In studies that could be vital to
an expanding field of industrial biotechnology, scientists at
the Center for Biosystems Research are learning to censor
what E.coli bacteria are ‘talking’ about.
Cell-to-cell cross talking by laboratory E. coli
strains engineered to produce antibiotics, industrial polymers
or other products in fermentation vessels can lead to stress
in the culture and severely limit product output. But scientists
with CBR and partners have begun to decipher and override
stress ‘talk’ among cells of recombinant E. coli. CBR is part of
the University of Maryland Biotechnology Institute (UMBI).
In separate fermentation experiments with strains of E. coli that
produce two important model products–interleukin-2 or organophosphorus
hydrolase–the team increased product output by three to four times. The
first product is an important drug in preventing cancer and HIV. The
latter is a detoxifying agent for biowarfare nerve agents.
The scientists achieved higher yields of both products by either
conditioning the bacterial cultures with high levels of molecules from a
signaling pathway called AI-2, or by splicing the LuxS gene for quorum
signaling into the recombinant E. coli.
"This is great," says team leader William Bentley, CBR research
professor. "We want to understand what are the receptor molecules on
these cells, to understand the communication circuitry," he adds. The
experimental techniques are applicable to boosting production of many
other products in E. coli cultures. However, the researchers say they
are only beginning to understand and control the communications
circuitry of E. coli and other bacterial cells.
E. coli is a common enteric bacterium that is one of the most highly
studied microbes, with its genome and basic physiology well known. It is
also one of the easiest vehicles for genetic engineering and a
workhorse for metabolic engineering, an emerging branch of industrial
biotechnology. Metabolic engineers study interactions of biological
molecules in order to improve the manufacture of cell products and
proteins for therapeutic and industrial value.
At CBR, Bentley heads one of a very few metabolic engineering
laboratories focused on non-pathogenic E. coli quorum sensing. "We first
learned that when you grow E. coli in a fermentor, the resulting
product per cell goes down to a third of what it was (on the laboratory
bench), but you still make more cells. Why don’t they make as much at
high cell density as low cell density? We decided to look at what
changes are going on."
The team also includes Matthew P. DeLisa, formerly of Bentley’s
laboratory and currently with Cornell University; and James J. Valdes,
U.S. Army Soldier & Biological Chemical Command, Aberdeen Proving
Scientists have known about chemical cross-talking among cells of
bacteria since 1970. Through such communication, bacteria gauge their
own population density and respond by altering their expression of
specific genes as a group, a process known as quorum sensing. In marine
microbes, quorum-sensing molecules speed up reproduction to form
bio-fouling films that may colonize on surfaces of boats or piers.
Pathogenic bacteria, such as those that cause cholera, salmonella, Lyme
disease, tuberculosis and pneumonia, become virulent. Still others such
as Anthrax may form spores when they reach a quorum, or make antibiotics
to fight off neighboring microbes.
But studies of cell-to-cell ‘talking’ in E. coli, so important to
industrial biotechnology, have lagged behind somewhat. Scientists at
Princeton University discovered signaling molecules in
E. coli in 1998.
Bentley’s team is investigating recombinant E. coli in the context of
"an emerging centralized role for quorum sensing where it overlaps with
signals for stress and starvation," he says. This new link between
quorum sensing, cellular metabolism and stress-responsive circuits
raises the possibility of targeting quorum pathways for improving
They observed that signaling in recombinant E. coli, decreased the
yields of a desired protein product. There had been no previously
published reports of experiments to use quorum-sensing signaling pathway
molecules or genes to improve recombinant protein yield in any
bacteria expression system.
The team began two years ago by mapping DNA transcription of four
quorum-regulated genes and 20 stress genes of E. coli. They found
significant regulatory overlap among several stress and starvation genes
and known quorum-sensing genes. "Because quorum-sensing signals turn
bacterial genes on and off, deciphering this language will enable us to
commandeer it for our own purposes, like controlling protein and
metabolic engineering, rewiring cells, you might say, to produce
important polymers or try to figure out the coordination of the
enzymatic pathways. This is just the very beginning."
Such biological processing (fermentation) to produce products is more
desirable than traditional industrial processing because of the use of
renewable resources such as E. coli bacteria. It is more energy
efficient and cleaner processing.