In 1969, molecular geneticist Max Delbrück accepted a Nobel Prize in Medicine for his work on the reproductive mechanisms and genetics of viruses that target Escherichia coli, a potentially deadly bacterium that has fascinated scientists since its formal description in 1885. “The riddle of life has been solved,” declared Delbrück. He envisioned that researchers would make great advancements in biology, chemistry, and medicine by illuminating E. coli’s dark secrets.
Forty years later, the scientific community has embraced the bacteria as a valuable research tool. To make it safer for laboratory use, researchers have tamed dangerous E. coli strains by removing their harmful genes. But this taming process has produced unexpected consequences, limiting E. coli’s usefulness in clinical and industrial applications.
Now, several groups are harnessing a combination of approaches—including sequencing and synthetic biology—to counter these unexpected consequences, manipulating E. coli more precisely than ever before in order to create better research tools. These scientists, like Delbrück, believe that there’s a potential for advancement locked within this small bacterium.
Biology’s unsung hero
“My wife was just saying, ‘Why would you ever modify E. coli?’” laughs Yong-Su Jin, assistant professor of microbial genomics at the University of Illinois at Urbana-Champaign (UIUC). Jin’s lab uses systems and synthetic approaches to understand the genetic and environmental factors that produce useful microbial strains for industrial production of fuels, chemicals, and nutraceuticals. “There is a very famous quotation—anything that happens in E. coli must happen in an elephant.”
The quote is attributed to French biologist Jacques Lucien Monod, who won the 1965 Nobel Laureate in Medicine for his discovery of the genetic control of enzyme and virus synthesis. To make this discovery, Monod used E. coli as a model system to demonstrate a process that occurs in all living cells. He saw E. coli as one of science’s greatest tools for learning about fundamental biological mechanisms.
Researchers first studied E. coli to document its virulent properties, but they quickly realized that its fast growth cycle, simplified prokaryotic system, and easily manipulated genome made it the perfect model organism for genetic modification and adaptation. Since then, non-virulent strains of E. coli have become a ubiquitous item in the toolbox of geneticists, chemists, pharmacologists, and industrialists.
“E. coli is a workhorse in biotechnology,” says Robert Landick, head of a laboratory in the Department of Biochemistry at the University of Wisconsin–Madison (UWM). His lab specializes in RNA polymerase (RNAP)—the enzyme in all living cells that is involved in the creation of long RNA chains, including messenger RNAs (mRNAs) and structural RNAs—and has recently begun characterizing the functional changes that result from insertions in bacterial RNAP genes. “It’s used by DuPont to make plastic for rugs, it’s used by the drug industry to make specialty chemicals, and, increasingly, synthetic biologists are using it to make ever more complicated drugs that are hard to make other ways.” These biotechnology applications produce more effective and affordable drugs and greener chemistry.
A minimal hurdle?
Before virulent E. coli strains are used for societal or health technologies, they are stripped of their virulent genes. But some of these mutated strains, which are selected for their industrial promise, come with some undesirable side effects—such as slow growth cycles or expensive culture conditions—which undermine the very qualities that make E. coli such a useful model system.
To be cost-effective, bacteria used in industry—whether pharmaceutical, agricultural, or clinical—must grow in minimal media. But the most popular E. coli strains grow prohibitively slow in minimal media and thus are of little value to the biotechnology industry.
“Everyone uses E. coli for preparing DNA, but the DH5α strain—which has mutations in recombinase and endonuclease, and so a very high and stable transformation efficiency—grows very poorly under minimal medium,” said Jin.
Landick made the same observation in his laboratory. Last year, researchers from the University of California, San Diego (UCSD) noticed small deletions in RNAP that were possibly hindering the growth of E. coli K-12 MG1655 in minimal medium. They contacted Landick to discuss their results.
“When they sequenced the genes in their adapted evolution cell lines, they discovered that the mutations, at least the most interesting ones, were actually small deletions in parts of the RNAP that my lab had previously studied,” says Landick.
Through laboratory-controlled adaptive evolution for beneficial traits, these two strains of E. coli had also developed negative mutations that prevented their successful growth in minimal media.
In order to reengineer the strains, Jin and Landick’s research teams have separately developed two new protocols to make E. coli better than before.
But before Landick’s team could make a better strain of E. coli, they needed to better understand the mutations. “From the structure-function standpoint that we came from, understanding the mutations was important because they appear in these two adjacent domains that modulate the catalytic center of E. coli RNAP but do not appear in other bacterial lineages,” says Landick.
In collaboration with the UCSD researchers, Landick’s lab examined the deletions in RNAP at the physiological level, at the molecular level, and at a systemic level to map exactly how one deletion can have such a great effect on an organism (1). According to Landick, multilayered approaches are emerging as an important tool for microbial gene expression studies. “Historically, it’s been not easy to do and people have tended to take a reductionist molecular approach,” says Landick, “but it’s now possible to make predictions that can be tested in future experiments that use comparable, multilayered approach.”
Landick’s team began with standard targeted sequencing of the domains of interest, and selected RNAP mutants were then grown on minimal medium with a higher metabolic rate and more efficient use of carbon than the slow-growing, preexisting strains. Kinetics assays demonstrated that the RNAP mutants increased their transcription elongation and shortened their pause periods; this corresponded to gene regulation data from chromatin immunoprecipitation followed by hybridization of immunoprecipitated DNA to a whole-genome tiling array (ChIP-chip), which showed up-regulation of genes that increased growth and down-regulation of genes that compete with growth, such as energetically costly motility genes.
The results provide a framework to address growth mutations in all E. coli RNAP domains. But Landick is enthusiastic about the process as well as the results. “The most interesting aspect to me is the ability to gain novel insights by combining experiments that were done to do adaptive evolution with molecular and biochemical studies with systems biology studies,” said Landick. “We’re going to see more cutting-edge science combining at these levels.”
“As a metabolic engineer, I wanted to know why [the DH5α strain] grows so slowly as compared to the other strains, especially since this strain has great potential as a recombinant host,” say Jin.
The answer was an interrupted catalysis that played a large role in retarding growth. “We did a genomic search and found there is a mutation in a gene responsible for purine biosynthesis,” said Jin. His lab set out to fix the mutation in purB (adenylosuccinate lyase) and reestablish efficient growth in minimal medium (2). They amplified and sequenced the open reading frame (ORF) of purB in E. coli DH5α to identify a point mutation that can be corrected by recombination with exogenous, wild-type purB.
Their research presents a simple way to address a growth mutation while maintaining the efficacy of a previously adapted bacterial strain. “It’s like dog breeding,” says Jin of his technique. “If you have a good strain, you use it for the next breeding. We have this gateway strain that grows very well in minimal medium, and we will continue to modify it to get what we want.”
Jin’s team aims to further modify the recombinant strain to produce human proteins necessary for drug therapy.
If Delbrück solved the riddle of life, then Landick and Jin are trying to solve a new puzzle: how to tailor life for specific applications. With a deeper understanding of how mutations affect growth rate and, more importantly, how to address those mutations to create stronger bacteria, the industrial uses of the two E. coli strains have just become exponential.
“E. coli is a wonderful model organism for understanding biology,” says Jin, “This strain already has done so many good things for human life, and we can modify this microorganism to help human life even more.”
(1) Conrad, TM, M Frazier, AR Joyce, B-K Cho, EM Knight, NE Lewis, R Landick, and BØ Palsson. RNA polymerase mutants found through adaptive evolution reprogram Escherichia coli for optimal growth in minimal media. Proceedings of the National Academy of Sciences doi: 10.1073/pnas.0911253107
(2) Jung, S-C, CL Smith, K-S Lee, M-E Hong, D-H Kweon, G. Stephanopoulos, and Y-S Jin. 2010. Restoration of growth phenotypes of Escherichia Coli DH5α in minimal media through reversal of a point mutation in purB. Applied Environmental Microbiology 76:18, 6307-6309. doi:10.1123/AEM.01210-10