be present. In addition, if a more energy-rich food is present, then the bacterium should use that food before it uses toluene or m-xylene. What happens when mixtures of compounds are around, such as at a contaminated site? My research will examine how environmental signals, including the presence or absence of oxygen, nitrate, toluene and m-xylene, actually affect the expression of benzylsuccinate synthase.

A lacZ fusion to a gene of interest is one way to test the expression pattern of that gene. There is a well-studied protein called b-galactosidase that converts a colorless compound called ONPG into a yellow compound. The gene that encodes b-galactosidase is lacZ. If you attach the lacZ gene to your gene of interest, and the construct is called a lacZ fusion, then b-galactosidase will be expressed whenever your protein of interest is expressed. But it's simple to test the presence of b-galactosidase because you can measure the color change from colorless to yellow. I will prepare a lacZ fusion to the benzylsuccinate synthase gene and grow the bacteria under different types of conditions, including the presence and absence of oxygen, nitrate, toluene and m-xylene, in order to easily quantify how different conditions affect the expression of benzylsuccinate synthase. Such experiments will allow us to better predict the success of bioremediation of gasoline at different contaminated sites, given a basic chemical composition of the water at the site.

Detecting specific microorganisms at a field site has become fast and cheap with the development of many DNA isolation and fingerprinting technologies. But the presence of an appropriate microorganism does not assure the success of bioremediation at a site. The relevant enzymes must also be expressed, which means that the environmental conditions must promote expression of those enzymes of interest. My research will improve our ability to predict the success of bioremediation at a gasoline-contaminated site given a simple chemical analysis of the water at that site. Furthermore, these regulatory studies will broaden our understanding of how bacterial cells detect environmental signals and how they integrate multiple signals into a few crucial responses.