RLBMicrobes

EXPLORING MICROBIAL LIFE AND BIODIVERSITY OF THE

RANCHO LA BREA TAR PITS

A COLLABORATION BETWEEN THE GEORGE C. PAGE MUSEUM AND

THE UNIVERSITY OF CALIFORNIA, RIVERSIDE

COLLABORATING SCIENTISTS

Dr. Jong Shik Kim – University of California, Riverside

Dr. David Crowley – University of California, Riverside

Dr. John Harris – George C. Page Museum

Christopher Shaw – George C. Page Museum

INTRODUCTION

New research at the Rancho La Brea Tar Pits has revealed hundreds of new bacterial species

that have never been described before. These exist in thriving microbial communities within the

asphalt-soil mixtures of the asphalt seeps. Because many of the bacteria in the tar are highly

specialized and cannot be easily grown in the laboratory, molecular biology methods are being

used to identify both the culturable and nonculturable bacteria in samples of the asphalt taken

from here in Hancock Park.

The bacteria are living microorganisms that have adapted to life in the asphalt and are able to

grow by producing enzymes that enable them to breakdown many different petroleum hydrocarbons

and use them to build their cells. The discovery of these microorganisms, has important applications

for biotechnology, including the possible production of new enzymes for use in manufacturing

chemicals from petroleum, surfactants that can be used to improve oil recovery, and for production

of medicines, polymers, and new types of biodegradable plastics. Some microorganisms that can be

isolated from the asphalt and grown in the laboratory may also have uses for treatment of oil wastes

and cleanup of contaminated soils.

Here we explain how the bacteria in the pits were identified using modern methods in molecular

biology.

HOW WE TELL MICROBES APART

Because the majority of bacteria in the asphalt are not easily cultured in the laboratory, the first test

was to see if the asphalt contained DNA, which is one of the signature molecules for life. The DNA

can then be analyzed to read the genetic code in certain sections (sequences) that are used to

identify and classify different organisms.

Typical Bacterial Cell

http://www.ou.edu/class/pheidole/General%20Bacteria.jpg

Today, organisms are classified on the basis of a highly conserved DNA sequence that is used

by all living organisms to produce proteins.

The DNA sequences for different organisms have changed slightly over billions of years as new

organisms have evolved. This information of DNA sequences has been used to construct a new

family tree for all of the living organisms on earth on the basis of their DNA similarities.

WHERE MICROBES FIT IN THE TREE OF LIFE

Biologists now recognize three domains of life and further classify living organisms into related

groups using the scheme:

Domain > Kingdom > Phylum > Class> Order > Family > Genus > Species

Note that most of the genetic biodiversity on earth is microbial, especially when compared to plants,

animals, and fungi which are only small twigs on the tree of life.

Universal Tree of Life Based on 16S rDNA.

Figure by Norman Pace, May 1997; Science 276: 734 - 740

HOW WE GET MICROBES FROM THE ASPHALT

1. To get DNA out of the asphalt, the asphalt sample is put in a tube with ceramic beads and

shaken at high speed in a machine called a bead beater. The bacteria are broken open, and their

DNA sticks to small glass particles that have a special sticky surface that is designed to bind DNA.

asphalt and DNA bead beater

2. The glass particles and DNA are washed in alcohol to remove the tar and other impurities. The

cleaned-up DNA is then stripped off the particles in water, purified, and used to make millions of

copies of particular sections (a process known as amplifying sequences). This takes place inside

a Polymerase Chain Reaction (PCR) machine.

Purifying the DNA PCR machine

DNA washing steps

using spin filters

3. In the PCR machine, millions of copies of the DNA are made in about an hour. The copied DNA

(16S ribosomal DNA genes) is like a long Social Security Number that can be used for identifying

different bacteria.

Red and blue segments are PCR primers that bind to specific regions of the DNA. Enzymes and

DNA molecule subunits are added to fill in the gaps between the primers. After each “cycle” the

amount of DNA doubles so that after 30 cycles there are millions of copies of the desired DNA

sequence.

HOW WE IDENTIFY THE MICROBES AND USEFUL GENES

Libraries containing all of the DNA sequences from the microorganisms in the tar pit samples are

produced by inserting short pieces of DNA from the Rancho La Brea bacteria into Escherichia coli,

the common human gut microorganism. Each E.coli cell contains a different piece of DNA so that

100,000 cells in a small tube contain a library of all the original DNA sequences. The E. coli

"clone library" can be kept in the freezer and studied any time later by growing colonies of cells on

agar plates .

Individual colonies produced from single cells on agar plates each contain unique DNA sequences.

These can be examined for the different types of genes they carry using different screening methods.

When something interesting is found, the DNA sequences are read by a "DNA sequencer" at a

genomics laboratory. For example, if one of the colonies of E. coli produces an antibiotic we can use

this gene to make new medicines. To identify the bacteria from the tar pits we read their ribosomal

DNA genes.

After the DNA sequence information for the ribosomal DNA is read, the deciphered

code is compared to a database to identify the microorganisms and their closest

relatives. We can then see where they fit into the tree of life (Figure is from Brock

Microbiology of organisms 2005).

BACTERIAL SPECIES DISCOVERED AT RANCHO LA BREA

Bacteria from the tar pits can also be identified by painting their DNA chromosomes with

fluorescent stains that target certain DNA sequences.

Using these stains, different groups of bacteria that are living on the tar particles can be

seen with a fluorescent microscope.

Here cells of the archaea are stained red, bacteria are green, and Pseudomonas (a genus

of bacteria) are blue. The bacterial colonies can be seen as clumps of different color cells

that are growing together, which suggests that they cooperate by producing different enzyme

mixtures to degrade the chemicals in the tar.

FUTURE RESEARCH QUESTIONS & HOW THIS MAY AFFECT OUR EVERYDAY LIFE

1. How do the microorganisms break down asphalt chemicals without oxygen?

Oil spills often happen in river sediments and ocean estuaries where there is very little

oxygen. Most of the bacteria that break down oil need oxygen in order to grow. The La Brea

microbes may help us to learn more about life without oxygen and new ways we can clean

up polluted soil and water.

2. Do the microorganisms work together in communities to degrade asphalt?

In the past, most microbes have been looked at in “pure cultures” where it is easier to

study their biochemistry and genetics. However, bacteria never work alone in nature and we

know that they can behave very differently in a community. We need to understand more

about their ecology to understand how biochemical processes work in nature.

3. Can we grow the bacteria from the asphalt in the lab so that we can study their

abilities to degrade asphalt and tar and collect any new chemicals they produce?

More than 95% of the bacteria in nature cannot be grown in the laboratory. In order to

use bacteria for biotechnology, it is important to be able to grow them in large “fermenters”

where we can purify the chemicals they make. This means figuring out the the right food

and conditions that they need in order to grow when we take them of the asphalt.

4. Can we isolate genes (DNA) for the enzymes, surfactants, polymers, and other

chemicals that are produced by these microorganisms for use in biotechnology?

Even if we cannot grow all of the different La Brea bacteria in the laboratory, we can still

use their genes to make new commercial products. Today, bacteria are used to make human

insulin to treat diabetes. In the same way, we can take the genes out of the bacteria that live

in the asphalt and put them into easily grown bacteria like E. coli to make new chemicals,

medicines, and biodegradable plastics. New surfactants (soaps) and enzymes from tar pit

bacteria may also be used by petrochemical companies to increase the amount of oil we

can recover from old oil wells and from oil shales, thereby giving us more oil and gasoline in

the future and more time to develop new energy sources.

5.How similar are these microorganisms to those from other asphalt seeps elsewhere in

the world?

This is a new “Golden Age” of microbiology where we are just beginning to learn about the

millions of species of microorganisms on Earth that recycle everything in nature and keep our

ecosystems working. At the same time, many places where microbes live are being changed

by human activities. Bacteria in the La Brea asphalt have evolved in isolation for thousands of

years. To protect this important resource of newly discovered biodiversity for the future, we

need to know if the Rancho La Brea bacteria only live here in Hancock Park, or if they

can be found in other places. By protecting unique microbial habitats we can appreciate, study

and use these special bacteria to improve our lives in the future – maybe even in ways we